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1.
Maternal poly(A)+RNA, histone mRNA, and actin mRNA exhibit unique spatial distributions in the different ooplasmic regions of ascidian eggs. These RNAs also appear to migrate with their respective ooplasms during the episode of extensive cytoplasmic rearrangement that occurs after fertilization, suggesting they are associated with a structural framework. The role of the cytoskeletal framework (CF) in determining the spatial distribution of maternal mRNA was tested by subjecting Triton X-100 extracted (Styela plicata) eggs and early embryos to in situ hybridization with poly(U) and cloned DNA probes. Grain counts indicated that substantial proportions of the egg poly(A)+RNA, histone mRNA, and actin mRNA were present in the CF and that there was no alteration in the extent of mRNA-CF interactions during the period between fertilization and the two-cell stage. Analysis of grain distributions indicated that poly(A)+RNA, histone mRNA, and actin mRNA were concentrated in the same regions of detergent-extracted eggs as they are in intact eggs. The proportions and spatial distribution of these RNAs in the CF were not affected when the actin cytoskeleton was destabilized by cytochalasin B or DNAse I. The data suggest that maternal mRNA is associated with the CF, that this association is responsible for mRNA rearrangement during ooplasmic segregation, and that mRNA-CF interactions are not dependent on the integrity of the actin cytoskeleton.  相似文献   

2.
Substrate-attached asynchronous HeLa cells were extracted with Triton X-100 and analysed by electron microscopy and two-dimensional gel electrophoresis. Such Triton cytoskeletons showed actin filament bundles, microtubules, intermediate filaments, and actin networks in the substrate-associated lamellae, and contained around 90 polypeptides (48 basic, 42 acidic; 52% of total actin, 99% of vimentin, 41% of α-actinin and 30% of β-tubulin).Cytoskeletons produced by further extraction in high and low salt buffers (L-H-L) showed only intermediate filaments, the nucleus and residual actin, and contained a total of 19 polypeptides (13 acidic, 6 basic). Of these, 12 corresponded to abundant acidic proteins in the 47,000 to 70,000 Mr region as determined by staining with Coomassie blue and labelling with a mixture of 14C-labelled amino acids. Using L-H-L extracted cytoplasts, and employing an actin depolymerising protein from slime moulds, seven abundant acidic IEF3 polypeptides were shown to be present in these intermediate filament-enriched, substrate-attached cytoplast cytoskeletons. These polypeptides (L-H-L cytoplast polypeptides) corresponded to vimentin (IEF 26, 54,000 Mrmr) and six polypeptides (IEF 12, 68,000 Mr; IEF 24, 56,000 Mr; IEF 31, 50,000 Mr; IEF 35, 49,000 Mr; IEF 36, 48,500 Mr and IEF 46, 43,500 Mr) not previously reported as present in cytoskeletons. Peptide analysis showed that these were not related as products of modification or proteolysis.Labelling of mitotic and interphase cells with [35S]methionine followed by one-dimensional peptide map analysis showed that IEF 24, 26 (vimentin), 31 and 36 are preferentially modified during mitosis. These modifications correspond to phosphorylations of IEF 26 (vimentin) and 31, and to an unknown type for IEF 24. IEF 36 is phosphorylated in interphase to yield IEF 37, and the latter is further phosphorylated in mitosis. These results suggest that modification of the L-H-L cytoplast polypeptides may be important in the reorganization of cytoskeletal elements that takes place during cell division.  相似文献   

