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1.
The formation and bipolar translocation of an ectoplasmic cytoskeleton of rings and meridional bands was studied in interphase zygotes of the glossiphoniid leech Theromyzon trizonare. Zygotes consisted of a peripheral organelle-rich ectoplasm and an internal yolk-rich endoplasm. After microinjection of labeled tubulin and/or actin, zygotes were examined by time-lapse video imaging, immunofluorescence and confocal microscopy. The rings and meridional bands were formed by condensation of a network of moving cytasters that represented ectoplasmic secondary centers of microtubule and actin filament nucleation. In some cases the network of cytasters persisted between the rings. The cytoskeleton had an outer actin layer and an inner microtubule layer that merged at the irregularly-shaped boundary zone. Bipolar translocation of the rings, meridional bands, or the network of cytasters led to accumulation of the cytoskeleton at both zygote poles. Translocation of the cytoskeleton was slowed or arrested by microinjected taxol or phalloidin, in a dose-dependent fashion. Results of drug treatment probably indicate differences in the degree and speed at which the cytoskeleton becomes stabilized. Moreover, drugs that selectively stabilized either microtubules or actin filaments stabilized and impaired movement of the entire cytoskeleton. Microtubule poisons and latrunculin-B failed to disrupt the cytoskeleton. It is concluded that the microtubule and actin cytoskeletons are dynamic, presumably cross-linked and resistant to depolymerizing drugs. They probably move along each other by a sliding mechanism that depends on the instability of microtubules and actin filaments. 相似文献
2.
Ooplasmic segregation in the late interphase zygote of the leech Theromyzon trizonare is accomplished by reorganization of an ectoplasmic cytoskeleton formed by polar rings and meridional bands. The dynamic properties of this cytoskeleton were explored by time-lapse confocal and video microscopy. Cytoskeleton assembly was investigated in zygotes pulse-labeled with microinjected fluorophore-tagged or biotin-tagged dimeric tubulin and G-actin. Cytoskeleton disassembly was studied by comparing the linear dimensions of the cytoskeleton at different time points during late interphase. The relative distributions of F- and-G-actin were determined after microinjection of rhodamine-labeled actin and fluorescein-labeled DNase I. Results showed that labeled precursors were readily incorporated into a network of microtubules or actin filaments. Bipolar translocation of the rings and meridional bands was accompanied by the rapid assembly and disassembly of microtubules and actin filaments. Because labeled microtubules and microfilaments gradually decreased, the rate of cytoskeleton disassembly was greater than the rate of cytoskeleton assembly. Hence, ooplasmic segregation was accompanied by the rapid turnover of cytoskeletal components. Co-distribution of F- and-G-actin during mid and late interphase may favor polymer-monomer interchange. We conclude that cytoskeleton reorganization during foundation of cytoplasmic domains can be conveniently studied in the live leech zygote after microinjection of labeled precursors. 相似文献
3.
Summary In order to define cytoskeletal domains of the mammalian photoreceptor, actin and tubulin were localized in adult retinae of mouse and human. For light-microscopic localization, actin was labeled using fluorescent phalloidin or monoclonal antibodies against actin, and tubulin was labeled using monoclonal antibodies against alpha- and beta-tubulin in an immunocytochemical method. Actin and tubulin were also localized by ultrastructural immunocytochemistry in the mouse. Filamentous actin was present in the retina at the outer limiting membrane and in synaptic terminals, especially of the cones, while globular actin was observed additionally in the inner segments. Müller cell cytoplasm and apical microvilli at the outer limiting membrane were also labeled for filamentous actin. Alpha- and beta-tubulin were evident throughout the photoreceptors, including the inner segments, but not in the synaptic terminals or at the outer limiting membrane. In the early postnatal retina of mouse, actin and tubulin were present at the ventricular surface. This pattern changed as photoreceptors fully elongated and as synaptogenesis occurred in the outer plexiform layer. 相似文献
4.
