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Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase
Abstract
Cell division, cell motility and the formation and maintenance of specialized structures in differentiated cells depend directly on the regulated dynamics of the actin cytoskeleton. To understand the mechanisms of these basic cellular processes, the signalling pathways that link external signals to the regulation of the actin cytoskeleton need to be characterized. Here we identify a pathway for the regulation of cofilin, a ubiquitous actin-binding protein that is essential for effective depolymerization of actin filaments. LIM-kinase 1, also known as KIZ, is a protein kinase with two amino-terminal LIM motifs that induces stabilization of F-actin structures in transfected cells. Dominant-negative LIM-kinasel inhibits the accumulation of the F-actin. Phosphorylation experiments in vivo and in vitro provide evidence that cofilin is a physiological substrate of LIM-kinase 1. Phosphorylation by LIM-kinase 1 inactivates cofilin, leading to accumulation of actin filaments. Constitutively active Rac augmented cofilin phosphorylation and LIM-kinase 1 autophosphorylation whereas phorbol ester inhibited these processes. Our results define a mechanism for the regulation of cofilin and hence of actin dynamics in vivo. By modulating the stability of actin cytoskeletal structures, this pathway should play a central role in regulating cell motility and morphogenesis.
... In this context, routine polymerization of G-actin into F-actin filaments along with the extrusion of pseudopodia (depolymerization of F-actin to G-actin) has been associated with cell motility [31]. This intricate process may be mediated by certain F-actin-linking proteins such as Cofilin1 [32][33][34], Gelsolin, and Profilin [35]. Cofilin1 is the primary regulatory protein for F-actin dynamics, orchestrating the polymerization/depolymerization processes [36]. ...
... In its unphosphorylated state (active form), Cofilin1 promotes the depolymerization of F-actin into G-actin. Phosphorylation by LIM domain kinase 1 (LIMK1) deactivates Cofilin1 [33,[37][38][39][40][41][42], inhibiting its depolymerization of F-actin. The severing mechanism is rapid, occurring at both the barbed and pointed ends, resulting in filament fragmentation and facilitating nucleation as required [39]. ...
... We observed significant alterations in the mechanical properties of HEK + P cells, whereas no discernible differences were observed between HEK and HEK-P cells. Moreover, the presence of decreased stress fiber length in HEK-P cells could be related to the basic functional remodeling of cytoskeletal components within the cells, which may involve other F-actin-linking proteins [32][33][34]. ...
... respectively. Once activated, LIM kinases inactivate the ADF/cofilin proteins by phosphorylating their Ser3, rendering them unable to sever actin polymers and inducing the accumulation of filamentous actin (F-actin), actin stress fibre formation, and impacting cytoskeleton dynamics [7,9,10]. Independently of their activity on the actin cytoskeleton, it has been shown that LIM kinases play a role in microtubule turnover by favouring free tubulin formation [11,12], but the molecular implication of the kinases in this process remains to be elucidated. ...
... Their most extensively described substrates are members of the actin depolymerising factor/cofilin (ADF/cofilin) family: cofilin1 (nonmuscle cofilin, or n-cofilin), cofilin2 (muscle cofilin, or m-cofilin), and destrin (also known as actin depolymerising factor, or ADF), usually regrouped under the term cofilin. Cofilin was discovered as the first substrate of LIMK1 and LIMK2 in 1998 [9,10] and 1999 [7], The structural aspects of LIMK regulation and pharmacology are more extensively described in the review of Chatterjee et al., which belongs to the Special Issue LIM Kinases: From Molecular to Pathological Features [23]. ...
... Cofilin was the first substrate of LIMK that was identified [9,10]. The other substrates of LIMKs have been described since then ( Figure 4). ...
LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) are serine/threonine and tyrosine kinases and the only two members of the LIM kinase family. They play a crucial role in the regulation of cytoskeleton dynamics by controlling actin filaments and microtubule turnover, especially through the phosphorylation of cofilin, an actin depolymerising factor. Thus, they are involved in many biological processes, such as cell cycle, cell migration, and neuronal differentiation. Consequently, they are also part of numerous pathological mechanisms, especially in cancer, where their involvement has been reported for a few years and has led to the development of a wide range of inhibitors. LIMK1 and LIMK2 are known to be part of the Rho family GTPase signal transduction pathways, but many more partners have been discovered over the decades, and both LIMKs are suspected to be part of an extended and various range of regulation pathways. In this review, we propose to consider the different molecular mechanisms involving LIM kinases and their associated signalling pathways, and to offer a better understanding of their variety of actions within the physiology and physiopathology of the cell.
... Previous studies [41] have found that cofilin1/2 phosphorylation is a prerequisite for cofilin/F-actin interaction and actin depolymerization. Therefore, we asked whether DNA-PKcs-dependent cofilin2 phosphorylation triggers F-actin depolymerization. ...
... Compared with its unphosphorylated form, p-cofilin2 Thr25 has increased affinity to F-actin. Similar to cofilin1 phosphorylation [41], p-cofilin2 Thr25 causes F-actin depolymerization into G-actin to promote actin cytoskeleton degradation. These findings identified cofilin2 phosphorylation and F-actin disassembly as novel downstream effectors of DNA-PKcs activation in endotoxemiainduced endothelial injury, thus elucidating the molecular basis by which LPS promotes endothelial cytoskeleton degradation. ...
The presence of endotoxemia is strongly linked to the development of endothelial dysfunction and disruption of myocardial microvascular reactivity. These factors play a crucial role in the progression of endotoxemic cardiomyopathy. Sepsis-related multiorgan damage involves the participation of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). However, whether DNA-PKcs contributes to endothelial dysfunction and myocardial microvascular dysfunction during endotoxemia remains unclear. Hence, we conducted experiments in mice subjected to lipopolysaccharide (LPS)-induced endotoxemic cardiomyopathy, as well as assays in primary mouse cardiac microvascular endothelial cells. Results showed that endothelial-cell-specific DNA-PKcs ablation markedly attenuated DNA damage, sustained microvessel perfusion, improved endothelial barrier function, inhibited capillary inflammation, restored endothelium-dependent vasodilation, and improved heart function under endotoxemic conditions. Furthermore, we show that upon LPS stress, DNA-PKcs recognizes a TQ motif in cofilin2 and consequently induces its phosphorylation at Thr²⁵. Phosphorylated cofilin2 shows increased affinity for F-actin and promotes F-actin depolymerization, resulting into disruption of the endothelial barrier integrity, microvascular inflammation, and defective eNOS-dependent vasodilation. Accordingly, cofilin2-knockin mice expressing a phospho-defective (T25A) cofilin2 mutant protein showed improved endothelial integrity and myocardial microvascular function upon induction of endotoxemic cardiomyopathy. These findings highlight a novel mechanism whereby DNA-PKcs mediates cofilin2Thr25 phosphorylation and subsequent F-actin depolymerization to contribute to endotoxemia-related cardiac microvascular dysfunction.
... Multiple Rho-effector protein kinases, including the ROCK, PAK and MRCK groups phosphorylate and activate the LIMKs 8-10 . Importantly, the LIMKs (and the TESKs which are related in their catalytic domains 11 ) appear unique in their ability to phosphorylate residue serine-3 of the actin depolymerizing factor, cofilin, which results in its inactivation 8,[11][12][13][14][15][16][17] . This unique high-fidelity kinase-substrate recognition therefore provides an essential link between Rho GTPase activation and suppression of actin severing, placing the LIMKs as central nodes in the many cellular processes for which elongation of actin filaments are required. ...
... Based on superposition of over 40 AlphaFold models of full-length LIMK1 and LIMK2 in different species, we found that the βA-βF-βD surface is almost completely surface exposed, with a small portion of the surface consistently found to interact with the adjacent LIM2 domain (residue L152 and residues of βA which makes an anti-parallel β-sheet interaction with the LIM2 domain). In these models, residue E206 (LIMK2) / E225 (LIMK1) is always surface exposed further supporting our finding that the βA-βF-βD surface has the potential to regulate the kinase, and also allows for an extended surface consistent with previous literature suggesting a role for the LIM domains in autoregulation 8,10,13,18,19 . The changes in kinase activity that we observe suggest that disruption of the surface that potentially mediates autoregulatory interactions between the PDZ domain and the kinase domain allows LIMK to reach a more "open" conformation. ...
LIM domain kinases (LIMK) are important regulators of actin cytoskeletal remodeling. These protein kinases phosphorylate the actin depolymerizing factor cofilin to suppress filament severing, and are key nodes between Rho GTPase cascades and actin. The two mammalian LIMKs, LIMK1 and LIMK2, contain consecutive LIM domains and a PDZ domain upstream of the C-terminal kinase domain. The roles of the N-terminal regions are not fully understood, and the function of the PDZ domain remains elusive. Here, we determine the 2.0 Å crystal structure of the PDZ domain of LIMK2 and reveal features not previously observed in PDZ domains including a core-facing arginine residue located at the second position of the ‘x-Φ-G-Φ’ motif, and that the expected peptide binding cleft is shallow and poorly conserved. We find a distal extended surface to be highly conserved, and when LIMK1 was ectopically expressed in yeast we find targeted mutagenesis of this surface decreases growth, implying increased LIMK activity. PDZ domain LIMK1 mutants expressed in yeast are hyperphosphorylated and show elevated activity in vitro. This surface in both LIMK1 and LIMK2 is critical for autoregulation independent of activation loop phosphorylation. Overall, our study demonstrates the functional importance of the PDZ domain to autoregulation of LIMKs.
... In addition, their immediate downstream signalling is inherently local as direct downstream effectors such as mDia1, Rock and Wasp and Wave require persistent binding for activation [27][28][29]. Furthermore, the activation of downstream actin effectors cofilin, formin and Arp2/3 was shown to be linked to f-actin binding [28,[30][31][32]. Correspondingly, association of these proteins to cortical f-actin is a measure of their activity at the actin cortex. ...
... In the context of cancer, cofilin activity at the cortex has been suggested to be a main factor in f-actin turnover thus playing a key role in cancer cell migration and invasion [40]. Cofilin becomes deactivated through phosphorylation mediated by Lim kinases [40] and phosphorylated cofilin was shown to not interact with f-actin [30]. Correspondingly, cortex-bound cofilin can be interpreted as active cofilin. ...
Epithelial-mesenchymal transition (EMT) is a key cellular transformation for many physiological and pathological processes ranging from cancer over wound healing to embryogenesis. Changes in cell migration, cell morphology and cellular contractility were identified as hallmarks of EMT. These cellular properties are known to be tightly regulated by the actin cytoskeleton. EMT-induced changes of actin-cytoskeletal regulation were demonstrated by previous reports of changes of actin cortex mechanics in conjunction with modifications of cortex-associated f-actin and myosin. However, at the current state, the changes of upstream actomyosin signalling that lead to corresponding mechanical and compositional changes of the cortex are not well understood. In this work, we show in breast epithelial cancer cells MCF-7 that EMT results in characteristic changes of the cortical association of Rho-GTPases Rac1, RhoA and RhoC and downstream actin regulators cofilin, mDia1 and Arp2/3. In the light of our findings, we propose that EMT-induced changes in cortical mechanics rely on two hitherto unappreciated signalling paths - i) an interaction between Rac1 and RhoC and ii) an inhibitory effect of Arp2/3 activity on cortical association of myosin II.
... Remarkably, Rac1 activity inhibits the actin-depolymerizing activity of Cofilin by the sequential activation of p21-activated kinase (PAK)/LIM kinase (LIMK), which modulates activity-dependent actin cytoskeleton remodeling and synaptic plasticity, and in turn, it is responsible for intrinsic active forgetting [4, [93][94][95][96]. For instance, Cofilin hyperactivation increases memory retention at 3 h similar to that observed with Rac1 inhibition [16]. ...
... This catalysis process requires the substrate determinants within LIMK's activation loop (a highly basic 11-amino-acid insertion from arginine 495 to arginine 506) and structural determinants in both the nitrogen-terminal regulatory (including the auto-inhibitory regulatory domain, amino acids 83-149, and the p21-binding domain, amino acids 67-86) and the carboxy-terminal catalytic domains (amino acids 232-544) of Pak [95]. Activated LIMK then phosphorylates the actin-regulatory protein cofilin at an N-terminal serine residue 3, thereby inhibiting its F-actin depolymerization activity and resulting in the accumulation of actin filaments [94,122]. The specific inhibition of Pak activity in its auto-inhibitory domain blocks LIMK-mediated actin cytoskeletal changes, and the inactivation of LIMK disturbs Rac1-dependent and Pak-dependent actin dynamics [95] (see Figure 3). ...
Animals are required to handle daily massive amounts of information in an ever-changing environment, and the resulting memories and experiences determine their survival and development, which is critical for adaptive evolution. However, intrinsic forgetting, which actively deletes irrelevant information, is equally important for memory acquisition and consolidation. Recently, it has been shown that Rac1 activity plays a key role in intrinsic forgetting, maintaining the balance of the brain's memory management system in a controlled manner. In addition, dysfunctions of Rac1-dependent intrinsic forgetting may contribute to memory deficits in neurological and neurodegenerative diseases. Here, these new findings will provide insights into the neurobiology of memory and forgetting, pathological mechanisms and potential therapies for brain disorders that alter intrinsic forgetting mechanisms.
... Y27632 (30 µM) did not suppress but caused rather an increase in transmittance with an increased ratio of large aggregates and decreased ratio of small aggregates stimulated by the combination ( Figure 6A). We confirmed that Y27632 reduced the phosphorylation of cofilin, a substrate of Rho-kinase [22] upregulated in platelets stimulated by the combination of ristocetin and CXCL12 at low doses ( Figure 6B). PRP was stimulated by 30 ng/mL of CXCL12 (A), 0.9 mg/mL of ristocetin (B) or their combination (C) for 60 s. ...
... Y27632 (30 µM) did not suppress but caused rather an increase in transmittance with an increased ratio of large aggregates and decreased ratio of small aggregates stimulated by the combination ( Figure 6A). We confirmed that Y27632 reduced the phosphorylation of cofilin, a substrate of Rho-kinase [22] The lysed platelets were subjected to Western blot analysis using antibodies against phospho-specific cofilin, total cofilin or GAPDH. ...
CXCL12, belonging to the CXC chemokine family, is a weak agonist of platelet aggregation. We previously reported that the combination of CXCL12 and collagen at low doses synergistically activates platelets via not CXCR7 but CXCR4, a specific receptor for CXCL12 on the plasma membrane. Recently, we reported that not Rho/Rho kinase, but Rac is involved in the platelet aggregation induced by this combination. Ristocetin is an activator of the von Willebrand factor that interacts with glycoprotein (GP) Ib/IX/V, which generates thromboxane A2 via phospholipase A2 activation, resulting in the release of the soluble CD40 ligand (sCD40L) from human platelets. In the present study, we investigated the effects of a combination of ristocetin and CXCL12 at low doses on human platelet activation and its underlying mechanisms. Simultaneous stimulation with ristocetin and CXCL12 at subthreshold doses synergistically induce platelet aggregation. A monoclonal antibody against not CXCR7 but CXCR4 suppressed platelet aggregation induced by the combination of ristocetin and CXCL12 at low doses. This combination induces a transient increase in the levels of both GTP-binding Rho and Rac, followed by an increase in phosphorylated cofilin. The ristocetin and CXCL12-induced platelet aggregation as well as the sCD40L release were remarkably enhanced by Y27362, an inhibitor of Rho-kinase, but reduced by NSC23766, an inhibitor of the Rac-guanine nucleotide exchange factor interaction. These results strongly suggest that the combination of ristocetin and CXCL12 at low doses synergistically induces human platelet activation via Rac and that this activation is negatively regulated by the simultaneous activation of Rho/Rho-kinase.
... Additionally, ROCK activates LIM kinase [38] , which in turn phosphorylates cofilin, thereby inactivating it [45,46] . Therefore, to interrogate our hypothesis further, p-MLC (Ser20) and p-cofilin (Ser3) were used as readouts for ROCK activity on fibronectin and laminin. ...
... These findings suggest that there may be myosin II-independent features of ROCK's contribution to the laminin phenotype. ROCK is known to activate specific formins [42] , LIM kinase (which inhibits cofilin) [38,45,46] , and ERM-domain containing proteins [40,41] . Of particular interest, the ERM protein ezrin links actin to the plasma membrane and must be removed before Arp2/3 complex can be recruited to form a lamellipodial protrusion [56] . ...