3.
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   

4.
Summary Ooplasmic segregation in ascidians includes the movement of the myoplasm, a pigmented cytoplasmic region thought to be involved in the determination of the embryonic muscle and mesenchyme cell lineages, into the vegetal hemisphere of the egg. A myoplasmic cytoskeletal domain (MCD), composed of a cortical actin network (the PML) and an underlying filamentous lattice extending deep into the cytoplasm, is present in this region. The MCD gradually recedes into the vegetal hemisphere during ooplasmic segregation. It has been proposed that the segregation of the myoplasm is mediated by the contraction of the PML. To test this possibility we have examined ooplasmic segregation in eggs in which the internal parts of the MCD were separated from the PML by centrifugal force. Transmission and scanning electron microscopy of eggs extracted with Triton X-100 showed that the PML remained intact when the internal portions of the MCD were displaced and stratified by centrifugation. When stratified eggs were fertilized there were no rearrangements of the visible cytoplasmic inclusions, but the cellular deformations and the recession of the PML characteristic of ooplasmic segregation occurred as usual. The results indicate that the recession of the PML occurs independently of the internal constituents of the MCD and suggest that PML contraction is the motive force for ooplasmic segregation.  相似文献   

5.
The core structures of microvilli from absorptive cells of the intestinal epithelium are primarily composed of calmodulin (Mr 16,000), actin (Mr 43,000), villin (Mr 95,000) and a protein of Mr 110,000. We have isolated this protein and raised antibodies against it. The antibodies interact specifically with villin and Mr 110,000 polypeptides present in isolated microvilli or brush borders. However, after absorption on an immobilized villin preparation, these antibodies still immunoprecipitate the Mr 110,000 protein but not villin. Thus, these two proteins appear to share some antigenic determinants but also contain other determinants specific for each protein. Immunolocalization studies have been performed using specific antibodies against the Mr 110,000 protein. Immunofluorescent studies on thin frozen sections of intestinal cells show that this protein is located in the brush border and at the basolateral faces of these polarized cells. Immunoferritin studies on rat brush borders demembranated with the detergent Triton X-100 show the association of the Mr 110,000 protein with core filaments of microvilli, as well as with some filaments localized in the terminal web network.Using sealed, right-side-out vesicles prepared from pig intestinal mucosa in the presence of Ca2+ and Mg2+, a polypeptide of Mr 140,000 was found to be a major component of the Triton X-100 insoluble pellet. This protein is a minor component of an equivalent pellet obtained from isolated microvilli prepared in the presence of EDTA. The significance of this Mr 140,000 polypeptide associated with the core residue of intestinal microvilli is discussed.  相似文献   

6.
The novel microtubule-interacting protein Mip-90 was originally isolated from HeLa cells by using affinity columns of agarose derivatized with peptides from the C-terminal regulatory domain on β-tubulin. Biochemical and immunocytochemical data have suggested that the association of Mip-90 with the microtubule system contributes to its cellular organization. Here we report the interaction patterns of Mip-90 with microtubules and actin filaments in interphase human fibroblasts. A polyclonal monospecific antibody against Mip-90 was used for immunofluorescence microscopy analysis to compare the distribution patterns of this protein with tubulin and actin. A detailed observation of fibroblasts revealed the colocalization of Mip-90 with microtubules and actin filaments. These studies were complemented with experiments using cytoskeleton-disrupting drugs which showed that colocalization patterns of Mip-90 with microtubules and actin filaments requires the integrity of these cytoskeletal components. Interestingly, a colocalization of Mip-90 with actin at the leading edge of fibroblasts grown under subconfluency was observed, suggesting that Mip-90 could play a role in actin organization, particularly at this cellular domain. Mip-90 interaction with actin polymers was further supportedin vitroby cosedimentation and immunoprecipitation experiments. The cosedimentation analysis indicated that Mip-90 bound to actin filaments with an association constantKa= 1 × 106M−1, while an stoichiometry Mip-90/actin of 1:12 mol/mol was calculated. Western blots of the immunoprecipitates revealed that Mip-90 associated to both actin and tubulin in fibroblasts extracts. These studies indicate that Mip-90, described as a microtubule-interacting protein, also bears the capacity to interact with the microfilament network, suggesting that it may play a role in modulating the interactions between these cytoskeletal filaments in nonneuronal cells.  相似文献   