Plant actin filament and microtubule interactions during anaphase--telophase transition: effects of antagonist drugs 总被引:5,自引:0,他引:5
A C Schmit A M Lambert 《Biology of the cell / under the auspices of the European Cell Biology Organization》1988,64(3):309-319
F-actin and microtubule co-distribution and interaction were studied during anaphase-telophase. Rapid and drastic changes in the cytoskeleton during these particular stages were studied in isolated plant endosperm cells of the blood lily. These wall-free cells can be considered as natural dividing protoplasts. As identified previously, an F-actin cytoskeletal network characterized the plant cortex and formed an elastic cage around the spindle, remaining throughout interphase, mitosis and cytokinesis. Actin was specifically labeled by fluorescent phalloidin and/or monoclonal antibodies. Gold-labelled secondary antibodies were used for ultrastructural observations and silver-enhancement was applied for video-enhanced microscopy. Microtubule and microfilament dynamics and interaction were studied using drug antagonists to actin (cytochalasins B, D) and to tubulin (colchicine). This permitted precise correlations to be made between chromosome movement inhibition and alteration in the actin/tubulin cytoskeleton. During anaphase chromosome migration, the cortical actin network was stretched along the microtubular spindle, while it remained homogeneous when anaphase was inhibited by colchicine. Cytochalasins did not inhibit chromosome movement but altered actin distribution. A new population of actin filaments appeared at the equator in late anaphase before the microtubular phragmoplast was formed and contributed to cell plate formation. Our conclusion is that F-actin-microtubule interaction may contribute to the regulatory mechanism of plant cytokinesis. 相似文献
5.
6.
Butler JH Hu S Brady SR Dixon MW Muday GK 《The Plant journal : for cell and molecular biology》1998,13(3):291-301
The N-1-naphthylphthalamic acid (NPA)-binding protein is part of the auxin efflux carrier, the protein complex that controls polar auxin transport in plant tissues. This study tested the hypothesis that the NPA-binding protein (NBP) is associated with the actin cytoskeleton in vitro and that an intact actin cytoskeleton is required for polar auxin transport in vivo. Cytoskeletal polymerization was altered in extracts of zucchini hypocotyls with reagents that stabilized either the polymeric or monomeric forms of actin or tubulin. Phalloidin treatment altered actin polymerization, as demonstrated by immunoblot analyses following native and denaturing electrophoresis. Phalloidin increased both filamentous actin (F-actin) and NPA-binding activity, while cytochalasin D and Tris decreased both F-actin and NPA-binding activity in cytoskeletal pellets. The microtubule stabilizing drug taxol increased pelletable tubulin, but did not alter either the amount of pelletable actin or NPA-binding activity. Treatment of etiolated zucchini hypocotyls with cytochalasin D decreased the amount of auxin transport and its regulation by NPA. These experimental results are consistent with an in vitro actin cytoskeletal association of the NPA-binding protein and with the requirement of an intact actin cytoskeleton for maximal polar auxin transport in vivo. 相似文献
7.
Yu JR Pyon JK Seo M Jung BS Cho SR Lee SH Hong ST 《The Korean journal of parasitology》2001,39(1):13-21
Pneumocystis carinii causes serious pulmonary infection in immunosuppressed patients. This study was undertaken to observe the cytoskeletal proteins of P. carinii by immuno-electron microscopy. P. carinii infection was experimentally induced by immunosuppression of Sprague-Dawley rats for seven weeks, and their lungs were used for the observations of this study. The gold particles localized actin, tropomyosin, and tubulin. The actin was irregularly scattered in the cytoplasm of the trophic forms but was much more concentrated in the inner space of the cell wall of the cystic forms called the inner electron-lucent layer. No significant amount of tropomyosin was observed in either trophic forms or cystic forms. The tubulin was distributed along the peripheral cytoplasm and filopodia of both the trophic and cystic forms rather than in the inner side of the cytoplasm. Particularly, in the cystic forms, the amount of tubulin was increased and located mainly in the inner electron-lucent layer of the cell wall where the actin was concentrated as well. The results of this study showed that the cell wall of P. carinii cystic forms is a structure whose inner side is rich in actin and tubulin. The location of the actin and tubulin in P. carinii suggests that the main role of these proteins is an involvement in the protection of cystic forms from the outside environment by maintaining rigidity of the cystic forms. 相似文献
8.
