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Actin and profilin do not interfere with each other's binding to Srv2. (A) The presence of profilin does not alter the affinity of Srv2 for G-actin. Addition of 1 M Srv2 or Srv2-201 to 0.2 M NBD-labeled ADP-G-actin resulted in a ~30% increase in the fluorescence. Addition of profilin (0-40 M) did not significantly reduce the NBD fluorescence signal, suggesting that profilin does not affect the binding of Srv2 or Srv2-201 to ADP-G-actin. Standard deviations are indicated by error bars. (B) Actin-binding does not change Srv2 affinity for profilin. Supernatant depletion pull-down assays were carried out with reactions containing 2 M profilin; the average of five independent assays is shown. Lane 1, profilin alone; lanes 2-6, 20 M GST-Srv2 on beads; lanes 3-6, variable concentrations of ADP-G-actin (1, 2, 4, 10 M). Addition of ADP- G-actin does not change the amount of profilin in the supernatant, demonstrating that actin monomers do not interfere with Srv2- profilin interaction. Results using Srv2-201 show that profilin does not bind indirectly to Srv2 through interaction with G-actin in this assay. Standard deviations are indicated by error bars.
Source publication
Profilin and cyclase-associated protein (CAP, known in yeast as Srv2) are ubiquitous and abundant actin monomer-binding proteins. Profilin catalyses the nucleotide exchange on actin monomers and promotes their addition to filament barbed ends. Srv2/CAP recycles newly depolymerized actin monomers from ADF/cofilin for subsequent rounds of polymerizat...
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Context 1
... Titration of the Srv2-201-ADP-G-actin complex yielded very similar results to those obtained with wild-type Srv2, suggesting further that the small decrease in fluorescence of Srv2-ADP-G-actin complexes after addition of high concentrations of profilin does not result from competitive interactions between profilin and actin for binding Srv2 (Fig. ...
Context 2
... profilin did not bind to Srv2 indirectly, through associations with ADP- actin, the same assay was carried out with Srv2-201, which could not deplete profilin from the supernatant. In this case, addition of actin did not decrease the amount of profilin in the supernatant, and only actin was detected in the pellet fractions as analyzed by SDS gels (Fig. 4B, and data not shown). Together, these experiments suggest that Srv2, profilin and ADP-G-actin form a ternary complex in which profilin and ADP-G-actin bind to Srv2 through separate interactions. However, further work is required to confirm the presence of this complex and to reveal the stoichiometry of these proteins in the ...
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Citations
... Microtubule is nucleated from the γ-tubulin ring complex and then assembled into hollow cylindrical polymers with α/β-tubulin heterodimers [17]. Although initially identified as an actin-sequestering protein, increasing evidence suggests that profilins bind to specific polyproline-rich regions in other actin-regulatory proteins [18], such as formins and Srv2 [19,20]. In recent years, profilin has been shown to contain specific residues that enable its direct interaction with microtubules [21]. ...
... The actin-binding site allows profilins to bind to ATP-G-actin, inhibits actin spontaneous nucleation, aids in the polymerization of ATP-G-actin to the positive end of F-actin, and also facilitates the conversion of ADP-G-actin to ATP-G-actin [22][23][24]. The PLP-binding site enables it to bind to specific polyproline-rich regions in other actin-regulatory proteins, such as formins, Srv2, and WASP [19,20,25,26]. The interaction of profilin with PLP-containing proteins is crucial for the spatial-temporal regulation of actin polymerization. ...
... Increasing evidence suggests that in addition to binding actin, profilins also bind to specific polyproline-rich regions in other actin-regulated proteins, and serve as hubs for controlling complex molecular interaction networks [18], such as formins and Srv2 [19,20]. Recently, new research has found that profilins promote the coordination of actin and microtubule systems and modulate microtubule dynamics formins-mediated indirect interaction [30]. ...
