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At least 883 records · Page 49Linked to original sources
PubMed2001

Centrosome migration into the Drosophila oocyte is independent of BicD and egl, and of the organisation of the microtubule cytoskeleton.

During early Drosophila oogenesis, one cell from a cyst of 16 germ cells is selected to become the oocyte, and accumulates oocyte-specific proteins and the centrosomes from the other 15 cells. Here we show that the microtubule cytoskeleton and the centrosomes follow the same stepwise restriction to one cell as other oocyte markers. Surprisingly, the centrosomes still localise to one cell after colcemid treatment, and in BicD and egl mutants, which abolish the localisation of all other oocyte markers and the polarisation of the microtubule cytoskeleton. In contrast, the centrosomes fail to migrate in cysts mutant for Dynein heavy chain 64C, which disrupts the fusome. Thus, centrosome migration is independent of the organisation of the microtubule cytoskeleton, and seems to depend instead on the polarity of the fusome.

Animals↗
PubMed2004

A role for the cytoskeleton in prolactin-dependent mammary epithelial cell differentiation.

The function of exocrine glands depends on signals within the extracellular environment. In the mammary gland, integrin-mediated adhesion to the extracellular matrix protein laminin co-operates with soluble factors such as prolactin to regulate tissue-specific gene expression. The mechanism of matrix and prolactin crosstalk and the activation of downstream signals are not fully understood. Because integrins organize the cytoskeleton, we analysed the contribution of the cytoskeleton to prolactin receptor activation and the resultant stimulation of milk protein gene expression. We show that the proximal signalling events initiated by prolactin (i.e. tyrosine phosphorylation of receptor and the associated kinase Jak2) do not depend on an intact actin cytoskeleton. However, actin networks and microtubules are both necessary for continued mammary cell differentiation, because cytoskeletal integrity is required to transduce the signals between prolactin receptor and Stat5, a transcription factor necessary for milk protein gene transcription. The two different cytoskeletal scaffolds regulate prolactin signalling through separate mechanisms that are specific to cellular differentiation but do not affect the general profile of protein synthesis.

Animals↗
PubMed2004

The lateral mobility of NHE3 on the apical membrane of renal epithelial OK cells is limited by the PDZ domain proteins NHERF1/2, but is dependent on an intact actin cytoskeleton as determined by FRAP.

The epithelial brush border (BB) Na+/H+ exchanger, NHE3, plays a major role in transcellular Na+ absorption in the renal proximal tubule. NHE3 activity is rapidly regulated by neurohumoral substances and growth factors via changes in its amount on the BB by a process partially involving vesicle trafficking. The PDZ domain-containing proteins, NHERF1/2, are scaffold proteins that link NHE3 to the actin cytoskeleton via their binding to both ezrin and NHE3. NHERF1/2 interact with both an internal C-terminal domain of NHE3 and the N-terminus of ezrin. We used fluorescence recovery after photobleaching (FRAP) to study the effect of NHERF1/2 on NHE3 mobility in the brush border of opossum kidney (OK) proximal tubule cells. A confocal microscope was used to allow the selective study of apical membrane versus intracellular NHE3. A chimera of NHE3-EGFP was transiently expressed in OK cells and its lateral diffusion in the apical membrane was measured with FRAP and confocal microscopy at 37 degrees C. The contribution of intracellular NHE3-EGFP to recovery on the OK surface not directly over the juxtanuclear area (non-JN) was negligible as exposure to the water soluble crosslinker BS3 (10 mM) at 4 degrees C resulted in no recovery of this component of surface NHE3-EGFP after photobleaching. The mobile fraction (Mf) of apical NHE3-EGFP was 47.5+/-2.2%; the effective diffusion coefficient (Deff) was (2.2+/-0.3) x10(-10) cm2/second. Overexpression of NHERF2 in OK cells decreased the Mf to 29.1+/-3.1% without changing Deff. In the truncation mutant, NHE3585-EGFP (aa 1-585), which lacks the NHERF1/2 binding domain, Mf increased to 66.4+/-2.2%, with no change in Deff, whereas NHE3660-EGFP, which binds NHERF1/2, had Mf (48.3+/-3.0%) and Deff both similar to full-length NHE3. These results are consistent with the PDZ domain proteins NHERF1 and NHERF2 scaffolding NHE3 in macromolecular complexes in the apical membrane of OK cells under basal conditions, which limits the lateral mobility of NHE3. It is probable that this is one of the mechanisms by which NHERF1/2 affects rapid regulation of NHE3 by growth factors and neurohumoral mediators. By contrast, disrupting the actin cytoskeleton by latrunculin B treatment (0.05 microM, 30 minutes) reduced the NHE3 Mf (21.9+/-4.5%) without altering the Deff. Therefore the actin cytoskeleton, independently of NHERF1/2 binding, is necessary for apical membrane mobility of NHE3.

