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Actin and microtubule cytoskeleton interactions
Abstract
Plant cytoskeleton consists of two major networks of protein polymers, actin microfilaments (AFs) and microtubules (MTs). These networks perform numerous functions that are essential for cell division and for maintaining the integrity of cytoplasm required for intracellular transport and cell shape. Besides the more or less indirect cooperation between AFs and MTs, their direct interactions through specific physically interacting proteins has been well described in yeast, nematodes, insect and animal cells. Recently, promising candidates for corresponding homologous proteins have been identified in plants, although there is still lack of functional evidence for these interactions. Here we summarize recent advances in our knowledge about the candidate proteins or protein complexes that interact with both AFs and MTs and their role in fundamental cellular and developmental processes.
... The cytoskeleton is a highly dynamic system comprising different groups of structural proteins including tubulin, actin, and intermediate filaments to form polymers and associated proteins with diverse regulatory functions. 68 Additionally, cytoskeletal proteins also have activities in cell 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 16 migration, mobility, apoptosis, and immunological synapse formation. 68 TCTP has been reported to be associated with cytoskeleton proteins and related cellular processes. ...
... 68 Additionally, cytoskeletal proteins also have activities in cell 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 16 migration, mobility, apoptosis, and immunological synapse formation. 68 TCTP has been reported to be associated with cytoskeleton proteins and related cellular processes. 17 Six chaperone proteins from the highly conserved HSP70 family were identified to be TCTP-interacting partners by MS (Fig. 2). ...
Translationally controlled tumor protein (TCTP) is a highly conserved housekeeping protein present in eukaryotic organisms. It is involved in regulating many fundamental processes and plays a critical role in tumor reversion and tumorigenesis. Increasing evidence suggests that TCTP plays a role in the regulation of cell fate determination and is a promising therapeutic target for cancer. To decipher the exact mechanisms by which TCTP functions and how all these functions are integrated, we analyzed the interactome of TCTP in HeLa cells by co-immunoprecipitation (IP) and mass spectrometry (MS). A total of 98 proteins were identified. We confirmed the in vitro and in vivo association of TCTP with six of the identified binding proteins using reciprocal IP and bimolecular fluorescence complementation (BiFC) analysis, respectively. Moreover, TCTP interacted with Y-box-binding protein 1 (YBX1), and their interaction was localized to the N-terminal region of TCTP and the 1–129 amino acid (aa) residues of YBX1. The YBX1 protein plays an important role in cell proliferation, RNA splicing, DNA repair, drug resistance and stress response to extracellular signals. These data suggest that the interaction of TCTP with YBX1 might cooperate and/or coordinate their functions in the control of diverse regulatory pathways in cancer cells. Taken together, our results not only reveal a large number of TCTP-associated proteins that possess pleiotropic functions, but also provide novel insights into the molecular mechanisms of TCTP in tumorigenesis.
... For example, microtubules generate the architectural identity of the cytoskeletal arrays that appear in the successive stages of M-phase and these are functionally supported by microfilaments (Claydon and Lloyd, 1985;Schmit and Lambert, 1987;Traas et al., 1987;Sano et al., 2005;Higaki et al., 2008), whereas in the tip-growing root hairs and pollen tubes, the microfilament array is essential and microtubules perform a supporting role (Ketelaar et al., 2003). Several proteins can potentially crosslink microfilaments and microtubules (for a review, see Petrasek and Schwarzerova, 2009;Deeks et al., 2010), facilitating their structural and functional cooperation. ...
... Microfilaments and microtubules have complementary roles in cells and are interconnected by a set of crosslinking proteins (Petrasek and Schwarzerova, 2009). Drug-induced depolymerisation of microtubules affects the organisation of microfilaments and vice versa, indicating that interplay between the different filament networks coordinates their dynamic behaviour (Collings, 2008). ...
... Next, the structures of the thick fibers in the No. 1 line (strongest sfGFP-TP fluorescence) were further assessed by analyzing the concentration-dependent effects of depolymerizing drugs on microtubules and actin filaments (other parts of the cytoskeleton). Although microtubules and actin filaments fulfill many functions independently, they also interact, and this crosstalk is important for plant growth [36][37][38][39]. For instance, microtubules and actin filaments form dynamic associations during interphase in plant cells [36]. ...
Studies on how exogenous molecules modulate properties of plant microtubules, such as their stability, structure, and dynamics, are important for understanding and modulating microtubule functions in plants. We have developed a Tau-derived peptide (TP) that binds to microtubules and modulates their properties by binding of TP-conjugated molecules in vitro. However, there was no investigation of TPs on microtubules in planta. Here, we generated transgenic Arabidopsis thaliana plants stably expressing TP-fused superfolder GFP (sfGFP-TP) and explored the binding properties and effects of sfGFP-TP on plant microtubules. Our results indicate that the expressed sfGFP-TP binds to the plant microtubules without inhibiting plant growth. A transgenic line strongly expressing sfGFP-TP produced thick fibrous structures that were stable under conditions where microtubules normally depolymerize. This study generates a new tool for analyzing and modulating plant microtubules.
... Another protein that may help metaxylem patterning is the ROP effector BDR1, which may recruit Wallin, a protein that promotes actin assembly, to the borders of the gap regions, a process that may help shape the borders of bordered pits [240]. In addition, actin networks can provide physical barriers to microtubules or cross-link with microtubules to redirect them [241,242], suggesting that the actin recruitment in metaxylem may also help create microtubule gaps. ...
... The dynamic network of cytoskeleton participates in defining the cell shape, cellular movement, cell expansion, maintaining the cell polarity, mitotic/meiotic cell division, intracellular trafficking and organization of cell organelles, resistance to mechanical forces, control exocytosis and endocytosis, preventing cell deformation, and several others (Boutt e, Vernhettes, & Satiat-Jeunemaitre, 2007;Shibaoka & Nagai, 1994). There are three types of protein fibers in the cytoskeleton of eukaryotes, viz., microtubules, intermediate filaments, and actin microfilaments; however, the existence of intermediate filaments has been poorly documented in plants, fungi, and most prokaryotes (Petrášek & Schwarzerová, 2009;Pollard & Goldman, 2017). Plant filamentous networks, therefore, constitute microtubules generated from heterodimeric tubulin proteins and globular-actin protein-generated actin microfilaments. ...
The cytoskeleton is the network of polymeric structures which is a highly dynamic component of the plant cells. Cytoskeletal networks have been indicated for almost every intracellular activity, from cell division to cell movement, cell morphogenesis, apoptosis, and cell signaling. In plants, this filamentous network is comprised of microtubules and actin filaments. Cytoskeletal elements are also involved in many signaling pathways, including abiotic stress responses. Deciphering the role of the cytoskeleton requires efficient omics technologies, advanced imaging methods, and molecular genetics. In this chapter, we discuss the structure of actin filaments and microtubules, the available methodologies to study these structures and finally the perception and cytoskeletal response to abiotic stresses such as salinity, drought, or temperature.
... Among good candidates for mechanotransduction is plant-specific phospholipase D delta (PLDd) (Ho et al. 2009;Cvrčkova 2013;Pleskot et al. 2013;Pejchar et al. 2020), a possible mediator in the cell wall-PM-cytoskeleton continuum (Marc et al. 1996;Gardiner et al. 2003) and also able to regulate the activity of both MTs and AFs (Petrasek and Schwarzerova 2009). In particular, PLDd binds to plant flotillin homolog (Ho et al. 2009), a lipid microdomain marker (Martin et al. 2005), constituting the sites where multimolecular signaling complexes containing G-proteins or kinases, flotillin, PLDd, MTs, actin filaments, Hsp70 and others are assembled and take part in cell signaling and vesicle trafficking (Martin et al. 2005;Ho et al. 2009;Tapken and Murphy 2015). ...
Plant cytoskeleton regulation has been studied using a new approach based on both (1) pharmacological analysis of tubulin and actin inhibitors and (2) mechanical stimulation achieved by using a slow-rotating (2 rpm) cli-nostat in combination with transcriptional analysis of genes encoding TUA6, ACT2, MAP65-1, CLASP, PLDd, FH4 and FH1 proteins in Arabidopsis thaliana seedling roots. The obtained data suggest feedback between the organization of microtubule (MT) and actin filament (AF) networks and the expression of the ACT2, TUA6, MAP65-1, CLASP and FH1/FH4 genes. Different regulation of feedback between MT/AF organization and TUA6, ACT2, MAP65-1, CLASP, FH4 and FH1 gene expression was noted during slow clinorotation, possibly due to altered mechanical impact on the cortical cytoskeleton. For the first time, the expression of the tubulin-associated gene MAP65-1 was shown to be dependent upon the organization of AFs. TUA6, MAP65-1, CLASP, FH1 and FH4 likely participate in mechanical signal transduction. Our work demonstrated that slow clinorotation is able to cause mechanical stress.
... Some of these drugs (Figure 9) are based on the oxetane ring such as paclitaxel (PTX, Taxol ® ) and cabazitaxel (Jevtana) acting via microtubule-targeting [74]. Microtubules are cytoskeletal elements that are necessary for many functions including intracellular transport, motility, morphogenesis, and cell division [75,76]. α-β tubulin heterodimers make up the microtubules by assembling in sequence to form the protofilaments of the tube [77]. ...
Pathological angiogenesis is a hallmark of cancer; accordingly, a number of anticancer FDA-approved drugs act by inhibiting angiogenesis via different mechanisms. However, the development process of the most potent anti-angiogenics has met various hurdles including redundancy, multiplicity, and development of compensatory mechanisms by which blood vessels are remodeled. Moreover, identification of broad-spectrum anti-angiogenesis targets is proved to be required to enhance the efficacy of the anti-angiogenesis drugs. In this perspective, a proper understanding of the structure activity relationship (SAR) of the recent anti-angiogenics is required. Various anti-angiogenic classes have been developed over the years; among them, the heterocyclic organic compounds come to the fore as the most promising, with several drugs approved by the FDA. In this review, we discuss the structure–activity relationship of some promising potent heterocyclic anti-angiogenic leads. For each lead, a molecular modelling was also carried out in order to correlate its SAR and specificity to the active site. Furthermore, an in silico pharmacokinetics study for some representative leads was presented. Summarizing, new insights for further improvement for each lead have been reviewed.
... Actin and tubulin were, in general, up-accumulated in both 2-year-old and 8-year-old EC compared to OC. Both make up the two main networks of polymers that comprise the cytoskeleton; they form the shape of the cell, maintain cytoplasmic integrity, respond to cell division, and establish routes for inter and intracellular transport (Petrášek and Schwarzerová 2009). There are various types of either actin or tubulin, and their expression may be tissue-specific: β-tubulin was expressed more in embryogenic Vitis vinifera cultures; however, one form of actin was up-accumulated in embryogenic and a different one was up-accumulated in non-embryogenic cultures (Zhang et al. 2009). ...
Formerly-embryogenic 8-year-old and 2-year-old embryogenic peach palm (Bactris gasipaes Kunth.) cultures were treated with 0, 4, 16, or 64 µM 5-azacytidine, an inhibitor of cytosine methylation, to evaluate its ability to restore embryogenic potential to old cultures and observe its effects on younger embryogenic tissue. Either 16 or 64 µM 5-azacytidine restored the ability of a small proportion of older cultures to produce somatic embryos, but the same concentration also caused somatic embryos from the 2-year-old embryogenic culture line to lose regulation, resulting in non-embryogenic cultures and fast-growing yellow callus. Two-year-old embryogenic tissue had slightly lower global methylation, but only 4 µM 5-azacytidine caused a drop in global methylation. Two-year-old and 5-azacytidine-treated 8-year-old embryogenic cultures both showed an up-accumulation of proteins involved in a number of cellular functions: cellular redox control, anaerobic fermentation, protein degradation, NO-related synthesis and storage, and several forms of epigenetic regulation, including the highly-conserved Argonaute 4 protein; and untreated 8-year-old cultures showed increased amounts of proteins involved in cell wall formation and rearrangement, defense-related peroxidases associated with the cell wall, and phospholipidase D. All three tissues showed proteins involved in hypoxia response. HPLC analysis of 8-year-old cultures with embryogenic and non-embryogenic 2-year-old cultures revealed that 8-year-old cultures lacked any detectable carbohydrates, whereas the younger ones contained measurable amounts of arabinose, ribose, and sucrose. Aging of in vitro cultures is, therefore, likely related to loss of metabolic ability due to constant hypoxia, leading to low vigor, inability to adapt to stress, and loss of embryogenic potential.
... In animal cells several actin-microtubule interactions are identified. These might involve either side interactions or tip-mediated interactions (reviewed in Petrásek and Schwarzerová, 2009). Also in plants there is accumulating evidence for actin-microtubule interactions regulating either cortical cytoskeletal organization or the assembly and progression of mitotic and cytokinetic cytoskeletal arrays. ...
... Interestingly, the OsMAR1-EYFP protein showed a punctuated pattern along the cytoskeleton signals, whereas empty EYFP protein occupied both the cytosol and nucleus in rice protoplasts (Fig. 1b). In plants, the cytoskeletons consist of two major networks of protein polymers, such as actin microfilaments and microtubules (Petrášek et al. 2009). It is well known that two cytoskeleton drugs, latrunculin (Lat) B and oryzalin, can depolymerize microtubules and actin microfilaments, respectively, (Milosavljevic et al. 2003;Hamada et al. 2014). ...