Fusarium head blight (FHB), caused by Fusarium graminearum species complexes (FGSG), is an epidemic disease in wheat and poses a serious threat to wheat production and security worldwide. Profilins are a class of actin-binding proteins that participate in actin depolymerization. However, the roles of profilins in plant fungal pathogens remain largely unexplored. Here, we identified FgPfn, a homolog to profilins in F. graminearum, and the deletion of FgPfn resulted in severe defects in mycelial growth, conidia production, and pathogenicity, accompanied by marked disruptions in toxisomes formation and deoxynivalenol (DON) transport, while sexual development was aborted. Additionally, FgPfn interacted with Fgα1 and Fgβ2, the significant components of microtubules. The organization of microtubules in the ΔFgPfn was strongly inhibited under the treatment of 0.4 μg/mL carbendazim, a well-known group of tubulin interferers, resulting in increased sensitivity to carbendazim. Moreover, FgPfn interacted with both myosin-5 (FgMyo5) and actin (FgAct), the targets of the fungicide phenamacril, and these interactions were reduced after phenamacril treatment. The deletion of FgPfn disrupted the normal organization of FgMyo5 and FgAct cytoskeleton, weakened the interaction between FgMyo5 and FgAct, and resulting in increased sensitivity to phenamacril. The core region of the interaction between FgPfn and FgAct was investigated, revealing that the integrity of both proteins was necessary for their interaction. Furthermore, mutations in R72, R77, R86, G91, I101, A112, G113, and D124 caused the non-interaction between FgPfn and FgAct. The R86K, I101E, and D124E mutants in FgPfn resulted in severe defects in actin organization, development, and pathogenicity. Taken together, this study revealed the role of FgPfn-dependent cytoskeleton in development, DON production and transport, fungicides sensitivity in F. graminearum.
... Bertling et al., 2007, Balcer, 2003Rust et al., 2020). We therefore concluded (which was not certified by peer review) is the author/funder. ...
Serum response factor (SRF) is a ubiquitously expressed transcription factor essential for brain development and function. SRF activity is controlled by two competing classes of coactivators, myocardin-related transcription factors (MRTF) and ternary complex factors (TCF), which introduce specificity into gene expression programs. To date, only few brain studies investigated upstream regulatory mechanisms, which mainly focused on TCF. Since an inhibitory function of monomeric actin towards MRTF-SRF signaling is well-established, we hypothesized a regulatory role for the key actin regulator ADF/cofilin. Surprisingly, ADF/cofilin was largely dispensable for neuronal MRTF-SRF activity. Instead, reporter assays combined with pharmacological and genetic approaches in isolated mouse neurons identified cyclase-associated protein 1 (CAP1) as an important regulator of this pathway. CAP1 promotes cytosolic MRTF retention and represses neuronal MRTF-SRF signaling via an actin-dependent mechanism that requires two specific protein domains. Further, deep RNA sequencing and mass spectrometry in mutant mice proved CAP1's in vivo relevance for this pathway in the cerebral cortex, and led to the identification of neuronal MRTF-SRF target genes. Together, we identified CAP1 as a novel and crucial repressor of neuronal MRTF-SRF signaling.
... proteinatlas.org). There is also evidence that adenylyl cyclase-associated protein 1 (CAP1) is involved in regulating functional activity of CFL1 [5] . Indeed, in vivo experiments of yeast have shown that CAP1 is also a physiological partner of profilin during actin reorganization [6] . ...
... CFL1 has been shown to function both independently and in conjunction with CAP1 [5] . CAP1 is involved in the regulation of actin filament remodeling by binding to both forms of actin (i.e., Gactin and F-actin), which determines its role in cancer progression [9][10][11][12] . ...
Circulating tumor cells (CTCs) play an important role in tumor metastases, which is positively correlated with an increased risk of death. Actin-binding proteins, including cofilin (CFL1), profilin 1 (PFN1), and adenylate cyclase-associated protein 1 (CAP1), are thought to be involved in tumor cell motility and metastasis, specifically in head and neck squamous cell carcinoma (HNSCC). However, currently, there are no published studies on CFL1, PFN1, and CAP1 in CTCs and leukocytes in HNSCC patients. We assessed serum levels of CFL1, PFN1, and CAP1 and the number of CTCs and leukocytes containing these proteins in blood from 31 HNSCC patients (T1-4N0-2M0). The analysis used flow cytometry and an enzyme-linked immunosorbent assay kit. We found that CAP1 + CTCs and CAP1 + leukocyte subpopulations were prevalent in these HNSCC patient samples, while the prevalence rates of CFL1 + and PFN1 + CTCs were relatively low. Patients with stage T2-4N1-2M0 had CFL1 + and PFN1 + CTCs with an elevated PFN1 serum level, compared with the T1-3N0M0 group. In summary, the PFN1 serum level and the relative number of PFN1 +CD326 + CTCs could be valuable prognostic markers for HNSCC metastases. The current study is the first to obtain data regarding the contents of actin-binding proteins (ABPs) in CTCs, and leukocytes in blood from HNSCC patients. This is also the first to assess the relationship between the number of CTCs subgroups and disease characteristics.
