Activated cofilin colocalises with Arp2/3 complex in apoptotic blebs during programmed cell death

Etoposide inhibits topoisomerase II and induces apoptosis in human epidermoid cancer cells (A431) and normal rat fibroblasts (NRK) as verified by apoptotic morphology and chromatin degradation. Here we examine changes in the localisation of actin, cofilin and the Arp2/3 complex during the apoptotic process in response to etoposide. Twenty-four hours after etoposide addition, a large number of cells of both lines exhibited nuclear and cytoplasmic fragmentation with the formation of numerous blebs typical of apoptosis. Etoposide exposure induces dissolution of stress fibres and an increase in actin and cofilin in membrane patches and apoptotic blebs. The actin is more peripherally located than the cofilin, similar to that reported for lamellipodia of highly motile keratocytes. By contrast, in control cells, cofilin is evenly distributed throughout the cytoplasm, though often enriched around the nucleus. The active form is inferred to be more peripherally localised and to be present in apoptotic blebs, since an antibody specific for phosphorylated cofilin did not stain the cell periphery nor apoptotic blebs. Although immunoblots of 2D gels demonstrate that the ratio of de-phosphorylated to phosphorylated cofilin does not change after etoposide treatment, this does not mean that there are no changes in the turnover of the active and inactive forms. Transfection of both cell lines with EGFP-containing constructs of wild-type cofilin and mutants resembling its activated (S3A) and inactivated (S3D) forms shows that the active form has a more peripheral localisation and is also present in the membrane blebs with a strong colocalisation with actin. We further show that Arp2/3 also localises in apoptotic blebs and discuss the role of these proteins in apoptosis by analogy with actin-based protrusive motility in lamellipodia.     

A tropomyosin 1 induced defect in cytokinesis can be rescued by elevated expression of cofilin

Cytokinesis in eukaryotic cells is mediated by the contractile ring, an actomyosin-based structure which provides the force required to separate daughter cells. Isoforms of the actin-binding protein tropomyosin are also localised to the contractile ring in both fission yeast and human astrocytes. Although tropomyosin is required for cytokinesis in yeast, its precise role in the contractile ring is unknown. In this study we find that increased expression of a single tropomyosin isoform, tropomyosin 1, in U373MG astrocytoma cells leads to multinucleated cells and mitotic spindle defects. Furthermore, cells expressing increased levels of tropomyosin 1 usually fail to complete cytokinesis and this is accompanied by reduced accumulation of actin depolymerising factor/cofilin in the contractile ring. Adenovirus mediated expression of cofilin is able to relieve the tropomyosin 1 induced effects on cytokinesis. We conclude that tropomyosin 1 and cofilin play antagonistic roles within the contractile ring and that the balance between tropomyosin 1 and cofilin expression is important for cytokinesis.     

Regulation of cell-matrix adhesion by OLA1, the Obg-like ATPase 1

Attachment of cells to the extracellular matrix induces clustering of membrane receptor integrins which in turn triggers the formation of focal adhesions (FAs). The adaptor/scaffold proteins in FAs provide linkage to actin cytoskeleton, whereas focal adhesion kinase (FAK) and other FA-associated kinases and phosphatases transduce integrin-mediated signaling cascades, promoting actin polymerization and progression of cell spreading. In this study, we explored the role of OLA1, a newly identified member of Obg-like ATPases, in regulating cell adhesion processes. We showed that in multiple human cell lines RNAi-mediated downregulation of OLA1 significantly accelerated cell adhesion and spreading, and conversely overexpression of OLA1 by gene transfection resulted in delayed cell adhesion and spreading. We further found that OLA1-deficient cells had elevated levels of FAK protein and decreased Ser3 phosphorylation of cofilin, an actin-binding protein and key regulator of actin filament dynamics, while OLA1-overexpressing cells exhibited the opposite molecular alterations in FAK and cofilin. These findings suggest that OLA1 plays an important negative role in cell adhesion and spreading, in part through the regulation of FAK expression and cofilin phosphorylation, and manipulation of OLA1 may lead to significant changes in cell adhesion and the associated phenotypes.     

Dual roles of tropomyosin as an F-actin stabilizer and a regulator of muscle contraction in Caenorhabditis elegans body wall muscle

Tropomyosin is a well-characterized regulator of muscle contraction. It also stabilizes actin filaments in a variety of muscle and non-muscle cells. Although these two functions of tropomyosin could have different impacts on actin cytoskeletal organization, their functional relationship has not been studied in the same experimental system. Here, we investigated how tropomyosin stabilizes actin filaments and how this function is influenced by muscle contraction in Caenorhabditis elegans body wall muscle. We confirmed the antagonistic role of tropomyosin against UNC-60B, a muscle-specific ADF/cofilin isoform, in actin filament organization using multiple UNC-60B mutant alleles. Tropomyosin was also antagonistic to UNC-78 (AIP1) in vivo and protected actin filaments from disassembly by UNC-60B and UNC-78 in vitro, suggesting that tropomyosin protects actin filaments from the ADF/cofilin-AIP1 actin disassembly system in muscle cells. A mutation in the myosin heavy chain caused greater reduction in contractility than tropomyosin depletion. However, the myosin mutation showed much weaker suppression of the phenotypes of ADF/cofilin or AIP1 mutants than tropomyosin depletion. These results suggest that muscle contraction has only minor influence on the tropomyosin's protective role against ADF/cofilin and AIP1, and that the two functions of tropomyosin in actin stability and muscle contraction are independent of each other.     