Macrophages are indispensable for proper immune surveillance and inflammatory regulation. They also exhibit dramatic phenotypic plasticity and are highly responsive to their local microenvironment, which includes the extracellular matrix (ECM). The present work demonstrates that two fibrous ECM glycoproteins, fibronectin (FN) and laminin (LAM), elicit distinct morphological and migratory responses to macrophages in 2D environments. Laminin 111 inhibits macrophage cell spreading, but drives them to migrate rapidly and less persistently compared to cells on fibronectin. Differential integrin engagement and ROCK/myosin II organization helps explain why macrophages alter their morphology and migration character on these two ECM components. The present study also demonstrates that laminin 111 exerts a suppressive effect toward fibronectin, as macrophages plated on a LAM/FN mixture adopt a morphology and migratory character almost identical to LAM alone. This suggests that distinct responses can be initiated in the cell downstream of receptor-ECM engagement, and that one component of the microenvironment may affect the ability to sense another. Overall, macrophages appear intrinsically poised to rapidly switch between distinct migratory modes based on their ECM environments. The role of ECM composition in dictating motile and inflammatory responses in 3D and in vivo contexts warrants further study.
... Collectively, these data suggested that in two different organs, liver and aorta, the expression of DIAPH1 was associated with the phosphorylation state of Cofilin 1. As roles for phosphorylation of Cofilin have been ascribed to the regulation of actin polymerization [27][28][29] , these findings pointed us to new directions for probing DIAPH1-dependent mechanisms in the regulation of the nuclear translocation of the transcription factors under study. a Representative Western Blots for the detection of DIAPH1, phosphorylated (Ser3) Cofilin and total Cofilin, ROCK1, phosphorylated (Thr508) LIMK1 and total LIMK1, phosphorylated (Ser978) SSH1 and total SSH1 on total liver lysates isolated from the indicated mice. ...
... Cofilin, an actin binding molecule, plays key roles in actin cytoskeleton organization; yet, its biology is complex. Cofilin may bind to F-actin and G-actin and has been implicated in both elongation vs. severing of actin filaments [27][28][29] . In general, the phosphorylation of Cofilin is linked to its inactivation 35 . ...
Atherosclerosis evolves through dysregulated lipid metabolism interwoven with exaggerated inflammation. Previous work implicating the receptor for advanced glycation end products (RAGE) in atherosclerosis prompted us to explore if Diaphanous 1 (DIAPH1), which binds to the RAGE cytoplasmic domain and is important for RAGE signaling, contributes to these processes. We intercrossed atherosclerosis-prone Ldlr−/− mice with mice devoid of Diaph1 and fed them Western diet for 16 weeks. Compared to male Ldlr−/− mice, male Ldlr−/−Diaph1−/− mice displayed significantly less atherosclerosis, in parallel with lower plasma concentrations of cholesterol and triglycerides. Female Ldlr−/−Diaph1−/− mice displayed significantly less atherosclerosis compared to Ldlr−/− mice and demonstrated lower plasma concentrations of cholesterol, but not plasma triglycerides. Deletion of Diaph1 attenuated expression of genes regulating hepatic lipid metabolism, Acaca, Acacb, Gpat2, Lpin1, Lpin2 and Fasn, without effect on mRNA expression of upstream transcription factors Srebf1, Srebf2 or Mxlipl in male mice. We traced DIAPH1-dependent mechanisms to nuclear translocation of SREBP1 in a manner independent of carbohydrate- or insulin-regulated cues but, at least in part, through the actin cytoskeleton. This work unveils new regulators of atherosclerosis and lipid metabolism through DIAPH1.
... Moreover, ADF/cofilin is inactivated by phosphorylation at serine-3, which prevents its association with actin (Arber et al., 1998). Many viruses have been reported to take advantage of insect cofilin to regulate the dynamics of F-actin during infection to promote virion entry into cells (Nie et al., 2021). ...
... We then ex- and extending the AAP after 24 h led to slightly higher transmission efficiencies. In general, cofilin/ADF activity is regulated by phosphorylation, which prevents the association of cofilin with actin (Arber et al., 1998), whereas dephosphorylation by phosphatases activates cofilin (Ambach et al., 2000). Recently, swine fever virus was reported to regulate the activity of cofilin to infect porcine kidney (PK-15) cells by inducing its phosphorylation and dephosphorylation through the epidermal growth factor receptor-phosphatidylinositol 3-kinase-mitogen-activated protein kinase-Ras homologue family member A-Ras-related C3 botulinum toxin substrate 1-cell division control protein 42 homologue (EGFR-PI3K-MAPK-RhoA-Rac1-Cdc42) signalling pathway (Cheng et al., 2021). ...
Hemipteran insects that transmit plant viruses in a persistent circulative manner acquire, retain and transmit viruses for their entire life. The mechanism enabling this persistence has remained unclear for many years. Here, we determined how wheat dwarf virus (WDV) persists in its leafhopper vector Psammotettix alienus. We found that WDV caused the up‐regulation of actin‐depolymerizing factor (ADF) at the mRNA and protein levels in the midgut cells of leafhoppers after experiencing a WDV acquisition access period (AAP) of 6, 12 or 24 h. Experimental inhibition of F‐actin depolymerization by jasplakinolide and dsRNA injection led to lower virus accumulation levels and transmission efficiencies, suggesting that depolymerization of F‐actin regulated by ADF is essential for WDV invasion of midgut cells. Exogenous viral capsid protein (CP) inhibited ADF depolymerization of actin filaments in vitro and in Spodoptera frugiperda 9 (Sf9) cells because the CP competed with actin to bind ADF and then blocked actin filament disassembly. Interestingly, virions colocalized with ADF after a 24‐h AAP, just as actin polymerization occurred, indicating that the binding of CP with ADF affects the ability of ADF to depolymerize F‐actin, inhibiting WDV entry. Similarly, the luteovirus barley yellow dwarf virus also induced F‐actin depolymerization and then polymerization in the gut cells of its vector Schizaphis graminum. Thus, F‐actin dynamics are altered by nonpropagative viruses in midgut cells to enable virus persistence in vector insects. Biphasic dynamics of F‐actin mediated by the ADF–CP interaction may play an important role in controlling virus entry and the virus accumulation level required for persistent transmission while maintaining the health of the vector.
... 31 When phosphorylated by Lim kinase, phosphorylated CFL1 (pCFL1) no longer binds F-actin, purportedly preventing the production of branched or G-actin. 32 Understanding actin dynamics is essential to determine how rod OS discs form and are maintained, and how constituent proteins are trafficked between the rod OS and inner segment (IS). ...
NUDC (nuclear distribution protein C) is a mitotic protein involved in nuclear migration and cytokinesis across species. Considered a cytoplasmic dynein (henceforth dynein) cofactor, NUDC was shown to associate with the dynein motor complex during neuronal migration. NUDC is also expressed in postmitotic vertebrate rod photoreceptors where its function is unknown. Here, we examined the role of NUDC in postmitotic rod photoreceptors by studying the consequences of a conditional NUDC knockout in mouse rods (rNudC−/−). Loss of NUDC in rods led to complete photoreceptor cell death at 6 weeks of age. By 3 weeks of age, rNudC−/− function was diminished, and rhodopsin and mitochondria were mislocalized, consistent with dynein inhibition. Levels of outer segment proteins were reduced, but LIS1 (lissencephaly protein 1), a well‐characterized dynein cofactor, was unaffected. Transmission electron microscopy revealed ultrastructural defects within the rods of rNudC−/− by 3 weeks of age. We investigated whether NUDC interacts with the actin modulator cofilin 1 (CFL1) and found that in rods, CFL1 is localized in close proximity to NUDC. In addition to its potential role in dynein trafficking within rods, loss of NUDC also resulted in increased levels of phosphorylated CFL1 (pCFL1), which would purportedly prevent depolymerization of actin. The absence of NUDC also induced an inflammatory response in Müller glia and microglia across the neural retina by 3 weeks of age. Taken together, our data illustrate the critical role of NUDC in actin cytoskeletal maintenance and dynein‐mediated protein trafficking in a postmitotic rod photoreceptor.
... We observed that both wild type and active, non-phosphorylatable Cofilin-1 mutant (Cofilin-1-S3A-GFP), exhibited a transient recruitment at the secondary ingression ( Fig. 1c arrowhead and Supplementary Fig. 1d). In contrast, the phosphomimetic mutant (Cofilin-1-S3E-GFP), which is inactive and had a lower affinity for actin [44][45][46][47] , did not localize to ICBs ( Supplementary Fig. 1e). This result suggested that Cofilin-1 recruitment at the secondary ingression was dependent on the binding of activated Cofilin-1 to F-actin. ...
Cell division is completed by the abscission of the intercellular bridge connecting the daughter cells. Abscission requires the polymerization of an ESCRT-III cone close to the midbody to both recruit the microtubule severing enzyme spastin and scission the plasma membrane. Here, we found that the microtubule and the membrane cuts are two separate events that are regulated differently. Using HeLa cells, we uncovered that the F-actin disassembling protein Cofilin-1 controls the disappearance of a transient pool of branched F-actin which is precisely assembled at the tip of the ESCRT-III cone shortly before the microtubule cut. Functionally, Cofilin-1 and Arp2/3-mediated branched F-actin favor abscission by promoting local severing of the microtubules but do not participate later in the membrane scission event. Mechanistically, we propose that branched F-actin functions as a physical barrier that limits ESCRT-III cone elongation and thereby favors stable spastin recruitment. Our work thus reveals that F-actin controls the timely and local disassembly of microtubules required for cytokinetic abscission.
... ROCKs are central regulators of the actin cytoskeleton downstream of the small GTPase RhoA. [1][2][3][4][5][6][7][8] The two ROCK isoforms, ROCK1 and ROCK2, are highly homologous with an overall amino acid sequence identity of 65%. [1][2][3] We previously found that ROCK1 is a key molecule in mediating apoptotic signaling in cardiomyocytes under pressure overload and in genetically induced pathological cardiac hypertrophy. ...
In this study, we investigated the roles of ROCK1 in regulating structural and functional features of caveolae located at the cell membrane of cardiomyocytes, adipocytes, and mouse embryonic fibroblasts (MEFs) as well as related physiopathological effects. Caveolae are small bulb‐shaped cell membrane invaginations, and their roles have been associated with disease conditions. One of the unique features of caveolae is that they are physically linked to the actin cytoskeleton that is well known to be regulated by RhoA/ROCKs pathway. In cardiomyocytes, we observed that ROCK1 deficiency is coincident with an increased caveolar density, clusters, and caveolar proteins including caveolin‐1 and ‐3. In the mouse cardiomyopathy model with transgenic overexpressing Gαq in myocardium, we demonstrated the reduced caveolar density at cell membrane and reduced caveolar protein contents. Interestingly, coexisting ROCK1 deficiency in cardiomyocytes can rescue these defects and preserve caveolar compartmentalization of β‐adrenergic signaling molecules including β1‐adrenergic receptor and type V/VI adenylyl cyclase. In cardiomyocytes and adipocytes, we detected that ROCK1 deficiency increased insulin signaling with increased insulin receptor activation in caveolae. In MEFs, we identified that ROCK1 deficiency increased caveolar and total levels of caveolin‐1 and cell membrane repair ability after mechanical or chemical disruptions. Together, these results demonstrate that ROCK1 can regulate caveolae plasticity and multiple functions including compartmentalization of signaling molecules and cell membrane repair following membrane disruption by mechanical force and oxidative damage. These findings provide possible molecular insights into the beneficial effects of ROCK1 deletion/inhibition in cardiomyocytes, adipocytes, and MEFs under certain diseased conditions.
... Since phosphorylation of Cofilin leads to autoinhibition of its activity 26 , we examined the relevance of the phosphorylation of Cofilin and TNF-induced apoptosis by pretreating cells with BMS-3, a LIMK inhibitor, to restrain the phosphorylation of Cofilin. We found a dosedependent decrease of PIcaspase-3/7 active HCT-116 cells pretreated with BMS-3 compared to controls (Fig. 7d), which indicated that phosphorylation of Cofilin promoted TNF-induced apoptosis. ...
TNF acts as one pathogenic driver for inducing intestinal epithelial cell (IEC) death and substantial intestinal inflammation. How the IEC death is regulated to physiologically prevent intestinal inflammation needs further investigation. Here, we report that EF-hand domain-containing protein D2 (EFHD2), highly expressed in normal intestine tissues but decreased in intestinal biopsy samples of ulcerative colitis patients, protects intestinal epithelium from TNF-induced IEC apoptosis. EFHD2 inhibits TNF-induced apoptosis in primary IECs and intestinal organoids (enteroids). Mice deficient of Efhd2 in IECs exhibit excessive IEC death and exacerbated experimental colitis. Mechanistically, EFHD2 interacts with Cofilin and suppresses Cofilin phosphorylation, thus blocking TNF receptor I (TNFR1) internalization to inhibit IEC apoptosis and consequently protecting intestine from inflammation. Our findings deepen the understanding of EFHD2 as the key regulator of membrane receptor trafficking, providing insight into death receptor signals and autoinflammatory diseases.
... One of the terminal effectors of Rho GTPases signaling cascades is the F-actin severing protein, cofilin (CFN), a ubiquitous actin-binding protein that is responsible for the effective depolymerization of actin filaments. Rho GTPases can activate the LIM Domain Kinase 1 (LIMK1), which in turn increases the CFN phosphorylation of serine residue (S) at position 3, leading to inactivation of actin-depolymerizing activity of CFN and polymerization of F-actin (41)(42)(43). To identify the effect of CFN on the NDV entry of cells, the phosphoryla tion status of CFN was examined during the NDV entry process. ...
As an intrinsic cellular mechanism responsible for the internalization of extracellular ligands and membrane components, caveolae-mediated endocytosis (CavME) is also exploited by certain pathogens for endocytic entry [e.g., Newcastle disease virus (NDV) of paramyxovirus]. However, the molecular mechanisms of NDV-induced CavME remain poorly understood. Herein, we demonstrate that sialic acid-containing gangliosides, rather than glycoproteins, were utilized by NDV as receptors to initiate the endocytic entry of NDV into HD11 cells. The binding of NDV to gangliosides induced the activation of a non-receptor tyrosine kinase, Src, leading to the phosphorylation of caveolin-1 (Cav1) and dynamin-2 (Dyn2), which contributed to the endocytic entry of NDV. Moreover, an inoculation of cells with NDV-induced actin cytoskeletal rearrangement through Src to facilitate NDV entry via endocytosis and direct fusion with the plasma membrane. Subsequently, unique members of the Rho GTPases family, RhoA and Cdc42, were activated by NDV in a Src-dependent manner. Further analyses revealed that RhoA and Cdc42 regulated the activities of specific effectors, cofilin and myosin regulatory light chain 2, responsible for actin cytoskeleton rearrangement, through diverse intracellular signaling cascades. Taken together, our results suggest that an inoculation of NDV-induced Src-mediated cellular activation by binding to ganglioside receptors. This process orchestrated NDV endocytic entry by modulating the activities of caveolae-associated Cav1 and Dyn2, as well as specific Rho GTPases and downstream effectors.
IMPORTANCE
In general, it is known that the paramyxovirus gains access to host cells through direct penetration at the plasma membrane; however, emerging evidence suggests more complex entry mechanisms for paramyxoviruses. The endocytic entry of Newcastle disease virus (NDV), a representative member of the paramyxovirus family, into multiple types of cells has been recently reported. Herein, we demonstrate the binding of NDV to induce ganglioside-activated Src signaling, which is responsible for the endocytic entry of NDV through caveolae-mediated endocytosis. This process involved Src-dependent activation of the caveolae-associated Cav1 and Dyn2, as well as specific Rho GTPase and downstream effectors, thereby orchestrating the endocytic entry process of NDV. Our findings uncover a novel molecular mechanism of endocytic entry of NDV into host cells and provide novel insight into paramyxovirus mechanisms of entry.