Main conclusion:
Our results suggest that a rice E3 ligase, OsMAR1, physically interacts with a cytosolic protein OCPI2 and may play an important role under salinity stress. Salt is an important abiotic stressor that negatively affects plant growth phases and alters development. Herein, we found that a rice gene, OsMAR1 (Oryza sativa microtubule-associated RING finger protein 1), encoding the RING E3 ligase was highly expressed in response to high salinity, water deficit, and ABA treatment. Fluorescence signals of its recombinant proteins were clearly associated with the microtubules in rice protoplasts. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) showed that OsMAR1 interacted with a cytosolic protein OCPI2 (O. sativa chymotrypsin protease inhibitor 2) and led to its degradation via the 26S proteasome. Heterogeneous overexpression of OsMAR1 in Arabidopsis showed retarded root growth compared with that of control plants, and then led to hypersensitivity phenotypes under high salinity stress. Taken together, OsMAR1 negatively regulates the salt-stress response via the regulation of the OCPI2 protein in rice.
... In this study, OsFH15, identified to the first one in rice, not only binds both AFs and MTs but also crosslinks these cytoskeletons (Figs 6,7 and Supplemental Figs 9,10). AFs and MTs, are mediated by specific bifunctional proteins or multiprotein complexes and often act physically in a coordinated manner 16 . In this report, OsFH15 interacted with both AFs and MTs (Figs 6,7 and Supplemental Figs 9,10), indicating that OsFH15 may be a potential bifunctional protein for these two cytoskeletal components in living plant cells. ...
Grain size is an important agronomic trait determining rice yield and is mainly restricted by spikelet hull size. However, it remains largely unknown how the spikelet hull size is regulated. In this study, OsFH15, a class I formin protein in Oryza sativa, was found to be able to regulate the size of cells and spikelet hull. OsFH15-Cas9 and OsFH15-RNAi mutants had decreased grain size with reduced cell length, cell width and cell area of inner epidermal cells of the lemma compared with wild-type plants. By contrast, OsFH15-overexpressed plants had increased grain size with larger cells, as well as more abundant microtubules (MTs) and actin filaments (AFs) arrays. OsFH15 was mainly expressed in shoot apical meristem (SAM), spikelets, spikelet hulls and seeds in rice. In vitro biochemical experiments showed that OsFH15 can efficiently nucleate actin polymerization with or without profilin, can cap the barbed end of AFs, and can bind and bundle both AFs and MTs. OsFH15 can also crosslink AFs with MTs, and preferentially bind MTs to AFs. These results demonstrated that OsFH15 played an important role in grain-size control by affecting cell expansion through regulating AFs and MTs.
... Accumulative evidences show that these processes not only rely on the specific properties of each type of filament, but also require a tight coordination between their organization and dynamics (Akhshi, Wernike, & Piekny, 2014;Huber, Boire, López, & Koenderink, 2015;Rodriguez et al., 2003). Indeed actin and microtubule crosstalk has been observed in various cell types in yeast, plants, and animals, illustrating their key role in basic cell functions (Akhshi et al., 2014;Petrásek & Schwarzerová, 2009). Cytoskeletal coordination is mediated by common signaling pathways that reciprocally control microtubule and actin networks. ...
Microtubule and actin cytoskeletons are key players in vital processes in cells. Although the importance of microtubule–actin interaction for cell development and function has been highlighted for years, the properties of these two cytoskeletons have been mostly studied separately. Thus we now need procedures to simultaneously assess actin and microtubule properties to decipher the basic mechanisms underlying microtubule–actin crosstalk. Here we describe an in vitro assay that allows the coassembly of both filaments and the real-time observation of their interaction by TIRF microscopy. We show how this assay can be used to demonstrate that tau, a neuronal microtubule-associated protein, is a bona fide actin–microtubule cross-linker. The procedure relies on the use of highly purified proteins and chemically passivated perfusion chambers. We present a step-by-step protocol to obtain actin and microtubule coassembly and discuss the major pitfalls. An ImageJ macro to quantify actin and microtubule interaction is also provided.
... Here, we discuss several candidate proteins capable of binding to actins and to MTs and thereby control their organization and functions. The list includes several kinesins, formins and MT-associated proteins [101,102]. ...
Interactions between actins and microtubules play an important role in many fundamental cellular processes in eukaryotes. Although several studies have shown actins and microtubules to be involved in specific cellular activities, little is known about how actins and microtubules contribute together to a given process. Preprophase band formation, which plays an essential role in plant division site determination, is a cellular process that lends itself to studies of actin-microtubule interactions and how they contribute to important cellular functions. Recently, we have analyzed microtubule-associated microfilaments during preprophase band formation in onion cotyledon epidermal cells using a combination of high-pressure freezing/freeze substitution and electron tomography. Quantitative analysis of our electron tomography data showed that relatively short single microfilaments form bridges between two adjacent microtubules in the process of narrowing of the preprophase microtubule band. Two types of microtubule-microfilament-microtubule connections are observed, and these microfilament-microtubule interactions suggest a direct role of F-actins in microtubule bundling. Based on these observations, we discuss how different actin-microtubule linkers might contribute to preprophase band narrowing and to other changes in microtubule organization in plant cells.
... Microtubules are essential components of the cytoskeleton that confers shape and function in eukaryotic cells. They also play a vital role in intracellular material transportation, organelle positioning, cell movement, signal transduction and cell division [1]. Microtubules should be ideally assembled using a special mode that is convenient for rapid reorganization and decomposition [2]. ...
A previous study found the key transcription factor of Litopenaeus vannamei PERK-eIF2α pathway cyclic AMP-dependent transcription factor 4 (LvATF4) was involved in the transcriptional regulation of white spot syndrome virus (WSSV) gene wsv023. Knocked-down expression of LvATF4 reduced the viral copy number and the cumulative mortality of WSSV-infected shrimp. These results suggested that wsv023 may be critical to WSSV infection but the precise function of wsv023 was still unknown. By using co-immunoprecipitation and pull-down assays, we show that wsv023 interacts with L. vannamei gamma complex-associated protein 2 (LvGCP2), which is the core protein of the γ-tubulin small complex. Knocked-down, the wsv023 gene significantly reduced the copy number of WSSV in L. vannamei muscle, as well as the cumulative mortality of infected shrimp. And PERK-eIF2α pathway inhibition also showed reduced virus copy number and abrogated shrimp mortality. Furthermore, overexpression of wsv023 inhibited the formation of microtubules in 293T cells. Flow cytometry revealed that WSSV infection similarly decreased the formation of microtubules in L. vannamei haemocytes. These findings suggested that wsv023 plays a role in microtubule organization in host cells, which in turn may be beneficial to WSSV.
... Further pharmacological experiments confirmed that the disruption of microfilaments with LatB increased the movement of AtHIR1 (Figure 5e-g). Considering the interaction and crosstalk between microtubules and microfilaments reported in previous studies (Petrasek and Schwarzerova, 2009), we hypothesized that the role of microfilaments in modulating AtHIR1 motility are a consequence of the changes in microtubule organization exerted by the disorganized actin filaments. The membrane microdomains and cytoskeleton can affect the movement of membrane proteins and other macromolecules (Li et al., 2013). ...
Arabidopsis hypersensitive induced reaction (AtHIR) proteins function in plant innate immunity. However, the underlying mechanisms by which AtHIRs participate in plant immunity remain elusive. Here, using VA-TIRFM and FLIM-FRET, we revealed that AtHIR1 is present in membrane microdomains and co-localizes with the membrane microdomain marker REM1.3. Single-particle tracking analysis revealed that membrane microdomains and the cytoskeleton, especially microtubules, restrict the lateral mobility of AtHIR1 at the plasma membrane and facilitate its oligomerization. Furthermore, protein proximity index measurements, fluorescence cross-correlation spectroscopy, and biochemical experiments demonstrated that the formation of the AtHIR1 complex upon pathogen perception requires intact microdomains and cytoskeleton. Taken together, these findings suggest that microdomains and the cytoskeleton constrain AtHIR1 dynamics, promote AtHIR1 oligomerization, and increase the efficiency of the interactions of AtHIR1 with components of the AtHIR1 complex in response to pathogens, thus providing valuable insight into the mechanisms of defense-related responses in plants. This article is protected by copyright. All rights reserved.
... The importance of microtubules and actin filaments in biological activities, such as vesicular and organelle transport, cell and nuclear migration, cell proliferation and division, makes them attractive targets for natural toxins in cancer research [9][10][11]. ...
Alkaloids, the largest group among the nitrogen-containing secondary metabolites of plants, usually interact with several molecular targets. In this study, we provide evidence that six cytotoxic alkaloids (sanguinarine, chelerythrine, chelidonine, noscapine, protopine, homoharringtonine), which are known to affect neuroreceptors, protein biosynthesis and nucleic acids, also interact with the cellular cytoskeleton, such as microtubules and actin filaments, as well. Sanguinarine, chelerythrine and chelidonine depolymerized the microtubule network in living cancer cells (Hela cells and human osteosarcoma U2OS cells) and inhibited tubulin polymerization in vitro with IC50 values of 48.41 ± 3.73, 206.39 ± 4.20 and 34.51 ± 9.47 μM, respectively. However, sanguinarine and chelerythrine did not arrest the cell cycle while 2.5 μM chelidonine arrested the cell cycle in the G₂/M phase with 88.27% ± 0.99% of the cells in this phase. Noscapine and protopine apparently affected microtubule structures in living cells without affecting tubulin polymerization in vitro, which led to cell cycle arrest in the G2/M phase, promoting this cell population to 73.42% ± 8.31% and 54.35% ± 11.26% at a concentration of 80 μM and 250.9 μM, respectively. Homoharringtonine did not show any effects on microtubules and cell cycle, while the known microtubule-stabilizing agent paclitaxel was found to inhibit tubulin polymerization in the presence of MAPs in vitro with an IC50 value of 38.19 ± 3.33 μM. Concerning actin filaments, sanguinarine, chelerythrine and chelidonine exhibited a certain effect on the cellular actin filament network by reducing the mass of actin filaments. The interactions of these cytotoxic alkaloids with microtubules and actin filaments present new insights into their molecular modes of action.
... Their organization and dynamics is modulated by actin binding proteins, such as formins, Arp2/3 complex, profilin, cofilin, myosin etc. (for review see Thomas et al., 2009). An increasing list of proteins interacting with both actin and MTs in plants was reported (for review see Petrasek and Schwarzerova, 2009). The existence of proteins interacting with both AF and MT is not surprising, since actin-cytoskeletal functions are fulfilled in a close collaboration with MT cytoskeleton (Collings, 2008;Smertenko et al., 2010;Sampathkumar et al., 2011), e.g., during plant cell division, in PPB and phragmoplast (Traas et al., 1987;Mineyuki, 1999;Sano et al., 2005;Wu and Bezanilla, 2014). ...
Microtubules (MTs) are involved in key processes in plant cells, including cell division, growth and development. MT-interacting proteins modulate MT dynamics and organization, mediating functional and structural interaction of MTs with other cell structures. In addition to conventional microtubule-associated proteins (MAPs) in plants, there are many other MT-binding proteins whose primary function is not related to the regulation of MTs. This review focuses on enzymes, chaperones, or proteins primarily involved in other processes that also bind to MTs. The MT-binding activity of these multifunctional MAPs is often performed only under specific environmental or physiological conditions, or they bind to MTs only as components of a larger MT-binding protein complex. The involvement of multifunctional MAPs in these interactions may underlie physiological and morphogenetic events, e.g., under specific environmental or developmental conditions. Uncovering MT-binding activity of these proteins, although challenging, may contribute to understanding of the novel functions of the MT cytoskeleton in plant biological processes.
... The recent demonstration that F-actin fragments are translocated along MTs (Sampathkumar et al., 2011) in jasplakinolide-treated interphase cells suggests an interesting mechanism for bringing F-actins attached to one MT into contact with a second MT, so that the MTs can become crosslinked by an F-actin bridge. Molecules capable of forming crosslinks between F-actins and MTs have an actin-binding domain at one end and a MT-binding motif at the other, or they can be composed of a protein complex with one subunit having an actin-binding motif and the second MT-binding motif (Petrasek and Schwarzerova, 2009). Candidate motor proteins for this activity include the kinesin-14 protein family with actin-binding, calponin-homology domains 16 (KCHs) (reviewed in Schneider and Persson 2015). ...