Background
TRPV4 and the cellular cytoskeleton have each been reported to influence cellular mechanosensitive processes as well as the development of mechanical hyperalgesia. If and how TRPV4 interacts with the microtubule and actin cytoskeleton at a molecular and functional level is not known.Methodology and Principal Findings
We investigated the interaction of TRPV4 with cytoskeletal components biochemically, cell biologically by observing morphological changes of DRG-neurons and DRG-neuron-derived F-11 cells, as well as functionally with calcium imaging. We find that TRPV4 physically interacts with tubulin, actin and neurofilament proteins as well as the nociceptive molecules PKCε and CamKII. The C-terminus of TRPV4 is sufficient for the direct interaction with tubulin and actin, both with their soluble and their polymeric forms. Actin and tubulin compete for binding. The interaction with TRPV4 stabilizes microtubules even under depolymerizing conditions in vitro. Accordingly, in cellular systems TRPV4 colocalizes with actin and microtubules enriched structures at submembranous regions. Both expression and activation of TRPV4 induces striking morphological changes affecting lamellipodial, filopodial, growth cone, and neurite structures in non-neuronal cells, in DRG-neuron derived F11 cells, and also in IB4-positive DRG neurons. The functional interaction of TRPV4 and the cytoskeleton is mutual as Taxol, a microtubule stabilizer, reduces the Ca2+-influx via TRPV4.Conclusions and Significance
TRPV4 acts as a regulator for both, the microtubule and the actin. In turn, we describe that microtubule dynamics are an important regulator of TRPV4 activity. TRPV4 forms a supra-molecular complex containing cytoskeletal proteins and regulatory kinases. Thereby it can integrate signaling of various intracellular second messengers and signaling cascades, as well as cytoskeletal dynamics. This study points out the existence of cross-talks between non-selective cation channels and cytoskeleton at multiple levels. These cross talks may help us to understand the molecular basis of the Taxol-induced neuropathic pain development commonly observed in cancer patients. 相似文献9.
J. POKORNÁ K. SCHWARZEROVÁ S. ZELENKOVÁ J. PETRÁEK I. JANOTOVÁ V. APKOVÁ & Z. OPATRNÝ 《Plant, cell & environment》2004,27(5):641-653
Cytoskeletal proteins assemble into dynamic polymers that play many roles in nuclear and cell division, signal transduction, and determination of cell shape and polarity. The distribution and dynamics of microtubules (MTs) and actin filaments (AFs) are determined, among other factors, by the location of their nucleation sites. Whereas the sites of microtubule nucleation in plants are known to be located under the plasma membrane and on the nuclear envelope during interphase, there is a striking lack of information about nucleation sites of AFs. In the studies reported herein, low temperature (0 °C) was used to de‐polymerize AFs and MTs in tobacco BY‐2 (Nicotiana tabacum L.) cells at interphase. The extent of de‐polymerization of cytoskeletal filaments in interphase cells during cold treatment and the subcellular distribution of nucleation sites during subsequent recovery at 25 °C were monitored by means of fluorescence microscopy. The results show that AFs re‐polymerized rapidly from sites located in the cortical region and on the nuclear envelope, similarly to the initiation sites of MTs. In contrast to MTs, however, complete reconstitution of AFs was preceded by the formation of transient actin structures including actin dots, rods, and filaments with a dotted signal. Immunoblotting of soluble and sedimentable protein fractions showed no changes in the relative amounts of free and membrane‐bound actin or tubulin. 相似文献
10.