... The actin cytoskeleton, crucial for epithelial junction remodeling and integrity, is regulated by actin-binding proteins (ABPs) 64 and phosphorylation 65 . For example, phosphorylation of TJ, AJ and desmosomal proteins [66][67][68][69] and phosphorylation of ABPs, such as cofilin (S3p) 70 , fimbrin (T103p) 71 and drebrin (S647p) 72 , can modulate apical junction complex assembly/disassembly. Intriguingly, we observed continuously decreasing changes in phosphorylation associated with actin filament-based processes, signaling by Rho GTPases and cell-cell junction assembly with age in the distal colon (Fig. 2f), suggesting that changes in phosphorylation may contribute more to cell-cell junction protein homeostasis in the distal colon. The mechanisms of phosphorylation dysregulation in cell-cell adhesion remain elusive and may be driven by age-related influences, such as shifts in cell-to-cell communication 73 , variations in kinase activities and other modulators, such as magnesium ions 74 . ...
The incidence of intestinal diseases increases with age, yet the mechanisms governing gut aging and its link to diseases, such as colorectal cancer (CRC), remain elusive. In this study, while considering age, sex and proximal–distal variations, we used a multi-omics approach in non-human primates (Macaca fascicularis) to shed light on the heterogeneity of intestinal aging and identify potential regulators of gut aging. We explored the roles of several regulators, including those from tryptophan metabolism, in intestinal function and lifespan in Caenorhabditis elegans. Suggesting conservation of region specificity, tryptophan metabolism via the kynurenine and serotonin (5-HT) pathways varied between the proximal and distal colon, and, using a mouse colitis model, we observed that distal colitis was more sensitive to 5-HT treatment. Additionally, using proteomics analysis of human CRC samples, we identified links between gut aging and CRC, with high HPX levels predicting poor prognosis in older patients with CRC. Together, this work provides potential targets for preventing gut aging and associated diseases.
... LIM kinases, specifically LIMK1 and LIMK2, represent serine/threonine kinases characterized by the presence of two zinc-finger motifs within their N-terminal regulatory domains [22]. Activated LIMK phosphorylates cofilin at the Ser3 residue, thereby inhibiting its depolymerization activity [24]. The effects of profilin and cofilin were not obvious, but P-LIMK1Thr508/P-LIMK2Thr505 and P-cofilin Ser3 decreased ( Figure 6B). ...
Background:
Glioma is one of the most aggressive malignant brain tumors and is characterized by invasive growth and poor prognosis. TBC1D1, a member of the TBC family, is associated with the development of various malignancies. However, the role of TBC1D1 in glioma-genesis remains unclear.
Methods:
The effect of TBC1D1 on the prognosis of glioma patients and related influencing factors were analyzed in the Chinese Glioma Genome Atlas (CGGA) and The Cancer Genome Atlas (TCGA) databases. Expression of TBC1D1 in glioma cell lines was detected by western blotting. Cell viability and proliferation were measured by EdU and Colony formation assays, respectively. Transwell and wound healing assays were performed to determine the cell migration and invasion capacities. Immunofluorescence was used to observe actin morphology in the cytoskeleton.
Results:
We discovered that high TBC1D1 expression in gliomas led to poor prognosis. Downregulation of TBC1D1 in glioma cells significantly inhibited multiple important functions, such as proliferation, migration, and invasion. We further demonstrated that the tumor-inhibitory effect of TBC1D1 might occur through the P-LIMK/cofilin pathway, destroying the cytoskeletal structure and affecting the depolymerization of F-actin, thereby inhibiting glioma migration.
Conclusion:
TBC1D1 affects the balance and integrity of the actin cytoskeleton via cofilin, thereby altering the morphology and aggressiveness of glioma cells. This study provides a new perspective on its role in tumorigenesis, thereby identifying a potential therapeutic target for the treatment of gliomas.
... We observed that both wild type and active, nonphosphorylable Cofilin-1 mutant (Cofilin-1-S3A-GFP), exhibited a transient recruitment at the secondary ingression ( Fig. 1c arrowhead and Supplementary Fig. 1d). In contrast, the phospho-mimetic mutant (Cofilin-1-S3E-GFP), which is inactive and had a lower affinity for actin [44][45][46][47], did not localize to ICBs ( Supplementary Fig. 1e). This result suggested that Cofilin-1 recruitment at the secondary ingression was dependent on the binding of activated Cofilin-1 to F-actin. ...
Cell division is completed by the abscission of the intercellular bridge connecting the daughter cells. Abscission requires the polymerization of an ESCRT-III cone close to the midbody to both recruit the microtubule severing enzyme spastin and scission the plasma membrane. Contrary to the common assumption, we found that the microtubule and the membrane cuts are two separate events and are regulated differently. We uncovered that the F-actin disassembling protein Cofilin-1 controls the disappearance of a transient pool of branched F-actin which is precisely assembled at the tip of the ESCRT-III cone just before the microtubule cut. Functionally, Cofilin-1 and Arp2/3-mediated branched F-actin favor abscission by promoting local severing of the microtubules but do not participate later in the membrane scission event. Mechanistically, branched F-actin functions as a physical barrier that limits ESCRT-III cone elongation and thereby favors stable spastin recruitment. Our work thus reveals that F-actin unexpectedly controls the timely and local disassembly of microtubules required for cytokinetic abscission.
... In addition to the generation of contractile forces by myosin-II, ROCK also controls actin filament stability and structural organization (Fig. 3). On the one hand, activation of LIM-kinase proteins (LIMK-1 and LIMK-2) by ROCKmediated phosphorylation leads to the inactivation of the actin-severing protein cofilin, which is critical to enable stress fiber formation [4,54,81]. Furthermore, ROCK also activates two important structural proteins at the cell cortex: ...
Cell contraction plays an important role in many physiological and pathophysiological processes. This includes functions in skeletal, heart, and smooth muscle cells, which lead to highly coordinated contractions of multicellular assemblies, and functions in non-muscle cells, which are often highly localized in subcellular regions and transient in time. While the regulatory processes that control cell contraction in muscle cells are well understood, much less is known about cell contraction in non-muscle cells. In this review, we focus on the mechanisms that control cell contraction in space and time in non-muscle cells, and how they can be investigated by light-based methods. The review particularly focusses on signal networks and cytoskeletal components that together control subcellular contraction patterns to perform functions on the level of cells and tissues, such as directional migration and multicellular rearrangements during development. Key features of light-based methods that enable highly local and fast perturbations are highlighted, and how experimental strategies can capitalize on these features to uncover causal relationships in the complex signal networks that control cell contraction.
... Small protein filamentins are a conserved family of actin-binding proteins that promote actin filament regeneration by cutting the original filament [36]. Filamentins are phosphorylated at Ser3 by LIMK or TESK, and thus their filamentous activity is inhibited [37][38][39]. Ezrin, radixin and moesin (ERM) proteins are involved in cell adhesion, membrane edge fluctuations, and microvilli formation [40,41]. Interacting cytosolic ERM proteins exist as monomers or dimers and can form intramolecular and intermolecular binding through their amino and carboxyl terminal domains [42]. ...
Background
Polypyrimidine tract binding protein 1 (PTBP1) has been found to play an important role in the occurrence and development of various tumors. At present, the role of PTBP1 in gastric cancer (GC) is still unknown and worthy of further investigation.
Methods
We used bioinformatics to analyze the expression of PTBP1 in patients with GC. Cell proliferation related experiments were used to detect cell proliferation after PTBP1 knockdown. Skeleton staining, scanning electron microscopy and transmission electron microscopy were used to observe the changes of actin skeleton. Proliferation and actin skeleton remodeling signaling pathways were detected by Western Blots. The relationship between PTBP1 and proliferation of gastric cancer cells was further detected by subcutaneous tumor transplantation. Finally, tissue microarray data from clinical samples were used to further explore the expression of PTBP1 in patients with gastric cancer and its correlation with prognosis.
Results
Through bioinformatics studies, we found that PTBP1 was highly expressed in GC patients and correlated with poor prognosis. Cell proliferation and cycle analysis showed that PTBP1 down-regulation could significantly inhibit cell proliferation. The results of cell proliferation detection related experiments showed that PTBP1 down-regulation could inhibit the division and proliferation of GC cells. Furthermore, changes in the morphology of the actin skeleton of cells showed that PTBP1 down-regulation inhibited actin skeletal remodeling in GC cells. Western Blots showed that PTBP1 could regulate proliferation and actin skeleton remodeling signaling pathways. In addition, we constructed PTBP1 Cas9-KO mouse model and performed xenograft assays to further confirm that down-regulation of PTBP1 could inhibit the proliferation of GC cells. Finally, tissue microarray was used to further verify the close correlation between PTBP1 and poor prognosis in patients with GC.
Conclusions
Our study demonstrates for the first time that PTBP1 may affect the proliferation of GC cells by regulating actin skeleton remodeling. In addition, PTBP1 is closely related to actin skeleton remodeling and proliferation signaling pathways. We suppose that PTBP1 might be a potential target for the treatment of GC.
Graphical abstract
... The enhanced activity of ROCK1 leads to the physical destruction of the nuclear membrane and the degradation of Lamin A [55]. LIMK1 inactivates cofilin, and caspase-activated LIMK1 may support the foaming of apoptotic membranes by inhibiting cofilin and promoting actin polymerization [60,61]. Then the nucleus invaginates moderately and blebs appear. ...
Apoptosis, a form of programmed cell death, is essential for growth and tissue homeostasis. Apoptotic bodies (ApoBDs) are a form of extracellular vesicles (EVs) released by dying cells in the last stage of apoptosis and were previously regarded as debris of dead cells. Recent studies unraveled that ApoBDs are not cell debris but the bioactive treasure left behind by the dying cells with an important role in intercellular communications related to human health and various diseases. Defective clearance of ApoBDs and infected-cells-derived ApoBDs are possible etiology of some diseases. Therefore, it is necessary to explore the function and mechanism of the action of ApoBDs in different physiological and pathological conditions. Recent advances in ApoBDs have elucidated the immunomodulatory, virus removal, vascular protection, tissue regenerative, and disease diagnostic potential of ApoBDs. Moreover, ApoBDs can be used as drug carriers enhancing drug stability, cellular uptake, and targeted therapy efficacy. These reports from the literature indicate that ApoBDs hold promising potential for diagnosis, prognosis, and treatment of various diseases, including cancer, systemic inflammatory diseases, cardiovascular diseases, and tissue regeneration. This review summarizes the recent advances in ApoBDs-related research and discusses the role of ApoBDs in health and diseases as well as the challenges and prospects of ApoBDs-based diagnostic and therapeutic applications.
Keywords: Apoptotic bodies, Diseases, Mesenchymal stem cells, Immunomodulation, Tissue regeneration
... Among these proteins, LIMK phosphorylates and thereby inactivates cofilin while dephosphorylation by SSH-1L activates cofilin. LIMK activity is stimulated by PAK, and SSH-1L activity via calciumcalcineurin [47][48][49][50]. In addition, cofilin is inhibited by binding to phosphatidylinositol-4,5bisphosphate (PIP 2 ). ...
Cellular actin dynamic is controlled by a plethora of actin binding proteins (ABPs), including actin nucleating, bundling, cross-linking, capping, and severing proteins. In this review, regulation of actin dynamics by ABPs will be introduced, and the role of the F-actin severing protein cofilin-1 and the F-actin bundling protein L-plastin in actin dynamics discussed in more detail. Since up-regulation of these proteins in different kinds of cancers is associated with malignant progression of cancer cells, we suggest the cryogenic electron microscopy (Cryo-EM) structure of F- actin with the respective ABP as template for in silico drug design to specifically disrupt the interaction of these ABPs with F-actin.
... Singla et al. suggested that macrophage growth factor initiation could activate cofilin and control actin dynamics by the SSH1 pathway [51]. LIM-kinase is an actin-binding protein that phosphorylates cofilin at Ser-3, reducing actin-binding and depolymerizing cofilin activities [52,53]. LIMK1/LIMK2 regulates cofilin phosphorylation activity; LIMK1 phosphorylates cofilin by ras-linked C3 botulinum substrate 1 (Rac1), while LIMK2 inactivates cofilin through Rho and cell division cycle 42 (CDC42) [54]. ...
Intracerebral hemorrhage (ICH) is a significant health concern associated with high mortality. Cofilin plays a crucial role in stress conditions, but its signaling following ICH in a longitudinal study is yet to be ascertained. In the present study, we examined the cofilin expression in human ICH autopsy brains. Then, the spatiotemporal cofilin signaling, microglia activation, and neurobehavioral outcomes were investigated in a mouse model of ICH. Human autopsy brain sections from ICH patients showed increased intracellular cofilin localization within microglia in the perihematomal area, possibly associated with microglial activation and morphological changes. Various cohorts of mice were subjected to intrastriatal collagenase injection and sacrificed at time points of 1, 3, 7, 14, 21, and 28 days. Mice suffered from severe neurobehavioral deficits after ICH, lasting for 7 days, followed by a gradual improvement. Mice suffered post-stroke cognitive impairment (PSCI) both acutely and in the chronic phase. Hematoma volume increased from day 1 to 3, whereas ventricle size increased from day 21 to 28. Cofilin protein expression increased in the ipsilateral striatum on days 1 and 3 and then decreased from days 7 to 28. An increase in activated microglia was observed around the hematoma on days 1 to 7, followed by a gradual reduction up to day 28. Around the hematoma, activated microglia showed morphological changes from ramified to amoeboid. mRNA levels of inflammatory [tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and interleukin-6 (IL-6) and anti-inflammatory markers [interleukin-10 (IL-10), transforming growth factor-β TGF-β, and arginase I (Arg1)] increased during the acute phase and decreased in the chronic phase. Blood cofilin levels increased on day 3 and matched the increase in chemokine levels. slingshot protein phosphatase 1 (SSH1) protein, which activates cofilin, was increased from day 1 to 7. These results suggest that microglial activation might be the sequel of cofilin overactivation following ICH, leading to widespread neuroinflammation and consequent PSCI.
... Actin-depolymerizing factor (ADF)/cofilin, a central regulator of actin dynamics, is a stimulus-responsive protein [8] which has been implicated in the regulation of actin dynamics in pollen tubes [9][10][11][12][13]. The activity of ADF/cofilin is subject to tight regulation by kinase-mediated phosphorylation [14][15][16][17], pH [18][19][20][21][22], and interactions with other binding partners, such as CAP1 [23,24], AIP1 [25][26][27], and Coronin [28], etc. Kinase-mediated phosphorylation represents a well-known inactivation mechanism of ADF/cofilin, with the phosphorylation occurring at Ser3 or Ser6 of ADFs/cofilins from different organisms [8,29]. This type of phosphoregulation has been reported for plant ADFs [30,31]. ...
As the stimulus-responsive mediator of actin dynamics, actin-depolymerizing factor (ADF)/cofilin is subject to tight regulation. It is well known that kinase-mediated phosphorylation inactivates ADF/cofilin. Here, however, we found that the activity of Arabidopsis ADF7 is enhanced by CDPK16-mediated phosphorylation. We found that CDPK16 interacts with ADF7 both in vitro and in vivo, and it enhances ADF7-mediated actin depolymerization and severing in vitro in a calcium-dependent manner. Accordingly, the rate of actin turnover is reduced in cdpk16 pollen and the amount of actin filaments increases significantly at the tip of cdpk16 pollen tubes. CDPK16 phosphorylates ADF7 at Serine128 both in vitro and in vivo, and the phospho-mimetic mutant ADF7S128D has enhanced actin-depolymerizing activity compared to ADF7. Strikingly, we found that failure in the phosphorylation of ADF7 at Ser128 impairs its function in promoting actin turnover in vivo, which suggests that this phospho-regulation mechanism is biologically significant. Thus, we reveal that CDPK16-mediated phosphorylation up-regulates ADF7 to promote actin turnover in pollen.
... Cofilin is an actin binding protein that severs f-actin at low concentrations and decorates it to form cofilactin at high concentrations (Nishida et al., 1984;Maciver et al., 1991;Andrianantoandro and Pollard, 2006). It is activated both by cellular stresses, such as energy depletion (Nishida et al., 1987;Meberg et al., 1998;Minamide et al., 2000), and by signaling pathways, particularly those found at the leading edge of the cell (Arber et al., 1998;Meberg et al., 1998;Yang et al., 1998;Chan et al., 2000;Zebda et al., 2000;Endo et al., 2003). Indeed, filaments resembling cofilactin have been observed in the cytoplasm of neuronal growth cones in two recent cryoET studies (Atherton et al., 2022;Hylton et al., 2022). ...
Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identity of these objects and what causes their accumulation has not been conclusively established. Here, we used cryogenic electron tomography (cryoET) of Drosophila S2 cell protrusions and found filaments inside the microtubule lumen, which resemble those reported recently in human HAP1 cells. The frequency of these filaments increased upon inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) with the small-molecule drug thapsigargin. Subtomogram averaging showed that the luminal filaments adopt a helical structure reminiscent of cofilin-bound actin (cofilactin). Consistent with this, cofilin was activated in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNAi knock-down of cofilin reduced the frequency of luminal filaments with cofilactin morphology. These results suggest that cofilin activation stimulates its accumulation on actin filaments inside the microtubule lumen.
... Cofilin-1 and drebrin are actin regulators involved in the signalling pathway mediating synaptic plasticity. Cofilin 1 binds to F-actin and exhibits pH-sensitive F-actin depolymerizing activity [33]. Drebrin is actin cytoskeleton-organizing protein that plays a role in the formation of cell projections [34]. ...
Background
According to the World Health Organization Report, depressive disorders affect about 10% of the population. The molecular mechanism of the pathogenesis of depression is still not well understood. The new findings point to phosphatases as potential targets for effective depression therapy. The aim of the present work was the development of a method that would enable the identification of mitogen-activated protein kinase phosphatase-1 (MKP-1) protein partners using a proteomic approach.
Methods
The research was carried out using the PC12 cell line, often used as a model for neurobiological research. The use of the procedure for efficient purification of protein complexes—tandem affinity purification (TAP) will facilitate the identification of proteins interacting with MKP-1, a potential goal of effective antidepressant therapy.
Results
Identified proteins belong to various groups: cytoskeletal, ribosomal, nucleic acid binding, chaperones, and enzymes and may potentially be involved in the molecular mechanism of depression.
Conclusions
The presented protocol for the purification of protein complexes is universal and can be successfully used in different mammalian cell lines. Proteins identified in the present work have been reported in the literature concerning studies on depressive disorders, which speaks in favour of their role in depression.
... Given our observation of disorganized F-actin in Ssh2 KO spermatids, we speculated that SSH2 acts as a modulator in actin remodeling through COFILIN dephosphorylation, which is essential for spermiogenesis. To pursue this further, we monitored the expression of COFILIN, phospho-COFILIN (p-COFILIN), and protein kinases that directly phosphorylate COFILIN (e.g., LIMK1 and LIMK2; Arber et al., 1998) by WB of testicular lysates of PD82 WT and Ssh2 KO testes. The Ssh2 KO mice had elevated levels of p-COFILIN compared to their WT littermates, whereas there were no significant differences in the levels of total COFILIN, LIMK1, or LIMK2 ( Figure 7A, Figure 7-figure supplement 1). ...
The acrosome is a membranous organelle positioned in the anterior portion of the sperm head and is essential for male fertility. Acrosome biogenesis requires the dynamic cytoskeletal shuttling of vesicles towards nascent acrosome which is regulated by a series of accessory proteins. However, much remains unknown about the molecular basis underlying this process. Here, we generated Ssh2 knock-out (KO) mice and HA-tagged Ssh2 knock-in (KI) mice to define the functions of Slingshot phosphatase 2 (SSH2) in spermatogenesis and demonstrated that as a regulator of actin remodeling, SSH2 is essential for acrosome biogenesis and male fertility. In Ssh2 KO males, spermatogenesis was arrested at the early spermatid stage with increased apoptotic index and the impaired acrosome biogenesis was characterized by defective transport/fusion of proacrosomal vesicles. Moreover, disorganized F-actin structures accompanied by excessive phosphorylation of COFILIN were observed in the testes of Ssh2 KO mice. Collectively, our data reveal a modulatory role for SSH2 in acrosome biogenesis through COFILIN-mediated actin remodeling and the indispensability of this phosphatase in male fertility in mice.
... Activation of Rac1/PAK1 signaling can phosphorylate cofilin, which facilitates retraction and stabilization of dendritic spines. 56,57 The signaling molecules that mediate dendritic spine morphogenesis may also be involved in the regulation of BCP. ...
Bone cancer pain (BCP) is severe chronic pain caused by tumor metastasis to the bones, often resulting in significant skeletal remodeling and fractures. Currently, there is no curative treatment. Therefore, insight into the underlying mechanisms could guide the development of mechanism-based therapeutic strategies for BCP. We speculated that Rac1/PAK1 signaling plays a critical role in the development of BCP. Tumor cells implantation (TCI) into the tibial cavity resulted in bone cancer-associated mechanical allodynia. Golgi staining revealed changes in the excitatory synaptic structure of WDR (Wide-dynamic range) neurons in the spinal cord, including increased postsynaptic density (PSD) length and thickness, and width of the cleft. Behavioral and western blotting test revealed that the development and persistence of pain correlated with Rac1/PAK1 signaling activation in primary sensory neurons. Intrathecal injection of NSC23766, a Rac1 inhibitor, reduced the persistence of BCP as well as reversed the remodeling of dendrites. Therefore, we concluded that activation of the Rac1/PAK1 signaling pathway in the spinal cord plays an important role in the development of BCP through remodeling of dendritic spines. Modulation of the Rac1/PAK1 pathway may be a potential strategy for BCP treatment.
... Au contraire, la phosphorylation de la cofiline au niveau de la Ser3 inhibe son association à l'actine, et par conséquent son activité(Moriyama et al., 1996). Une augmentation de la phosphorylation de la cofiline à la Ser3 se traduit donc par une stabilisation et une accumulation de F-actine dans les cellules(Arber et al., 1998, Niwa et al., 2002. Différentes sérine kinases assurent la phosphorylation à cette position : les LIM Kinases (LIMKs) 1 et 2 et les protéines kinases testiculaires (TESK) 1 et 2. Les kinases de la cofiline les plus importantes chez les mammifères sont les LIMK, et plus particulièrement la LIMK1, très étudiée pour sa fonction dans le système nerveux(Cuberos et al., 2015) (Figure 23). ...
Les épines dendritiques sont de petites protrusions membranaires qui portent la partie postsynaptique des synapses excitatrices. Les épines sont des structures extrêmement dynamiques : elles subissent des changements de forme et de fonction qui dépendent d’une réorganisation dynamique du cytosquelette d’actine. En effet, la dynamique de l’actine régule la forme de l’épine ainsi que la force de transmission synaptique. L’actine influence cette activité synaptique à travers le contrôle du nombre et de la localisation des récepteurs AMPA (AMPARs), qui assurent la transmission excitatrice rapide. Parmi les protéines qui régulent la dynamique de l’actine, la cofiline, une protéine qui dépolymérise l’actine, favorise le renouvellement dynamique de l’actine. L’inactivation de la cofiline par sa phosphorylation par la LIMK augmente la stabilité de l’actine, ce qui conduit à des altérations de forme d’épine et à un défaut de recrutement des AMPARs pendant la plasticité synaptique. Des anomalies de la dynamique des épines et de leur fonction sont une caractéristique de la plupart des pathologies neuropsychiatriques.La maladie de Huntington (MH) est une pathologie neurodégénérative et génétique caractérisée par la dysfonction et la dégénérescence des neurones du striatum et du cortex adultes. Les symptômes apparaissent à l’âge adulte et incluent des manifestations motrices, cognitives et psychiatriques. La MH est causée par la mutation du gène qui code pour la protéine huntingtine (HTT). Jusqu’à présent, la plupart des études se sont concentrées sur le gain de nouvelles fonctions toxiques de la HTT mutée. Cependant, on considère désormais que les évènements conduisant à la manifestation clinique de la MH sont en partie dus à la modification des fonctions de la HTT normale. La compréhension des fonctions normales de cette protéine est donc cruciale pour élucider les mécanismes cellulaires à l’origine de la MH.Nous montrons que la perte de HTT pendant le développement altère la morphologie des épines dendritiques et l’activité des synapses chez le jeune animal. Plus précisément, la HTT est présente dans le compartiment postsynaptique, et sa déplétion de façon cellule-autonome augmente le nombre d’épines matures et réduit la transmission synaptique excitatrice dépendante des AMPARs. Les AMPARs sont en effet affectés par la perte de HTT, puisque cette dernière réduit leur expression postsynaptique et limite leur recyclage à la suite d’une stimulation synaptique. Le cytosquelette d’actine est par ailleurs plus stable dans les épines déplétées en HTT, et ceci est associé à une hyperactivité de la voie moléculaire RAC1-LIMK-Cofiline. Ces résultats apportent de nouvelles informations quant aux fonctions moléculaires de la HTT dans la régulation de la morphologie des épines et de la physiologie de la synapse, processus qui pourrait être altéré dans le contexte de la MH.
... Branching of dendrites has for example been shown to be regulated by NOMA-GAP, a negative regulator of the Rho family member, Cdc42 and the downstream actin severing protein, Cofilin . Inhibition of cofilin by Cdc42-PAK-LIMK stabilizes pre-existing filaments and blocks the growth of new ones (Arber et al., 1998;Simo & Cooper, 2012). ...
Dendritische Defekte vermitteln Störungen der Erregbarkeit, Modulation und Plastizität von Neuronen, die zur Entwicklung neurodegenerativer Krankheiten führen können. Eine Mutation des Transkriptionsregulators Zeb2 führt zur Entwicklung des Mowat-Wilson-Syndroms, einer Erkrankung, die mit kognitiven Störungen und einem erkennbaren Gesichtsphänotyp einhergeht. Obwohl kognitive Defekte häufig mit Defekten bei der Bildung des dendritischen Baums in Verbindung gebracht werden, wurde die Rolle von Zeb2 bei der dendritischen Entwicklung bisher nicht untersucht. Hier zeige ich, dass Zeb2-defiziente Neuronen in den oberen neokortikalen Schichten einen abnormalen dendritische Baum aufweisen. Außerdem führt der Verlust von Zeb2 zu einer Fehlorientierung des apikalen Dendriten in einer nicht senkrechten Ausrichtung zur Pia. Darüber hinaus habe ich die Signalwege analysiert, die an der Regulierung der Morphologie des Dendritenbaum stromabwärts von Zeb2 beteiligt sind, und zwar durch Deep Sequencing des Transkriptoms von Zeb2-defizienten und Wildtyp-Neokortices sowie durch Massenspektrometrie-Screens auf Veränderungen in der Expression von Zelloberflächenproteinen nach dem Verlust von Zeb2. Für die vielversprechenden Kandidaten habe ich ein in situ-Hybridisierungs-Screening bei E15,5 durchgeführt. Eine Reihe von Genen, die an der neuronalen Morphologie und an Membranproteinen beteiligt sind, darunter Neuropilin1 und Cadherin6, werden in Gehirnen mit Zeb2-Mangel überexprimiert. Insbesondere habe ich die Rolle der neuen nachgeschalteten Zielgene Nrp1, Cdh6 und Wnt5a analysiert. Ich verwendete shRNA von Nrp1, Cdh6 und Wnt5a in neuronale Zellkultur, um zu zeigen, dass Nrp1 und Wnt5a eine erhöhte dendritische Komplexität in den Zeb2-defizienten neuronalen Zellen fördern. Die Überexpression von Nrp1 in Neuronen der oberen Schicht in vivo mittels in utero Elektroporation ist ausreichend, um die dendritische Komplexität zu fördern. Darüber hinaus zeige ich durch in utero Elektroporation einer shRNA gegen Nrp1 in Zeb2-defiziente Tiere, dass die Unterdrückung von Nrp1 durch Zeb2 für die Unterdrückung exzessiver Verzweigungen während der Entwicklung erforderlich ist. Für die Ausrichtung des Dendritenbaum ist sie jedoch nicht erforderlich. Zusammengenommen zeigen diese Daten die wichtige Rolle des Zeb2-Gens bei der Entwicklung des korrekten Dendritenbaum von Neuronen und der Ausrichtung des apikalen Dendriten.
... Cofilin-1 phosphorylation controls the interactions of cofilin-1 and actin and thus the mechanics of the actin regulatory machinery 74 . The phosphorylation of cofilin-1 on Ser3 by TESK and LIMK was previously described to markedly attenuates cofilin-1/actin interactions 16,75,76 , while dephosphorylation at this site restores the affinity of cofilin-1 for actin 77 . Cofilin phosphocycling at this residue clearly drives a diverse variety of actindependent processes, ranging from actin-based motility 78 to the pathogenesis of neurodegenerative diseases 79,80 . ...
Mutations in the lamin A/C gene ( LMNA ) cause dilated cardiomyopathy associated with increased activity of ERK1/2 in the heart. We recently showed that ERK1/2 phosphorylates cofilin-1 on threonine 25 (phospho(T25)-cofilin-1) that in turn disassembles the actin cytoskeleton. Here, we show that in muscle cells carrying a cardiomyopathy-causing LMNA mutation, phospho(T25)-cofilin-1 binds to myocardin-related transcription factor A (MRTF-A) in the cytoplasm, thus preventing the stimulation of serum response factor (SRF) in the nucleus. Inhibiting the MRTF-A/SRF axis leads to decreased α-tubulin acetylation by reducing the expression of ATAT1 gene encoding α-tubulin acetyltransferase 1. Hence, tubulin acetylation is decreased in cardiomyocytes derived from male patients with LMNA mutations and in heart and isolated cardiomyocytes from Lmna p.H222P/H222P male mice. In Atat1 knockout mice, deficient for acetylated α-tubulin, we observe left ventricular dilation and mislocalization of Connexin 43 (Cx43) in heart. Increasing α-tubulin acetylation levels in Lmna p.H222P/H222P mice with tubastatin A treatment restores the proper localization of Cx43 and improves cardiac function. In summary, we show for the first time an actin-microtubule cytoskeletal interplay mediated by cofilin-1 and MRTF-A/SRF, promoting the dilated cardiomyopathy caused by LMNA mutations. Our findings suggest that modulating α-tubulin acetylation levels is a feasible strategy for improving cardiac function.
... The spatial and temporal regulation of F-actin mediates multiple cellular signaling responses including cell division, motility and phagocytosis. The evolutionary conserved LIM domain kinase 1 (LIMK1) and Cofilin are essential for the formation of actin filaments [26,27]. Cofilin, also called actin-depolymerizing factor (ADF), promotes rapid F-actin turnover by severing actin filaments [28]. ...
Stress-induced cell death, mainly apoptosis, and its subsequent tissue repair is interlinked although our knowledge of this connection is still very limited. An intriguing finding is apoptosis-induced proliferation (AiP), an evolutionary conserved mechanism employed by apoptotic cells to trigger compensatory proliferation of their neighboring cells. Studies using Drosophila as a model organism have revealed that apoptotic caspases and c-Jun N-terminal kinase (JNK) signaling play critical roles to activate AiP. For example, the initiator caspase Dronc, the caspase-9 ortholog in Drosophila, promotes activation of JNK leading to release of mitogenic signals and AiP. Recent studies further revealed that Dronc relocates to the cell cortex via Myo1D, an unconventional myosin, and stimulates production of reactive oxygen species (ROS) to trigger AiP. During this process, ROS can attract hemocytes, the Drosophila macrophages, which further amplify JNK signaling cell non-autonomously. However, the intrinsic components connecting Dronc, ROS and JNK within the stressed signal-producing cells remain elusive. Here, we identified LIM domain kinase 1 (LIMK1), a kinase promoting cellular F-actin polymerization, as a novel regulator of AiP. F-actin accumulates in a Dronc-dependent manner in response to apoptotic stress. Suppression of F-actin polymerization in stressed cells by knocking down LIMK1 or expressing Cofilin, an inhibitor of F-actin elongation, blocks ROS production and JNK activation, hence AiP. Furthermore, Dronc and LIMK1 genetically interact. Co-expression of Dronc and LIMK1 drives F-actin accumulation, ROS production and JNK activation. Interestingly, these synergistic effects between Dronc and LIMK1 depend on Myo1D. Therefore, F-actin remodeling plays an important role mediating caspase-driven ROS production and JNK activation in the process of AiP.
