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Results for “Cortactin”

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At least 19 recordsLinked to original sources

PubMed2006

Transgenic mice with mammary gland targeted expression of human cortactin do not develop (pre-malignant) breast tumors: studies in MMTV-cortactin and MMTV-cortactin/-cyclin D1 bitransgenic mice.

BACKGROUND: In human breast cancers, amplification of chromosome 11q13 correlates with lymph node metastasis and increased mortality. To date, two genes located within this amplicon, CCND1 and EMS1, were considered to act as oncogenes, because overexpression of both proteins, respectively cyclin D1 and cortactin, correlated well with 11q13 amplification. Cyclin D1 is involved in cell cycle regulation and the F-actin-binding protein cortactin in cytoskeletal dynamics and cell migration. To study the role of cortactin in mammary gland tumorigenesis, we examined mouse mammary tumor virus (MMTV)-cortactin transgenic mice and MMTV-cortactin/-MMTV-cyclin D1 bitransgenic mice. METHODS: MMTV-cortactin transgenic mice were generated and intercrossed with previously described MMTV-cyclin D1 transgenic mice. Immunohistochemical, Northern and Western blot analyses were performed to study the expression of human transgene cortactin during mammary gland development and in mammary tumors. For tumor incidence studies, forced-bred, multiparous mice were used to enhance transgene expression in the mammary gland. Microscopical examination was performed using haematoxylin and eosin staining. RESULTS: Mammary gland tumors arose stochastically (incidence 21%) with a mean age of onset at 100 weeks. This incidence, however, did not exceed that of aged-matched control FVB/N mice (38%), which unexpectedly, also developed spontaneous mammary gland tumors. We mimicked 11q13 amplification by generating MMTV-cortactin/-MMTV-cyclin D1 bitransgenic mice but did not observe any synergistic effect of cortactin on cyclin D1-induced mammary hyperplasias or carcinomas, nor development of distant metastasis. CONCLUSION: From this study, we conclude that development of (pre-malignant) breast tumors in either wild type or MMTV-cyclin D1 mice was not augmented due to mammary gland targeted overexpression of human cortactin.

Animals↗

PubMed1998

Isolation and characterization of cortactin isoforms and a novel cortactin-binding protein, CBP90.

BACKGROUND: Cortactin is a major phosphotyrosyl protein in pp60v-src-transformed chicken embryo cells. Cortactin binds to actin filament (F-actin) through a unique region which consists of six tandem 37 amino acid repeats, named cortactin repeats. Furthermore cortactin has one src homology 3 (SH3) domain. RESULTS: In this study we have isolated two new isoforms of cortactin from the rat brain using a polymerase chain reaction (PCR) method, and have named the original cortactin as cortactin-A and the newly isolated forms as cortactin-B and -C. Cortactin-A, -B and -C had six, five, and four cortactin repeats, respectively. All the isoforms were able to bind to F-actin, but only cortactin-A demonstrated an F-actin-crosslinking activity. In addition, cortactin-A was able to bind along the side of F-actin. Next, using a blot overlay assay with glutathione S-transferase (GST)-cortactin-A, we identified a cortactin-A-binding protein with an Mr of approximately 90 kDa in rat brain and named it CBP90 (cortactin-binding protein with an Mr of approximately 90 KDa). CBP90 was purified from rat brain and its cDNA was cloned from a rat brain cDNA library. The deduced amino acid sequence of CBP90 had no significant similarity to any other protein, but it had a proline-rich domain at the C-terminal region. CBP90 was able to bind to all the cortactin isoforms. A deletion mutant analysis of cortactin-A and CBP90 revealed that the SH3 domain of cortactin-A was able to bind to the proline-rich region of CBP90. A Western blot analysis with an anti-CBP90 antibody indicated that, among the rat tissues examined, CBP90 was exclusively expressed in brain. Furthermore, its subcellular distribution and developmental expression patterns were similar to those of cortactin. CONCLUSION: These results suggest that cortactin interacts with CBP90 and plays a role in regulation of the actin cytoskeleton in brain.

Actins↗

PubMed2005

Inducible phosphorylation of cortactin is not necessary for cortactin-mediated actin polymerisation.