The cytoskeleton has a unique property such that changes of conformation result in polymerization into a filamentous form. alphaB-Crystallin, a small heat shock protein (sHsp), has chaperone activities for various substrates, including proteins constituting the cytoskeleton, such as actin; intermediate filament; and tubulin. However, it is not clear whether the "alpha-crystallin domain" common to sHsps also has chaperone activity for the protein cytoskeleton. To investigate the possibility that the C-terminal alpha-crystallin domain of alpha-crystallin has the aggregation-preventing ability for tubulin, we constructed an N-terminal domain deletion mutant of alphaB-crystallin. We characterized its structural properties and chaperone activities. Far-ultraviolet (UV) circular dichroism measurements showed that secondary structure in the alpha-crystallin domain of the deletion mutant is maintained. Ultracentrifuge analysis of molecular masses indicated that the deletion mutant formed smaller oligomers than did the full-length protein. Chaperone activity assays demonstrated that the N-terminal domain deletion mutant suppressed heat-induced aggregation of tubulin well. Comparison of chaperone activities for 2 other substrates (citrate synthase and alcohol dehydrogenase) showed that it was less effective in the suppression of their aggregation. These results show that alphaB-crystallin recognizes a variety of substrates and especially that alpha-crystallin domain binds free cytoskeletal proteins. We suggest that this feature would be advantageous in its functional role of holding or folding multiple proteins denatured simultaneously under stress conditions. 相似文献
11.
Goebel J Forrest K Wills-Karp M Roszman TL 《Journal of receptor and signal transduction research》2006,26(1-2):87-106
Few data exist on the modulation of cytokine receptor signaling by the actin or tubulin cytoskeleton. Therefore, we studied interleukin-2 receptor (IL-2R) signaling in phytohemagglutinine (PHA)-pretreated human T cells in the context of alterations in the cytoskeletal system induced by cytochalasin D (CyD), jasplaklinolide (Jas), taxol (Tax), or colchicine (Col). We found that changes in cytoskeletal tubulin polymerization altered the strength of several IL-2-triggered signals. Moreover, Tax-induced tubulin hyperpolymerization augmented the surface expression of the IL-2R ss -chain and enhanced the association of the IL-2R beta -chain with cytoskeletal tubulin. The IL-2R beta-chain, in turn, was constitutively associated with tubulin and, more weakly, actin. To exclude the possibility that these associations are artifacts caused by PHA, we confirmed them in T cells from TCR-transgenic DO 11.10 mice stimulated with their nominal antigen. We conclude that altered polymerization of cytoskeletal components, especially tubulin, is accompanied by modulation of IL-2 signaling at the receptor level. 相似文献
12.
N-WASP regulates the epithelial junctional actin cytoskeleton through a non-canonical post-nucleation pathway 总被引:1,自引:0,他引:1
Kovacs EM Verma S Ali RG Ratheesh A Hamilton NA Akhmanova A Yap AS 《Nature cell biology》2011,13(8):934-943
N-WASP is a major cytoskeletal regulator that stimulates Arp2/3-mediated actin nucleation. Here, we identify a nucleation-independent pathway by which N-WASP regulates the cytoskeleton and junctional integrity at the epithelial zonula adherens. N-WASP is a junctional protein whose depletion decreased junctional F-actin content and organization. However, N-WASP (also known as WASL) RNAi did not affect junctional actin nucleation, dominantly mediated by Arp2/3. Furthermore, the junctional effect of N-WASP RNAi was rescued by an N-WASP mutant that cannot directly activate Arp2/3. Instead, N-WASP stabilized newly formed actin filaments and facilitated their incorporation into apical rings at the zonula adherens. A major physiological effect of N-WASP at the zonula adherens thus occurs through a non-canonical pathway that is distinct from its capacity to activate Arp2/3. Indeed, the junctional impact of N-WASP was mediated by the WIP-family protein, WIRE, which binds to the N-WASP WH1 domain. We conclude that N-WASP-WIRE serves as an integrator that couples actin nucleation with the subsequent steps of filament stabilization and organization necessary for zonula adherens integrity. 相似文献
13.