... 62 Various proteins and miRNA (microRNA)s are involved in regulating the activity of LIMK. LIMKs could be activated by effector kinases of Ras homology (Rho), 63 Rac, 57,58 and cell division control protein 42 (CDC42) proteins, 64 including Rho-associated kinase1/2 (ROCK1/2), 65,66 p21-activated protein kinase1/2/4 (PAK1/2/4), 67 and myotonic dystrophin-related CDC42-binding kinase-α. 68 To achieve this, these effector kinases phosphorylate the conserved threonine residues in the LIMK domain (Threonine508(Thr508) in LIMK1 or Thr505 in LIMK2), which are the primary factors tuning the activity of LIMKs. ...
Cofilin, as a depolymerization factor of actin filaments, has been widely studied. Evidences show that cofilin has a role in actin structural reorganization and dynamic regulation. In recent years, several studies have demonstrated a regulatory role for cofilin in the migration and invasion mediated by cell dynamics and epithelial to mesenchymal transition (EMT)/EMT-like process, apoptosis, radiotherapy resistance, immune escape, and transcriptional dysregulation of malignant tumor cells, particularly glioma cells. On this basis, it is practical to evaluate cofilin as a biomarker for predicting tumor metastasis and prognosis. Targeting cofilin regulating kinases, Lin11, Isl-1 and Mec-3 kinases (LIM kinases/LIMKs) and their major upstream molecules inhibits tumor cell migration and invasion and targeting cofilin-mediated mitochondrial pathway induces apoptosis of tumor cells represent effective options for the development of novel anti-malignant tumor drug, especially anti-glioma drugs. This review explores the structure, general biological function, and regulation of cofilin, with an emphasis on the critical functions and prospects for clinical therapeutic applications of cofilin in malignant tumors represented by glioma.
... LIMK1 and LIMK2 are two isoforms of serine/threonine kinases and their activation is caused by phosphorylation at threonine 508 by ROCK [9,10]. The downstream targets of LIMK1 and LIMK2 are members of factors of depolymerisation of the actin-(ADF) family called cofilin [11]. In its inactivated state, cofilin is phosphorylated at serine 3 by LIMK1 or LIMK2, and dephosphorylation caused by inhibiting LIMK1/2 leads to the activation of cofilin, and consequently to actin reorganization and mitochondrial apoptosis in cancer cells [12,13]. ...
Ph+ (BCR::ABL+) B-ALL was considered to be high risk, but recent advances in BCR::ABL-targeting TKIs has shown improved outcomes in combination with backbone chemotherapy. Nevertheless, new treatment strategies are needed, including approaches without chemotherapy for elderly patients. LIMK1/2 acts downstream from various signaling pathways, which modifies cytoskeleton dynamics via phosphorylation of cofilin. Upstream of LIMK1/2, ROCK is constitutively activated by BCR::ABL, and upon activation, ROCK leads to the phosphorylation of LIMK1/2, resulting in the inactivation of cofilin by its phosphorylation and subsequently abrogating its apoptosis-promoting activity. Here, we demonstrate the anti-leukemic effects of a novel LIMK1/2 inhibitor (LIMKi) CEL_Amide in vitro and in vivo for BCR::ABL-driven B-ALL. The IC50 value of CEL_Amide was ≤1000 nM in BCR::ABL+ TOM-1 and BV-173 cells and induced dose-dependent apoptosis and cell cycle arrest in these cell lines. LIMK1/2 were expressed in BCR::ABL+ cell lines and patient cells and LIMKi treatment decreased LIMK1 protein expression, whereas LIMK2 expression was unaffected. As expected, CEL_Amide exposure caused specific activating downstream dephosphorylation of cofilin in cell lines and primary cells. Combination experiments with CEL_Amide and BCR::ABL TKIs imatinib, dasatinib, nilotinib, and ponatinib were synergistic for the treatment of both TOM-1 and BV-173 cells. CDKN2Ako/BCR::ABL1+ B-ALL cells were transplanted in mice, which were treated with combinations of CEL_Amide and nilotinib or ponatinib, which significantly prolonged their survival. Altogether, the LIMKi CEL_Amide yields activity in Ph+ ALL models when combined with BCR::ABL-targeting TKIs, showing promising synergy that warrants further investigation.
... They act directly on actin-associated proteins and thus affect actin polymerization or turnover. For example, Rho guanosine triphosphate enzyme (GTPase) activates p21-activated kinase (PAK) and LIM domain kinase 1 sequentially, which inhibits the activity of cofilin via phosphorylating its serine residue and ultimately disturbs the rearrangement of actin (Arber et al., 1998;Edwards et al., 1999;Yang et al., 1998). ...
Although forgetting was once regarded as a passive decline in memory and an occasional source of embarrassment, recent research suggests that it is an active biological process of removing outdated or irrelevant memories via activation of specific genes and signal transduction pathways. Rho family G proteins are known to have a role in synaptic plasticity mediated by the actin cytoskeleton. However, the current study reveals that another Rho guanosine triphosphate enzyme (GTPase), RAC-2, facilitates the occurrence of forgetting in Caenorhabditis elegans independent of actin dynamics. Functioning downstream of RAC-2 in the same signaling pathway, JNK-1 and its phosphorylated protein are required to positively regulate forgetting. The pan-neuronal rescue of RAC-2 or JNK-1, instead of AWC neuron-specific expression, reverses the delayed forgetting caused by the rac-2 mutation, which indicates that the involvement of RAC-2/JNK-1 in more than AWCs must be required. In summary, our work elucidates the action of the Rho GTPase RAC-2 and downstream JNK-1 as a potential novel pathway in forgetting in C. elegans.
... Phosphorylation of LIMK1 inhibits actin depolymerization through inactivation of cofilin, resulting in actin filament stabilization. 56,57 Interestingly, MYBPH may inhibit the phosphorylation of LIM kinases through interaction with Rho-associated coiled-coil containing protein kinases, and muscles from patients with ALS with high levels of MYBPH also accumulated nonphosphorylated LIMK1, which may underlie disruption of actin-myosin interactions. 48 Our findings show higher expression of both MYBPH and LIMK1 genes within gastrocnemius muscles of patients with PAD with progression, suggesting destabilization of actin may be taking place within these muscles. ...
Background
Peripheral artery disease (PAD) is associated with gastrocnemius muscle abnormalities. However, the biological pathways associated with gastrocnemius muscle dysfunction and their associations with progression of PAD are largely unknown. This study characterized differential gene and microRNA (miRNA) expression in gastrocnemius biopsies from people without PAD compared with those with PAD. Participants with PAD included those with and without PAD progression.
Methods and Results
mRNA and miRNA sequencing were performed to identify differentially expressed genes, differentially expressed miRNAs, mRNA‐miRNA interactions, and associated biological pathways for 3 sets of comparisons: (1) PAD progression (n=7) versus non‐PAD (n=7); (2) PAD no progression (n=6) versus non‐PAD; and (3) PAD progression versus PAD no progression. Immunohistochemistry was performed to determine gastrocnemius muscle fiber types and muscle fiber size. Differentially expressed genes and differentially expressed miRNAs were more abundant in the comparison of PAD progression versus non‐PAD compared with PAD with versus without progression. Among the top significant cellular pathways in subjects with PAD progression were muscle contraction or development, transforming growth factor‐beta, growth/differentiation factor, and activin signaling, inflammation, cellular senescence, and notch signaling. Subjects with PAD progression had increased frequency of smaller Type 2a gastrocnemius muscle fibers in exploratory analyses.
Conclusions
Humans with PAD progression exhibited greater differences in the number of gene and miRNA expression, biological pathways, and Type 2a muscle fiber size compared with those without PAD. Fewer differences were observed between people with PAD without progression and control patients without PAD. Further study is needed to confirm whether the identified transcripts may serve as potential biomarkers for diagnosis and progression of PAD.
... Various reports have delineated the regulators of retarded endocytosis. For example, LIMK1 regulates actin dynamics by inhibiting the activity of actin depolymerization factor cofilin [120], thus inhibiting EGFR endocytosis [121]. Moreover, amphiregulin (AR) and TNK2 promote sustained activation and conduction of downstream signaling pathways by maintaining phosphorylated EGFR on the cell membrane [122][123][124][125], leading to drug resistance to gefitinib in breast cancer cells [126,127]. ...
Epidermal growth factor receptor (EGFR) is highly expressed in certain cancer types and is involved in regulating the biological characteristics of cancer progression, including proliferation, metastasis, and drug resistance. Various medicines targeting EGFR have been developed and approved for several cancer types, such as lung and colon cancer. To date, however, EGFR inhibitors have not achieved satisfactory clinical results in breast cancer, which continues to be the most serious malignant tumor type in females. Therefore, clarifying the underlying mechanisms related to the ineffectiveness of EGFR inhibitors in breast cancer and developing new EGFR-targeted strategies (e.g., combination therapy) remain critical challenges. Various studies have demonstrated aberrant expression and maintenance of EGFR levels in breast cancer. In this review, we summarize the regulatory mechanisms underlying EGFR protein expression in breast cancer cells, including EGFR mutations, amplification, endocytic dysfunction, recycling acceleration, and degradation disorders. We also discuss potential therapeutic strategies that act directly or indirectly on EGFR, including reducing EGFR protein expression, treating the target protein to mediate precise clearance, and inhibiting non-EGFR signaling pathways. This review should provide new therapeutic perspectives for breast cancer patients with high EGFR expression.
The acrosome is a membranous organelle positioned in the anterior portion of the sperm head and is essential for male fertility. Acrosome biogenesis requires the dynamic cytoskeletal shuttling of vesicles toward nascent acrosome which is regulated by a series of accessory proteins. However, much remains unknown about the molecular basis underlying this process. Here, we generated Ssh2 knockout (KO) mice and HA-tagged Ssh2 knock-in (KI) mice to define the functions of Slingshot phosphatase 2 (SSH2) in spermatogenesis and demonstrated that as a regulator of actin remodeling, SSH2 is essential for acrosome biogenesis and male fertility. In Ssh2 KO males, spermatogenesis was arrested at the early spermatid stage with increased apoptotic index and the impaired acrosome biogenesis was characterized by defective transport/fusion of proacrosomal vesicles. Moreover, disorganized F-actin structures accompanied by excessive phosphorylation of COFILIN were observed in the testes of Ssh2 KO mice. Collectively, our data reveal a modulatory role for SSH2 in acrosome biogenesis through COFILIN-mediated actin remodeling and the indispensability of this phosphatase in male fertility in mice.
The eukaryotic cytoskeleton is a complex scaffold consisting of actin filaments, intermediate filaments, and microtubules. Though fungi and plants lack intermediate filaments, the dynamic structural network of actin filaments and microtubules regulates cell shape, division, polarity, and vesicular trafficking in both. However, the specialized functions of the cytoskeleton during plant-fungus interactions remain elusive. Recent reports demonstrate that the plant cytoskeleton responds to signal cues and pathogen invasion through remodeling, thereby coordinating immune receptor trafficking, membrane microdomain formation, aggregation of organelles, and transport of defense compounds. Emerging evidence also suggests that cytoskeleton remodeling further regulates host immunity by triggering salicylic acid signaling, reactive oxygen species generation, and pathogenesis-related gene expression. Interestingly, during host invasion, fungi undergo systematic cytoskeleton remodeling, which is crucial for successful host penetration and colonization. Furthermore, phytohormones act as an essential regulator of plant cytoskeleton dynamics and are frequently targeted by fungal effectors to disrupt the host's growth-defense balance. In this review, we comprehensively discussed recent advances in the understanding of cytoskeleton dynamics during plant-fungus interaction and provided novel insights explaining the phytohormone relationship with cytoskeleton remodeling upon pathogen attack. We also highlight the importance of fungal cytoskeleton rearrangements during host colonization and provide directions for future investigations in this field.
Macrophages are indispensable for proper immune surveillance and inflammatory regulation. They also exhibit dramatic phenotypic plasticity and are highly responsive to their local microenvironment, which includes the extracellular matrix (ECM). The present work demonstrates that two fibrous ECM glycoproteins, fibronectin (FN) and laminin (LAM), elicit distinct morphological and migratory responses from macrophages in 2D environments. Laminin 111 inhibits macrophage cell spreading, but drives them to migrate rapidly and less persistently compared to cells on fibronectin. Differential integrin engagement and ROCK/myosin II organization helps explain why macrophages alter their morphology and migration character on these two ECM components. The present study also demonstrates that laminin 111 exerts a suppressive effect toward fibronectin, as macrophages plated on a LAM/FN mixture adopt a morphology and migratory character almost identical to LAM alone. This suggests that distinct responses can be initiated downstream of receptor‐ECM engagement, and that one component of the microenvironment may affect the cell's ability to sense another. Overall, macrophages appear intrinsically poised to rapidly switch between distinct migratory characters based on their ECM environments. The role of ECM composition in dictating motile and inflammatory responses in 3D and in vivo contexts warrants further study.
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Cofilin acts as a key regulator of actin cytoskeletal remodeling via stimulating actin filament disassembly. Cofilin is inactivated by Ser-3 phosphorylation and reactivated by cofilin-phosphatase Slingshot-1 (SSH1). SSH1 is activated upon binding to F-actin, and this activation is inhibited by its phosphorylation at Ser-937 and Ser-978 and the subsequent binding of 14-3-3 proteins. In this study, we identified MARK3 (also named Par-1a and C-TAK1) as a kinase responsible for Ser-937/Ser-978 phosphorylation of SSH1. MARK3-mediated phosphorylation promoted SSH1 binding to 14-3-3 proteins and suppressed its F-actin-assisted cofilin-phosphatase activity. When Jurkat cells were stimulated with SDF-1α, actin filaments formed multidirectional F-actin-rich lamellipodia around the cells in the initial stage, and thereafter, they were rearranged as a single polarized lamellipodium to the direction of cell migration. Upon SDF-1α stimulation, SSH1 was translocated into F-actin-rich lamellipodia, but its Ser-937/Ser-978 non-phosphorylatable mutant SSH1(2SA) was retained at the location of the original cortical F-actin. Knockdown of MARK3 or overexpression of SSH1(2SA), similar to SSH1 knockdown, impaired the conversion of multiple lamellipodia to a single polarized lamellipodium. These results indicate that MARK3-mediated Ser-937/Ser-978 phosphorylation is required for SSH1 liberation from F-actin and translocation to lamellipodia, and hence, facilitates the formation of a single polarized lamellipodium for directional cell migration. Our results suggest that the phosphorylation-dephosphorylation cycle of SSH1 is crucial for its localization to lamellipodia via promoting the dissociation-reassociation cycle of SSH1 to F-actin, and thereby the stimulus-induced lamellipodium formation to the direction of cell movement.
We investigated the cytotoxic effect of Pelargonium sidoides extract on Madin–Darby canine kidney (MDCK) cells. P. sidoides extract decreased the cell viability in a dose dependent manner (> 0.2%). The extract of P. sidoides decreased the mitochondrial action potential, increased the number of reactive oxygen species (ROS) inside the cell, and caused nicotinamide adenine dinucleotide hydride (NADH) to be released, all of which are signs of mitochondrial dysfunction. The results of unbiased mRNA sequencing showed that 0.3% P. sidoides extract upregulates the apoptosis-related gene (BBC3). This finding was supported by immunoblot analysis of apoptosis signal pathways, which included Bcl-2, Bax, cytochrome C (CytC), cleaved caspase 3 (CC3), cleaved caspase 7 (CC7), cleaved caspase 9 (CC9) and cleaved PARP (CP). It is interesting to note that the elevated levels of Bax, CytC, CC3, CC7, and CC9, as well as CP, were suppressed by N-acetyl-L-cysteine (NAC) pretreatment, which points to ROS-mediated apoptosis. The small GTPases, RhoA, and Rac1/cdc42-GTP-bound active form were all lowered when P. sidoides extract was used. Also, RhoA-related cytoskeleton signals (ROCK, p-LIMK1/2, p-cofilin) and Rac1/cdc42-related signals (N-WASP, WAVE-2) were inhibited by P. sidoides extract. NAC or RhoA/Rac1/cdc42 activator pretreatment reduced P. sidoides extract-induced actin destabilization. In this work, P. sidoides extract promotes apoptosis by causing mitochondrial dysfunction and cytoskeleton disassembly.