Cortactin is an SH3 domain-containing protein that contributes to the formation of dynamic cortical actin-associated structures, such as lamellipodia and membrane ruffles. It was originally identified as a substrate for the protein kinase Src; however, the role of tyrosine phosphorylation in the translocation of cortactin to the cell periphery and in the subsequent actin polymerisation is still unclear. Recently, two serine/threonine kinases, Pak1 and Erk, have been implicated in the regulation of cortactin. Therefore, we systematically investigated whether phosphorylation on either tyrosine or serine/threonine residues is necessary for cortactin function. In COS7 cells over-expressing Vav2 or treated with EGF, we could not detect tyrosine phosphorylation, although cortactin was translocated to cell periphery and induced membrane ruffle formation. In addition, the selective MEK inhibitor, PD98059, did not influence in vivo the ability of cortactin to bind to and induce membrane ruffles upon Vav2 over-expression or short-term EGF treatment. Finally, using a constitutively active Pak1 mutant, Pak1 T423E, we showed that Pak1 is not capable of phosphorylating cortactin either in vitro or in COS7 cells. These results suggest that cortactin-mediated actin polymerisation at cell periphery requires only Rac activation but neither tyrosine nor serine/threonine phosphorylation.

Actins↗

PubMed2006

Cortactin is essential for F-actin assembly in enteropathogenic Escherichia coli (EPEC)- and enterohaemorrhagic E. coli (EHEC)-induced pedestals and the alpha-helical region is involved in the localization of cortactin to bacterial attachment sites.

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are important human pathogens. Upon attachment to host cells, EPEC and EHEC are able to induce actin polymerization, which accumulates, forming a pedestal-like structure beneath the attached bacteria. Using siRNA, we show here that EPEC- and EHEC-induced pedestals are dependent on cortactin, an F-actin-binding protein found in the mammalian cell cortex. Knock-down of cortactin by siRNA resulted in a dramatic reduction of the pedestal formation induced by both pathogens. We also show that disruption of the Src homology 3 (SH3) domain of cortactin, or its downregulation by specific point mutations, negatively affects pedestal formation, suggesting that this domain is important for regulation of F-actin assembly by EPEC and EHEC. Green fluorescent protein (GFP) fused with the SH3 domain (GFP-SH3), proline-rich region (GFP-PRR) or alpha-helical region of cortactin markedly reduced the amount of F-actin at the bacterial attachment sites. Interestingly, neither GFP-SH3 nor GFP-PRR was recruited to the vicinity of the bacterial adherence sites; however, GFP fused to the alpha-helical region was efficiently recruited and colocalized with the attached bacteria. These results demonstrate that cortactin is a requirement for pedestal formation and suggest a novel function for the predicted alpha-helical region of cortactin in actin assembly induced by EPEC and EHEC.

Actins↗

PubMed2003

Alternative splicing of the actin binding domain of human cortactin affects cell migration.

Cortactin is a filamentous actin (F-actin)-binding protein that regulates cytoskeletal dynamics by activating the Arp2/3 complex; it binds to F-actin by means of six N-terminal "cortactin repeats". Gene amplification of 11q13 and consequent overexpression of cortactin in several human cancers is associated with lymph node metastasis. Overexpression as well as tyrosine phosphorylation of cortactin has been reported to enhance cell migration, invasion, and metastasis. Here we report the identification of two alternative splice variants (SV1 and SV2) that affect the cortactin repeats: SV1-cortactin lacks the 6th repeat (exon 11), whereas SV2-cortactin lacks the 5th and 6th repeats (exons 10 and 11). SV-1 cortactin is found co-expressed with wild type (wt)-cortactin in all tissues and cell lines examined, whereas the SV2 isoform is much less abundant. SV1-cortactin binds F-actin and promotes Arp2/3-mediated actin polymerization equally well as wt-cortactin, whereas SV2-cortactin shows reduced F-actin binding and polymerization. Alternative splicing of cortactin does not affect its subcellular localization or growth factor-induced tyrosine phosphorylation. However, cells that overexpress SV1- or SV2-cortactin show significantly reduced cell migration when compared with wt-cortactin-overexpressing cells. Thus, in addition to overexpression and tyrosine phosphorylation, alternative splicing of the F-actin binding domain of cortactin is a new mechanism by which cortactin influences cell migration.