Actins↗
PubMed2005

Ste20/GCK kinase Nak1/Orb3 polarizes the actin cytoskeleton in fission yeast during the cell cycle.

Polar growth is a crucial process during cell morphogenesis. The microtubule and actin cytoskeletons, and vesicular transport are tightly regulated to direct cellular growth and to generate specific cell forms. We demonstrate here that the Ste20-related protein kinase Nak1/Orb3 is required in fission yeast to polarize the actin cytoskeleton at the tips of the cells and for cell separation, and so is involved in controlling both cell shape and late stages of cytokinesis. The localization of the Nak1/Orb3 kinase to the cell tips, a medial ring and the spindle-pole bodies changes during the cell cycle, and the accumulation of F-actin at the cell tips is dependent on Nak1/Orb3 kinase. The phosphorylation of Nak1/Orb3 is periodic during the cell cycle and could be part of a mechanism that relocalizes a constitutively active kinase from the cell tips to the middle of the cell, thereby coordinating reorganization of the actin cytoskeleton and regulation of cell separation with cell-cycle progression.

Actins↗
PubMed2006

Architecture of the vimentin cytoskeleton is modified by perturbation of the GTPase ARF1.

Intermediate filaments are required for proper membrane protein trafficking. However, it remains unclear whether perturbations in vesicular membrane transport result in changes in the architecture of the vimentin cytoskeleton. We find that treatment of cells with Brefeldin A, an inhibitor of specific stages of membrane transport, causes changes in the organization of vimentin filaments. These changes arise from movement of pre-existing filaments. Brefeldin A treatment also leads to alterations in the microtubule cytoskeleton. However, this effect is not observed in cells lacking intermediate filaments, indicating that microtubule bundling is downstream of perturbations in the vimentin cytoskeleton. Brefeldin A-induced changes in vimentin architecture are probably mediated through its effects on ADP-ribosylation factor 1 (ARF1). Expression of a dominant-negative mutant of ARF1 induces BFA-like modifications in vimentin morphology. The BFA-dependent changes in vimentin architecture occurred concurrently with the release of the ARF1-regulated adaptor complexes AP-3 and AP-1 from membranes and adaptor redistribution to vimentin networks. These observations indicate that perturbation of the vesicular membrane transport machinery lead to reciprocal changes in the architecture of vimentin networks.

ADP-Ribosylation Factor 1↗
PubMed1992

A high molecular mass phosphoprotein defined by a novel monoclonal antibody is closely associated with the intermicrotubule cross bridges in the Trypanosoma brucei cytoskeleton.

The main component of the cell body cytoskeleton of Trypanosoma brucei is the highly organised array of stable, subpellicular microtubules on the cytoplasmic face of the plasma membrane. Although several microtubule associated proteins (MAPs) have been shown to be associated with this array, the mechanisms by which individual microtubules interact with one another and with the membrane are still largely undetermined. In this study we have used the T. brucei cytoskeleton as a complex immunogen for the production of monoclonal antibodies to define novel cytoskeletal antigens. Screening by immunofluorescence enabled the selection of an antibody, WCB-1, which detects an antigen associated specifically with the subpellicular microtubules and not with the flagellum microtubules. The antigen (WCB210) was shown to have a relative molecular mass of 210,000 by western blotting. Immunogold studies showed the epitope to be located on the membrane-facing side of the subpellicular cage; it appears to be closely associated with the cross-bridges lying between the microtubules. Unlike many MAPs this protein was shown not to be heat stable and is predicted to be a roughly globular monomer. Even though WCB210 is a very minor component of the cytoskeleton it is heavily phosphorylated. It is possible that this protein is involved in regulation of the subpellicular microtubule crossbridges by interaction with other proteins.