7.
The mammalian neurofilament triplet proteins (210, 160 and 68 × 103Mr proteins) are resolved by anion exchange chromatography in the presence of urea. Upon dialysis against physiological buffers at 37 °C only the 68 × 103Mr protein shows self-assembly into morphologically normal intermediate-sized filaments. Addition of 210 × 103Mr protein to 68 × 103Mr protein leads to shorter filaments, which upon embedding reveal a rough surface and whisker-like protrusions that are not present on the smooth surface of filaments assembled from 68 × 103Mr protein alone. Certain emerging principles of neurofilament structure are discussed, emphasizing a possible relation between neurofilaments and other intermediate-sized filaments.  相似文献   

8.
We have purified from a membrane fraction of bovine brain a calmodulin-binding protein (calspectin) that shares a number of properties with erythrocyte spectrin: It has a heterodimeric structure with Mr 240 000 and 235 000 and binds to (dimeric form) or crosslinks (tetrameric form) F-actin. We show that calspectin (tetramer) is capable of inducing the polymerization of G-actin to actin filaments by increasing nucleation under conditions where actin alone polymerizes at a much slower rate. Thus, brain calspectin behaves in the same manner as erythrocyte spectrin, supporting the idea that, in conjunction with actin oligomers it comprises the cytoskeletal meshwork underlying the cytoplasmic surface of the nerve cell.  相似文献   

9.
Following the second polar body formation (PBF), the Tubifex egg undergoes ooplasmic segregation consisting of two steps, i.e., centrifugal migration of membranous organelles forming a subcortical ooplasmic layer and then movements of these organelles along the egg surface. The present investigation was undertaken to examine the microfilament organization in eggs during these ooplasmic rearrangements. Microfilaments throughout the egg are identified as actin by their reversible heavy meromyosin binding. Before the second PBF, a distinct network of actin filaments is present in the endoplasmic region. It is disorganized during the second PBF; short actin filaments are caused to aggregate with membranous organelles. Following the second PBF, similar short filaments become localized in the subcortical layer but not in the underlying yolky region. However, it is not until 50-60 min after the second PBF that an elaborate actin network is established in the subcortical layer. The cortex contains a sheet-like lattice of actin filaments. It is thickest around the animal pole, and tapes toward the equator of the egg. At about 90 min after the second PBF, this polarized distribution of cortical filaments becomes more pronounced as the result of their movements. Chronologically, subcortical actin network formation and cortical reorganization correspond to the later portion of the first step and the earlier portion of the second step of ooplasmic segregation, respectively. These findings are discussed in terms of ooplasmic movements and rearrangements.  相似文献   

10.
Cadmium is a toxic metal with pleiotropic effects on cell death and survival. The mesangial cell is particularly responsive to Cd's effects on kinase signaling pathways and cytoskeletal dynamics. Here we show that CaMK‐II is a participant in the cytoskeletal effects of Cd2+. A major mesangial cell isoform, CaMK‐IIδ, was identified in pellets of DNase I pull‐downs and cytosolic immunoprecipitates of G‐actin. CaMK‐IIδ was also present in Triton X‐100‐insoluble cytoskeletal preparations and translocated to the cytoskeleton in a concentration‐dependent manner in Cd‐treated cells. Translocation was suppressed by KN93, an inhibitor of CaMK‐II phosphorylation. In vitro actin polymerization studies indicated that recombinant CaMK‐IIδ sequestered actin monomer. Cytoskeletal preparations from Cd‐treated cells decrease the rate of polymerization, but KN93 co‐treatment prevents this effect. Over‐expressed CaMK‐IIδ also translocated to the cytoskeleton upon Cd exposure, and this was prevented by KN93. Conversely, siRNA silencing of CaMK‐IIδ increases the effect of cytoskeletal extracts on actin polymerization, and abrogates the effect of Cd. The actin capping and severing protein, gelsolin, translocates to the cytoskeleton in the presence of Cd2+, dependent upon the phosphorylation of CaMK‐II, and is recovered together with actin and CaMK‐IIδ in G‐actin pull‐downs and F‐actin sedimentation. Translocation is accompanied by generation of a 50 kDa gelsolin fragment whose appearance is prevented by KN93 and CaMK‐IIδ silencing. We conclude that cytoskeletal effects of Cd in mesangial cells are partially mediated by Cd‐dependent activation of CaMK‐IIδ, binding of CaMK‐IIδ and gelsolin to actin filaments, and cleavage of gelsolin. J. Cell. Physiol. 228: 78–86, 2013. © 2012 Wiley Periodicals, Inc.  相似文献   