Actins↗

PubMed2004

Actin depolymerization-induced tyrosine phosphorylation of cortactin: the role of Fer kinase.

The F-actin-binding protein cortactin is an important regulator of cytoskeletal dynamics, and a prominent target of various tyrosine kinases. Tyrosine phosphorylation of cortactin has been suggested to reduce its F-actin cross-linking capability. In the present study, we investigated whether a reciprocal relationship exists, i.e. whether the polymerization state of actin impacts on the cortactin tyrosine phosphorylation. Actin depolymerization by LB (latrunculin B) induced robust phosphorylation of C-terminal tyrosine residues of cortactin. In contrast, F-actin stabilization by jasplakinolide, which redistributed cortactin to F-actin-containing patches, prevented cortactin phosphorylation triggered by hypertonic stress or LB. Using cell lines deficient in candidate tyrosine kinases, we found that the F-actin depolymerization-induced cortactin phosphorylation was mediated by the Fyn/Fer kinase pathway, independent of Src and c-Abl. LB caused modest Fer activation and strongly facilitated the association between Fer and cortactin. Interestingly, the F-actin-binding region within the cortactin N-terminus was essential for the efficient phosphorylation of C-terminal tyrosine residues. Investigating the structural requirements for the Fer-cortactin association, we found that (i) phosphorylation-incompetent cortactin still bound to Fer; (ii) the isolated N-terminus associated with Fer; and (iii) the C-terminus alone was insufficient for binding. Thus the cortactin N-terminus participates in the Fer-cortactin interaction, which cannot be fully due to the binding of the Fer Src homology 2 domain to C-terminal tyrosine residues of cortactin. Taken together, F-actin stabilization prevents cortactin tyrosine phosphorylation, whereas depolymerization promotes it. Depolymerization-induced phosphorylation is mediated by Fer, and requires the actin-binding domain of cortactin. These results define a novel F-actin-dependent pathway that may serve as a feedback mechanism during cytoskeleton remodelling.

Actins↗

PubMed2006

CXCL12 induces tyrosine phosphorylation of cortactin, which plays a role in CXC chemokine receptor 4-mediated extracellular signal-regulated kinase activation and chemotaxis.

CXC chemokine receptor 4 (CXCR4) plays a role in the development of immune and central nervous systems as well as in cancer growth and metastasis. CXCR4-initiated signaling cascades leading to cell proliferation and chemotaxis are critical for these functions. The present study demonstrated that stimulation of CXCR4 by its ligand, CXCL12, induced transient translocation of cortactin from endosomal compartments to the cell periphery where it colocalized with CXCR4 followed by internalization of CXCR4 together with cortactin into endosomes. Cortactin was co-immunoprecipitated with CXCR4 in response to CXCL12 treatment in a time-dependent manner. Ligand stimulation induced phosphorylation of cortactin at tyrosine 421, and the phosphorylation was both c-Src- and dynamin-dependent. Cortactin overexpression promoted CXCR4 internalization and recycling. However, overexpression of a cortactin mutant in which tyrosine 421 was replaced with alanine (cortactin-Y421A) or knockdown of cortactin with RNA interference (RNAi) reduced CXCR4 internalization in response to CXCL12. CXCR4-mediated activation of extracellular signal-regulated kinases 1 and 2 was significantly prolonged by overexpression of wild-type cortactin but not by the cortactin-Y421A mutant and was inhibited by cortactin knockdown with RNAi. Moreover, CXCL12-induced chemotaxis was enhanced by cortactin overexpression, reduced by overexpression of the cortactin-Y421A mutant, and blocked by cortactin knockdown with RNAi. These data provide strong evidence for an important role of cortactin in CXCR4 signaling and trafficking as well in the receptor-mediated cell migration.

Cell Line↗

PubMed2005

Effects of tyrosine phosphorylation of cortactin on podosome formation in A7r5 vascular smooth muscle cells.