Animals↗
PubMed1994

Calponin is localised in both the contractile apparatus and the cytoskeleton of smooth muscle cells.

Calponin and caldesmon are two thin filament-binding proteins found in smooth muscle that have both been attributed a role in modulating the interaction of actin and myosin. Using high-resolution dual-label immunocytochemistry we have determined the distribution of calponin relative to the contractile and cytoskeletal compartments of the smooth muscle cell. We show, using chicken gizzard smooth muscle, that calponin occurs in the cytoskeleton, with beta-cytoplasmic actin, filamin and desmin, as well as in the contractile apparatus, with myosin and caldesmon. According to the observed labelling intensities, calponin was more concentrated in the cytoskeleton and it was additionally localised in the cytoplasmic dense bodies as well as in the adhesion plaques at the cell surface, which both harbour the beta-cytoplasmic isoform of actin. It is probable that these results explain earlier conflicting reports on the composition of smooth muscle thin filaments and suggest that calponin, together with a Ca(2+)-receptor protein, could just as likely serve a role in the cytoskeleton of smooth muscle as in the contractile apparatus.

Animals↗
PubMed1995

Temperature sensitivity of the keratin cytoskeleton and delayed spreading of keratinocyte lines derived from EBS patients.

Point mutations in the keratin intermediate filament genes for keratin 5 or keratin 14 are known to cause hereditary skin blistering disorders such as epidermolysis bullosa simplex, in which epidermal keratinocytes are extremely fragile and the skin blisters on mild trauma. We show that in 2 phenotypically diverse cases of epidermolysis bullosa simplex, the keratin mutations result in a thermoinstability of the intermediate filament cytoskeleton which can be reproducibly demonstrated even in the presence of tissue culture-induced keratins and in conditions where filament fragility is not otherwise obvious. SV40-T antigen and HPV16 (E6--E7) immortalised keratinocyte cell lines were examined, established from control and epidermolysis bullosa simplex-affected individuals with either severe (Dowling-Meara) or mild (Weber-Cockayne) forms of the disease. In standard tissue culture conditions no significant and consistent abnormality of the keratin cytoskeleton could be demonstrated. However after thermal stress a reduced stability of the keratin filaments was demonstrable in the epidermolysis bullosa simplex cell lines, with filaments breaking into aggregates similar to those seen in skin from EBS patients. These aggregates were maximal at 15 minutes after heat shock and the filament network structure was substantially reversed by 60 minutes. Differences were also seen in the cells during respreading after replating: cells containing mutant keratins were slower to respread than controls and fine aggregates were seen at the cell margins in the Dowling-Meara derived cell line. Such delays in restoring the normal intermediate filament network after physiological processes involving cytoskeleton remodelling may render the cells vulnerable to cytolysis in vivo if physically challenged during this time window. The steady reduction in the mitotic index of the epidermis during the first few years of life could then explain the clinical improvement which is frequently observed in growing children.

Cell Line↗
PubMed1996

Transport of protein kinase C alpha into the nucleus requires intact cytoskeleton while the transport of a protein containing a canonical nuclear localization signal does not.

Protein kinase C undergoes a redistribution from the cytosol into the nucleus upon various stimuli. Since protein kinase C does not contain any known nuclear localization signal, the exact pathway and mechanism of the translocation into the nucleus is not known. We used immunofluorescence microscopy to investigate the role of the cytoskeleton in this process, and to detect the subcellular distribution of protein kinase C alpha in NIH 3T3 fibroblasts. In these cells protein kinase C alpha is translocated into the nucleus after stimulation with phorbol ester. We observed that cells treated with the cytoskeleton disrupting agents cytochalasin B or colchicine do not show the nuclear translocation of protein kinase C alpha after stimulation. In contrast, the nuclear accumulation of a nuclear localization signal containing reporter protein in an in vitro nuclear transport assay is not affected by these drugs. This observation has been confirmed for intact cells by microinjection experiments: cells which have been incubated with cytochalasin B or colchicine prior to microinjection of the reporter protein show the same accumulation in the nucleus as untreated cells. Our data show that intact cytoskeleton plays an important role in the translocation of protein kinase C alpha into the nucleus but not in the nuclear import of a karyophilic reporter protein.