The preprophase band (PPB) is a cytokinetic apparatus that determines the site of cell division in plants. It originates as a broad band of microtubules (MT) in G2 and narrows to demarcate the future division site during late prophase. Studies with fluorescent probes have shown that PPBs contain F-actin during early stages of their development, but become actin-depleted in late prophase. Although this suggests that actins contribute to the early stages of PPB formation, how actins contribute to PPB-MT organization remains unsolved. To address this question, we have used electron tomography to investigate the spatial relationship between microfilaments (MFs) and MTs at different stages of PPB assembly in onion cotyledon epidermal cells. We demonstrate that the PPB actins observed by fluorescence microscopy correspond to short single MFs. A majority of the MFs were bound to MTs, with a subset forming MT-MF-MT bridging structures. During the later stages of PPB assembly the MF-mediated links between MTs were displaced by MT-MT linkers as the PPB MT arrays matured into tightly packed MT bundles. Based on these observations, we propose that the primary function of actins during PPB formation is to mediate the initial bundling of the PPB MTs.
... It consists of two major networks of protein polymers, microfilaments (MFs) and microtubules (MTs). During the cell cycle arrays of MFs and MTs undergo dramatic architectural changes, specifically at the entry into and exit from mitosis (Petrasek and Schwarzerova, 2009). To better understand the changes that occur in cytoskeleton during cell cycle, we studied the effects of protein kinase (staurosporine (ST), olomoucine (OM)) and protein phosphatase (NSC) inhibitors on cytoskeletal components in primary roots of Pisum sativum. ...
... Actin dynamics itself was also reported to be modulated by MTs in protoplasts isolated from tobacco BY-2 cells [60]. Several proteins were reported to interact with both AFs and MTs [61]. Interestingly, proteins associated with the cytoskeleton are emerging as an important group of cytoskeleton crosstalk mediators. ...
Arp2/3 complex plays a fundamental role in the nucleation of actin filaments (AFs) in yeasts, plants, and animals. In plants, the aberrant shaping and elongation of several types of epidermal cells observed in Arp2/3 complex knockout plant mutants suggest the importance of Arp2/3-mediated actin nucleation for various morphogenetic processes. Here we show that ARPC2, a core Arp2/3 complex subunit, interacts with both actin filaments (AFs) and microtubules (MTs). Plant GFP-ARPC2 expressed in Nicotiana tabacum BY-2 cells, leaf epidermal cells of Nicotiana benthamiana and root epidermal cells of Arabidopsis thaliana decorated MTs. The interaction with MTs was demonstrated by pharmacological approach selectively interfering with either AFs or MTs dynamics as well as by the in vitro co-sedimentation assays. A putative MT-binding domain of tobacco NtARPC2 protein was identified using the co-sedimentation of several truncated NtARPC2 proteins with MTs. Newly identified MT-binding ability of ARPC2 subunit of Arp2/3 complex may represent a new molecular mechanism of AFs and MTs interaction.
... The cytoskeleton is an essential cell compartment for the successful completion of most of the structural changes mentioned above. The plant cytoskeleton is a dynamic filamentous network, consisting of actin filaments and microtubules associated with various proteins (Collings 2008;Petrášek and Schwarzerová 2009). This complex system has diverse roles in the life of a cell: it takes part in the formation of cell shape, sets up and maintains cell polarity, transports organelles, and coordinates cell division (Kost and Chua 2002). ...
The improvement of androgenic induction efficiency of anther cultures is an important goal for plant biotechnology. Although n-butanol has been proven to enhance the androgenic induction, the structural background of this effect has not been investigated in detail. In the present study, the cytological and ultrastructural alterations triggered by two treatments that improve androgenic induction, n-butanol (0.2 % n-butanol for 6 h) and cold pretreatment (7 °C for 10 days) were studied in maize anther cultures. Both treatments increased the frequency of responding microspores, and the highest embryo yield (20.9 embryos per 100 plated anthers compared to 0.5/100 anthers in control) was achieved when a combination of both treatments was applied. To study the effect of the treatments on the cytoskeleton, we labeled microtubules using indirect immunofluorescence and actin filaments by rhodamine phalloidin. Cold pretreatment increased the quantity of actin filaments, whereas the microtubule network remained unaffected. In contrast, n-butanol treatment triggered the reversible depolymerization of microtubules, without having any effect on the actin network. Transmission electron microscopy revealed that n-butanol induced the formation of irregular cell walls. Autophagy-related structures were present during the early development of embryogenic microspores following both treatments, but autophagy was only sustained after fourteen days in microspore-derived structures treated with n-butanol. The results support the concept that the androgenic developmental switch is assisted by cytoskeletal rearrangements, which may facilitate androgenic induction through the promotion of symmetric divisions. The longer duration of autophagic processes may also play a role in the elevated embryo induction after n-butanol treatment.
... Though much less is known about their function, microtubules also contribute to some polar expansion phenomena [62,63]. Their roles may include the organization of CesA complexes into organized clusters that yield specific cellulose microfibril orientations necessary for polar growth and/or coordination between cellulose synthesis and delivery of proteins and other molecules in polar growth zones [1]. ...
Polar expansion is a widespread phenomenon in plants spanning all taxonomic groups from the Charophycean Green Algae to pollen tubes in Angiosperms and Gymnosperms. Current data strongly suggests that many common features are shared amongst cells displaying polar growth mechanics including changes to the structural features of localized regions of the cell wall, mobilization of targeted secretion mechanisms, employment of the actin cytoskeleton for directing secretion and in many cases, endocytosis and coordinated interaction of multiple signal transduction mechanisms prompted by external biotic and abiotic cues. The products of polar expansion perform diverse functions including delivery of male gametes to the egg, absorption, anchorage, adhesion and photo-absorption efficacy. A comparative analysis of polar expansion dynamics is provided with special emphasis on those found in early divergent plants.
... PLDα1-derived PA binds to MAP65-1 to regulate microtubule polymerization and bundling under salt stress, but its role in plant-pathogen interaction has not yet been tested . PLDδ has been identified as a cortical microtubule-binding protein (Gardiner et al., 2001(Gardiner et al., , 2003Dhonukshe et al., 2003), and both actin 7 and β-tubulin are co-sedimented with GFP-PLDδ in a pull-down assay from transgenic Arabidopsis suspension cells, suggesting that PLDδ is involved in interactions with both actin filaments and microtubule cytoskeletons, which form a cytoskeletal network (Andreeva et al., 2009;Ho et al., 2009;Petrasek and Schwarzerova, 2009) (Fig. 1). Tubulin binding in turn may activate PLD activity (Chae et al., 2005). ...
Phospholipase Ds (PLDs) and PLD-derived phosphatidic acids (PAs) play vital roles in plant hormonal and environmental responses and various cellular dynamics. Recent studies have further expanded the functions of PLDs and PAs into plant-microbe interaction. The molecular diversities and redundant functions make PLD-PA an important signalling complex regulating lipid metabolism, cytoskeleton dynamics, vesicle trafficking, and hormonal signalling in plant defence through protein-protein and protein-lipid interactions or hormone signalling. Different PLD-PA signalling complexes and their targets have emerged as fast-growing research topics for understanding their numerous but not yet established roles in modifying pathogen perception, signal transduction, and downstream defence responses. Meanwhile, advanced lipidomics tools have allowed researchers to reveal further the mechanisms of PLD-PA signalling complexes in regulating lipid metabolism and signalling, and their impacts on jasmonic acid/oxylipins, salicylic acid, and other hormone signalling pathways that essentially mediate plant defence responses. This review attempts to summarize the progress made in spatial and temporal PLD/PA signalling as well as PLD/PA-mediated modification of plant defence. It presents an in-depth discussion on the functions and potential mechanisms of PLD-PA complexes in regulating actin filament/microtubule cytoskeleton, vesicle trafficking, and hormonal signalling, and in influencing lipid metabolism-derived metabolites as critical signalling components in plant defence responses. The discussion puts PLD-PA in a broader context in order to guide future research.
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... However, observations on wound healing using APM in V. utricularis (Satoh et al. 2000) and protoplast formation in E. verticillata and B. forbeseii (La Claire 1987) indicated no effect on contraction. Ruling out any effect of MT degradation on protoplast contraction would mean that no possible actin-MT interaction exists which recent studies have proven otherwise (Petrášek & Schwarzerová 2009). For instance, crosslinking of MTs and AFs in plants was found to be facilitated by proteins called formins (Deeks et al. 2010). ...
Ultrastructure and cytoskeletal properties of the coenocytic green alga, Valonia, were described using light field, immunofluorescence and electron microscopy to investigate the dynamics among cell wall, cell membrane, and protoplasm during cell regeneration. Protoplasts were artificially induced in three species by cutting thalli and extruding the protoplasm. Protoplasts contracted and formed irregularly shaped masses within 30 minutes concomitant with bundling of actin filaments (AFs), convolution of cortical microtubules (CMTs) and formation of a thin enveloping membrane composed of polysaccha-rides. Size affected survival rates: protoplasts less than 10 μm in diameter displayed lower viability than larger protoplasts. A new cell wall was produced within 24 hours simultaneous with CMT and AF depolymerization. AFs were reduced to granular structures and aggregates that repolymerized by 48 hours. Concurrently, new CMTs polymerized and attained a parallel arrangement. Actin-and microtubule-destabilizing agents had variable effects on protoplast contraction indicating a minor role of intact cytoskeletons in this process; however, resulting cells exhibited abnormal protoplasm distri-bution and cell deformation after three days. Rhizoids began to form after 7 days on untreated cells which subsequently produced lateral branch cells that eventually developed into mature thalli.
Background
Epothilone B (EpoB) is a microtubule‐stabilizing agent with neuroprotective properties.
Objectives
This study examines the regenerative properties of ANA supplemented with EpoB on a sciatic nerve deficit in male Wistar rats.
Methods
For this purpose, the 10 mm nerve gap was filled with acellular nerve allografts (ANAs) containing EpoB at 0.1, 1, and 10 nM concentrations. The sensorimotor recovery was evaluated up to 16 weeks after the operation. Real‐time PCR, histomorphometry analysis, and electrophysiological evaluation were also used to evaluate the process of nerve regeneration.
Results
ANA/EpoB (0.1 nM) significantly improved sensorimotor recovery in rats compared to ANA, ANA/EpoB (1 nM), and ANA/EpoB (10 nM) groups. This led to reduced muscle atrophy, improved sciatic functional index, and thermal paw withdrawal reflex latency, indicating nerve regeneration and target organ reinnervation. The electrophysiological and histomorphometry findings also confirmed the ANA/EpoB regenerative properties (0.1 nM). EpoB only enhanced ANA regenerative properties at 0.1 nM, with no therapeutic effects at higher concentrations.
Conclusion
Totally, we concluded that ANA loaded with 0.1 nM EpoB can effectively reconstruct the transected sciatic nerve in rats, likely by enhancing axonal sprouting and extension.
Plant cytoskeleton consisting of microtubules (MTs) and actin filaments (AFs) plays diverse irreplaceable roles in plant development and response to environmental stimuli. The cytoskeleton forms a highly dynamic and tightly regulated network which offers microenvironments for intra- and inter-cellular communication and physiological events. Plant endomembrane system is composed of membrane-enclosed compartments including the endoplasmic reticulum (ER), the Golgi apparatus, the endosome, the vacuole, etc. These compartments communicate with each other and are essential for cellular activities such as vesicle trafficking, proliferation, immunity and stress response. The ER is the platform for protein synthesis and the initiation site of secretory pathway. Maintenance of plant ER morphology and dynamics mainly depends on actin cytoskeleton. The ER-Cplasma membrane (PM) contact site (EPCS) is a common feature in eukaryotic organisms which is implicated in endomembrane dynamics, cytoskeleton organization and intercellular communication. EPCSs also function as signaling platforms response to environmental stimuli. Cortical cytoskeleton composed of cortical MTs (cMTs) and cortical AFs (cAFs) cooperates with the PM, cell wall and phytohormone to regulate plant cell division, morphogenesis and defense. The interplay between the cellular structures and signals is strictly controlled by diverse factors and signaling pathways. In brief, the endomembrane system cooperates with cytoskeleton network for regulation of plant development and environmental stimuli response and adaption. Here, we summarize the current knowledge to highlight the synergism of plant cytoskeleton network and endomembrane system in regulation of plant development and defense.
Salt stress simultaneously causes ionic toxicity, osmotic stress and oxidative stress, which directly impact plant growth and development. Plants have developed numerous strategies to adapt to saline environments. Whereas some of these strategies have been investigated and exploited for crop improvement, much remains to be understood, including how salt stress is perceived by plants and how plants coordinate effective responses to the stress. It is, however, clear that the plant cell wall is the first contact point between external salt and the plant. In this context, we have achieved significant advances in our understanding of halotropism, cell wall synthesis and integrity surveillance, as well as salt-related cytoskeletal rearrangements. Indeed, molecular mechanisms underpinning some of these processes have recently been elucidated. In this review, we aim to provide insights into how plants respond and adapt to salt stress, with a special focus on primary cell wall biology in the model plant Arabidopsis thaliana.
Rice (Oryza sativa) is one of our main food crops, feeding around 3.5 billion people worldwide. An increasing number of studies note the importance of the cytoskeleton, including actin filaments and microtubules, on rice development and environmental responses. Yet, reliable in vivo cytoskeleton markers are lacking in rice, which critically limits our knowledge of cytoskeletal functions in living cells. Therefore, we generated bright fluorescent marker lines of the actin and microtubule cytoskeletons in rice, suitable for live cell imaging in a wide variety of rice tissues. Using these lines, we show that actin bundles and microtubules engage and co-function during pollen grain development, how the cytoskeletal components are coordinated during root cell development and that the actin cytoskeleton is robust and facilitates microtubule responses during salt stress. Hence, we conclude that our cytoskeletal marker lines, highlighted by our exciting findings of cytoskeletal associations and dynamics, will substantially further future investigations in rice biology.