Cortactin, a predominant substrate of Src family kinases, plays an important role in Arp2/3-dependent actin polymerization in lamellipodia and membrane ruffles and was recently shown to be enriched in podosomes induced by either c-Src or phorbol ester. However, the mechanisms by which cortactin regulates podosome formation have not been determined. In this study, we showed that cortactin is required for podosome formation, using siRNA knockdown of cortactin expression in smooth muscle A7r5 cells. Treatment with phorbol ester or expression of constitutively active c-Src induced genesis of cortactin-containing podosomes as well as increase in phosphorylation of cortactin at Y421 and Y466, the Src phosphorylation sites on cortactin. The Src kinase inhibitor SU-6656 significantly inhibited formation of podosomes induced by phorbol ester and phosphorylation of cortactin, whereas PKCalpha inhibitor did not affect podosome formation in c-Src-transfected cells. Unexpectedly, expression of cortactin mutants containing Y421F, Y421D, Y466F, or Y466D mutated sites did not affect podosome formation or cortactin translocation to podosomes, although endogenous tyrosine-phosphorylated cortactin at Y421 and Y466 was present in podosomes. Our data indicate that 1) PKCalpha acts upstream of Src in phosphorylation of cortactin and podosome formation in smooth muscle cells; 2) expression of cortactin is essential for genesis of podosomes; 3) phosphorylation at Y421 and Y466 is not required for translocation of cortactin to podosomes, although phosphorylation at these sites appears to be enriched in podosomes; and 4) tyrosine phosphorylation of cortactin may be involved in regulation of stability and turnover of podosomes, rather than targeting this protein to the site of podosome formation.

Animals↗

PubMed1998

Identification of a novel cortactin SH3 domain-binding protein and its localization to growth cones of cultured neurons.

Cortactin is an actin-binding protein that contains several potential signaling motifs including a Src homology 3 (SH3) domain at the distal C terminus. Translocation of cortactin to specific cortical actin structures and hyperphosphorylation of cortactin on tyrosine have been associated with the cortical cytoskeleton reorganization induced by a variety of cellular stimuli. The function of cortactin in these processes is largely unknown in part due to the lack of information about cellular binding partners for cortactin. Here we report the identification of a novel cortactin-binding protein of approximately 180 kDa by yeast two-hybrid interaction screening. The interaction of cortactin with this 180-kDa protein was confirmed by both in vitro and in vivo methods, and the SH3 domain of cortactin was found to direct this interaction. Since this protein represents the first reported natural ligand for the cortactin SH3 domain, we designated it CortBP1 for cortactin-binding protein 1. CortBP1 contains two recognizable sequence motifs within its C-terminal region, including a consensus sequence for cortactin SH3 domain-binding peptides and a sterile alpha motif. Northern and Western blot analysis indicated that CortBP1 is expressed predominately in brain tissue. Immunofluorescence studies revealed colocalization of CortBP1 with cortactin and cortical actin filaments in lamellipodia and membrane ruffles in fibroblasts expressing CortBP1. Colocalization of endogenous CortBP1 and cortactin was also observed in growth cones of developing hippocampal neurons, implicating CortBP1 and cortactin in cytoskeleton reorganization during neurite outgrowth.

3T3 Cells↗

PubMed2002

Interaction of cortactin and N-WASp with Arp2/3 complex.

BACKGROUND: Dynamic actin assembly is required for diverse cellular processes and often involves activation of Arp2/3 complex. Cortactin and N-WASp activate Arp2/3 complex, alone or in concert. Both cortactin and N-WASp contain an acidic (A) domain that is required for Arp2/3 complex binding. RESULTS: We investigated how cortactin and the constitutively active VCA domain of N-WASp interact with Arp2/3 complex. Structural studies showed that cortactin is a thin, elongated monomer. Chemical crosslinking studies demonstrated selective interaction of the Arp2/3 binding NTA domain of cortactin (cortactin NTA) with the Arp3 subunit and VCA with Arp3, Arp2, and ARPC1/p40. Cortactin NTA and VCA crosslinking to the Arp3 subunit were mutually exclusive; however, cortactin NTA did not inhibit VCA crosslinking to Arp2 or ARPC1/p40, nor did it inhibit activation of Arp2/3 complex by VCA. We conducted an experiment in which a saturating concentration of cortactin NTA modestly lowered the binding affinity of VCA for Arp2/3; the results of this experiment provided further evidence for ternary complex formation. Consistent with a common binding site on Arp3, a saturating concentration of VCA abolished binding of cortactin to Arp2/3 complex. CONCLUSIONS: Under certain circumstances, cortactin and N-WASp can bind simultaneously to Arp2/3 complex, accounting for their synergy in activation of actin assembly. The interaction of cortactin NTA with Arp2/3 complex does not inhibit Arp2/3 activation by N-WASp, despite competition for a common binding site located on the Arp3 subunit. These results suggest a model in which cortactin may bridge Arp2/3 complex to actin filaments via Arp3 and N-WASp activates Arp2/3 complex by binding Arp2 and/or ARPC1/p40.