Cortical GABAergic interneurons are critical components of neural networks. They provide local and long-range inhibition and help coordinate network activities involved in various brain functions, including signal processing, learning, memory and adaptative responses. Disruption of cortical GABAergic interneuron migration thus induces profound deficits in neural network organization and function, and results in a variety of neurodevelopmental and neuropsychiatric disorders including epilepsy, intellectual disability, autism spectrum disorders and schizophrenia. It is thus of paramount importance to elucidate the specific mechanisms that govern the migration of interneurons to clarify some of the underlying disease mechanisms. GABAergic interneurons destined to populate the cortex arise from multipotent ventral progenitor cells located in the ganglionic eminences and pre-optic area. Post-mitotic interneurons exit their place of origin in the ventral forebrain and migrate dorsally using defined migratory streams to reach the cortical plate, which they enter through radial migration before dispersing to settle in their final laminar allocation. While migrating, cortical interneurons constantly change their morphology through the dynamic remodeling of actomyosin and microtubule cytoskeleton as they detect and integrate extracellular guidance cues generated by neuronal and non-neuronal sources distributed along their migratory routes. These processes ensure proper distribution of GABAergic interneurons across cortical areas and lamina, supporting the development of adequate network connectivity and brain function. This short review summarizes current knowledge on the cellular and molecular mechanisms controlling cortical GABAergic interneuron migration, with a focus on tangential migration, and addresses potential avenues for cell-based interneuron progenitor transplants in the treatment of neurodevelopmental disorders and epilepsy.
Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identities of these objects and mechanisms for their accumulation have not been conclusively established. Here, we used cryogenic electron tomography of Drosophila S2 cell protrusions and found filaments inside the microtubule lumen, which resemble those reported recently in human HAP1 cells. The frequency of these filaments increased upon inhibition of the sarco/endoplasmic reticulum Ca ²⁺ ATPase with the small molecule drug thapsigargin. Subtomogram averaging showed that the luminal filaments adopt a helical structure reminiscent of cofilin‐bound actin (cofilactin). Consistent with this, we observed cofilin dephosphorylation, an activating modification, in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNA interference knock‐down of cofilin reduced the frequency of luminal filaments with cofilactin morphology. These results suggest that cofilin activation stimulates its accumulation on actin filaments inside the microtubule lumen.
Signaling from the Rho family small GTPases controls a wide range of signaling outcomes. Key among the downstream effectors for many of the Rho GTPases are the p21-activated kinases, or PAK group. The PAK family comprises two types, the type I PAKs (PAK1, 2 and 3) and the type II PAKs (PAK4, 5 and 6), which have distinct structures and mechanisms of regulation. In this review, we discuss signal transduction from Rho GTPases with a focus on the type II PAKs. We discuss the role of PAKs in signal transduction pathways and selectivity of Rho GTPases for PAK family members. We consider the less well studied of the Rho GTPases and their PAK-related signaling. We then discuss the molecular basis for kinase domain recognition of substrates and for regulation of signaling. We conclude with a discussion of the role of PAKs in cross talk between Rho family small GTPases and the roles of PAKs in disease.
The second volume of Behavioral Genetics of the Mouse provides a comprehensive overview of the major genetically modified mouse lines used to model human neurobehavioral disorders; from disorders of perception, of autonomous and motor functions to social and cognitive syndromes, drug abuse and dependence as well as neurodegenerative pathologies. Mouse models obtained with different types of genetic manipulations (i.e. transgenic, knockout/in mice) are described in their pathological phenotypes, with a special emphasis on behavioral abnormalities. The major results obtained with many of the existing models are discussed in depth highlighting their strengths and limitations. A lasting reference, the thorough reviews offer an easy entrance into the extensive literature in this field, and will prove invaluable to students and specialists alike.
Plasma membrane deformation is a crucial process for cells to perform functions such as cell migration, trafficking, and division. These functions are mediated by the cellular actin cytoskeleton, shaping the plasma membrane into protrusions or invaginations and initiating movement. Two different protrusion morphologies, namely lamellipodia and filopodia, are the key factors in determining the direction of cell movement. The architecture of these structures is regulated by the small guanosine triphosphatases of the Rho family, nucleation-promoting factors of the Wiskott–Aldrich syndrome protein family, Arp2/3 complex, and focal adhesion proteins. These proteins regulate the protrusion formation through a spatio-temporal interplay of actin assembly and disassembly at the leading edge of cells.
The enzyme chorismate synthase was purified in milligram quantities from an overproducing strain of Escherichia coli. The amino acid sequence was deduced from the nucleotide sequence of the aroC gene and confirmed by determining the N-terminal amino acid sequence of the purified enzyme. The complete polypeptide chain consists of 357 amino acid residues and has a calculated subunit Mr of 38,183. Cross-linking and gel-filtration experiments show that the enzyme is tetrameric. An improved purification of chorismate synthase from Neurospora crassa is also described. Cross-linking and gel-filtration experiments on the N. crassa enzyme show that it is also tetrameric with a subunit Mr of 50,000. It is proposed that the subunits of the N. crassa enzyme are larger because they contain a diaphorase domain that is absent from the E. coli enzyme.
An in vitro method by which reagents, cells, and Toxoplasma gondii trophozoites are conserved (micromethod) was developed
to quantitate the effect of antimicrobial agents on T. gondii. Sulfadoxine alone had no effect on T. gondii in vitro when
evaluated with a macromethod, the new micromethod, or visual inspection of Giemsa-stained preparations. Sulfadoxine combined
with pyrimethamine inhibited T. gondii more than did pyrimethamine alone, but the combination of sulfadoxine plus pyrimethamine
was slightly less active than was the combination of sulfadiazine plus pyrimethamine. Neither clindamycin nor metronidazole,
alone or in combination with sulfadiazine or pyrimethamine and sulfadiazine, had any effect on intracellular T. gondii. Brief
exposure (10 min before and during challenge) to clindamycin had no effect on extracellular T. gondii when clindamycin was
studied alone or with sulfadiazine or pyrimethamine plus sulfadiazine. Cyclosporin A inhibited T. gondii replication at concentrations
of ca. greater than or equal to 2 micrograms/ml.
Many of the ribosomal protein (RP) genes in both bacterial and chloroplast genomes occur, for reasons not yet understood, in operons that include nonribosomal genes. Here we report such an operon organization in a cyanobacterium (Synechocystis PCC6803) involving the genes for four RPs that are important in the GTPase function of the ribosome and the aroC gene encoding chorismate synthase, a key enzyme in the shikimate pathway for biosynthesis of aromatic amino acids and cell wall components. The Synechocystis aroC encodes a 362-amino-acid residue protein which is 52, 60, and 68% identical to two eubacterial (both 52%), yeast, and a higher plant (Corydalis) chorismate synthase, respectively. The gene was overexpressed in Escherichia coli, and the gene product was shown to cross-react with antibodies to Corydalis chorismate synthase; it also complemented an aroC-lacking E. coli strain. The Synechocystis rpl1 and rpl11 genes encode polypeptides of 237 and 141 amino acid residues, respectively, also with high sequence identities to the corresponding RP sequences from other eubacteria and higher plant chloroplasts. The gene order is shown to be: rpl11-86bp spacer-rpl1-460bp spacer-rpl10-87-bp spacer-rpl12-206bp spacer-aroC. Southern and Northern blot analyses of Synechocystis DNA and RNA, respectively, revealed a single cluster of these genes per genome which is transcribed from a common promoter to an unusually long, approximately 9500-nucleotide transcript. Several constructs of the cyanobacterial aroC and rpl12 genes were made and expressed in E. coli to examine the mechanisms for their very differential expression from a polycistronic mRNA (e.g. four copies L12/ribosome; chorismate synthase, a non-abundant protein). These results present the first biochemical/molecular genetic evidence of shikimate pathway in the cyanobacterial group.
Actin depolymerizing factor (ADF) occurs naturally in two forms, one of which contains a phosphorylated Ser and does not bind
G-actin or depolymerize F-actin. Removal of this phosphate in vitro by alkaline phosphatase restores full F-actin depolymerizing activity. To identify the phosphorylation site, [P]pADF was purified and digested with endoproteinase Lys-C. The digest contained only one P-labeled peptide. Further digestion with endoproteinase Asp-N and mass spectrometric analysis showed that this peptide came
from the N terminus of ADF. Alkaline phosphatase treatment of one Asp-N peptide (mass 753) converted it to a peptide of mass
673, demonstrating that this peptide contains the phosphate group. Tandem mass spectrometric sequence analysis of this peptide
identified the phosphorylated Ser as the encoded Ser3 (Ser2 in the processed protein). HeLa cells, transfected with either chick wild-type ADF cDNA or a cDNA mutated to code for Ala
in place of Ser or Thr, express and phosphorylate the exogenous ADF. Cells also expressed high levels of mutant ADF when Ser3 was deleted or converted to either Ala or Glu. However, none of these mutants was phosphorylated, confirming that Ser3 in the encoded ADF is the single in vivo regulatory site.
Activities of all chorismate synthases (CS) so far analyzed are absolutely dependent upon reduced flavin. For monofunctional CSs, which represent the only class of CSs that have yet been cloned, the flavin must be reduced either (photo-)chemically or by a separable flavin reductase (FR) for in vitro activity. Neurospora crassa CS, in contrast, possesses an intrinsic FR activity and represents the only firmly established member of a bifunctional class of CSs. To better understand this bifunctional protein, a cDNA from an N. crassa expression library encoding a 46.4-kDa protein was cloned by complementation of the CS-deficient Escherichia coli strain AB2849. The deduced amino acid sequence was highly similar (79%) to a previously isolated Saccharomyces cerevisiae CS. The N. crassa sequence was unequivocally shown to encode the bifunctional CS/FR by analysis of the purified protein expressed in E. coli. Based on sequence comparisons with known monofunctional CSs, two regions of 18 internal residues and 29 C-terminal residues unique to N. crassa CS were deleted, and the constructs were also expressed in E. coli. The presence of these regions was found not essential for complementation of the CS- phenotype of E. coli strain AB2849. Although a 3.5-fold decline in specific activity of the purified CS from cells expressing the C-terminal deletion construct was observed, bifunctional activity was not eliminated. These data strongly suggest that the domain(s) responsible for reduction of flavin lie(s) within regions in which homology is also shared among monofunctional CSs.
Actin depolymerizing factor (ADF) is an 18.5-kD protein with pH-dependent reciprocal F-actin binding and severing/depolymerizing activities. We previously showed developing muscle down-regulates ADF (J. R. Bamburg and D. Bray. 1987. J. Cell Biol. 105: 2817-2825). To further study this process, we examined ADF expression in chick myocytes cultured in vitro. Surprisingly, ADF immunoreactivity increases during the first 7-10 d in culture. This increase is due to the presence of a new ADF species with higher relative molecular weight which reacts identically to brain ADF with antisera raised against either brain ADF or recombinant ADF. We have purified both ADF isoforms from myocytes and have shown by peptide mapping and partial sequence analysis that the new isoform is structurally related to ADF. Immunoprecipitation of both isoforms from extracts of cells prelabeled with [32P]orthophosphate showed that the new isoform is radiolabeled, predominantly on a serine residue, and hence is called pADF. pADF can be converted into a form which comigrates with ADF on 1-D and 2-D gels by treatment with alkaline phosphatase. pADF has been quantified in a number of cells and tissues where it is present from approximately 18% to 150% of the amount of unphosphorylated ADF. pADF, unlike ADF, does not bind to G-actin, or affect the rate or extent of actin assembly. Four ubiquitous protein kinases failed to phosphorylate ADF in vitro suggesting that ADF phosphorylation in vivo is catalyzed by a more specific kinase. We conclude that the ability to regulate ADF activity is important to muscle development since myocytes have both pre- and posttranslational mechanisms for regulating ADF activity. The latter mechanism is apparently a general one for cell regulation of ADF activity.
(6S)-6-Fluoroshikimic acid inhibited the growth of Escherichia coli B on minimal medium (MIC, 0.25 micrograms ml-1), and it
protected mice challenged intraperitoneally with the same organism (50% protective dose, 0.06 mg kg of body weight-1). We
propose that inhibitors of bacterial aromatic biosynthesis have the potential for use in human medicine.
We describe the phenotypic and molecular characterization of twinstar (tsr), an essential gene in Drosophila melanogaster. Two P-element induced alleles of tsr (tsr1 and tsr2) result in late larval or pupal lethality. Cytological examination of actively dividing tissues in these mutants reveals defects in cytokinesis in both mitotic (larval neuroblast) and meiotic (larval testis) cells. In addition, mutant spermatocytes show defects in aster migration and separation during prophase/prometaphase of both meiotic divisions. We have cloned the gene affected by these mutations and shown that it codes for a 17-kD protein in the cofilin/ADF family of small actin severing proteins. A cDNA for this gene has previously been described by Edwards et al. (1994). Northern analysis shows that the tsr gene is expressed throughout development, and that the tsr1 and tsr2 alleles are hypomorphs that accumulate decreased levels of tsr mRNA. These findings prompted us to examine actin behavior during male meiosis to visualize the effects of decreased twinstar protein activity on actin dynamics in vivo. Strikingly, both mutants exhibit abnormal accumulations of F-actin. Large actin aggregates are seen in association with centrosomes in mature primary spermatocytes. Later, during ana/telophase of both meiotic divisions, aberrantly large and misshaped structures appear at the site of contractile ring formation and fail to disassemble at the end of telophase, in contrast with wild-type. We discuss these results in terms of possible roles of the actin-based cytoskeleton in centrosome movement and in cytokinesis.
We previously isolated human cDNA coding for LIMK1 (LIM motif-containing protein kinase-1), a putative protein kinase containing two LIM motifs at the N terminus and an unusual protein kinase domain at the C terminus. In the present study, we isolated human cDNA encoding LIMK2, a second member of a LIMK family, with a domain structure similar to LIMK1 and 50% overall amino acid identity with LIMK1. The protein kinase domains of LIMK1 and LIMK2 are unique in that they contain an unusual sequence motif Asp-Leu-Asn-Ser-His-Asn in subdomain VIB and a highly basic insert between subdomains VII and VIII. Expression patterns of LIMK1 and LIMK2 mRNAs in human tissues differ significantly. Chromosomal localization of human LIMK1 and LIMK2 genes was assigned to 7q11.23 and 22q12, respectively, by fluorescence in situ hybridization. The Myc epitope-tagged LIMK1 and LIMK2 proteins transiently expressed in COS cells exhibited serine/threonine-specific kinase activity toward myelin basic protein and histone in in vitro kinase assay. Immunofluorescence and subcellular fractionation analysis revealed that Myc-tagged LIMK1 and LIMK2 were localized mainly in the cytoplasm. The "native" LIMK1 protein endogenously expressed in A431 epidermoid carcinoma cells also exhibited serine/threonine kinase activity. The specific activity of native LIMK1 from A431 cells was apparently much higher than that of "recombinant" LIMK1 ectopically expressed in COS cells, hence, it is likely that there is a mechanism, by which native LIMK1 is activated. A 140-kDa tyrosine-phosphorylated protein (pp140) was co-immunoprecipitated with native LIMK1 form A431 cell lysates; therefore, pp140 may be a LIMK1-associated protein involved in the regulation of LIMK1 function.
The intracompartmental sorting and functional consequences of ectopic expression of the six vertebrate actin isoforms was investigated in different types of cultured cells. In transfected fibroblasts all isoactin species associated with the endogenous microfilament cytoskeleton, even though cytoplasmic actins also showed partial localization to peripheral submembranous sites. Functional and structural studies were performed in neonatal and adult rat cardiomyocytes. All the muscle isoactin constructs sorted preferentially to sarcomeric sites and, to a lesser extent, also to stress-fiber-like structures. The expression of muscle actins did not interfere with cell contractility, and did not disturb the localization of endogenous sarcomeric proteins. In sharp contrast, ectopic expression of the two cytoplasmic actin isoforms resulted in rapid cessation of cellular contractions and induced severe morphological alterations characterized by an exceptional outgrowth of filopodia and cell flattening. Quantitative analysis in neonatal cardiomyocytes indicated that the levels of accumulation of the different isoactins are very similar and cannot be responsible for the observed isoproteins-specific effects. Structural analysis revealed a remodeling of the cytoarchitecture including a specific alteration of sarcomeric organization; proteins constituting the sarcomeric thin filaments relocated to nonmyofibrillar sites while thick filaments and titin remained unaffected. Experiments with chimeric proteins strongly suggest that isoform specific residues in the carboxy-terminal portion of the cytoplasmic actins are responsible for the dominant negative effects on function and morphology.