11.
Intracellular Ca2+ transients occur at fertilization in the eggs of all animal species and are thought to be critical for the initiation of several events in egg activation. The rho family of small GTPases are known to organize and maintain the actin filament-dependent cytoskeleton, and rho is involved in the control mechanism of cytokinesis. In the ascidian Ciona savignyi, the first step of ooplasmic segregation observed just after fertilization is cortical contraction with egg deformation, mediated by the cortical actin filaments. C3 exoenzyme, a rho-specific inhibitor, did not affect the pattern of [Ca2+]i transients in the ascidian egg, but inhibited ooplasmic segregation and cytokinesis at the first cleavage. Injection of inositol 1,4,5-trisphosphate or treatment of Ca2+ ionophore induced deformation of the egg and extrusion of the first polar body, but these phenomena did not occur in the C3 exoenzyme-injected egg. These results suggest that rho proteins are involved in egg deformation, ooplasmic segregation and cytokinesis downstream of the [Ca2+]i transients.  相似文献   

12.
In this study isolated cortical regions of both penetrated and nonpenetrated Syrian hamster eggs were examined in whole mounts and platinum replicas of detergent-extracted cortical patches. Two types of cytoskeletal organization were observed in the egg cortex: Loose networks (LN regions) with integrated localized dense networks (LDN regions). Decoration with heavy meromyosin and labeling with antiactin/protein G gold both indicate that the cortical cytoskeleton consists mainly of a LN of actin microfilaments and several types of nonactin filaments, whereas LDN regions dispersed within the LN were comprised of nonactin filaments. Cortical patches and replicas of eggs incubated with sperm for 10-15 min provide evidence that cortical microfilaments may be intimately associated with penetrating spermatozoa. The results of this investigation provide the first high resolution view of the cortical cytoskeletal domain of a mammalian egg and suggest that actin microfilaments might play a role in sperm penetration of the egg cortex.  相似文献   

13.
We have examined the structure of actin-binding molecules in solution and interacting with actin filaments. At physiological ionic strength, actin-binding protein has a Mr value of 540 × 103 as determined by direct and indirect hydrodynamic measurements. It is an asymmetrical dimer composed of 270 × 103 dalton subunits. Viewed in the electron microscope after negative staining or low angle shadowing, actin-binding protein molecules assume a broad range of conformations varying from closed circular structures to fully extended strands 162 nm in contour length. All configurations are apparently derived from the same structure which consists of two monomer chains connected end-to-end. The radius of gyration determined from the electron microscopic images was 21.3 nm in agreement with the value of 17.6 nm calculated from hydrodynamic assays. The average axial ratio from hydrodynamic measurements was 17:1, whereas fully extended dimer molecules in the electron microscope would have an axial ratio of 54:1. All of these observations indicate that actin-binding protein dimers are extremely flexible. The flexibility parameter λ (Landau &; Lifshits, 1958) for actinbinding protein is 0.18 nm?1.As determined by sedimentation, actin-binding protein binds to actin filaments with a Ka value of 2 × 106m?1 and a capacity of one dimer to 14 actin monomers in filaments. After incubation of high concentrations (molar ratio to actin ≥ 1:10) of actin-binding protein with actin filaments, long filament bundles are visible in the electron microscope. Under these conditions, actin-binding protein molecules decorate the actin filaments in the bundles at regular 40 nm intervals or once every 15 monomers, approximately equivalent to the binding capacity measured by sedimentation. Low concentrations of actin-binding protein (molar ratio to actin ≥ 1:50) which promote the gelation of actin filaments in solution, did not detectably alter the isotropy of the actin filaments. Direct visualization of actinbinding protein molecules between actin filaments in the electron microscope showed that dimers are sufficient for crossbridging of actin filaments and that actinbinding protein dimers are bipolar, composed of monomers connected head-to-head and having actin-binding sites located on the free tails.We conclude that actin-binding protein is a dimer at physiological ionic strength. Each dimer has two actin filament binding sites and is therefore sufficient to gel actin filaments in solution. The length and flexibility of the actin-binding protein subunits render this molecule structurally suited for the crosslinking of large helical filaments into isotropic networks.  相似文献   