Actin-Related Protein 2↗

PubMed2006

A role for cortactin in Listeria monocytogenes invasion of NIH 3T3 cells, but not in its intracellular motility.

Cortactin is an F-actin binding protein that binds to the Arp2/3 complex, stimulates its actin nucleation activity, and inhibits actin filament debranching. Using RNA interference directed against cortactin, we explored the importance of cortactin for several processes involving dynamic actin assembly. Silencing cortactin expression was efficiently achieved in HeLa and NIH 3T3 cells, with less than 5% of cortactin expression in siRNA-treated cells. Surprisingly, endocytosis in HeLa and NIH 3T3 cells, and cell migration rates, were not altered by RNAi-mediated cortactin silencing. Listeria utilizes actin-based motility to move within and spread among mammalian host cells; its actin-clouds and tails recruit cortactin. We explored the role of cortactin during the Listeria life cycle in cortactin "knockdown" NIH 3T3 cells. Interestingly, cortactin siRNA-treated cells showed a significant reduction in the efficiency of the bacteria invasion in NIH 3T3 cells. However, cortactin depletion did not interfere with assembly of Listeria actin clouds or actin tails, or Listeria intracellular motility or speed. Therefore, our findings suggest that cortactin plays a role in Listeria internalization, but not in the formation of actin clouds and tails, or in bacteria intracellular motility.

Actins↗

PubMed2004

Cortactin modulates cell migration and ring canal morphogenesis during Drosophila oogenesis.

Cortactin is a Src substrate that interacts with F-actin and can stimulate actin polymerization by direct interaction with the Arp2/3 complex. We have isolated complete loss-of-function mutants of the single Drosophila cortactin gene. Mutants are viable and fertile, showing that cortactin is not an essential gene. However, cortactin mutants show distinct defects during oogenesis. During oogenesis, Cortactin protein is enriched at the F-actin rich ring canals in the germ line, and in migrating border cells. In cortactin mutants, the ring canals are smaller than normal. A similar phenotype has been observed in Src64 mutants and in mutants for genes encoding Arp2/3 complex components, supporting that these protein products act together to control specific processes in vivo. Cortactin mutants also show impaired border cell migration. This invasive cell migration is guided by Drosophila EGFR and PDGF/VEGF receptor (PVR). We find that accumulation of Cortactin protein is positively regulated by PVR. Also, overexpression of Cortactin can by itself induce F-actin accumulation and ectopic filopodia formation in epithelial cells. We present evidence that Cortactin is one of the factors acting downstream of PVR and Src to stimulate F-actin accumulation. Cortactin is a minor contributor in this regulation, consistent with the cortactin gene not being essential for development.

Actins↗

PubMed2005

Proteolysis of cortactin by calpain regulates membrane protrusion during cell migration.