Two cDNAs, isolated from a Xenopus laevis embryonic library, encode proteins of 168 amino acids, both of which are 77% identical to chick cofilin and 66% identical to chick actin-depolymerizing factor (ADF), two structurally and functionally related proteins. These Xenopus ADF/cofilins (XADs) differ from each other in 12 residues spread throughout the sequence but do not differ in charge. Purified GST-fusion proteins have pH-dependent actin-depolymerizing and F-actin-binding activities similar to chick ADF and cofilin. Similarities in the developmental and tissue specific expression, embryonic localization, and in the cDNA sequence of the noncoding regions, suggest that the two XACs arise from allelic variants of the pseudotetraploid X. laevis. Immunofluorescence localization of XAC in oocyte sections with an XAC-specific monoclonal antibody shows it to be diffuse in the cortical cytoplasm. After fertilization, increased immunostaining is observed in two regions: along the membrane, particularly that of the vegetal hemisphere, and at the interface between the cortical and animal hemisphere cytoplasm. The cleavage furrow and the mid-body structure are stained at the end of first cleavage. Neuroectoderm derived tissues, notochord, somites, and epidermis stain heavily either continuously or transiently from stages 18-34. A phosphorylated form of XAC (pXAC) was identified by 2D Western blotting, and it is the only species found in oocytes. Dephosphorylation of >60% of the pXAC occurs within 30 min after fertilization. Injection of one blastomere at the 2 cell stage, either with constitutively active XAC or with an XAC inhibitory antibody, blocked cleavage of only the injected blastomere in a concentration-dependent manner without inhibiting nuclear division. The cleavage furrow of eggs injected with constitutively active XAC completely regressed. Blastomeres injected with neutralized antibody developed normally. These results suggest that XAC is necessary for cytokinesis and that its activity must be properly regulated for cleavage to occur.
We explicitly acknowledge key intellectual contributions from Jody Rosenblatt (depolymerization) and Julie Theriot (protrusion, Listeria motility). Owing to space restrictions, we were unable to cite all relevant publications. We attempted to fairly represent different laboratories, systems, and opinions and apologize to authors whose papers we did not include. We thank Matt Welch and Jody Rosenblatt for helpful comments on the manuscript. This work was funded by grant GM48027 from the National Institutes of Health and a fellowship from the Packard Foundation to T. J. Mitchison, and a senior postdoctoral fellowship from the American Cancer Society to L. P. Cramer.
Signal transduction processes in T-cells and other cell types alter the phosphorylation state of cofilin, an actin-binding
phosphoprotein. Whether reversible phosphorylation is responsible for the regulation of the functional activities of cofilin
is not clear at present. Here we have identified the phosphoacceptor site of cofilin and analyzed the role of cofilin phosphorylation
with respect to its subcellular localization. Site-directed mutagenesis studies show that phosphorylation occurs exclusively
on Ser-3. Expression of non-phosphorylatable mutant cofilin proteins in NIH3T3 cells and determination of their subcellular
localization by confocal laser scanning microscopy reveal that non-phosphorylated cofilin accumulates within nuclei. This
analysis shows that the subcellular localization of cofilin depends on the phosphorylation state of Ser-3.
Protozoan parasites of the phylum Apicomplexa contain three genetic elements: the nuclear and mitochondrial genomes characteristic of virtually all eukaryotic cells and a 35-kilobase circular extrachromosomal DNA. In situ hybridization techniques were used to localize the 35-kilobase DNA of Toxoplasma gondii to a discrete organelle surrounded by four membranes. Phylogenetic analysis of the tufA gene encoded by the 35-kilobase genomes of coccidians T. gondii and Eimeria tenella and the malaria parasite Plasmodium falciparum grouped this organellar genome with cyanobacteria and plastids, showing consistent clustering with green algal plastids. Taken together, these observations indicate that the Apicomplexa acquired a plastid by secondary endosymbiosis, probably from a green alga.
In contrast to the slow rate of depolymerization of pure actin in vitro, populations of actin filaments in vivo turn over rapidly. Therefore, the rate of actin depolymerization must be accelerated by one or more factors in the cell. Since the actin dynamics in Listeria monocytogenes tails bear many similarities to those in the lamellipodia of moving cells, we have used Listeria as a model system to isolate factors required for regulating the rapid actin filament turnover involved in cell migration. Using a cell-free Xenopus egg extract system to reproduce the Listeria movement seen in a cell, we depleted candidate depolymerizing proteins and analyzed the effect that their removal had on the morphology of Listeria tails. Immunodepletion of Xenopus actin depolymerizing factor (ADF)/cofilin (XAC) from Xenopus egg extracts resulted in Listeria tails that were approximately five times longer than the tails from undepleted extracts. Depletion of XAC did not affect the tail assembly rate, suggesting that the increased tail length was caused by an inhibition of actin filament depolymerization. Immunodepletion of Xenopus gelsolin had no effect on either tail length or assembly rate. Addition of recombinant wild-type XAC or chick ADF protein to XAC-depleted extracts restored the tail length to that of control extracts, while addition of mutant ADF S3E that mimics the phosphorylated, inactive form of ADF did not reduce the tail length. Addition of excess wild-type XAC to Xenopus egg extracts reduced the length of Listeria tails to a limited extent. These observations show that XAC but not gelsolin is essential for depolymerizing actin filaments that rapidly turn over in Xenopus extracts. We also show that while the depolymerizing activities of XAC and Xenopus extract are effective at depolymerizing normal filaments containing ADP, they are unable to completely depolymerize actin filaments containing AMPPNP, a slowly hydrolyzible ATP analog. This observation suggests that the substrate for XAC is the ADP-bound subunit of actin and that the lifetime of a filament is controlled by its nucleotide content.
Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical-chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADP-bound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi-bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics.
Actin is among the most thoroughly studied of proteins. It was first identified half a century ago as the major component of thin filaments in muscle. Work in the 1960s and 1970s showed that actin is also present in nonmuscle cells as well as in plants and protozoa. The actin-based cytoskeleton appears to be ubiquitous among eukaryotes, and indeed the invention of the actin cytoskeleton may have been a key step in the earliest history of the eukaryotic lineage. A densely written summary of the most important known properties of actin fills a good-sized volume (23). But there are major discrepancies between the well-characterized in vitro behavior of purified actin and the apparent behavior of actin filaments inside of intact, living cells.
Actin depolymerizing factor (ADF) is an 18.5-kD protein with pH-dependent reciprocal F-actin binding and severing/depolymerizing activities. We previously showed developing muscle down-regulates ADF (J. R. Bamburg and D. Bray. 1987. J. Cell Biol. 105: 2817-2825). To further study this process, we examined ADF expression in chick myocytes cultured in vitro. Surprisingly, ADF immunoreactivity increases during the first 7-10 d in culture. This increase is due to the presence of a new ADF species with higher relative molecular weight which reacts identically to brain ADF with antisera raised against either brain ADF or recombinant ADF. We have purified both ADF isoforms from myocytes and have shown by peptide mapping and partial sequence analysis that the new isoform is structurally related to ADF. Immunoprecipitation of both isoforms from extracts of cells prelabeled with [32P]orthophosphate showed that the new isoform is radiolabeled, predominantly on a serine residue, and hence is called pADF. pADF can be converted into a form which comigrates with ADF on 1-D and 2-D gels by treatment with alkaline phosphatase. pADF has been quantified in a number of cells and tissues where it is present from approximately 18% to 150% of the amount of unphosphorylated ADF. pADF, unlike ADF, does not bind to G-actin, or affect the rate or extent of actin assembly. Four ubiquitous protein kinases failed to phosphorylate ADF in vitro suggesting that ADF phosphorylation in vivo is catalyzed by a more specific kinase. We conclude that the ability to regulate ADF activity is important to muscle development since myocytes have both pre- and posttranslational mechanisms for regulating ADF activity. The latter mechanism is apparently a general one for cell regulation of ADF activity.
Two cDNAs, isolated from a Xenopus laevis embryonic library, encode proteins of 168 amino acids, both of which are 77% identical to chick cofilin and 66% identical to chick actin-depolymerizing factor (ADF), two structurally and functionally related proteins. These Xenopus ADF/cofilins (XADs) differ from each other in 12 residues spread throughout the sequence but do not differ in charge. Purified GST-fusion proteins have pH-dependent actin-depolymerizing and F-actin-binding activities similar to chick ADF and cofilin. Similarities in the developmental and tissue specific expression, embryonic localization, and in the cDNA sequence of the noncoding regions, suggest that the two XACs arise from allelic variants of the pseudotetraploid X. laevis. Immunofluorescence localization of XAC in oocyte sections with an XAC-specific monoclonal antibody shows it to be diffuse in the cortical cytoplasm. After fertilization, increased immunostaining is observed in two regions: along the membrane, particularly that of the vegetal hemisphere, and at the interface between the cortical and animal hemisphere cytoplasm. The cleavage furrow and the mid-body structure are stained at the end of first cleavage. Neuroectoderm derived tissues, notochord, somites, and epidermis stain heavily either continuously or transiently from stages 18-34. A phosphorylated form of XAC (pXAC) was identified by 2D Western blotting, and it is the only species found in oocytes. Dephosphorylation of >60% of the pXAC occurs within 30 min after fertilization. Injection of one blastomere at the 2 cell stage, either with constitutively active XAC or with an XAC inhibitory antibody, blocked cleavage of only the injected blastomere in a concentration-dependent manner without inhibiting nuclear division. The cleavage furrow of eggs injected with constitutively active XAC completely regressed. Blastomeres injected with neutralized antibody developed normally. These results suggest that XAC is necessary for cytokinesis and that its activity must be properly regulated for cleavage to occur.
The intracompartmental sorting and functional consequences of ectopic expression of the six vertebrate actin isoforms was investigated in different types of cultured cells. In transfected fibroblasts all isoactin species associated with the endogenous microfilament cytoskeleton, even though cytoplasmic actins also showed partial localization to peripheral submembranous sites. Functional and structural studies were performed in neonatal and adult rat cardiomyocytes. All the muscle isoactin constructs sorted preferentially to sarcomeric sites and, to a lesser extent, also to stress-fiber-like structures. The expression of muscle actins did not interfere with cell contractility, and did not disturb the localization of endogenous sarcomeric proteins. In sharp contrast, ectopic expression of the two cytoplasmic actin isoforms resulted in rapid cessation of cellular contractions and induced severe morphological alterations characterized by an exceptional outgrowth of filopodia and cell flattening. Quantitative analysis in neonatal cardiomyocytes indicated that the levels of accumulation of the different isoactins are very similar and cannot be responsible for the observed isoproteins-specific effects. Structural analysis revealed a remodeling of the cytoarchitecture including a specific alteration of sarcomeric organization; proteins constituting the sarcomeric thin filaments relocated to nonmyofibrillar sites while thick filaments and titin remained unaffected. Experiments with chimeric proteins strongly suggest that isoform specific residues in the carboxy-terminal portion of the cytoplasmic actins are responsible for the dominant negative effects on function and morphology.
6-Fluoroshikimates have interesting antibiotic properties. The enzymatic preparation of (6R)- and (6S)-fluoroshikimates from 3-fluorophosphoenolpyruvate is described. The multistep enzymatic conversion is followed by UV and 19F NMR spectroscopy.
Much progress has been achieved in the last decade in terms of development of laboratory techniques, reagents and in vivo models. They have undoubtedly contributed to better and more accurate investigations. Despite a concerted effort by many investigators, however, breakthroughs have been minimal. The development of adequate in vitro and in vivo techniques for drug screening, and the intensified and systematic screening, has so far not resulted in the discovery of an effective therapy. The reason for the failure may well be due to the unique biological niche the parasite occupies (discussed at length in the first chapter in this volume). Its location beneath the cell membrane, but outside the cell cytoplasm, may prove a crucial element that needs to be considered when designing new therapeutic approaches. Laboratory investigations on two drugs currently used against chronic Cryptosporidium parvum in acquired immune deficiency syndrome (AIDS) are discussed. This chapter also provides information and the rationale for work in progress in our laboratory that relates to the development of novel approaches for control of the disease. This includes the identification of molecular targets of parasite origin for drug design, and studies on the structure-activity relationships of partially effective drugs with a view to synthesize more effective derivatives. Other investigations attempt to establish the role of secretory antibody, and the merit of repeated mucosal immunizations as a means of providing protection to individuals with AIDS who are at risk of developing chronic C. parvum infection.
We describe here the cloning, characterization and analysis of the regulation of the ARO2 gene of Saccharomyces cerevisiae , the first reported study of a eukaryotic gene encoding chorismate synthase (E.C. 4.6.1.4). The gene contains an ORF of 1128 bp, encoding a protein with a calculated molecular mass of 40.8 kDa. ARO2 is regulated under the ‘general control system’ for amino acid biosynthesis by the transcriptional activator GCN4 which binds in vitro at three sites within the AR02 promoter.
The AR02 gene product is highly similar to its Escherichia coli counterpart, with a 47% identity distributed over the entire length of the peptide. We therefore suggest that the S. cerevisiae chorismate synthase, in contrast to the Neurospora crassa enzyme, but like other chorismate synthases, is a monofunctional peptide, solely possessing chorismate synthase activity.
The Mycobacterium tuberculosis shikimate pathway genes designated aroB and aroQ encoding 3-dehydroquinate synthase and 3-dehydroquinase, respectively were isolated by molecular cloning and their nucleotide sequences determined. The deduced dehydroquinate synthase amino acid sequence from M. tuberculosis showed high similarity to those of equivalent enzymes from prokaryotes and filamentous fungi. Surprisingly, the deduced M. tuberculosis 3-dehydroquinase amino acid sequence showed no similarity to other characterised prokaryotic biosynthetic 3-dehydroquinases (bDHQases). A high degree of similarity was observed, however, to the fungal catabolic 3-dehydroquinases (cDHQases) which are active in the quinic acid utilisation pathway and are isozymes of the fungal bDHQases. This finding indicates a common ancestral origin for genes encoding the catabolic dehydroquinases of fungi and the biosynthetic dehydroquinases present in some prokaryotes. Deletion of genes encoding shikimate pathway enzymes represents a possible approach to generation of rationally attenuated strains of M. tuberculosis for use as live vaccines.
Three possible mechanisms of resistance to sulfadoxine were investigated in resistant Plasmodium falciparum: drug uptake, metabolism and alternate pathways. Uptake of [35S] sulfadoxine was markedly reduced in resistant plasmodia. By Thin Layer Radiochromatography it could be demonstrated that plasmodia do not metabolize sulfadoxine to pharmacologically inactive forms. Metabolism of sulfadoxine to the toxic analog of dihydropteroate is reduced in resistant plasmodia. Para-aminobenzoic acid (pABA) is not an essential nutrient for sulfonamide-resistant plasmodia. Instead, they seem to be able to synthesize pABA de novo. Four enzymes of the respective biosynthetic chain were demonstrated in isolated plasmodia: 3-deoxy-D-arabino-heptulosonate-7-phosphate synthetase (EC 4.2.1.15), shikimate dehydrogenase (EC 1.1.1.25), shikimate kinase (EC 2.7.1.71) and pABA synthetase. We conclude that these three effects account for the reduced sulfonamide stress observed in the resistant parasite.