3T3 Cells↗
PubMed1997

Neurofibromatosis 2 tumor suppressor protein colocalizes with ezrin and CD44 and associates with actin-containing cytoskeleton.

Neurofibromatosis 2 (NF2) protein (merlin; schwannomin) is a tumor suppressor involved in tumorigenesis of NF2-associated and sporadic schwannomas and meningiomas. The protein shares the domain structure of three homologous proteins: ezrin, radixin and moesin (ERM). ERM proteins function as membrane organizers and may act as linkers between plasma membrane molecules, such as CD44 and ICAM-2, and the cytoskeleton. We analyzed the distribution and effects of transfected NF2 protein in COS-1, CHO and 293 cells, and endogenous NF2 protein in U251 glioma cells. The distribution was compared to ezrin, CD44 and F-actin. Both transfected and endogenous NF2 protein localized underneath the plasma membrane in a pattern typical of an ERM protein. In COS-1 transfectants, NF2 protein typically codistributed with ezrin but, in cells with poorly developed actin cytoskeleton, it replaced ezrin in filopodia and ruffling edges. NF2 protein colocalized with CD44, which in transfected cells accumulated into restructured cell membrane protrusions. The association of CD44 and NF2 protein was further suggested by binding of CD44 from cellular lysates to recombinant NF2 protein. Interaction between NF2 protein and the actin-containing cytoskeleton was indicated by partial colocalization, by cytochalasin B-induced coclustering, and by retention of NF2 protein in the detergent-insoluble fraction. Transfected NF2 protein induced morphogenic changes. The cells contained restructured membrane extensions and blebs, and CHO cells expressing NF2 protein were more elongated than control transfectants. In conclusion, NF2 protein possesses functional properties of an ERM family member.

Actins↗
PubMed1998

SRC binding to the cytoskeleton, triggered by growth cone attachment to laminin, is protein tyrosine phosphatase-dependent.

The interaction of the non-receptor tyrosine kinase, Src, with the cytoskeleton of adhesion sites was studied in nerve growth cones isolated from fetal rat brain. Of particular interest was the role of protein tyrosine phosphatases in the regulation of Src-cytoskeleton binding. Growth cones were found to contain a high level of protein tryrosine phosphatase activity, most of it membrane-associated and forming large, multimeric and wheat germ agglutinin-binding complexes. The receptor tyrosine phosphatase PTPalpha seems to be the most prevalent species among the membrane-associated enzymes. As seen by immunofluorescence, PTPalpha is present throughout the plasmalemma of the growth cone including filopodia, and it forms a punctate pattern consistent with that of integrin beta1. For adhesion site analysis, isolated growth cones were either plated onto the neurite growth substratum, laminin, or kept in suspension. Plating growth cones on laminin triggered an 8-fold increase in Src binding to the adherent cytoskeleton. This effect was blocked completely with the protein tyrosine phosphatase inhibitor, vanadate. Growth cone plating also increased the association with adhesion sites of tyrosine phosphatase activity (14-fold) and of PTPalpha immunoreactivity (6-fold). Vanadate blocked the enzyme activity but not the recruitment of PTPalpha to the adhesion sites. In conjunction with our previous results on growth cones, these data suggest that integrin binding to laminin triggers the recruitment of PTPalpha (and perhaps other protein tyrosine phosphatases) to adhesion sites, resulting in de-phosphorylation of Src's tyr 527. As a result Src unfolds, becomes kinase-active, and its SH2 domain can bind to an adhesion site protein. This implies a critical role for protein tyrosine phosphatase activity in the earliest phases of adhesion site assembly.

Animals↗
PubMed1998

E-cadherin mediated cell adhesion recruits SAP97 into the cortical cytoskeleton.