The formation of diatom silica structures (valves and girdle bands) is controlled by the cytoskele‐ ton. This mini‐review covers the evidence for the role of microtubules and actin microfilaments in this process, as revealed by inhibiting these cytoskeletal elements in diatoms with various symmetry types including centric, as well as raphid and araphid pennates. The degree to which diatoms are affected by inhibitors varies between species; exact morphological abnormalities induced in the valve depend on the stage of valve formation during which inhibitors are administered. These abnormalities are documented and classified. Inhibitors of microtubule polymerization can significantly affect the structure of deposited silica, implying an effect on micromorphogenesis. We also discuss potential applications of cytoskeleton inhibitors in studying the interplay of microtubules and actin microfilaments during valve morphogenesis.
Actin filaments and microtubules are cytoskeletal polymers that form specific three-dimensional arrays to execute essential cellular activities. While these two cytoskeletal systems are often studied separately, accruing evidence indicates that they are interdependent for their organization and function. Here, we use examples of conserved biological processes to discuss how actin-microtubule crosstalk contributes to both plant and animal cell biology.
The eukaryotic cytoplasm is a complex, organized and highly dynamic environment, whose dynamic organization is dependent on a network of filaments and associated proteins. These networks are established, accepted and well studied. The nucleus is no less complex, organized or dynamic, but the existence of an equivalent nuclear network in is not universally accepted and has proved difficult to study. Theoretically there seems an almost overwhelming requirement for a nucleoskeleton, which is needed to provide a structural framework to organize the genome, as well as other subnuclear components, into functionally distinct regions. Such organization must be, and is, highly dynamic so that it can change during development and in response to changing requirements of the cell. Unfortunately, filamentous structures in the nucleus are difficult to detect amongst all the other fibrous material (the chromatin), which has to be removed in order to visualize the putative nucleoskeleton. Therefore, although such structures can be prepared from both plants and animals, their in vivo relevance has remained contentious. Nucleoskeletal filaments have the appearance of intermediate filaments and in fact animal cells have a clearly defined intermediate filament network at the nuclear periphery: the nuclear lamina. Plant cells, however, have no proteins that are clear equivalents of the lamins, or indeed any other intermediate filament protein. In this review we discuss the evidence for nuclear intermediate filament‐like proteins and other potential nucleoskeleton components in plants and discuss their possible roles in plant nuclear organization.
The cortical cytoskeleton, consisting of microtubules and actin filaments, plays a crucial role in the shaping and synthesis of the cell wall. Microtubules and actin filaments engage in cross-talk in plant cells, and this interplay is mediated by a number of molecular interactors and signaling pathways. This work is focused on the interconnected role of actin and microtubules during cell wall formation. Proteins possibly involved in the cross-talk between microtubules and actin filaments for cell wall assembly control are described, and pathways connecting their function in specialized cells with complex shapes are discussed. These include Arabidopsis trichomes, interdigitating epidermal pavement cells, and xylem vessel cells. Mutual interactions between microtubules and actin filaments in these cells are based on restrictive cooperation, often controlled by overlapping regulatory pathways, rather than direct cross-link between both cytoskeletons. A specialized formation of the cytoskeletal structure, the preprophase band, is further discussed as an example of direct microtubules and actin filaments cross-linking.
Since 1999, various forward- and reverse-genetic approaches have uncovered nearly 200 genes required for symbiotic nitrogen fixation (SNF) in legumes. These discoveries advanced our understanding of the evolution of SNF in plants and its relationship to other beneficial endosymbioses, signaling between plants and microbes, control of microbial infection of plant cells, control of plant cell division leading to nodule development, autoregulation of nodulation, intracellular accommodation of bacteria, nodule oxygen homeostasis, control of bacteroid differentiation, metabolism and transport supporting symbiosis, and control of nodule senescence. This review catalogs and contextualizes all of the plant genes currently known to be required for SNF in two model legume species, Medicago truncatula and Lotus japonicus, and two crop species, Glycine max (soybean) and Phaseolus vulgaris (common bean). We also consider, briefly, the future of SNF genetics in the era of pan genomics and genome editing.
The laterally heterogeneous plant plasma membrane (PM) induces its finely controlled compartmentalization into specialized areas including membrane-ordered domains. Recently, PM spatial distribution has emerged with a key role in cell responses to environmental challenges. To monitor the local level of membrane order, BY-2 tobacco suspension cell PMs were labelled with an environment-sensitive probe (di-4-ANEPPDHQ) to evaluate membrane packing. To identify mechanisms and cell components involved in short-term (one hour) maintenance of the ordered domain organization in steady-state cell PMs, four experimental models were compared: modulation of the cytoskeleton or the cell wall integrity of tobacco BY-2 cells; and formation of giant vesicles using either a lipid mixture of tobacco BY-2 cell PMs or the original lipids and proteins combination of tobacco BY-2 cell PM. Whereas inhibiting phosphorylation or disrupting either the cytoskeleton or the cell wall had no observable effect, we observed that lipids and proteins significantly modified both the abundance and spatial distribution of ordered domains. This indicates the involvement of intrinsic membrane components on the local physical state of the plant PM. Our findings thus support a major role for the "lipid raft" model, defined as sterol-dependent ordered assemblies of specific lipids and proteins in plant PM organization.
The actin cytoskeleton plays an essential role in several biological processes in plants, including cell division, cell expansion, organelle movement, vesicle trafficking, and the establishment of polar cell growth. To function properly, actin has to undergo continuous rounds of dynamic remodeling as the plant is presented with a constant stream of endogenous and exogenous signals. Remodeling of the actin cytoskeleton in plants is modulated by a multitude of highly conserved actin-binding proteins (ABPs). In recent years, additional proteins that interact directly or indirectly with actin have been uncovered. Although the precise roles of these newly described proteins have yet to be fully understood, initial studies suggest that they could confer actin functionalities and remodeling mechanisms that are distinct from those found in other eukaryotes. In this chapter, we briefly highlight some of the recent advances toward understanding how the actin cytoskeleton modulates plant growth, form, and adaptation to the environment. We focus primarily on live cell actin tools and on new insights about plant actin and ABP function culminating from the use of such tools. We also discuss some recently discovered plant proteins that function in actin-mediated biological processes that are unique to plants.
Evolutionarily conserved and pleiotropic, the translationally controlled tumor protein (TCTP) is a housekeeping protein present in eukaryotic organisms. It plays an important role in regulating many fundamental processes, such as cell proliferation, cell death, immune responses, and apoptosis. As a result of the pioneer work by Adam Telerman and Robert Amson, the critical role of TCTP in tumor reversion was revealed. Moreover, TCTP has emerged as a regulator of cell fate determination and a promising therapeutic target for cancers. The multifaceted action of TCTP depends on its ability to interact with different proteins. Through this interaction network, TCTP regulates diverse physiological and pathological processes in a context-dependent manner. Complete mapping of the entire sets of TCTP protein interactions (interactome) is essential to understand its various cellular functions and to lay the foundation for the rational design of TCTP-based therapeutic approaches. So far, the global profiling of the interacting partners of TCTP has rarely been performed, but many interactions have been identified in small-scale studies in a specific biological system. This chapter, based on information from protein interaction databases and the literature, illustrates current knowledge of the TCTP interactome.
Cucurbitacins, a class of toxic tetracyclic triterpenoids in Cucurbitaceae, modulate many molecular targets. Here we investigated the interactions of cucurbitacin B, E and I with cytoskeletal proteins such as microtubule and actin filaments. The effects of cucurbitacin B, E and I on microtubules and actin filaments were studied in living cells (Hela and U2OS) and in vitro using GFP markers, immunofluorescence staining and in vitro tubulin polymerization assay. Cucurbitacin B, E and I apparently affected microtubule structures in living cells and cucurbitacin E inhibited tubulin polymerization in vitro with IC 50 value of 566.91 ± 113.5 µM. Cucurbitacin E did not affect the nucleation but inhibited the growth phase and steady state during microtubule assembly in vitro . In addition, cucurbitacin B, E and I all altered mitotic spindles and induced the cell cycle arrest at G2/M phase. Moreover, they all showed potent effects on actin cytoskeleton by affecting actin filaments through the depolymerization and aggregation. The interactions of cucubitacin B, E and I with microtubules and actin filaments present new insights into their modes of action.
The actin cytoskeleton is a dynamic filamentous structure composed of actin and
its associated proteins. In non-motile plant cells, the actin cytoskeleton powers
diverse intracellular motility events and has been implicated in numerous fundamental
physiological cellular processes, including cell division, cytokinesis
and cell morphogenesis. Normally, these actin-based functions are carried out by
the filamentous form of actin. Therefore, one central question in this field is how
actin polymerizes to form diverse dynamic networks in plant cells. Despite lots
of promising progresses have been made on this topic in recent years, many
questions remain to be answered. In this chapter, we will briefly describe the
basic information of the actin cytoskeleton, followed by the description of the
organization and dynamics as well as the regulation of the actin cytoskeleton in
plants.
GhKCH2 belongs to a group of plant-specific kinesins (KCHs) containing an actin-binding calponin homology (CH) domain in the N-terminus. Previous studies revealed that the GhKCH2 CH domain (GhKCH2-CH) had a higher affinity for F-actin (
K
d
= 0.42 ± 0.02 µ
M
) than most other CH-domain-containing proteins. To understand the underlying mechanism, prokaryotically expressed GhKCH2-CH (amino acids 30–166) was purified and crystallized. Crystals were grown by the sitting-drop vapour-diffusion method using 0.1
M
Tris–HCl pH 7.0, 20%(
w
/
v
) PEG 8000 as a precipitant. The crystals diffracted to a resolution of 2.5 Å and belonged to space group
P
2
1
, with unit-cell parameters
a
= 41.57,
b
= 81.92,
c
= 83.00 Å, α = 90.00, β = 97.31, γ = 90.00°. Four molecules were found in the asymmetric unit with a Matthews coefficient of 2.22 Å
3
Da
−1
, corresponding to a solvent content of 44.8%.
The generation of asymmetry, at both cellular and tissue level, is one of the most essential capabilities of all eukaryotic organisms. It mediates basically all multicellular development ranging from embryogenesis and de novo organ formation till responses to various environmental stimuli. In plants, the awe-inspiring number of such processes is regulated by phytohormone auxin and its directional, cell-to-cell transport. The mediators of this transport, PIN auxin transporters, are asymmetrically localized at the plasma membrane, and this polar localization determines the directionality of intercellular auxin flow. Thus, auxin transport contributes crucially to the generation of local auxin gradients or maxima, which instruct given cell to change its developmental program. Here, we introduce and discuss the molecular components and cellular mechanisms regulating the generation and maintenance of cellular PIN polarity, as the general hallmarks of cell polarity in plants.
The cytoskeleton in pollen tubes has been intensively studied, because of its abundance and prominent roles and because the pollen tube is an excellent experimental system for cell biological studies. Pollen actin microfilaments (MFs) exist as multiple distinct populations, each participating in a specific cellular trafficking or organization process. Consequently, MFs are essential for pollen tube growth and are tightly regulated in response to various signals. Pollen microtubules (MTs) are non-essential and less characterized, but recent studies have implicated MTs in vesicle trafficking and cell wall construction in pollen tubes. This review summarizes recent advances in understanding the organization and regulation of both MFs and MTs and discusses their roles in cellular trafficking and the modulation of pollen-tube tip growth.
Plant-pathogen interactions can result in dramatic visual changes in the host, such as galls, phyllody, pseudoflowers, and altered root-system architecture, indicating that the invading microbe has perturbed normal plant growth and development. These effects occur on a cellular level but range up to the organ scale, and they commonly involve attenuation of hormone homeostasis and deployment of effector proteins with varying activities to modify host cell processes. This review focuses on the cellular-reprogramming mechanisms of filamentous and bacterial plant pathogens that exhibit a biotrophic lifestyle for part, if not all, of their lifecycle in association with the host. We also highlight strategies for exploiting our growing knowledge of microbial host reprogramming to study plant processes other than immunity and to explore alternative strategies for durable plant resistance. Expected final online publication date for the Annual Review of Cell and Developmental Biology Volume 31 is October 06, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
The article highlights data about a recently discovered group of regulatory proteins that are able to bind both actin filaments and microtubules and to take part in plant cell growth. Structure and functional activity of formins AtFH1, AtFH4, and AtFH14; phospholipase PLDδ, and kinesin class KCH14 are discussed. Database search of homologues for the above-mentioned proteins has been conducted among various groups of plants.