14.
Adenovirus (Ad) endocytosis via αv integrins requires activation of the lipid kinase phosphatidylinositol-3-OH kinase (PI3K). Previous studies have linked PI3K activity to both the Ras and Rho signaling cascades, each of which has the capacity to alter the host cell actin cytoskeleton. Ad interaction with cells also stimulates reorganization of cortical actin filaments and the formation of membrane ruffles (lamellipodia). We demonstrate here that members of the Rho family of small GTP binding proteins, Rac and CDC42, act downstream of PI3K to promote Ad endocytosis. Ad internalization was significantly reduced in cells treated with Clostridium difficile toxin B and in cells expressing a dominant-negative Rac or CDC42 but not a H-Ras protein. Viral endocytosis was also inhibited by cytochalasin D as well as by expression of effector domain mutants of Rac or CDC42 that impair cytoskeletal function but not JNK/MAP kinase pathway activation. Thus, Ad endocytosis requires assembly of the actin cytoskeleton, an event initiated by activation of PI3K and, subsequently, Rac and CDC42.  相似文献   

15.
Internodal cells of Chara corallina Klein ex. Wild have been studied to determine the number of actin isoforms they contain and whether actin occurs at locations in the cortical cytoplasm outside the filament bundles. A monoclonal antibody to chicken actin is specific for actin in numerous animal cells but binds to two Chara proteins after their separation by two-dimensional polyacrylamide gel electrophoresis. One protein resembles known actins in relative molecular mass (43,000-Mr) and isoelectric point (5.5) while the other is distinctly different (58,000-Mr, isoelectric point = 4.8). Because it is indetectable in cells whose actin bundles have been extracted, the 43,000-Mr protein is assigned to the bundles and concluded to be rare or absent in the remaining cortical cytoplasm. The 58,000-Mr protein, in contrast, does not extract with the actin bundles. It was localized within the chloroplasts by immunofluorescence and by the dependence of proteolysis on the permeabilization of the chloroplast envelope.  相似文献   

16.
17.
The ability to control the assembly and disassembly dynamics of actin filaments is an essential property of the cellular cytoskeleton. While many different proteins are known which accelerate the polymerization of monomers into filaments or promote their disintegration, much less is known on mechanisms which guarantee the kinetic stability of the cytoskeletal filaments. Previous studies indicate that cross-linking molecules might fulfill these stabilizing tasks, which in addition facilitates their ability to regulate the organization of cytoskeletal structures in vivo. The effect of depolymerization factors on such structures or the mechanism which leads finally to their disintegration remain unknown. Here, we use multiple depolymerization methods in order to directly demonstrate that cross-linking and bundling proteins effectively suppress the actin depolymerization in a concentration dependent manner. Even the actin depolymerizing factor cofilin is not sufficient to facilitate a fast disintegration of highly cross-linked actin networks unless molecular motors are used simultaneously. The drastic modification of actin kinetics by cross-linking molecules can be expected to have wide-ranging implications for our understanding of the cytoskeleton, where cross-linking molecules are omnipresent and essential.  相似文献   