Calpain 2 regulates membrane protrusion during cell migration. However, relevant substrates that mediate the effects of calpain on protrusion have not been identified. One potential candidate substrate is the actin binding protein cortactin. Cortactin is a Src substrate that drives actin polymerization by activating the Arp2/3 complex and also stabilizes the cortical actin network. We now provide evidence that proteolysis of cortactin by calpain 2 regulates membrane protrusion dynamics during cell migration. We show that cortactin is a calpain 2 substrate in fibroblasts and that the preferred cleavage site occurs in a region between the actin binding repeats and the alpha-helical domain. We have generated a mutant cortactin that is resistant to calpain proteolysis but retains other biochemical properties of cortactin. Expression of the calpain-resistant cortactin, but not wild-type cortactin, impairs cell migration and increases transient membrane protrusion, suggesting that calpain proteolysis of cortactin limits membrane protrusions and regulates migration in fibroblasts. Furthermore, the enhanced protrusion observed with the calpain-resistant cortactin requires both the Arp2/3 binding site and the Src homology 3 domain of cortactin. Together, these findings suggest a novel role for calpain-mediated proteolysis of cortactin in regulating membrane protrusion dynamics during cell migration.

Actins↗

PubMed1995

Translocation of cortactin (p80/85) to the actin-based cytoskeleton during thrombin receptor-mediated platelet activation.

Cortactin (p80/85) was discovered as a src kinase substrate and an actin filament binding protein. We investigated translocation of cortactin to the cytoskeleton during thrombin receptor-mediated platelet activation. Only a few percent of total cortactin (minor cortactin pool) translocates to the cytoskeleton as early as 5 s after platelet activation, while about 40% of total cortactin (major cortactin pool) is thereafter recovered in the cytoskeleton during platelet aggregation. Pretreatment of platelets with cytochalasin D suppresses completely this translocation, indicating that the translocation is dependent on actin polymerization. Inhibition of platelet aggregation by a tetrapeptide with the sequence RGDS, chelator of extracellular Ca2+, or a nonstirring condition results in marked suppression of translocation of the major cortactin pool. These results suggest that a minor cortactin pool translocates to the cytoskeleton independent of GPII-bIIIa (alpha IIb beta 3 integrin) engagement, and a major pool requires GPIIbIIIa-mediated signals into the cell for the translocation. Methyl 2,5-hydroxycinamate, a tyrosine kinase inhibitor, inhibits tyrosine phosphorylation of cortactin without affecting its translocation, indicating that tyrosine phosphorylation is not essential for the translocation. Morphological studies reveal that cortactin is colocalized with filamentous actin in aggregated platelets and that it is localized at the cell peripheries along actin filaments in spread platelets. Taking these together, we have demonstrated in this paper that the translocation of cortactin is associated with the reorganization of the actin-based cytoskeleton during platelet activation, particularly with platelet aggregation.

Actins↗

PubMed2003

Cortactin tyrosine phosphorylation requires Rac1 activity and association with the cortical actin cytoskeleton.

Cortactin is an F-actin binding protein that activates actin-related protein 2/3 complex and is localized within lamellipodia. Cortactin is a substrate for Src and other protein tyrosine kinases involved in cell motility, where its phosphorylation on tyrosines 421, 466, and 482 in the carboxy terminus is required for cell movement and metastasis. In spite of the importance of cortactin tyrosine phosphorylation in cell motility, little is known regarding the structural, spatial, or signaling requirements regulating cortactin tyrosine phosphorylation. Herein, we report that phosphorylation of cortactin tyrosine residues in the carboxy terminus requires the aminoterminal domain and Rac1-mediated localization to the cell periphery. Phosphorylation-specific antibodies directed against tyrosine 421 and 466 were produced to study the regulation and localization of tyrosine phosphorylated cortactin. Phosphorylation of cortactin tyrosine 421 and 466 was elevated in response to Src, epidermal growth factor receptor and Rac1 activation, and tyrosine 421 phosphorylated cortactin localized with F-actin in lamellipodia and podosomes. Cortactin tyrosine phosphorylation is progressive, with tyrosine 421 phosphorylation required for phosphorylation of tyrosine 466. These results indicate that cortactin tyrosine phosphorylation requires Rac1-induced cortactin targeting to cortical actin networks, where it is tyrosine phosphorylated in hierarchical manner that is closely coordinated with its ability to regulate actin dynamics.

Actins↗

PubMed2006

Cortactin has an essential and specific role in osteoclast actin assembly.