A novel role for the nuclear membrane protein emerin in association of the centrosome to the outer nuclear membrane 总被引:8,自引:0,他引:8
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Salpingidou G Smertenko A Hausmanowa-Petrucewicz I Hussey PJ Hutchison CJ 《The Journal of cell biology》2007,178(6):897-904
The type II inner nuclear membrane protein emerin is a component of the LINC complex that connects the nuclear lamina to the actin cytoskeleton. In emerin-null or -deficient human dermal fibroblasts we find that the centrosome is detached from the nucleus. Moreover, following siRNA knockdown of emerin in wild-type fibroblasts, the centrosome also becomes detached from the nucleus. We show that emerin interacts with tubulin, and that nocadozole-treated wild-type cells phenocopy the detached centrosome characteristic of emerin-null/deficient cells. We also find that a significant fraction of emerin is located at the outer nuclear membrane and peripheral ER, where it interacts directly with the centrosome. Our data provide the first evidence in mammalian cells as to the nature of the linkage of the centrosome, and therefore the tubulin cytoskeleton, with the outer nuclear membrane. 相似文献
14.
Jens Goebel Kathy Forrest Marsha Wills-Karp Thomas L. Roszman 《Journal of receptor and signal transduction research》2013,33(1-2):87-106
Few data exist on the modulation of cytokine receptor signaling by the actin or tubulin cytoskeleton. Therefore, we studied interleukin-2 receptor (IL-2R) signaling in phytohemagglutinine (PHA)-pretreated human T cells in the context of alterations in the cytoskeletal system induced by cytochalasin D (CyD), jasplaklinolide (Jas), taxol (Tax), or colchicine (Col). We found that changes in cytoskeletal tubulin polymerization altered the strength of several IL-2-triggered signals. Moreover, Tax-induced tubulin hyperpolymerization augmented the surface expression of the IL-2R β -chain and enhanced the association of the IL-2R γ -chain with cytoskeletal tubulin. The IL-2R β -chain, in turn, was constitutively associated with tubulin and, more weakly, actin. To exclude the possibility that these associations are artifacts caused by PHA, we confirmed them in T cells from TCR-transgenic DO11.10 mice stimulated with their nominal antigen. We conclude that altered polymerization of cytoskeletal components, especially tubulin, is accompanied by modulation of IL-2 signaling at the receptor level. 相似文献
15.
Acetylated alpha-tubulin in microtubules during mouse fertilization and early development 总被引:6,自引:0,他引:6
alpha-Tubulin in the microtubules of mouse oocytes and embryos is acetylated in a specific spatial and temporal sequence. In the unfertilized oocyte, a monoclonal antibody to the acetylated form of alpha-tubulin is bound predominantly at the poles of the arrested metaphase meiotic spindle. The labeling intensity of the spindle microtubules is weaker as observed by immunofluorescence using oocytes double-labeled for total tubulin and acetylated alpha-tubulin, and as measured by immuno high-voltage electron microscopy (immunoHVEM) with colloidal gold; cytasters are not acetylated. At meiotic anaphase, the spindle becomes labeled, and by telophase and during second polar body formation only the meiotic midbody is acetylated. The sperm axoneme retains its acetylation after incorporation though the interphase microtubules are not detected. First mitosis follows a pattern similar to that observed at the second meiosis and during interphase only the mitotic midbodies are acetylated. After treatment with cold, colcemid, or griseofulvin, the remaining stable microtubules are acetylated, but immunoHVEM observations suggest that these fibers might not have been acetylated prior to microtubule disruption. Taxol stabilization does not alter acetylation patterns. Acetylated microtubules are not necessarily old microtubules since acetylated fibers are observed at 30 sec after cold recovery. These results show the presence of acetylated microtubules during meiosis and mitosis and demonstrate a cell-cycle-specific pattern of acetylation, with acetylated microtubules found at the centrosomes at metaphase, an increase in spindle labeling at anaphase, and the selective deacetylation of all but midbody microtubules at telophase. 相似文献
16.