... To test the relevance of these domains in spines, we expressed myc-tagged CAP1 variants (WT-CAP1, CAP1-HFD, and CAP1-CARP) together with the volume marker GFP and either Cre-mut or Cre in CAP1 flx/flx neurons (Fig. 4A). These experiments also included a CAP1 variant with mutations in a proline-rich domain (CAP1-P1) that reportedly disrupted interaction with the ABP profilin [5,37]. Myc antibody staining confirmed successful triple transfection of CAP1 flx/flx neurons (Fig. S9), and it revealed localization of all mutant variants and of myc-WT-CAP1 in spines from CTR and CAP1-KO neurons (Fig. 4B). ...
The vast majority of excitatory synapses are formed on small dendritic protrusions termed dendritic spines. Dendritic spines vary in size and density that are crucial determinants of excitatory synaptic transmission. Aberrations in spine morphogenesis can compromise brain function and have been associated with neuropsychiatric disorders. Actin filaments (F-actin) are the major structural component of dendritic spines, and therefore, actin-binding proteins (ABP) that control F-actin dis-/assembly moved into the focus as critical regulators of brain function. Studies of the past decade identified the ABP cofilin1 as a key regulator of spine morphology, synaptic transmission, and behavior, and they emphasized the necessity for a tight control of cofilin1 to ensure proper brain function. Here, we report spine enrichment of cyclase-associated protein 1 (CAP1), a conserved multidomain protein with largely unknown physiological functions. Super-resolution microscopy and live cell imaging of CAP1-deficient hippocampal neurons revealed impaired synaptic F-actin organization and dynamics associated with alterations in spine morphology. Mechanistically, we found that CAP1 cooperates with cofilin1 in spines and that its helical folded domain is relevant for this interaction. Moreover, our data proved functional interdependence of CAP1 and cofilin1 in control of spine morphology. In summary, we identified CAP1 as a novel regulator of the postsynaptic actin cytoskeleton that is essential for synaptic cofilin1 activity.
... In addition, the CARP domain, which has nucleotide exchange activity, is in proximity and available to capture newly depolymerized ADP-actin and promote rapid conversion to ATP-actin. It would be interesting to determine whether binding of other proteins to WH2 or prolinerich region in the flexible linker (23,(40)(41)(42) or phosphorylation of CAP (43,44) alters the structure and function of the CAP-actin complex. Thus, our structural model provides mechanistic insight in the function of CAP in the regulation of actin turnover. ...
Cyclase-associated protein (CAP) is a conserved actin-binding protein that regulates multiple aspects of actin dynamics, including polymerization, depolymerization, filament severing, and nucleotide exchange. CAP has been isolated from different cells and tissues in an equimolar complex with actin, and previous studies have shown that a CAP-actin complex contains six molecules each of CAP and actin. Intriguingly, here, we successfully isolated a complex of Xenopus cyclase-associated protein 1 (XCAP1) with actin from oocyte extracts which contained only four molecules each of XCAP1 and actin. This XCAP1-actin complex remained stable as a single population of 340 kDa species during hydrodynamic analyses using gel filtration or analytical ultracentrifugation. Examination of the XCAP1-actin complex by high-speed atomic force microscopy revealed a tripartite structure: one middle globular domain and two globular arms. The two arms were observed in high and low states. The arms at the high state were spontaneously converted to the low state by dissociation of actin from the complex. However, when extra G-actin was added, the arms at the low state were converted to the high state. Based on the known structures of the N-terminal helical-folded domain and C-terminal CARP domain, we hypothesize that the middle globular domain corresponds to a tetramer of the N-terminal helical-folded domain of XCAP1, and that each arm in the high state corresponds to a hetero-tetramer containing a dimer of the C-terminal CARP domain of XCAP1 and two G-actin molecules. This novel configuration of a CAP-actin complex should help to understand how CAP promotes actin filament disassembly.
... In addition, the CARP domain, which has nucleotide exchange activity, is in proximity and available to capture newly depolymerized ADP-actin and promote rapid conversion to ATP-actin. It would be interesting to determine whether binding of other proteins to WH2 or proline-rich region in the flexible linker (22,(38)(39)(40) or phosphorylation of CAP (41,42) alters the structure and function of the CAP-actin complex. Thus, our structural model provides mechanistic insight in the function of CAP in the regulation of actin turnover. ...