Microtubules and actin filaments function coordinately in many cellular processes(1-3). Although much of this Coordination is mediated by proteins that statically bridge the two cytoskeletal networks(4-6), kinesin-14 motor; with an actin binding caiponin homology domain (KCHs) have been discovered as putatively dynamic crosslinkers piants(7,8). OsKCH1, KCH from rice, interacts with both71:microtubules and actin filaments in vivo and in vitro However, remained unclear whether this interaction binding rethces or even abniishos the motor's molity rnicrotuimiosY) Here, we directly show in vitro that Osi(Ci"ri is a non-processive minusend -directed motor that transports actin filaments along microtubules: interestingly we observe two distinct transport velocities dependent on the relative orientation of the actin filaments with respect to the microtuhules: in torsional cortmliance rneznAiremontL on molecules reveal low flexibility in 0.sKCH1,. We suggest that the orientation-dependent transport velocities emerge from OsKall's low torsional compliance combined with an inherently oriented binding to the actin filament. Together, our results imply a C1 ntral role of OsIKCH1 in the our orientation of actin filaments along microtubules, and thus a contribution to the organization of the cytoskeicial architecture.
Multicellular organisms have to generate asymmetries in cells and tissues to create different organs. Moreover, several responses to environmental factors are directional and hence require an equal directional response. In plants, such a challenge is accomplished by a multitude of polarly localized proteins that are involved in embryonic and post-embryonic development and dynamic polar responses to the environment. The phytohormone auxin and its polar cell to cell transport play a key role in several of those events providing a mean to coordinate cell and tissue polarities through regulating the polar localization of plasma membrane localized PIN auxin transporters. In this chapter, we discuss the crosstalk between cell trafficking and polarity as a way to integrate external as well as internal signals into asymmetry generation and directional responses in the context of PIN localizations and auxin-dependent processes.
Cell morphogenesis is of great importance for plant cells to fulfill their biological functions. Immobile plant cells achieve their final shapes through polarized cell growth, which is under precise developmental and environmental regulation. The pollen tube system is one of the most-studied models for cell morphogenesis in plants. This chapter highlights recent advances evaluating F-actin-based molecular mechanisms that underlie pollen tube growth, and emphasizes the ROP-dependent regulation of actin organization and dynamics as well as feedback regulation of Rho of Plant (ROP) activity by the actin cytoskeleton. ROP signaling to F-actin is critical to polar cell growth in different cell types, including tip growth in pollen tubes.
The cytoskeleton is a protein-based intracellular superstructure that evolved early after the appearance of bacterial prokaryotes. Eventually cytoskeletal proteins and their macromolecular assemblies were established in eukaryotes and assumed critical roles in cell movements, intracellular organization, cell division and cell differentiation. In biomedicine the small-molecules targeting cytoskeletal elements are in the frontline of anticancer research with plant-derived cytoskeletal drugs such as Vinca alkaloids and toxoids, being routinely used in the clinical practice. Moreover, plants are also major material, food and energy resources for human activities ranging from agriculture, textile industry, carpentry, energy production and new material development to name some few.
Phospholipase D (PLD), in combination with the cytoskeleton, plays a key role in plant signal transduction. One isotype of the multigene Arabidopsis PLD family, AtPLDδ, has been implicated in binding microtubules, although the molecular details of the mechanism and identities of potential interaction partners are unclear. We constructed a GFP-AtPLDδ reporter gene, stably transformed it into an Arabidopsis suspension cell line, and used epitope-tagged affinity pull-down assays to isolate a complex of co-purifying proteins. Mass spectrometry analysis of the complex revealed a set of proteins including β-tubulin, actin 7, HSP70, clathrin heavy chain, ATP synthase subunits, and a band 7-4/flotillin homologue. Sequence alignments with defined tubulin- and actin-binding regions from human HsPLD2 revealed highly homologous regions in all 12 AtPLD isotypes, suggesting direct interactions of AtPLDδ with tubulin and actin, while interactions with the remaining partners are likely to be mediated by the cytoskeleton. We propose that AtPLDδ acts through a complex of cytoskeletal and partner proteins to modulate fundamental cellular processes such as cytoskeletal rearrangements, vesicular trafficking, assembly of Golgi apparatus, mitosis and cytokinesis.
Interactions between microtubules and actin are a basic phenomenon that underlies many fundamental processes in which dynamic cellular asymmetries need to be established and maintained. These are processes as diverse as cell motility, neuronal pathfinding, cellular wound healing, cell division and cortical flow. Microtubules and actin exhibit two mechanistic classes of interactions — regulatory and structural. These interactions comprise at least three conserved 'mechanochemical activity modules' that perform similar roles in these diverse cell functions.
Organelle movement is an evident feature of pollen tubes and is essential for the process of tube growth because it enables
the proper distribution of organelles and the accumulation of secretory vesicles in the tube apex. Organelles move along the
actin filaments through dynamic interactions with myosin but other proteins are probably responsible for control of this activity.
The role of microtubules and microtubule-based motors is less clear and somewhat enigmatic. Nevertheless, the pollen tube
is an excellent cell model in which to study and analyse the molecular mechanisms that drive and control organelle motility
in relation to plant cell expansion. Current knowledge and the main scientific discoveries in this field of research over
the last 20 years are summarized here. Future prospects in the study of the molecular mechanisms that mediate organelle transport
and vesicle accumulation during pollen tube elongation are also discussed.
The involvement of myosin XI in generating the motive force for cytoplasmic streaming in plant cells is becoming evident. For a comprehensive understanding of the physiological roles of myosin XI isoforms, it is necessary to elucidate the properties and functions of each isoform individually. In tobacco cultured BY-2 cells, two types of myosins, one composed of 175 kDa heavy chain (175 kDa myosin) and the other of 170 kDa heavy chain (170 kDa myosin), have been identified biochemically and immunocytochemically. From sequence analyses of cDNA clones encoding heavy chains of 175 kDa and 170 kDa myosin, both myosins have been classified as myosin XI. Immunocytochemical studies using a polyclonal antibody against purified 175 kDa myosin heavy chain showed that the 175 kDa myosin is distributed throughout the cytoplasm as fine dots in interphase BY-2 cells. During mitosis, some parts of 175 kDa myosin were found to accumulate in the pre-prophase band (PPB), spindle, the equatorial plane of a phragmoplast and on the circumference of daughter nuclei. In transgenic BY-2 cells, in which an endoplasmic reticulum (ER)-specific retention signal, HDEL, tagged with green fluorescent protein (GFP) was stably expressed, ER showed a similar behaviour to that of 175 kDa myosin. Furthermore, this myosin was co-fractionated with GFP-ER by sucrose density gradient centrifugation. From these findings, it was suggested that the 175 kDa myosin is a molecular motor responsible for translocating ER in BY-2 cells.
Formin proteins, characterized by the presence of conserved formin homology (FH) domains, play important roles in cytoskeletal regulation via their abilities to nucleate actin filament formation and to interact with multiple other proteins involved in cytoskeletal regulation. The C-terminal FH2 domain of formins is key for actin filament interactions and has been implicated in playing a role in interactions with microtubules. Inverted formin 1 (INF1) is unusual among the formin family in having the conserved FH1 and FH2 domains in its N-terminal half, with its C-terminal half being composed of a unique polypeptide sequence. In this study, we have examined a potential role for INF1 in regulating microtubule structure. INF1 associates discretely with microtubules, and this association is dependent on a novel C-terminal microtubule-binding domain. INF1 expressed in fibroblast cells induced actin stress fiber formation, coalignment of microtubules with actin filaments, and the formation of bundled, acetylated microtubules. Endogenous INF1 showed an association with acetylated microtubules, and knockdown of INF1 resulted in decreased levels of acetylated microtubules. Our data suggests a role for INF1 in microtubule modification and potentially in coordinating microtubule and F-actin structure.
The microtubule network is thought to be used for long-range transport of cellular components in animal cells whereas the actin network is proposed to be used for short-range transport, although the mechanism(s) by which this transport is coordinated is poorly understood. For example, in sea urchins long-range Ca2+-regulated transport of exocytotic vesicles requires a microtubule-based motor, whereas an actin-based motor is used for short-range transport. In neurons, microtubule-based kinesin motor proteins are used for long-range vesicular transport but microtubules do not extend into the neuronal termini, where actin filaments form the cytoskeletal framework, and kinesins are rapidly degraded upon their arrival in neuronal termini, indicating that vesicles may have to be transferred from microtubules to actin tracks to reach their final destination. Here we show that an actin-based vesicle-transport motor, MyoVA, can interact directly with a microtubule-based transport motor, KhcU. As would be expected if these complexes were functional, they also contain kinesin light chains and the localization of MyoVA and KhcU overlaps in the cell. These results indicate that cellular transport is, in part, coordinated through the direct interaction of different motor molecules.
We have discovered evidence for a physical interaction between a class V myosin, Myo2p, and a kinesin-related protein, Smy1p, in budding yeast. These proteins had previously been linked by genetic and colocalization studies, but we had been unable to determine the nature of their association. We now show by two-hybrid analysis that a 69-amino acid region of the Smy1p tail interacts with the globular portion of the Myo2p tail. Deletion of this myosin-binding region of Smy1p eliminates its ability to colocalize with Myo2p and to overcome the myo2-66 mutant defects, suggesting that the interaction is necessary for these functions. Further insights about the Smy1p-Myo2p interaction have come from studies of a new mutant allele, myo2-2, which causes a loss of Myo2p localization. We report that Smy1p localization is also lost in the myo2-2 mutant, demonstrating that Smy1p localization is dependent on Myo2p. We also found that overexpression of Smy1p partially restores myo2-2p localization in a myosin-binding region-dependent manner. Thus, overexpression of Smy1p can overcome defects in both the head and tail domains of Myo2p (caused by the myo2-66 and myo2-2 alleles, respectively). We propose that Smy1p enhances some aspect of Myo2p function, perhaps delivery or docking of vesicles at the bud tip.
Spatial control of cytokinesis in plant cells depends on guidance of the cytokinetic apparatus, the phragmoplast, to a cortical "division site" established before mitosis. Previously, we showed that the Tangled1 (Tan1) gene of maize is required for this process during maize leaf development (Cleary, A.L., and L.G. Smith. 1998. Plant Cell. 10:1875-1888.). Here, we show that the Tan1 gene is expressed in dividing cells and encodes a highly basic protein that can directly bind to microtubules (MTs). Moreover, proteins recognized by anti-TAN1 antibodies are preferentially associated with the MT-containing cytoskeletal structures that are misoriented in dividing cells of tan1 mutants. These results suggest that TAN1 protein participates in the orientation of cytoskeletal structures in dividing cells through an association with MTs.
The organization of microtubule arrays in the plant cell cortex involves interactions with the plasma membrane, presumably through protein bridges. We have used immunochemistry and monoclonal antibody 6G5 against a candidate bridge protein, a 90-kD tubulin binding protein (p90) from tobacco BY-2 membranes, to characterize the protein and isolate the corresponding gene. Screening an Arabidopsis cDNA expression library with the antibody 6G5 produced a partial clone encoding phospholipase D (PLD), and a full-length gene was obtained by sequencing a corresponding expressed sequence tag clone. The predicted protein of 857 amino acids contains the active sites of a phospholipid-metabolizing enzyme and a Ca(2+)-dependent lipid binding domain and is identical to Arabidopsis PLD delta. Two amino acid sequences obtained by Edman degradation of the tobacco p90 are identical to corresponding segments of a PLD sequence from tobacco. Moreover, immunoprecipitation using the antibody 6G5 and tobacco BY-2 protein extracts gave significant PLD activity, and PLD activity of tobacco BY-2 membrane proteins was enriched 6.7-fold by tubulin-affinity chromatography. In a cosedimentation assay, p90 bound and decorated microtubules. In immunofluorescence microscopy of intact tobacco BY-2 cells or lysed protoplasts, p90 colocalized with cortical microtubules, and taxol-induced microtubule bundling was accompanied by corresponding reorganization of p90. Labeling of p90 remained along the plasma membrane when microtubules were depolymerized, although detergent extraction abolished the labeling. Therefore, p90 is a specialized PLD that associates with membranes and microtubules, possibly conveying hormonal and environmental signals to the microtubule cytoskeleton.
CHO1 is a kinesin-like protein of the mitotic kinesin-like protein (MKLP)1 subfamily present in central spindles and midbodies in mammalian cells. It is different from other subfamily members in that it contains an extra approximately 300 bp in the COOH-terminal tail. Analysis of the chicken genomic sequence showed that heterogeneity is derived from alternative splicing, and exon 18 is expressed in only the CHO1 isoform. CHO1 and its truncated isoform MKLP1 are coexpressed in a single cell. Surprisingly, the sequence encoded by exon 18 possesses a capability to interact with F-actin, suggesting that CHO1 can associate with both microtubule and actin cytoskeletons. Microinjection of exon 18-specific antibodies did not result in any inhibitory effects on karyokinesis and early stages of cytokinesis. However, almost completely separated daughter cells became reunited to form a binulceate cell, suggesting that the exon 18 protein may not have a role in the formation and ingression of the contractile ring in the cortex. Rather, it might be involved directly or indirectly in the membrane events necessary for completion of the terminal phase of cytokinesis.