18.
Dynamic assembly and disassembly of the actin cytoskeleton has been implicated in the regulation of pollen germination and subsequent tube growth. It is widely accepted that actin filaments are arrayed into distinct structures within different regions of the pollen tube. Maintenance of the equilibrium between monomeric globular actin (G‐actin) and filamentous actin (F‐actin) is crucial for actin assembly and array construction, and the local concentration of G‐actin thus directly impacts actin assembly. The localization and dynamics of G‐actin in the pollen tube, however, remain to be determined conclusively. To address this question, we created a series of fusion proteins between green fluorescent protein (GFP) and the Arabidopsis reproductive actin ACT11. Expression of a fusion protein with GFP inserted after methionine at position 49 within the DNase I‐binding loop of ACT11 (GFPMet49–ACT11) rescued the phenotypes in act11 mutants. Consistent with the notion that the majority of actin is in its monomeric form, GFPMet49–ACT11 and GFP fusion proteins of four other reproductive actins generated with the same strategy do not obviously label filamentous structures. In further support of the functionality of these fusion proteins, we found that they can be incorporated into filamentous structures in jasplakinolide (Jasp)‐treated pollen tubes. Careful observations showed that G‐actin is distributed uniformly in the pollen tube and is rapidly redistributed via cytoplasmic streaming during pollen tube growth. Our study suggests that G‐actin is readily available in the cytoplasm to support continuous actin polymerization during rapid pollen tube growth.  相似文献   

19.
Yeast protein, Bee1, exhibits sequence homology to Wiskott-Aldrich syndrome protein (WASP), a human protein that may link signaling pathways to the actin cytoskeleton. Mutations in WASP are the primary cause of Wiskott-Aldrich syndrome, characterized by immuno-deficiencies and defects in blood cell morphogenesis. This report describes the characterization of Bee1 protein function in budding yeast. Disruption of BEE1 causes a striking change in the organization of actin filaments, resulting in defects in budding and cytokinesis. Rather than assemble into cortically associated patches, actin filaments in the buds of Δbee1 cells form aberrant bundles that do not contain most of the cortical cytoskeletal components. It is significant that Δbee1 is the only mutation reported so far that abolishes cortical actin patches in the bud. Bee1 protein is localized to actin patches and interacts with Sla1p, a Src homology 3 domain–containing protein previously implicated in actin assembly and function. Thus, Bee1 protein may be a crucial component of a cytoskeletal complex that controls the assembly and organization of actin filaments at the cell cortex.  相似文献   

20.
Annelid embryos are comprised of yolk-deficient animal and yolk-filled vegetal blastomeres. This "unipolar" organization along the animal-vegetal axis (in terms of ooplasmic distribution) is generated via selective segregation of yolk-free, clear cytoplasm to the animal blastomeres. The pathway that leads to the unipolar organization is different between polychaetes and clitellates (i.e., oligochaetes and hirudinidans). In polychaetes, the clear cytoplasm domain, which is established through ooplasmic segregation at the animal side of the egg, is simply cut up by unequal equatorial cleavage. In clitellates, localization of clear cytoplasm to animal blastomeres is preceded by unification of the initially separated polar domains of clear cytoplasm, which result from bipolar ooplasmic segregation. In this article, I have reviewed recent studies on cytoskeletal mechanisms for ooplasmic localization during early annelid development. Annelid eggs accomplish ooplasmic rearrangements through various combinations of three cytoskeletal mechanisms, which are mediated by actin microfilaments, microtubules and mitotic asters, respectively. One of the unique features of annelid eggs isthat a homologous process is driven by distinct cytoskeletal elements. Annelid eggs may provide an intriguing system to investigate not only mechanical aspects of ooplasmic segregation but also evolutionary divergence of cytoskeletal mechanisms that operate in a homologous process.  相似文献   

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