Cyclase-associated protein (CAP) is a conserved actin-binding protein that regulates multiple aspects of actin filament dynamics, including polymerization, depolymerization, filament severing, and nucleotide exchange. Intriguingly, CAP has been isolated from different cells and tissues as an equimolar complex with actin, and previous studies have shown that a CAP-actin complex contains six molecules each of CAP and actin. Here, we successfully isolated a complex of Xenopus cyclase-associated protein 1 (XCAP1) and actin from oocyte extracts and demonstrated that the complex contained four molecules each of XCAP1 and actin. The XCAP1-actin complex remained stable as a single population of 340 kDa in hydrodynamic analysis using gel filtration or analytical ultracentrifugation. Examination of the XCAP1-actin complex by high-speed atomic force microscopy revealed a tripartite structure: a middle globular domain and two globular arms. The two arms were connected with the middle globular domain by a flexible linker and observed in two states with different heights, presumably representing the presence or absence of G-actin. We hypothesize that the middle globular domain corresponds to a tetramer of the N-terminal helical-folded domain of XCAP1, and that each arm in the high state corresponds to a hetero-tetramer containing a dimer of the C-terminal CARP domain of XCAP1 and two G-actin molecules. This novel configuration of a CAP-actin complex may represent a functionally important aspect of this complex.
... Interestingly, human CAP also interacts with SH3 domain of the tyrosine kinase abelson murine leukemia viral oncogene homolog 1 (ABL) through its P1 region (Freeman et al., 1996). The functional relevance of CAP's interaction with Profilin or ABL remained elusive, also because P1 mutations only caused very subtle effects in yeast (Bertling et al., 2007). However, genetic studies in D. melanogaster revealed at least functional interaction of CAP with Profilin or ABL, e.g., in determining cell morphology or in growth cone function (Wills et al., 1999(Wills et al., , 2002Benlali et al., 2000). ...
... Biochemical assays confirm the requirement of complete mouse and yeast C-CAP segment for nucleotide exchange of ADF/Cofilin-bound ADP-G-actin, implying that WH2 and CARP domains must be connected for efficient activity in G-actin recharging (Chaudhry et al., 2014;Jansen et al., 2014). In the next step, the only domain of CAP that has comparably higher affinity to ATP-G-actin, i.e., WH2 domain (Chaudhry et al., 2010), might release the polymerization competent G-actin to the surrounding or transfer it to ATP-G-actin-binding proteins such as Profilin (Bertling et al., 2007;Makkonen et al., 2013). Future studies will show which of those mechanisms occurs in vivo. ...
... Briefly, studies in S. cerevisiae revealed a role for CAP in RAS2-cAMP signaling (Fedor-Chaiken et al., 1990;Field et al., 1990), which may depend on CAP's stimulatory effect on post-translational RAS2 modification (Lila and Drubin, 1997). Furthermore, an actin regulatory activity of CAP was first described in yeast (Vojtek et al., 1991;Balcer et al., 2003;Mattila et al., 2004;Bertling et al., 2007). Apart from S. cerevisiae, cellular CAP functions have been studied in the soil-dwelling amoeba Dictyostelium (D.) discoideum. ...
Cyclase-associated protein (CAP) has been discovered three decades ago in budding yeast as a protein that associates with the cyclic adenosine monophosphate (cAMP)-producing adenylyl cyclase and that suppresses a hyperactive RAS2 variant. Since that time, CAP has been identified in all eukaryotic species examined and it became evident that the activity in RAS-cAMP signaling is restricted to a limited number of species. Instead, its actin binding activity is conserved among eukaryotes and actin cytoskeleton regulation emerged as its primary function. However, for many years, the molecular functions as well as the developmental and physiological relevance of CAP remained unknown. In the present article, we will compile important recent progress on its molecular functions that identified CAP as a novel key regulator of actin dynamics, i.e., the spatiotemporally controlled assembly and disassembly of actin filaments (F-actin). These studies unraveled a cooperation with ADF/Cofilin and Twinfilin in F-actin disassembly, a nucleotide exchange activity on globular actin monomers (G-actin) that is required for F-actin assembly and an inhibitory function towards the F-actin assembly factor INF2. Moreover, by focusing on selected model organisms, we will review current literature on its developmental and physiological functions, and we will present studies implicating CAP in human pathologies. Together, this review article summarizes and discusses recent achievements in understanding the molecular, developmental and physiological functions of CAP, which led this protein emerge as a novel CAPt’n of actin dynamics.