The microtubule cytoskeleton is a dynamic filamentous structure involved in many key processes in plant cell morphogenesis including nuclear and cell division, deposition of cell wall, cell expansion, organelle movement and secretion. The principal microtubule protein is tubulin, which associates to form the wall of the tubule. In addition, various associated proteins bind microtubules either to anchor, cross-link or regulate the microtubule network within cells. Biochemical, molecular biological and genetic approaches are being successfully used to identify these microtubule-associated proteins (MAPs) in plants, and we describe recent progress on three of these proteins.
A phospholipase D (PLD) was shown recently to decorate microtubules in plant cells. Therefore, we used tobacco BY-2 cells expressing the microtubule reporter GFP-MAP4 to test whether PLD activation affects the organization of plant microtubules. Within 30 min of adding n-butanol, a potent activator of PLD, cortical microtubules were released from the plasma membrane and partially depolymerized, as visualized with four-dimensional confocal imaging. The isomers sec- and tert-butanol, which did not activate PLD, did not affect microtubule organization. The effect of treatment on PLD activation was monitored by the in vivo formation of phosphatidylbutanol, a specific reporter of PLD activity. Tobacco cells also were treated with mastoparan, xylanase, NaCl, and hypoosmotic stress as reported activators of PLD. We confirmed the reports and found that all treatments induced microtubule reorganization and PLD activation within the same time frame. PLD still was activated in microtubule-stabilized (taxol) and microtubule-depolymerized (oryzalin) situations, suggesting that PLD activation triggers microtubular reorganization and not vice versa. Exogenously applied water-soluble synthetic phosphatidic acid did not affect the microtubular cytoskeleton. Cell cycle studies revealed that n-butanol influenced not just interphase cortical microtubules but also those in the preprophase band and phragmoplast, but not those in the spindle structure. Cell growth and division were inhibited in the presence of n-butanol, whereas sec- and tert-butanol had no such effects. Using these novel insights, we propose a model for the mechanism by which PLD activation triggers microtubule reorganization in plant cells.
In plants, it is unclear how dispersed cortical microtubules are nucleated, polarized and organized in the absence of centrosomes. In Arabidopsis thaliana cells, expression of a fusion between the microtubule-end-binding protein AtEB1a and green fluorescent protein (GFP) results in labelling of spindle poles, where minus ends gather. During interphase, AtEB1a-GFP labels the microtubule plus end as a comet, but also marks the minus end as a site from which microtubules can grow and shrink. These minus-end nucleation sites are mobile, explaining how the cortical array can redistribute during the cell cycle and supporting the idea of a flexible centrosome in plants.
Actin filaments and microtubules are two major cytoskeletal systems involved in wide cellular processes, and the organizations of their filamentous networks are regulated by a large number of associated proteins. Recently, evidence has accumulated for the functional cooperation between the two filament systems via associated proteins. However, little is known about the interactions of the kinesin superfamily proteins, a class of microtubule-based motor proteins, with actin filaments. Here, we describe the identification and characterization of a novel kinesin-related protein named DdKin5 from Dictyostelium. DdKin5 consists of an N-terminal conserved motor domain, a central stalk region, and a C-terminal tail domain. The motor domain showed binding to microtubules in an ATP-dependent manner that is characteristic of kinesin-related proteins. We found that the C-terminal tail domain directly interacts with actin filaments and bundles them in vitro. Immunofluorescence studies showed that DdKin5 is specifically enriched at the actin-rich surface protrusions in cells. Overexpression of the DdKin5 protein affected the organization of actin filaments in cells. We propose that a kinesin-related protein, DdKin5, is a novel actin-bundling protein and a potential cross-linker of actin filaments and microtubules associated with specific actin-based structures in Dictyostelium.
Involvement of conservative molecular modules and cellular mechanisms in the widely diversified processes of eukaryotic cell morphogenesis leads to the intriguing question: how do similar proteins contribute to dissimilar morphogenetic outputs. Formins (FH2 proteins) play a central part in the control of actin organization and dynamics, providing a good example of evolutionarily versatile use of a conserved protein domain in the context of a variety of lineage-specific structural and signalling interactions.
In order to identify possible plant-specific sequence features within the FH2 protein family, we performed a detailed analysis of angiosperm formin-related sequences available in public databases, with particular focus on the complete Arabidopsis genome and the nearly finished rice genome sequence. This has led to revision of the current annotation of half of the 22 Arabidopsis formin-related genes. Comparative analysis of the two plant genomes revealed a good conservation of the previously described two subfamilies of plant formins (Class I and Class II), as well as several subfamilies within them that appear to predate the separation of monocot and dicot plants. Moreover, a number of plant Class II formins share an additional conserved domain, related to the protein phosphatase/tensin/auxilin fold. However, considerable inter-species variability sets limits to generalization of any functional conclusions reached on a single species such as Arabidopsis.
The plant-specific domain context of the conserved FH2 domain, as well as plant-specific features of the domain itself, may reflect distinct functional requirements in plant cells. The variability of formin structures found in plants far exceeds that known from both fungi and metazoans, suggesting a possible contribution of FH2 proteins in the evolution of the plant type of multicellularity.
Proper spindle positioning and orientation are essential for asymmetric cell division and require microtubule-actin filament (F-actin) interactions in many systems. Such interactions are particularly important in meiosis, where they mediate nuclear anchoring, as well as meiotic spindle assembly and rotation, two processes required for asymmetric cell division. Myosin-10 proteins are phosphoinositide-binding, actin-based motors that contain carboxy-terminal MyTH4 and FERM domains of unknown function. Here we show that Xenopus laevis myosin-10 (Myo10) associates with microtubules in vitro and in vivo, and is concentrated at the point where the meiotic spindle contacts the F-actin-rich cortex. Microtubule association is mediated by the MyTH4-FERM domains, which bind directly to purified microtubules. Disruption of Myo10 function disrupts nuclear anchoring, spindle assembly and spindle-F-actin association. Thus, this myosin has a novel and critically important role during meiosis in integrating the F-actin and microtubule cytoskeletons.
A novel kinesin, GhKCH1, has been identified from cotton (Gossypium hirsutum) fibers. GhKCH1 has a centrally located kinesin catalytic core, a signature neck peptide of minus end-directed kinesins, and a unique calponin homology (CH) domain at its N terminus. GhKCH1 and other CH domain-containing kinesins (KCHs) belong to a distinct branch of the minus end-directed kinesin subfamily. To date the KCH kinesins have been found only in higher plants. Because the CH domain is often found in actin-binding proteins, we proposed that GhKCH1 might play a role in mediating dynamic interaction between microtubules and actin microfilaments in cotton fibers. In an in vitro actin-binding assay, GhKCH1's N-terminal region including the CH domain interacted directly with actin microfilaments. In cotton fibers, GhKCH1 decorated cortical microtubules in a punctate manner. Occasionally GhKCH1 was found to be associated with transverse-cortical actin microfilaments, but never with axial actin cables in cotton fibers. Localization of GhKCH1 on cortical microtubules was independent of the integrity of actin microfilaments. Thus, GhKCH1 may play a role in organizing the actin network in coordination with the cortical microtubule array. These data also suggest that flowering plants may employ unique KCHs to coordinate actin microfilaments and microtubules during cell growth.
A key event in plant morphogenesis is the establishment of a division plane. A plant-specific microtubular preprophase band (PPB) accurately predicts the line of cell division, whereas the phragmoplast, another plant-specific array, executes cell division by maintaining this predicted line. Although establishment of these specific arrays apparently involves intracellular repolarization events that focus cellular resources to a division site, it still remains unclear how microtubules position the cell division planes. Here we study GFP-AtEB1 decorated microtubule plus-ends to dissect events at the division plane.
Early mitotic events included guided growth of endoplasmic microtubules (EMTs) towards the PPB site and their coincident localization with endocytic vesicles. Consequently, an endosomal belt lay in close proximity to the microtubular PPB at its maturation and was maintained during spindle formation. During cytokinesis, EMTs radiated from the former spindle poles in a geometrical conformation correlating with cell-plate navigation and tilt-correction. Naphthylphtalamic acid (NPA), an inhibitor of polar auxin efflux, caused abnormal PPBs and shifted division planes.
Our observations reveal a spatio-temporal link between microtubules and intracellular polarization essential for localized endocytosis and precise establishment of the division plane in plants. Additionally, they implicate the growth regulator, auxin, in this important cellular event.
In a previous study on Arabidopsis thaliana suspension cells transiently infected with the microtubule end binding protein AtEB1a-green fluorescent protein (GFP), we reported that interphase microtubules grow from multiple sites dispersed over the cortex, with plus ends forming the characteristic comet-like pattern. In this study, AtEB1a-GFP was used to study the transitions of microtubule arrays throughout the division cycle of cells lacking a defined centrosome. During division, the dispersed origin of microtubules was replaced by a more focused pattern with the plus end comets growing away from sites associated with the nuclear periphery. The mitotic spindle then evolved in two quite distinct ways depending on the presence or absence of the preprophase band (PPB): the cells displaying outside-in as well as inside-out mitotic pathways. In those cells possessing a PPB, the fusion protein labeled material at the nuclear periphery that segregated into two polar caps, perpendicular to the PPB, before nuclear envelope breakdown (NEBD). These polar caps then marked the spindle poles upon NEBD. However, in the population of cells without PPBs, there was no prepolarization of material at the nuclear envelope before NEBD, and the bipolar spindle only emerged clearly after NEBD. Such cells had variable spindle orientations and enhanced phragmoplast mobility, suggesting that the PPB is involved in a polarization event that promotes early spindle pole morphogenesis and subsequent positional stability during division. Astral-like microtubules are not usually prominent in plant cells, but they are clearly seen in these Arabidopsis cells, and we hypothesize that they may be involved in orienting the division plane, particularly where the plane is not determined before division.
The ability to establish cell polarity is crucial to form and function of an individual cell. Polarity underlies critical processes during cell development, such as cell growth, cell division, cell differentiation and cell signalling. Interphase cytoplasmic microtubules in tip‐growing fission yeast cells have been shown to play a particularly important role in regulating cell polarity. By placing proteins that serve as spatial cues in the cell cortex of the expanding tip, microtubules determine the site where excocytosis, and therefore growth, takes place. Transport and the targeting of exocytotic vesicles to the very tip depend on the actin cytoskeleton. Recently, endoplasmic microtubules have been identified in tip‐growing root hairs, which are an experimental system for plant cell growth. Here, we review the data that demonstrate involvement of microtubules in hair elongation and polarity of the model plants Medicago truncatula and Arabidopsis thaliana . Differences and similarities between the microtubule organization and function in these two species are discussed and we compare the observations in root hairs with the microtubule‐based polarity mechanism in fission yeast. Contents
Summary
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I.
Introduction
711
II.
Root hair tip growth
712
III.
Microtubule organization and dynamics in elongating root hairs
713
IV.
Microtubules and their role in root hair cell polarity
714
V.
Microtubules and their putative role in targeting polarity markers to the very tip of elongating root hairs: what we might learn from Schizosaccharomyces pombe
716
VI.
Conclusion
717
References
717
Kinesins and kinesin-like proteins (KLPs) constitute a large family of microtubule-based motors that play important roles in many fundamental cellular and developmental processes. To date, a number of kinesins or KLPs have been identified in plants including Arabidopsis thaliana. Here, a polyclonal antibody against AtKP1 (kinesin-like protein 1 in A. thaliana) was raised by injection the expressed AtKP1 specific C-terminal polypeptides in rabbits, and immunoblot analysis was conducted with the affinity-purified anti-AtKP1 antibody. The results indicated that this antibody recognized the AtKP1 fusion proteins expressed in E. coli and proteins of ~125 kDa in the soluble fractions of Arabidopsis extracts. The molecular weight was consistent with the calculated molecular weight based on deduced amino acids sequence of AtKP1. To acquire the subcellular localization of the protein, AtKP1 in Arabidopsis root cells was observed by indirect immunofluorescence microscopy. AtKP1 was localized to particle-like organelles in interphase or dividing cells, but not to mitotic microtubule arrays. Relatively more AtKP1 was found in isolated mitochondria fraction on immunoblot of the subcellular fractions. The AtKP1 protein could not be released following a 0.6 M KI washing, indicating that AtKP1 is tightly bind to mitochondria and might function associated with this kind of organelles.
The cytoskeleton plays important roles in plant cell shape determination by influencing the patterns in which cell wall materials are deposited. Cortical microtubules are thought to orient the direction of cell expansion primarily via their influence on the deposition of cellulose into the wall, although the precise nature of the microtubule-cellulose relationship remains unclear. In both tip-growing and diffusely growing cell types, F-actin promotes growth and also contributes to the spatial regulation of growth. F-actin has been proposed to play a variety of roles in the regulation of secretion in expanding cells, but its functions in cell growth control are not well understood. Recent work highlighted in this review on the morphogenesis of selected cell types has yielded substantial new insights into mechanisms governing the dynamics and organization of cytoskeletal filaments in expanding plant cells and how microtubules and F-actin interact to direct patterns of cell growth. Nevertheless, many important questions remain to be answered.