Osteoclasts are essential for bone dynamics and calcium homeostasis. The cells form a tight seal on the bone surface, onto which they secrete acid and proteases to resorb bone. The seal is associated with a ring of actin filaments. Cortactin, a c-Src substrate known to promote Arp2/3-mediated actin assembly in vitro, is expressed in osteoclasts and localizes to the sealing ring. To address the role of cortactin and actin assembly in osteoclasts, we depleted cortactin by RNA interference. Cortactin-depleted osteoclasts displayed a complete loss of bone resorption with no formation of sealing zones. On nonosteoid surfaces, osteoclasts flatten with a dynamic, actin-rich peripheral edge that contains podosomes, filopodia, and lamellipodia. Cortactin depletion led to a specific loss of podosomes, revealing a tight spatial compartmentalization of actin assembly. Podosome formation was restored in cortactin-depleted cells by expression of wild-type cortactin or a Src homology 3 point mutant of cortactin. In contrast, expression of a cortactin mutant lacking tyrosine residues phosphorylated by Src did not restore podosome formation. Cortactin was found to be an early component of the nascent podosome belt, along with dynamin, supporting a role for cortactin in actin assembly.

Actin-Related Protein 2↗

PubMed2005

Cortactin and phagocytosis in isolated Sertoli cells.

BACKGROUND: Cortactin, an actin binding protein, has been associated with Sertoli cell ectoplasmic specializations in vivo, based on its immunolocalization around the heads of elongated spermatids, but not previously identified in isolated Sertoli cells. In an in vitro model of Sertoli cell-spermatid binding, cortactin was identified around debris and dead germ cells. Based on this observation, we hypothesized that this actin binding protein may be associated with a non-junction-related physiological function, such as phagocytosis. The purpose of this study was to identify the presence and distribution of cortactin in isolated rat Sertoli cells active in phagocytic activity following the addition of 0.8 microm latex beads. RESULTS: Sertoli cell monocultures were incubated with or without follicle stimulating hormone (FSH; 0.1 microg/ml) in the presence or absence of cytochalasin D (2 microM), as an actin disrupter. Cortactin was identified by standard immunostaining with anti-cortactin, clone 4F11 (Upstate) after incubation times of 15 min, 2 hr, and 24 hr with or without beads. Cells exposed to no hormone and no beads appeared to have a ubiquitous distribution of cortactin throughout the cytoplasm. In the presence of cytochalasin D, cortactin immunostaining was punctate and distributed in a pattern similar to that reported for actin in cells exposed to cytochalasin D. Sertoli cells not exposed to FSH, but activated with beads, did not show cortactin immunostaining around the phagocytized beads at any of the time periods. FSH exposure did not alter the distribution of cortactin within Sertoli cells, even when phagocytic activity was upregulated by the presence of beads. CONCLUSION: Results of this study suggest cortactin is not associated with peripheralized actin at junctional or phagocytic sites. Further studies are necessary to clarify the role of cortactin in Sertoli cells.

Animals↗

PubMed2000

Association of cortactin with dynamic actin in lamellipodia and on endosomal vesicles.

We have used fluorescent protein tagging to study the localization and dynamics of the actin-binding protein cortactin in living NIH 3T3 fibroblast cells. Cortactin was localized to active lamellipodia and to small cytoplasmic spots. Time-lapse imaging revealed that these cortactin labeled structures were very dynamic. In the lamellipodia, cortactin labeled structures formed at the leading edge and then moved toward the cell center. Experiments with green fluorescent protein (GFP)-tagged actin showed that cortactin movement was coincident with the actin retrograde flow in the lamellipodia. Cytoplasmic cortactin spots also contained F-actin and were propelled by actin polymerization. Arp3, a component of the arp2/3 complex which is a key regulator of actin polymerization, co-localized with cortactin. Cytoplasmic cortactin-labeled spots were found to be associated with endosomal vesicles. Association was asymmetric and approximately half of the endosomes were associated with cortactin spots. Time-lapse imaging suggested that these cortactin and F-actin-containing spots propelled endosomes. Actin polymerization based propulsion may be a common mechanism for endomembrane trafficking in the same manner as used in the plasma membrane protrusions. As cortactin is known to interact with membrane-associated signaling proteins it could have a role in linking signaling complexes with dynamic actin on endosomes and in lamellipodia.

3T3 Cells↗