... We therefore concluded that CAP1's interaction with ADF/cofilin-actin complexes is crucial in growth cones (Kotila et al., 2019;Moriyama and Yahara, 2002;Quintero-Monzon et al., 2009;Shekhar et al., 2019). Instead, CAP1's nucleotide exchange activity on G-actin that depends on its CARP and WH2 domains as well as C-terminal four aa residues seems to be dispensable in growth cones, very much alike CAP1's interaction with actin regulators such as profilin or Abl1 that depends on PP1 (Bertling et al., 2007;Chaudhry et al., 2010;Kotila et al., 2018;Makkonen et al., 2013;Mattila et al., 2004;Nomura and Ono, 2013). Indeed, we confirmed the relevance of the CAP1-cofilin1 interaction in growth cones in a genetic approach, in which i) we found similar defects in growth cone actin dynamics upon inactivation of either CAP1, cofilin1 or both ABPs, ii) we found a similar increase in (which was not certified by peer review) is the author/funder. ...
Neuron connectivity depends on growth cones that navigate axons through the developing brain. Growth cones protrude and retract actin-rich structures to sense guidance cues. These cues control local actin dynamics and steer growth cones towards attractants and away from repellents, thereby directing axon outgrowth. Hence, actin binding proteins (ABPs) moved into the focus as critical regulators of neuron connectivity. We found cyclase-associated protein 1 (CAP1), an ABP with unknown brain function, abundant in growth cones. Super-resolution microscopy and live cell imaging combined with pharmacological approaches on hippocampal neurons from gene-targeted mice revealed a crucial role for CAP1 in actin dynamics that is critical for growth cone morphology and function. Growth cone defects in mutant neurons compromised neuron differentiation and was associated with impaired neuron connectivity in CAP1 mutant brains. Mechanistically, we found that CAP1 and cofilin1 synergistically control growth cone actin dynamic and morphology. Together, we identified CAP1 as a novel actin regulator in growth cone that is relevant for neuron connectivity.
... Only two proteins are known to catalyze nucleotide exchange on actin monomers, Cyclase-Associated Protein (CAP) and Profilin. Historically Profilin has been depicted as the key driver of actin monomer recycling because it is able to bind the "aged" ADP-bound actin monomers that are released from depolymerizing actin filaments (Bertling et al., 2007;Goldschmidt-Clermont et al., 1992;Kotila et al., 2018;Mockrin and Korn, 1980;Vinson et al., 1998;Wolven et al., 2000). New evidence suggests that CAP can do this more efficiently than Profilin in mammalian systems, and there are organisms where Profilin is not known to catalyze this nucleotide exchange (Kotila et al., 2018;Ono, 2013). ...
Actin and microtubules play essential roles in aberrant cell processes that define and converge in cancer including: signaling, morphology, motility, and division. Actin and microtubules do not directly interact, however shared regulators coordinate these polymers. While many of the individual proteins important for regulating and choreographing actin and microtubule behaviors have been identified, the way these molecules collaborate or fail in normal or disease contexts is not fully understood. Decades of research focus on Profilin as a signaling molecule, lipid-binding protein, and canonical regulator of actin assembly. Recent reports demonstrate that Profilin also regulates microtubule dynamics and polymerization. Thus, Profilin can coordinate both actin and microtubule polymer systems. Here we reconsider the biochemical and cellular roles for Profilin with a focus on the essential cytoskeletal-based cell processes that go awry in cancer. We also explore how the use of model organisms has helped to elucidate mechanisms that underlie the regulatory essence of Profilin in vivo and in the context of disease.
... Decoration occurs preferentially from the pointed ends, since cofilin has a higher affinity towards ADP-actin 9 . Although cofilin bound monomers have a decreased nucleotide exchange rate 12 , the actin binding protein CAP1 acts as a nucleotide exchange factor and thus replenishes the active monomer pool in the prescence of ATP 13,14 alongside profilin [15][16][17] . Additionally, CAP1 increases the overall disassembling activity of cofilin 18 . ...
The dynamics of actin networks is modulated by a machinery consisting of actin binding proteins that control the turnover of filaments in space and time. To study this complex orchestration, in vitro reconstitution approaches strive to project actin dynamics in ideal, minimal systems. To this extent we reconstitute a self-supplying, dense network of globally treadmilling filaments. In this system we analyze growth and intrinsic turnover by means of FRAP measurements and thereby demonstrate how the depletion of monomers and actin binding partners modulate the dynamics in active actin networks. The described effects occur only in dense networks, as single filament dynamics are unable to produce depletion effects to this extent. Furthermore, we demonstrate a synergistic relationship between the nucleators formin and Arp2/3 when branched networks and formin-induced networks are colocalized. As a result, the formin-enhanced filament turnover depletes cofilin at the surface and thus protects the dense, Arp2/3 polymerized network from debranching. Ultimately, these results may be key for understanding the maintenance of the two contradicting requirements of network stability and dynamics in cells.