The actin-nucleation factors Spire and Cappuccino (Capu) regulate the onset of ooplasmic streaming in Drosophila melanogaster. Although this streaming event is microtubule-based, actin assembly is required for its timing. It is not understood how the interaction of microtubules and microfilaments is mediated in this context. Here, we demonstrate that Capu and Spire have microtubule and microfilament crosslinking activity. The spire locus encodes several distinct protein isoforms (SpireA, SpireC and SpireD). SpireD was recently shown to nucleate actin, but the activity of the other isoforms has not been addressed. We find that SpireD does not have crosslinking activity, whereas SpireC is a potent crosslinker. We show that SpireD binds to Capu and inhibits F-actin/microtubule crosslinking, and activated Rho1 abolishes this inhibition, establishing a mechanistic basis for the regulation of Capu and Spire activity. We propose that Rho1, cappuccino and spire are elements of a conserved developmental cassette that is capable of directly mediating crosstalk between microtubules and microfilaments.
Expression of a functional yellow fluorescent protein fusion to cellulose synthase (CESA) in transgenic Arabidopsis plants allowed the process of cellulose deposition to be visualized in living cells. Spinning disk confocal microscopy revealed
that CESA complexes in the plasma membrane moved at constant rates in linear tracks that were aligned and were coincident
with cortical microtubules. Within each observed linear track, complex movement was bidirectional. Inhibition of microtubule
polymerization changed the fine-scale distribution and pattern of moving CESA complexes in the membrane, indicating a relatively
direct mechanism for guidance of cellulose deposition by the cytoskeleton.
Adenomatous polyposis coli (APC) protein is a large tumor suppressor that is truncated in most colorectal cancers. The carboxyl-terminal
third of APC protein mediates direct interactions with microtubules and the microtubule plus-end tracking protein EB1. In
addition, APC has been localized to actin-rich regions of cells, but the mechanism and functional significance of this localization
have remained unclear. Here we show that purified carboxyl-terminal basic domain of human APC protein (APC-basic) bound directly
to and bundled actin filaments and associated with actin stress fibers in microinjected cells. Actin filaments and microtubules
competed for binding to APC-basic, but APC-basic also could cross-link actin filaments and microtubules at specific concentrations,
suggesting a possible role in cytoskeletal cross-talk. APC interactions with actin in vitro were inhibited by its ligand EB1, and co-microinjection of EB1 prevented APC association with stress fibers. Point mutations
in EB1 that disrupted APC binding relieved the inhibition in vitro and restored APC localization to stress fibers in vivo, demonstrating that EB1-APC regulation is direct. Because tumor formation and metastasis involve coordinated changes in the
actin and microtubule cytoskeletons, this novel function for APC and its regulation by EB1 may have direct implications for
understanding the molecular basis of tumor suppression.
The organization of microtubule arrays in the plant cell cortex involves interactions with the plasma membrane, presumably through protein bridges. We have used immunochemistry and monoclonal antibody 6G5 against a candidate bridge protein, a 90-kD tubulin binding protein (p90) from tobacco BY-2 membranes, to characterize the protein and isolate the corresponding gene. Screening an Arabidopsis cDNA expression library with the antibody 6G5 produced a partial clone encoding phospholipase D (PLD), and a full-length gene was obtained by sequencing a corresponding expressed sequence tag clone. The predicted protein of 857 amino acids contains the active sites of a phospholipid-metabolizing enzyme and a Ca²+-dependent lipid binding domain and is identical to Arabidopsis PLDδ. Two amino acid sequences obtained by Edman degradation of the tobacco p90 are identical to corresponding segments of a PLD sequence from tobacco. Moreover, immunoprecipitation using the antibody 6G5 and tobacco BY-2 protein extracts gave significant PLD activity, and PLD activity of tobacco BY-2 membrane proteins was enriched 6.7-fold by tubulin-affinity chromatography. In a cosedimentation assay, p90 bound and decorated microtubules. In immunofluorescence microscopy of intact tobacco BY-2 cells or lysed protoplasts, p90 colocalized with cortical microtubules, and taxol-induced microtubule bundling was accompanied by corresponding reorganization of p90. Labeling of p90 remained along the plasma membrane when microtubules were depolymerized, although detergent extraction abolished the labeling. Therefore, p90 is a specialized PLD that associates with membranes and microtubules, possibly conveying hormonal and environmental signals to the microtubule cytoskeleton.
The cytoskeleton, which mainly consists of microtubules (MTs) and actin microfilaments (MFs), plays various significant roles that are indispensable for eukaryotic viability, including determination of cell shape, cell movement, nuclear division, and cytokinesis. In animal cells, MFs appear to be of more importance than MTs, except for spindle formation in nuclear division. In contrast, higher plants have a rigid cell wall around their cells, and have thus evolved elegant systems of MTs to control the direction of cellulose microfibrils (CMFs) deposited in the cell wall, and to divide centrifugally in a physically limited space. Dynamic changes in MTs during cell cycle progression in higher plant cells have been observed over several decades, including cortical MTs (CMTs) during interphase, preprophase bands (PPBs) from late G2 phase to prophase, spindles from prometaphase to anaphase, and phragmoplasts at telophase. The MFs also show some changes not as obvious as MT dynamics. However, questions regarding the process of formation of these arrays, and the precise mechanisms by which they fulfill their roles, remain unsolved. In this article, we present an outline of the changes in the cytoskeleton based on our studies with highly-synchronized tobacco BY-2 cells. Some candidate molecules that could play roles in cytoskeletal dynamics are discussed. We also hope to draw attention to recent attempts at visualization of cytoskeletons with molecular techniques, and to some examples of genetic approaches in this field.
Flowering plant genomes lack flagellar and cytoplasmic dyneins as well as the proteins that make up the dynactin complex. The mechanisms for organizing the Golgi apparatus, establishing spindle poles, and moving nuclei, vesicles, and chromosomes in flowering plants must be fundamentally different from those in other systems where these processes are dependent upon dynein and dynactin.
The growth of pollen tubes is characterized by intense secretory activity in the tip region. This process of vesicle-mediated secretion and tip growth is strongly influenced by calcium gradients. The cytoskeletal apparatus is also critically involved, as it is required for the translocation of organelles along the tube (a prerequisite for tube extension) and for the transport of the generative/sperm cells. The microtubules and actin filaments probably have distinct functions that relate to different, but related, cytological events within the pollen tubes. Both systems, as well as cytoskeleton-based motor proteins, are necessary for the proper development and growth of the pollen tubes. Different approaches have allowed the roles of several cytoskeletal components to be deciphered, and it is now possible to speculate how they might interact.
Plant cells divide in two by constructing a new cell wall (cell plate) between daughter nuclei after mitosis. Golgi-derived vesicles are transported to the equator of a cytoskeletal structure called a phragmoplast, where they fuse together to form the cell plate. Orientation of new cell walls involves actindependent guidance of phragmoplasts and associated cell plates to cortical sites established prior to mitosis. Recent work has provided new insights into how actin filaments and other proteins in the phragmoplast and cell plate contribute to cytokinesis. Newly discovered mutations have identified a variety of genes required for cytokinesis or its spatial regulation.
Interaction and cross-talk between microtubules and actin microfilaments are important for the cell axis and polarity during
plant cell growth and development, but little is known about the molecular components of this interaction. Plant kinesins
with a calponin-homology domain (KCHs) were recently identified and associated with a putative role in microtubule–microfilament
cross-linking. KCHs belong to a distinct branch of the minus end-directed kinesin subfamily and so far have only been identified
in land plants including the mosses. Here we report the identification of a new KCH from rice (Oryza sativa), OsKCH1, and show that OsKCH1 is associated with cortical microtubules and actin microfilaments in vivo. Furthermore, OsKCH1 is shown to bind to micro-tubules and actin microfilaments in vitro in a domain-dependent way. Additionally, this unique type of kinesin is shown to oligomerize both in vivo and in vitro. These findings are discussed
with respect to a general role for KCHs as linkers between actin filaments and microtubules in both cell elongation and division.
We reported previously that the protein SB401 from Solanum berthaultii binds to and bundles both microtubules and F-actin. In the current study, we investigated the regulation of SB401 activity by its phosphorylation. Our experimental results showed that the phosphorylation of SB401 by casein kinase II (CKII) downregulates the activities of SB401, namely the bundling of microtubules and enhancement of the polymerization of tubulin. However, phosphorylation of SB401 had no observable effect on its bundling of F-actin. Further investigation using extract of potato pollen indicated that a CKII-like kinase may exist in potato pollen. Antibodies against CKII alpha recognized specifically a major band from the pollen extract and the pollen extract was able to phosphorylate the SB401 protein in vitro. The CKII-like kinase showed a similar ability to downregulate the bundling of microtubules. Our experiments demonstrated that phosphorylation plays an important role in the regulation of SB401 activity. We propose that this phosphorylation may regulate the effects of SB401 on microtubules and the actin cytoskeleton.
The preprophase bands of microtubules and F-actin are primary markers of the division site for most plant cells. After preprophase band breakdown, the division site has been considered to be 'negatively' memorized by the local absence of cortical actin filaments. However, there have been reports of cortical F-actin at the division site of mitotic plant cells, calling into question its distribution and possible role there. In this article, previous and recent data on this issue are reviewed. It is proposed that the division site of mitotic plant cells is not devoid of F-actin but is traversed by scarce cortical actin filaments. The description of the division site as an 'actin exclusion zone' might therefore be attributed to a failure to preserve and/or image the notoriously sensitive actin filaments. Accordingly, the 'actin-depleted zone' should be considered as a site with fewer actin filaments than the rest of the cortical cytoplasm. Taking into account recent molecular data on division site components, a possible role for the scarcity of cortical actin filaments in establishing a zone of minimum mobility is also proposed.
A major breakthrough was the recent discovery that cellulose synthases really do move along the plasma membrane upon tracks provided by the underlying cortical microtubules. It emphasized the cytoplasmic contribution to cell wall organization. A growing number of microtubule-associated proteins has been identified and shown to affect the way that microtubules are ordered, with downstream effects on the pattern of growth. The dynamic properties of microtubules turn out to be key in understanding the behaviour of the global array and good progress has been made in deciphering the rules by which the array is self-organized.
The completed genome from the model plant Arabidopsis thaliana reveals the presence of a diverse multigene family of formin-like sequences, comprising more than 20 isoforms. This review highlights recent findings from biochemical, cell biological and reverse-genetic analyses of this family of actin nucleation factors. Important advances in understanding cellular function suggest major roles for plant formins during cytokinesis and cell expansion. Biochemical studies on a subset of plant formins emphasize the need to examine molecular mechanisms outside of mammalian and yeast systems. Notably, a combination of solution-based assays for actin dynamics and timelapse, single-filament imaging with TIRFM provide evidence for the first non-processive formin (AtFH1) in eukaryotes. Despite these advances it remains difficult to generate a consensus view of plant formin activities and cellular functions. One limitation to summarizing formin properties relates to the enormous variability in domain organization among the plant formins. Generating homology-based predictions that depend on conserved domains outside of the FH1 and FH2 will be virtually impossible for plant formins. A second major drawback is the lack of facile techniques for examining dynamics of individual actin filaments within live plant cells. This constraint makes it extremely difficult to bridge the gap between biochemical characterization of particular formin and its specific cellular function. There is promise, however, that recent technical advances in engineering appropriate fluorescent markers and new fluoresence imaging techniques will soon allow the direct visualization of cortical actin filament dynamics. The emergence of other model systems for studying actin cytoskeleton in vivo, such as the moss Physcomitrella patens, may also enhance our knowledge of plant formins.
The polarized growth of cells as diverse as fungal hyphae, pollen tubes, algal rhizoids and root hairs is characterized by a highly localized regulation of cell expansion confined to the growing tip. In apically growing plant cells, a tip-focused [Ca2+]c gradient and the cytoskeleton have been associated with growth. Although actin has been established to be essential for the maintenance of elongation, the role of microtubules remains unclear. To address whether the microtubule cytoskeleton is involved in root hair growth and orientation, we applied microtubule antagonists to root hairs of Arabidopsis. In this report, we show that depolymerizing or stabilizing the microtubule cytoskeleton of these apically growing root hairs led to a loss of directionality of growth and the formation of multiple, independent growth points in a single root hair. Each growing point contained a tip-focused gradient of [Ca2+]c. Experimental generation of a new [Ca2+]c gradient in root hairs pre-treated with microtubule antagonists, using the caged-calcium ionophore Br-A23187, was capable of inducing the formation of a new growth point at the site of elevated calcium influx. These data indicate a role for microtubules in regulating the directionality and stability of apical growth in root hairs. In addition, these results suggest that the action of the microtubules may be mediated through interactions with the cellular machinery that maintains the [Ca2+]c gradient at the tip.
In diverse cell types, microtubule (MT) and actin filament networks cooperate functionally during a wide variety of processes, including vesicle and organelle transport, cleavage furrow placement, directed cell migration, spindle rotation, and nuclear migration. The mechanisms by which MTs and actin filaments cooperate to mediate these different processes can be grouped into two broad categories: coordinated MT- and actin-based transport to move vesicles, organelles, and cell fate determinants; and targeting and capture of MT ends at cortical actin sites. Over the past several years, a growing number of cellular factors that bridge these cytoskeletal systems have been identified. These include 'hetero-motor' complexes (physically associated myosin and kinesin), myosin-CLIP170 complexes, formin homology (FH) proteins, dynein and the dynactin complex, Kar9p, coronin, Kelch repeat-containing proteins, and ERM proteins.