Members of the SAP family of synapse-associated proteins have recently emerged as central players in the molecular organization of synapses. In this study, we have examined the mechanism that localizes one member, SAP97, to sites of cell-cell contact. Utilizing epithelial CACO-2 cells and fibroblast L-cells as model systems, we demonstrate that SAP97 is associated with the submembranous cortical cytoskeleton at cell-cell adhesion sites. Furthermore, we show that its localization into this structure is triggered by E-cadherin. Although SAP97 can be found in an E-cadherin/catenin adhesion complex, this interaction seems to be mediated by the attachment of SAP97 to the cortical cytoskeleton. Our results are consistent with a model in which SAP97 is recruited to sites of cell-cell contact via an E-cadherin induced assembly of the cortical cytoskeleton.

Actins↗
PubMed1999

Rac GTPases localize at sites of actin reorganization during dynamic remodeling of the cytoskeleton of normal embryonic fibroblasts.

Rac GTP-binding proteins are implicated in the dynamic organization of the actin cytoskeleton, and the mechanisms utilized for this purpose are not understood yet. In this paper we have analysed the effects of the expression of Rac proteins on the organization of the cytoskeleton, and their subcellular distribution in chicken embryo fibroblasts. In these cells, overexpression of wild-type Rac GTPases induces disassembly of stress fibers, and production of long, highly branched actin-rich protrusions, with consequent dramatic changes in cell morphology. The formation of these protrusions is mediated by adhesion to the substrate, and is prevented by incubation with anti-(beta)1 function-blocking antibodies. Rac-mediated cell shape changes require a wild-type GTPase, since expression of constitutively active V12-Rac proteins affects actin organization differently in these cells, without causing alterations in their morphology. Localization studies performed on ventral plasma membranes from fibroblasts transfected with wild-type or mutant GTPases show codistribution of Rac along stress fibers, before their disassembly and the formation of the actin-rich protrusions. These data show a link between Rac protein distribution, and their effects on the actin cytoskeleton. Altogether, our results are indicative of an active role of Rac proteins in stress fiber disassembly, and show that Rac, which can cycle its bound nucleotide, produces unique dynamic effects on actin organization.

Actins↗
PubMed2000

Trafficking and signaling through the cytoskeleton: a specific mechanism.

A specific mechanism for the intracellular translocation of nonvesicle-associated proteins is proposed. This movement machinery is based on the assumption that the cytoskeleton represents an interconnected network of filamentous macromolecules, which extends over the entire cytoplasm. Diffusion along the filaments provides an efficient way for movement and with this, for signal transduction, between various intracellular compartments. We calculate the First Passage Time (FPT), the average time it takes a signaling molecule, diffusing along the cytoskeleton, to arrive from the cell surface to the nucleus for the first time. We compare our results with the FPT of free diffusion and of diffusion in the permeating cytoplasm. The latter is hindered by intracellular organelles and the cytoskeleton itself. We find that for filament concentrations even below physiological values, the FPT along cytoskeletal filaments converges to that for free diffusion. When filaments are considered as obstacles, the FPT grows steadily with filament concentration. At realistic filament concentrations the FPT is insensitive to local modifications in the cytoskeletal network, including bundle formation. We conclude that diffusion along cytoskeletal tracks is a reliable alternative to other established ways of intracellular trafficking and signaling, and therefore provides an additional level of cell function regulation.

Actin Cytoskeleton↗
PubMed2000

The actin cytoskeleton and plasma membrane connection: PtdIns(4,5)P(2) influences cytoskeletal protein activity at the plasma membrane.

The co-ordination of rearrangements of the actin cytoskeleton depends on its tight connection to the plasma membrane. Phosphatidylinositol 4,5-bisphosphate is thought to transmit signals originating at the plasma membrane to the underlying actin cytoskeleton. This lipid binds to, and influences the activity of, several actin-associated proteins in vitro that regulate the architecture of the actin cytoskeleton. Signalling intermediates in this process include focal adhesion molecules such as vinculin and members of two families of proteins, ERM and WASP. These proteins interact with phosphatidylinositol 4,5-bisphosphate and appear to be regulated by interplay between small GTPases and phosphatidylinositol 4,5-bisphosphate metabolism, and thus link the plasma membrane with cytoskeletal remodelling.

Actins↗
PubMed2000

Live dynamics of GFP-calponin: isoform-specific modulation of the actin cytoskeleton and autoregulation by C-terminal sequences.