Degradation of HMG-CoA reductase-induced membranes in the fission yeast, Schizosaccharomyces pombe 总被引:1,自引:0,他引:1
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《The Journal of cell biology》1995,131(1):81-94
Although the overall structures of flagellar and cytoplasmic microtubules are understood, many details have remained a matter of debate. In particular, studies of the arrangement of tubulin subunits have been hampered by the low contrast of the tubulin subunits. This problem can now be addressed by the kinesin decoration technique. We have shown previously that the recombinant kinesin head domain binds to beta-tubulin, thus enhancing the contrast between alpha- and beta- tubulin in the electron microscope; this allows one to study the arrangement of tubulin dimers. Here we describe the lattices of the four different types of microtubules in eukaryotic flagellar axonemes (outer doublet A and B, central pair C1 and C2). They could all be labeled with kinesin head with an 8-nm axial periodicity (the tubulin dimer repeat), and all of them showed the B-surface lattice. This lattice is characterized by a 0.92-nm stagger between adjacent protofilaments. The B-lattice was observed on the axonemal microtubules as well as on extensions made by polymerizing porcine brain tubulin onto axonemal microtubules in the proximal and distal directions. This emphasizes that axonemal microtubules serve as high fidelity templates for seeding microtubules. The presence of a B-lattice implies that there must be a helical discontinuity ("seam") in the wall. This discontinuity is now placed near protofilaments A1 and A2 of the A- tubule, close to the inner junction between A- and B-microtubules. The two junctions differ in structure: the protofilaments of the inner junction (A1-B10) are staggered roughly by half a dimer, those of the outer junction (A10-B1) are roughly in register. Of the two junctions the inner one appears to have the stronger bonds, whereas the outer one is more labile and opens up easily, generating "composite sheets" with chevron patterns from which the polarity can be deduced (arrow in the plus direction). Decorated microtubules have a clear polarity. We find that all flagellar microtubules have the same polarities. The orientation of the dimers is such that the plus end terminates with a crown of alpha subunits, the minus end terminates with beta subunits which thus could be in contact with gamma-tubulin at the nucleation centers. 相似文献
17.
The parasitic protozoan Entamoeba histolytica relies on a very dynamic cytoskeleton in order to invade and survive in host tissues. Identification of cytoskeletal elements is key to understanding these processes. Here we present the characterization of EhLimA, the first LIM protein of E. histolytica. EhLimA consists of a single LIM domain at its N terminus and exhibits the highest degree of homology with DdLimE from Dictyostelium discoideum. Immunofluorescence localization of EhLimA using anti-EhLimA antibodies revealed that EhLimA is highly concentrated at the plasma membrane of cells. Silencing or overexpression of the EhLimA gene did not have a significant effect on the growth or morphology of the parasite. EhLimA associates with the cytoskeleton as demonstrated by the enrichment of the protein in cytoskeleton fractions as well as in pull-down assays that revealed that cytoskeleton association involves interaction with actin. EhLimA binding to actin was shown to be dependent on the N-terminal LIM domain of EhLimA, as removal of even half of the LIM domain resulted in almost complete inhibition of the binding to actin. We also found that a portion of EhLimA floats to the lower-density regions of a sucrose gradient together with portions of the Gal-lectin light subunit and actin. Treatment of cells with the cholesterol-sequestering agent digitonin resulted in increased solubility of EhLimA. These results indicate that in addition to cytoskeletal association, EhLimA may also associate with lipid rafts in the parasite plasma membrane and suggest that EhLimA may be part of the molecular system connecting the actin cytoskeleton to membrane rafts. 相似文献
18.