Interaction between actin filaments (AFs) and microtubules (MTs) has been reported in various plant cells, and the presence of a factor(s) connecting these two cytoskeletal networks has been suggested, but its molecular entity has not been elucidated yet. We obtained a fraction containing MT-binding polypeptides, which induced bundling of AFs and of MTs. A 190 kDa polypeptide which associated with AFs was selectively isolated from the fraction. This polypeptide was thought to have an ability to bind to both AFs and MTs. We raised a monoclonal antibody against the 190 kDa polypeptide. Immunostaining demonstrated the association of the 190 kDa polypeptide with AF bundles and with MT bundles formed in vitro. Immunocytochemical studies throughout the cell cycle revealed that the 190 kDa polypeptide was localized in the nucleus before nuclear envelope breakdown, and in the spindle and the phragmoplast during cell division. After the re-formation of the nuclear envelope, the 190 kDa polypeptide was sequestered to the daughter nuclei. Using the antibody, we succeeded in cloning a cDNA encoding the 190 kDa polypeptide.
The actin and microtubule cytoskeletons play key roles in cell polarity, spindle orientation and nuclear movement. Recent work in fungal systems has identified potential "functional links" between these cytoskeletal systems. This review discusses molecular mechanisms through which these links may be established.
Mitochondrion movement and positioning was studied in elongating cultured cells of tobacco (Nicotiana tabacum L.), containing mitochondria‐localized green fluorescent protein. In these cells mitochondria are either actively moving
in strands of cytoplasm transversing or bordering the vacuole, or immobile positioned in the cortical layer of cytoplasm.
Depletion of the cell's ATP stock with the uncoupling agent DNP shows that the movement is much more energy demanding than
the positioning. The active movement is F‐actin based. It is inhibited by the actin filament disrupting drug latrunculin B,
the myosin ATPase inhibitor 2,3‐butanedione 2‐monoxime and the sulphydryl‐modifying agent N‐ethylmaleimide. The microtubule disrupting drug oryzalin did not affect the movement of mitochondria itself, but it slightly
stimulated the recruitment of cytoplasmic strands, along which mitochondria travel. The immobile mitochondria are often positioned
along parallel lines, transverse or oblique to the cell axis, in the cortical cytoplasm of elongated cells. This positioning
is mainly microtubule based. After complete disruption of the F‐actin, the mitochondria parked themselves into conspicuous
parallel arrays transverse or oblique to the cell axis or clustered around chloroplasts and around patches and strands of
endoplasmic reticulum. Oryzalin inhibited all positioning of the mitochondria in parallel arrays.
The control of cell growth and polarity depends on a dynamic actin cytoskeleton that has the ability to reorganize in response to developmental and environmental stimuli. In animals and fungi, formins are just one of the four major classes of poly-L-proline-containing (PLP) proteins that form part of the signal-transduction cascade that leads to rearrangement of the actin cytoskeleton. Analysis of the Arabidopsis genome sequence indicates that, unlike animals and fungi, formins are the only class of conserved profilin-binding PLP proteins in plants. Moreover, plant formins show significant structural differences compared with their animal and fungal counterparts, raising the possibility that plant formins are subject to novel mechanisms of control or perform unique roles in plants.
A group of microtubule-associated proteins called +TIPs (plus end tracking proteins), including EB1 family proteins, label growing microtubule ends specifically in diverse organisms and are implicated in spindle dynamics, chromosome segregation, and directing microtubules toward cortical sites. Here, we report three new EB1-like proteins from Arabidopsis and provide the intracellular localization for AtEB1, which differs from all known EB1 proteins in having a very long acidic C-terminal tail. In marked contrast to other EB1 proteins, the GFP-AtEB1 fusion protein localizes not only to microtubule plus ends but also to motile, pleiomorphic tubulovesicular membrane networks that surround other organelles and frequently merge with the endoplasmic reticulum. AtEB1 behavior thus resembles that of +TIPs, such as the cytoplasmic linker protein CLIP-170, that are known to associate with and pull along membrane tubules in animal systems but for which homologs have not been identified in plants. In addition, though EB1 proteins are believed to stabilize microtubules, a different behavior is observed for AtEB1 where instead of stabilizing a microtubule it localizes to already stabilized regions on a microtubule. The dual localization pattern of AtEB1 suggests links between microtubule plus end dynamics and endomembrane organization during polarized growth of plant cells.
The mode of cytokinesis, especially in determining the site of cell division, is not well understood in higher-plant cells. The division site appears to be predicted by the preprophase band of microtubules that develop with the phragmosome, an intracellular structure of the cytoplasm suspending the nucleus and the mitotic apparatus in the center. As the preprophase band disappears during mitosis, it is thought to leave some form of "memory" on the plasma membrane to guide the growth of the new cell plate at cytokinesis. However, the intrinsic nature of this "memory" remains to be clarified. In addition to microtubules, microfilaments also dynamically change forms during cell cycle transition from the late G2 to the early G1 phase. We have studied the relationships between microtubules and microfilaments in tobacco BY-2 cells and transgenic BY-2 cells expressing a fusion protein of green-fluorescent protein and tubulin. At the late G2 phase, microfilaments colocalize with the preprophase band of microtubules. However, an actin-depleted zone which appears at late prometaphase is observed around the chromosomes, especially at metaphase, but also throughout anaphase. To study the functions of the actin-depleted zone, we disrupted the microfilament structures with bistheonellide A, a novel macrolide that depolymerizes microfilaments very rapidly even at low concentrations. The division planes became disorganized when the drug was added to synchronized BY-2 cells before the appearance of the actin-depleted zone. In contrast, the division planes appeared smooth, as in control cells, when the drug was added after the appearance of the actin-depleted zone. These results suggest that the actin-depleted zone may participate in the demarcation of the division site at the final stage of cell division in higher plants.
Cytoskeletal motor proteins are ATPases that use the energy released from ATP hydrolysis to move along the cytoskeletal elements of microtubules and actin microfilaments. Found among all eukaryotic organ- isms, kinesins are microtubule-based motor proteins with a conserved kinesin motor domain, and myosins are actin microfilament-based motor proteins with a conserved myosin motor domain. Cytoskeletal mo- tor proteins directly contribute to the organization of various cytoskeletal arrays during cell division and cell growth in plant tissues. They are also responsible for the motility of molecules and organelles, and the segregation of genetic materials during mitosis and meiosis. In the genome of the model plant Arabidopsis (Arabidopsis thaliana), there are at least 61 genes encod- ing kinesins and 17 genes encoding myosins. Most Arabidopsis kinesins and all myosins are evolution- arily divergent from their counterparts in animals and fungi. Little is known about the functions of most plant kinesins and myosins. Arabidopsis kinesins form a number of subfamilies. The mitotic kinesins in the BIMC/Kinesin-5 and the NCD/Kinesin-14 subfami- lies appear to be similar to those in fungi and animals. Others, however, are very divergent, as their non- motor sequences are unique to plants. Some of Arabi- dopsis kinesins are associated with microtubules, mitochondria, Golgi stacks, and vesicles. They affect microtubule organization, organelle distribution, and vesicle transport, respectively. Ultimately, Arabidopsis kinesins contribute directly or indirectly to cell di- vision and cell growth in various tissues. Arabidopsis myosins are classified into two subfamilies: class VIII and class XI. The class XI myosins are associated with various organelles/vesicles. Functions of Arabidopsis myosins are still elusive. Future efforts will be devoted to deciphering not only the functions of these motors by molecular genetics but also the molecular mecha- nisms underlying how these roles are played. INTRODUCTION OF CYTOSKELETAL MOTORS
Coordinating growth and communication between adjacent cells is a critical yet poorly understood aspect of tissue development and organ morphogenesis. We report a Rho GTPase signaling network underlying the jigsaw puzzle appearance of Arabidopsis leaf pavement cells, in which localized outgrowth in one cell is coordinated with localized inhibition of outgrowth of the adjacent cell to form interdigitating lobes and indentations. Locally activated ROP2, a Rho-related GTPase from plants, activates RIC4 to promote the assembly of cortical actin microfilaments required for localized outgrowth. Meanwhile, ROP2 inactivates another target RIC1, whose activity promotes well-ordered cortical microtubules. RIC1-dependent microtubule organization not only locally inhibits outgrowth but in turn suppresses ROP2 activation in the indentation zones. Thus, outgrowth-promoting ROP2 and outgrowth-inhibiting RIC1 pathways antagonize each other. We propose that the counteractivity of these two pathways demarcates outgrowing and indenting cortical domains, coordinating a process that gives rise to interdigitations between adjacent pavement cells.
Microtubules regulate actin-based processes such as cell migration and cytokinesis, but molecular mechanisms are not understood. In the fission yeast Schizosaccharomyces pombe, microtubule plus ends regulate cell polarity in part by transporting the kelch repeat protein tea1p to cell ends. Here, we identify tea4p, a SH3 domain protein that binds directly to tea1p. Like tea1p, tea4p localizes to growing microtubule plus ends and to cortical sites at cell ends, and it is necessary for the establishment of bipolar growth. Tea4p binds directly to and recruits the formin for3p, which nucleates actin cable assembly. During "new end take off" (NETO), formation of a protein complex that includes tea1p, tea4p, and for3p is necessary and sufficient for the establishment of cell polarity and localized actin assembly at new cell ends. Our results suggest a molecular mechanism for how microtubule plus ends regulate the spatial distribution of actin assembly.
Cell polarization is a major event of the cell cycle and underlies the function of most cells. Cell polarity is often achieved through the coordinated organization of the microtubule and act in cytoskeletons. Dramatic changes in cell polarization occur during the cell cycle and are subject to regulation by cell cycle controls. Cells of the fission yeast Schizosaccharomyces pombe grow by tip extension in a cell cycle-controlled manner. During G2 phase, these cells exhibit a transition in cell polarization known as New End Take Off (NETO), in which monopolar cells initiate bipolar growth. Dynamic microtubules contribute to this process by depositing at cell ends the microtubule plus end proteins tea1p and tea4p, which are necessary for NETO. We discuss here how these proteins may recruit for 3p, a formin responsible for actin nucleation, as well as two other actin binding proteins, bud6p and sla2p, to initiate cell polarization at the new end of the cell. Thus, the study of NETO is revealing a mechanism by which the plus ends of microtubules regulate the spatial organization of actin.
Significant recent progress toward understanding directional expansion in diffusely growing plant cells concerns actin. Tools for imaging actin, including both live-cell reporters and fixation protocols, have been improved. Proteins that interact with actin have been identified and their functions probed biochemically and genetically. Specifically, members of the actin-related protein2/3 (ARP2/3) complex and the Wiskott-Aldrich syndrome Verprolin-homologous (WAVE) complex have been identified. These proteins have salient functions in shaping trichomes and leaf pavement cells. Additionally, two targets of a rho-of-plants (ROP) G-protein have been discovered that exert opposing regulatory action on actin and microtubules, a pathway that appears to be responsible for establishing the undulating shapes of pavement cells. Finally, several mutants of the fragile fiber class have revealed a link between actin organization, cell wall synthesis, and phosphoinositol signaling.
We purified an 84 kDa polypeptide from the MAP (microtubule-associated protein) fraction of tobacco BY-2 cultured cells. LC/MS/MS (liquid chromatography-tandem mass spectrometry) analysis revealed that this polypeptide is a tobacco homolog of AtDRP3 (Arabidopsis thaliana dynamin-related protein 3). Electron microscopy revealed that NtDRP3 (Nicotiana tabacum dynamin-related protein 3) assembles to form a filamentous structure. When GDP was added to the NtDRP3 fraction, the filaments disappeared and many particles appeared. Biochemical analysis revealed that NtDRP3 could bind to and bundle both microtubules and actin filaments in vitro.
The pollen tube exhibits cytoplasmic streaming of organelles, which is dependent on the actin-myosin system. Although microtubule-based motors have also been identified in the pollen tube, many uncertainties exist regarding their role in organelle transport. As part of our attempt to understand the role of microtubule-based movement in the pollen tube of tobacco, we investigated the cooperation between microtubules and actin filaments in the transport of mitochondria and Golgi vesicles, which are distributed differently in the growing pollen tube. The analysis was performed using in vitro motility assays in which organelles move along both microtubules and actin filaments. The results indicated that the movement of mitochondria and Golgi vesicles is slow and continuous along microtubules but fast and irregular along actin filaments. In addition, the presence of microtubules in the motility assays forces organelles to use lower velocities. Actin- and tubulin-binding tests, immunoblotting and immunogold labeling indicated that different organelles bind to identical myosins but associate with specific kinesins. We found that a 90 kDa kinesin (previously known as 90 kDa ATP-MAP) is associated with mitochondria but not with Golgi vesicles, whereas a 170 kDa myosin is distributed on mitochondria and other organelle classes. In vitro and in vivo motility assays indicate that microtubules and kinesins decrease the speed of mitochondria, thus contributing to their positioning in the pollen tube.