Weeds, which compete with crops for soil nutrients and harbor pests that attack corps, routinely lead to significant losses in crop yield. The chemical method of weed control is the major method used in modern-day food production. Several chemicals kill weeds, but many of these lack selectivity to distinguish between the crop and the weed. Analogues of active molecules have been synthesized to identify selective herbicides; however, most analogues are either inactive as herbicides or do not show the desired selectivity. A second approach is to identify compounds that either stimulate the metabolism of the herbicide in the treated plant or interfere with the activity of the herbicide by some other mechanism. Such compounds, referred to as herbicide antidotes (or safeners), have to display the selectivity for crops to be commercially useful. Hence, identification of safeners is difficult. An alternative approach is to identify plant genotypes that display tolerance and to transfer the genes for tolerance to commercial cultivars by conventional breeding techniques. While useful, this approach is limited in scope because such genotypes are not always available. With the advent of plant genetic engineering technology, it is now possible to engineer selective tolerance to herbicides by transfer of appropriate genes to crop plants. Plant processes inhibited by herbicides include biosynthesis of essential compounds, photosynthesis, and cell division. Several herbicides inhibit either photosynthesis or amino acid biosynthesis. The reactions of photosynthesis and essential amino acid biosynthesis are unique to plants and microbes, and therefore their inhibitors are potentially nontoxic to animals. Several key differences exist between photosynthesis (PS) and amino acid (AA) biosynthesis inhibitors. The PS inhibitors primarily exert their effects on leaves, the major tissue of plants involved in photosynthesis. Death of nonphotosynthetic tissue is therefore a secondary event. Herbicides such as paraquat and diquat are reduced by electrons derived from photosynthesis. During their reoxidation, they generate activated oxygen such as hydroxyl radicals, which cause membrane damage. These herbicides are therefore catalytic. The AA inhibitors initially block amino acid biosynthesis at the rapidly growing regions of the plant. This is followed by inhibition in other tissues. Death of the plant occurs slowly and may take several days. However, since most cells of the plant are killed, these herbicides are systemic. In general, lower doses of AA herbicides are sufficient to inhibit plant growth. Selective tolerance of crops to either class of herbicides is generally associated with metabolism of the herbicide by the corps. Our knowledge of plant genes and enzymes responsible for amino acid biosynthesis has improved significantly due to interest in engineering herbicide tolerance to crop species. Our understanding of the nature of interaction(s) of herbicides with their target enzymes as well as the chemical mechanisms of the reactions catalyzed by these enzymes has been enhanced due to these efforts. It is expected that several new inhibitors will be synthesized based on these studies that may surpass the capabilities of the current-day herbicides. In this article, we review the current understanding of plant enzymes and genes involved in the biosynthesis of amino acids. In addition, we review the current status of genetic engineering of tolerance to a variety of AA herbicides in plants.
Natural isolates of Plasmodium falciparum represent a genetically heterogeneous population of parasites. To obtain stable strains of the parasites for long term experiments, a rapid and definitive method of cloning was developed using micropipets and a micromanipulator. Homogeneous parasite clones prepared by this technique were characterized and compared with the parent isolates during 4 years of continuous culture. The process of phenotypic dominance and selection of drug resistance which occur in nature was also simulated in vitro by evaluating population dynamics of two cocultured isolates of P. falciparum.
The broadspectrum herbicide glyphosate (N-[phosphonomethyl]-glycine), which causes the accumulation of shikimic acid in plant tissues, inhibits the enzymatic conversion of shikimic acid to anthranilic acid in a cell-free extract of 50% at 5 to 7 μM concentrations. Of the four enzymes involved in the transformation, only 5-enolpyruvylshikimic acid-3-phosphate synthase is inhibited by the herbicide.
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The previous investigation (Abe et al. (1989) J. Biochem. 106, 696-702) suggested that cofilin is deeply involved in the regulation of actin assembly in developing skeletal muscle. In this study, to examine further the function of cofilin in living myogenic cells in culture, recombinant cofilin having extra Cys residues at the N terminus was produced in Escherichia coli and was labeled with tetramethylrhodamine-iodoacetamide (IATMR). When the cofilin labeled with IATMR (IATMR-cofilin) was introduced into myogenic cells, actin filaments in the cytoplasm or nascent myofibrils were promptly disrupted, and many cytoplasmic rods which contained both IATMR-cofilin and actin were generated. Sarcomeric myofibrillar structures were not disrupted but tropomyosin was dissociated from the structures by the exogenous cofilin, and the IATMR-cofilin became localized in I-band regions. 24 hours after the injection, however, the actin-cofilin rods disappeared completely and the IATMR-cofilin became diffused in the cytoplasm as endogenous cofilin. Concomitantly, actin filaments were recovered and tropomyosin was re-associated with sarcomeric I-bands. At this point, the IATMR-cofilin in the cells still retained the functional activity to form intranuclear actin-cofilin rods in response to stimulation by DMSO just as endogenous cofilin. FITC-labeled actin introduced into myogenic cells at first failed to assemble into filamentous structures in the presence of the exogenous cofilin, but was gradually incorporated into myofibrils with time. The drastic effects of the exogenous cofilin on actin assembly were suppressed by phosphatidylinositol 4,5-bisphosphate (PIP2). These results indicate that the exogenous cofilin is active and alters actin dynamics remarkably in muscle cells, but its activity in the cytoplasm gradually becomes regulated by the action of some factors including PIP2-binding.
Ultrastructural evidence is presented for the presence of plastid-like organelles in Toxoplasma gondii, Sarcocystis muris, Babesia ovis, and Plasmodium falciparum. In addition, it was shown that merozoites of T. gondii contain protochlorophyllidae a and traces of chlorophyll a bound to the photosynthetic reaction centers I PS I and PS II. A psbA gene was isolated from merozoites of S. muris by the polymerase chain reaction (PCR). Partial sequencing of the PCR product revealed that the herbicide-binding region is highly conserved. Therefore, it is likely that the sensitivity of apicomplexans to the herbicide toltrazuril depends on the interaction of the herbicide with the D1 protein of the photosynthetic reaction center of the parasite's organelles.
The olfactory epithelium is the only neuronal tissue capable of generating new neurons during adult life and hence must express genes responsible for this phenomenon. Therefore, we have used mRNA from immortalized olfactory epithelial cells to search for novel protein tyrosine kinases by polymerase chain reaction, using as primers conserved sequences from the catalytic domain of known kinase genes. A full-length complementary DNA clone corresponding to one such polymerase chain reaction product was isolated and sequenced. This complementary DNA, designated Kiz-1, encodes a protein containing two prominent domains; the NH2-terminal region contains a cysteine/histidine-rich moiety previously identified as a zinc-finger domain in proteins of the LIM family, while the COOH-terminus contains a kinase domain. Kiz-1 is expressed mainly in the brain of adult mice but also in a range of cultured cell lines, regardless of their tissue of origin. Immunohistochemical studies on adult mouse brain demonstrated that Kiz-1 is expressed exclusively in neurons, not in astrocytes or oligodendrocytes. In the developing embryo, however, Kiz-1 is expressed in all tissues. In COS cells transfected with Kiz-1 complementary DNA and in the immortalized olfactory epithelial cells, Kiz-1 was found mainly in the cytoplasm, but in neurons of the adult brain, it resided also in the nucleus. Two Kiz-1 mRNA species are expressed in cell lines as well as in the murine and human brain. One transcript lacks a region of 60 nucleotides, which lies within the catalytic domain of the kinase and is encoded by a separate exon. Our results suggest that Kiz-1 may play distinct roles in dividing cells and in differentiated neurons.
A sensitive, continuous, spectrophotometric assay for chorismate synthase has been developed utilizing photoreduced flavin mononucleotide (FMNH2) as a cofactor under anaerobic conditions. The assay monitors directly the formation of chorismate from 5-enolpyruvylshikimate-3-phosphate (EPSP) at 275 nm with a precision of +/- 2 microM product. The assay conditions have been optimized with respect to FMNH2 (cofactor), EPSP (substrate) and enzyme concentrations, buffer type, and pH. The potential of the assay for detailed steady-state kinetic studies to elucidate the mechanism of action of this commercially important enzyme is also demonstrated.
Peptides accounting for 157 residues of the bifunctional shikimate pathway enzyme, dehydroquinase/shikimate dehydrogenase, of Pisum sativum were sequenced. Three of the peptides were homologous to regions in Escherichia coli dehydroquinase and two to E. coli shikimate dehydrogenase. The pea dehydroquinase activity was inhibited by treatment with dehydroquinate plus sodium borohydride, establishing it as a type I dehydroquinase. Synthetic oligonucleotides designed from the amino acid sequence were used as PCR primers to amplify fragments of P. sativum cDNA. DNA sequence analysis showed that these amplified products were derived from dehydroquinase/shikimate dehydrogenase cDNA. The complete amino acid sequence of the dehydroquinase domain has been defined; it is homologous to all other type I dehydroquinases and is N-terminal.
In common with other Apicomplexan parasites, Plasmodium falciparum carries two extrachromosomal DNAs, one of which, the 6 kb element, is undoubtedly mitochondrial. The second, generally referred to as the 35 kb circle, is of unknown provenance, but the nature and organization of its genetic content makes a mitochondrial association unlikely and the molecule has features reminiscent of plastid genomes. We now report the occurrence on the circle of an open reading frame specifying a predicted 470 amino acid protein that shares more than 50% identity with a gene currently known only on the plastome of red algae. This high degree of conservation confirms the 35 kb circle's plastid ancestry, and we speculate that it may have originated from the rhodoplast of an ancient red algal endosymbiont in the progenitor of the Apicomplexa.
By low-stringency screening of a human hepatoma HepG2 cell cDNA library, using the genomic fragment of chick c-sea receptor tyrosine kinase as a probe, we isolated overlapping cDNAs encoding a novel protein kinase, which we termed LIM-kinase (LIMK).* The predicted open reading frame encodes a 647-amino-acid polypeptide containing a putative protein kinase structure in the C-terminal half. In addition, LIMK has two repeats of cysteine-rich LIM/double zinc finger motif at the most N-terminus. To our knowledge, this is the first protein kinase seen to contain the LIM motif(s) in the molecule. Although the protein kinase domain of LIMK has highly conserved sequence elements of protein kinases, phylogenetic analysis revealed that LIMK cannot be classified into any subfamily of known protein kinases. Northern blot analysis revealed that the single species of LIMK mRNA of 3.3 kb was expressed in various human epithelial and hematopoietic cell lines. In rat tissues, LIMK mRNA was expressed in the brain, at the highest level. LIM is suggested to be involved in protein-protein interactions by binding to another LIM motif. As the LIM domain is frequently present in the homeodomain-containing transcriptional regulators and oncogenic nuclear proteins, LIMK may be involved in developmental or oncogenic processes through interactions with these LIM-containing proteins.
In tomato (Lycopersicon esculentum L. cv. UC82b) two distinct genes (designated LeCS1 and LeCS2) code for chorismate synthase. The corresponding cDNAs have been isolated and characterized. The deduced amino acid sequences are 88% identical. Both genes encode chorismate synthases with putative plastid-specific N-terminal transit peptides. The two genes are predominantly expressed in flowers and roots and, to a lesser extent, in stems, leaves, and cotyledons, but the steady-state levels of LeCS1-specific transcripts are consistently higher than those of the LeCS2-specific transcripts.
The DNA sequences of a portion of the 5-enolpyruvyl shikimate phosphate synthase domain of the arom gene, encoding the pentafunctional AROM protein, were determined from isolates of Pneumocystis carinii from five mammalian host species (rat, human, ferret, rabbit and mouse). High levels of genetic divergence were found among P. carinii derived from different host species, 7-22% at the DNA sequence level, and 7-26% at the derived amino acid sequence level. Two separate and distinct sequences were isolated from infected ferret lungs. Low levels of divergence were seen in human-derived organisms.
The LIM motif defines a double zinc finger structure found in proteins involved in cell fate determination and growth control. LIM proteins, which include LIM homeo domain, LIM kinase, focal adhesion, and LIM-only proteins, usually contain two or more LIM motifs clustered at their amino- or carboxy-terminal end. At present, the mode of action of the LIM domain is not clear. In this study we have analyzed the binding properties of LIM motifs in the cellular environment. We show that MLP, CRP, and betaCRP define a subclass of LIM-only proteins with unique dual subcellular localization in the nucleus and along actin-based filaments in the cytosol. A double MLP construct that accumulated nearly exclusively along actin filaments promoted myogenic differentiation efficiently, arguing for a functional role of cytoskeleton-associated MLP. Binding of MLP to the actin cytoskeleton is specifically attributable to its second LIM motif. An additional LIM motif potentiates binding. Potentiating LIM motifs can be interchanged, resulting in differential targeting of interacting proteins. To analyze LIM-LIM interactions in situ, this property was exploited to develop a hybrid interaction approach based on the relocalization of LIM-containing constructs to the actin cytoskeleton. These experiments revealed the existence of marked selectivity in the interactions of single LIM motifs, and among LIM domains from different LIM-homeo domain and LIM-only proteins. Furthermore, the analysis suggested that the LIM motif has two interacting interfaces. On the basis of these findings, we propose that LIM motifs function as specific adapter elements to promote the assembly and targeting of multiprotein complexes.
A novel EPSP synthase inhibitor 4 has been designed and synthesized to probe the configurational details of glyphosate recognition in its herbicidal ternary complex with enzyme and shikimate 3-phosphate (S3P). A kinetic evaluation of the new 3-dephospho analog 12, as well as calorimetric and (31)P NMR spectroscopic studies of enzyme-bound 4, now provides a more precise quantitative definition for the molecular interactions of 4 with this enzyme. The very poor binding, relative to 4, displayed by the 3-dephospho analog 12 is indicative that 4 has a specific interaction with the S3P site. A comparison of Ki(calc) for 12 versus the Ki(app) for 4 indicates that the 3-phosphate group in 4 contributes about 4.8 kcal/mol to binding. This compares well with the 5.2 kcal/mol which the 3-phosphate group in S3P contributes to binding. Isothermal titration calorimetry demonstrates that 4 binds to free enzyme with an observed Kd of 0.53 +/- 0.04 microM. As such, 4 binds only 3-fold weaker than glyphosate and about 150-fold better than N-methylglyphosate. Consequently, 4 represents the most potent N-alkylglyphosate derivative identified to date. However, the resulting thermodynamic binding parameters clearly demonstrate that the formation of EPSPS x 4 is entropy driven like S3P. The binding characteristics of 4 are fully consistent with a primary interaction localized at the S3P subsite. Furthermore, (31)P NMR studies of enzyme-bound 4 confirm the expected interaction at the shikimate 3-phosphate site. However, the chemical shift observed for the phosphonate signal of EPSPS x 4 is in the opposite direction than that observed previously when glyphosate binds with enzyme and S3P. Therefore, when 4 occupies the S3P binding site, there is incomplete overlap at the glyphosate phosphonate subsite. As a glyphosate analog inhibitor, the potency of 4 most likely arises from predominant interactions which occur outside the normal glyphosate binding site. Consequently, 4 is best described as an S3P-based substrate-analog inhibitor. These combined results corroborate the previous kinetic model [Gruys, K. J., Marzabadi, M. R., Pansegrau, P. D., & Sikorski, J. A. (1993) Arch. Biochem. Biophys. 304, 345-351], which suggested that 4 interacts well with the S3P subsite but has little, if any, interaction at the expected glyphosate phosphonate or phosphoenolpyruvate-Pi subsites.
Rho, Rac and Cdc42 are three Ras-related GTP-binding proteins that control the assembly and disassembly of the actin cytoskeleton in response to extracellular signals. During the past year, numerous candidate downstream targets for these GTPases have been identified using affinity chromatography and yeast two-hybrid techniques. These techniques have revealed that Rho regulates the myosin light chain phosphatase and that Rho and Rac control the synthesis of phosphatidylinositol 4,5-bisphosphate, two activities that might help to explain the effects of these GTPases on the actin cytoskeleton.
Actin dynamics in lamellipodia are driven by continuous cycles of actin polymerization, retrograde flow, and depolymerization. In the past year, advances have been made in identifying signaling pathways that regulate actin-filament uncapping and polymerization, in determining the role of myosin motor proteins in retrograde flow, and in evaluating the role of severing proteins in actin depolymerization. Both Listeria monocytogenes and Saccharomyces cerevisiae have emerged as powerful model organisms for studying actin dynamics in cells.
MLP is a LIM-only protein of terminally differentiated striated muscle cells, where it accumulates at actin-based structures involved in cytoarchitecture organization. To assess its role in muscle differentiation, we disrupted the MLP gene in mice. MLP (-/-) mice developed dilated cardiomyopathy with hypertrophy and heart failure after birth. Ultrastructural analysis revealed dramatic disruption of cardiomyocyte cytoarchitecture. At birth, these hearts were not hypertrophic, but already abnormally soft, with cell-autonomous and MLP-sensitive alterations in cytoarchitecture. Thus, MLP promotes proper cardiomyocyte cytoarchitecture, whose perturbation can lead to dilated cardiomyopathy. In vivo analysis revealed that MLP-deficient mice reproduce the morphological and clinical picture of dilated cardiomyopathy and heart failure in humans, providing the first model for this condition in a genetically manipulatable organism.