长期以来,人们认为细胞骨架仅为真核生物所特有的结构,但近年来的研究发现它也存在于细菌等原核生物中。目前已经在细菌中发现的FtsZ、MreB和CreS依次与真核细胞骨架蛋白中的微管蛋白、肌动蛋白丝及中间丝类似。FtsZ能在细胞分裂位点装配形成Z环结构,并通过该结构参与细胞分裂的调控;MreB能形成螺旋丝状结构,其主要功能有维持细胞形态、调控染色体分离等;CreS存在于新月柄杆菌中,它在细胞凹面的细胞膜下面形成弯曲丝状或螺旋丝状结构,该结构对维持新月柄杆菌细胞的形态具有重要作用。 相似文献
19.
Microtubule dynamics in nerve cells: analysis using microinjection of biotinylated tubulin into PC12 cells 总被引:27,自引:21,他引:6
To study microtubule (MT) dynamics in nerve cells, we microinjected biotin-labeled tubulin into the cell body of chemically fused and differentiated PC12 cells and performed the immunofluorescence or immunogold procedure using an anti-biotin antibody followed by secondary antibodies coupled to fluorescent dye or colloidal gold. Incorporation of labeled subunits into the cytoskeleton of neurites was observed within minutes after microinjection. Serial electron microscopic reconstruction revealed that existing MTs in PC12 neurites incorporated labeled subunits mainly at their distal ends and the elongation rate of labeled segments was estimated to be less than 0.3 micron/min. Overall organization of MTs in the nerve cells was different from that in undifferentiated cells such as fibroblasts. Namely, we have not identified any MT-organizing centers from which labeled MTs are emanating in the cell bodies of the injected cells. Stereo electron microscopy revealed that some fully labeled segments seemed to start in the close vicinity of electron dense material within the neurites. This suggests new nucleation off some structures in the neurites. We have also studied the overall pattern of the incorporation of labeled subunits which extended progressively from the proximal part of the neurites toward their tips. To characterize the mechanism of tubulin incorporation, we have measured mean density of gold labeling per unit length of labeled segments at different parts of the neurites. The results indicate access of free tubulin subunits into the neurites and local incorporation into the neurite cytoskeleton. Our results lead to the conclusion that MTs are not static polymers but dynamic structures that continue to elongate even within the differentiated nerve cell processes. 相似文献
20.
Formin family proteins coordinate actin filaments and microtubules. The mechanisms by which formins bind and regulate the actin cytoskeleton have recently been well defined. However, the molecular mechanism by which formins coordinate actin filaments and microtubules remains poorly understood. We demonstrate here that Isoform-Ib of the Formin-1 protein (Fmn1-Ib) binds to microtubules via a protein domain that is physically separated from the known actin-binding domains. When expressed at low levels in NIH3T3 fibroblasts, Fmn1-Ib protein localizes to cytoplasmic filaments that nocodazole disruption confirmed as interphase microtubules. A series of progressive mutants of Fmn1-Ib demonstrated that deletion of exon-2 caused dissociation from microtubules and a stronger association with actin membrane ruffles. The exon-2-encoded peptide binds purified tubulin in vitro and is also sufficient to localize GFP to microtubules. Exon-2 does not contain any known formin homology domains. Deletion of exon 5, 7, 8, the FH1 domain or FH2 domain did not affect microtubule binding. Thus, our results indicate that exon-2 of Fmn1-Ib encodes a novel microtubule-binding peptide. Since formin proteins associate with actin filaments through the FH1 and FH2 domains, binding to interphase microtubules through this exon-2-encoded domain provides a novel mechanism by which Fmn1-Ib could coordinate actin filaments and microtubules. 相似文献