The calponin family of F-actin-, tropomyosin- and calmodulin-binding proteins currently comprises three genetic variants. Their functional roles implicated from in vitro studies include the regulation of actomyosin interactions in smooth muscle cells (h1 calponin), cytoskeletal organisation in non-muscle cells (h2 calponin) and the control of neurite outgrowth (acidic calponin). We have now investigated the effects of calponin (CaP) isoforms and their C-terminal deletion mutants on the actin cytoskeleton by time lapse video microscopy of GFP fusion proteins in living smooth muscle cells and fibroblasts. It is shown that h1 CaP associates with the actin stress fibers in the more central part of the cell, whereas h2 CaP localizes to the ends of stress fibres and in the motile lamellipodial protrusions of spreading cells. Cells expressing h2 CaP spread more efficiently than those expressing h1 CaP and expression of GFP h1 CaP resulted in reduced cell motility in wound healing experiments. Notably, expression of GFP h1 CaP, but not GFP h2 CaP, conferred increased resistance of the actin cytoskeleton to the actin polymerization antagonists cytochalasin B and latrunculin B, as well as to the protein kinase inhibitors H7-dihydrochloride and rho-kinase inhibitor Y-27632. These data point towards a dual role of CaP in the stabilization and regulation of the actin cytoskeleton in vivo. Deletion studies further identify an autoregulatory role for the unique C-terminal tail sequences in the respective CaP isoforms.

Actins↗
PubMed2000

The Saccharomyces cerevisiae SDA1 gene is required for actin cytoskeleton organization and cell cycle progression.

The organization of the actin cytoskeleton is essential for several cellular processes. Here we report the characterization of a Saccharomyces cerevisiae novel gene, SDA1, encoding a highly conserved protein, which is essential for cell viability and is localized in the nucleus. Depletion or inactivation of Sda1 cause cell cycle arrest in G(1) by blocking both budding and DNA replication, without loss of viability. Furthermore, sda1-1 temperature-sensitive mutant cells arrest at the non-permissive temperature mostly without detectable structures of polymerized actin, although a normal actin protein level is maintained, indicating that Sda1 is required for proper organization of the actin cytoskeleton. To our knowledge, this is the first mutation shown to cause such a phenotype. Recovery of Sda1 activity restores proper assembly of actin structures, as well as budding and DNA replication. Furthermore we show that direct actin perturbation, either in sda1-1 or in cdc28-13 cells released from G(1) block, prevents recovery of budding and DNA replication. We also show that the block in G(1) caused by loss of Sda1 function is independent of Swe1. Altogether our results suggest that disruption of F-actin structure can block cell cycle progression in G(1) and that Sda1 is involved in the control of the actin cytoskeleton.

Actins↗
PubMed2001

Inhibition of PYK2-induced actin cytoskeleton reorganization, PYK2 autophosphorylation and focal adhesion targeting by FAK.

Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2) are structurally related tyrosine kinases. They are implicated in regulating actin cytoskeleton organization, a process critical for cell migration, mitosis and tumor metastasis. In this paper, we demonstrate that, although both PYK2 and FAK were expressed and colocalized at focal adhesions in fibroblasts, microinjection of PYK2, but not FAK, in Swiss 3T3 fibroblastic cells led to reorganization of focal adhesions and cell rounding. PYK2-mediated actin cytoskeleton reorganization required the PYK2 N terminus, the focal adhesion targeting (FAT) domain, catalytic activity and autophosphorylation. Remarkably, FAK suppressed PYK2-mediated reorganization of focal adhesions and cell rounding. In addition, FAK inhibited PYK2 autophosphorylation and focal adhesion targeting, which might contribute to FAK-mediated suppression of PYK2's phenotypes. Further analyses demonstrated that the inhibition of PYK2 autophosphorylation required the FAK N terminus but not FAK tyrosine phosphorylation. The FAK FAT domain seemed to be critical for FAK-mediated suppression of PYK2 focal adhesion targeting. Taken together, these results demonstrate that FAK could inhibit PYK2 autophosphorylation, focal adhesion targeting and actin cytoskeleton reorganization, suggesting that the balance between FAK and PYK2 tyrosine kinases is important for regulating cellular morphology, cell migration and cell growth.

Actins↗