Non-muscle tropomyosin (Tpm3) is crucial for asymmetric cell division and maintenance of cortical integrity in mouse oocytes

Tropomyosins are actin-binding cytoskeletal proteins that play a pivotal role in regulating the function of actin filaments in muscle and non-muscle cells; however, the roles of non-muscle tropomyosins in mouse oocytes are unknown. This study investigated the expression and functions of non-muscle tropomyosin (Tpm3) during meiotic maturation of mouse oocytes. Tpm3 mRNA was detected at all developmental stages in mouse oocytes. Tpm3 protein was localized at the cortex during the germinal vesicle and germinal vesicle breakdown stages. However, the overall fluorescence intensity of Tpm3 immunostaining was markedly decreased in metaphase II oocytes. Knockdown of Tpm3 impaired asymmetric division of oocytes and spindle migration, considerably reduced the amount of cortical actin, and caused membrane blebbing during cytokinesis. Expression of a constitutively active cofilin mutant and Tpm3 overexpression confirmed that Tpm3 protects cortical actin from depolymerization by cofilin. The data indicate that Tpm3 plays crucial roles in maintaining cortical actin integrity and asymmetric cell division during oocyte maturation, and that dynamic regulation of cortical actin by Tpm3 is critical to ensure proper polar body protrusion.     

Myoblast attachment and spreading are regulated by different patterns by ubiquitous calpains

The calcium-dependent proteolytic system is a large family of well-conserved ubiquitous and tissue-specific proteases, known as calpains, and an endogenous inhibitor, calpastatin. Ubiquitous calpains are involved in many physiological phenomena, such as the cell cycle, muscle cell differentiation, and cell migration. This study investigates the regulation of crucial steps of cell motility, myoblast adhesion and spreading, by calpains. Inhibition of each ubiquitous calpain isoform by antisense strategy pinpointed the involvement of each of these proteases in myoblast adhesion and spreading. Moreover, the actin cytoskeleton and microtubules were observed in transfected cells, demonstrating that each ubiquitous calpain could be involved in the actin fiber organization. C2C12 cells with reduced mu- or m-calpain levels have a rounded morphology and disorganized stress fibers, but no modification in the microtubule cytoskeleton. Antisense strategy directed against MARCKS, a calpain substrate during C2C12 migration, showed that this protein could play a role in stress fiber polymerization. A complementary proteomic analysis using C2C12 cells over-expressing calpastatin indicated that two proteins were under-expressed, while six, which are involved in the studied phenomena, were overexpressed after calpain inhibition. The possible role of these proteins in adhesion, spreading, and migration was discussed.     

Calpain Regulates Actin Remodeling during Cell Spreading

Previous studies suggest that the Ca²⁺-dependent proteases, calpains, participate in remodeling of the actin cytoskeleton during wound healing and are active during cell migration. To directly test the role that calpains play in cell spreading, several NIH-3T3– derived clonal cell lines were isolated that overexpress the biological inhibitor of calpains, calpastatin. These cells stably overexpress calpastatin two- to eightfold relative to controls and differ from both parental and control cell lines in morphology, spreading, cytoskeletal structure, and biochemical characteristics. Morphologic characteristics of the mutant cells include failure to extend lamellipodia, as well as abnormal filopodia, extensions, and retractions. Whereas wild-type cells extend lamellae within 30 min after plating, all of the calpastatin-overexpressing cell lines fail to spread and assemble actin-rich processes. The cells genetically altered to overexpress calpastatin display decreased calpain activity as measured in situ or in vitro. The ERM protein ezrin, but not radixin or moesin, is markedly increased due to calpain inhibition. To confirm that inhibition of calpain activity is related to the defect in spreading, pharmacological inhibitors of calpain were also analyzed. The cell permeant inhibitors calpeptin and MDL 28, 170 cause immediate inhibition of spreading. Failure of the intimately related processes of filopodia formation and lamellar extension indicate that calpain is intimately involved in actin remodeling and cell spreading.     

The Yeast V159N Actin Mutant Reveals Roles for Actin Dynamics In Vivo

Actin with a Val 159 to Asn mutation (V159N) forms actin filaments that depolymerize slowly because of a failure to undergo a conformational change after inorganic phosphate release. Here we demonstrate that expression of this actin results in reduced actin dynamics in vivo, and we make use of this property to study the roles of rapid actin filament turnover. Yeast strains expressing the V159N mutant (act1-159) as their only source of actin have larger cortical actin patches and more actin cables than wild-type yeast. Rapid actin dynamics are not essential for cortical actin patch motility or establishment of cell polarity. However, fluid phase endocytosis is defective in act1-159 strains. act1-159 is synthetically lethal with cofilin and profilin mutants, supporting the conclusion that mutations in all of these genes impair the polymerization/ depolymerization cycle. In contrast, act1-159 partially suppresses the temperature sensitivity of a tropomyosin mutant, and the loss of cytoplasmic cables seen in fimbrin, Mdm20p, and tropomyosin null mutants, suggesting filament stabilizing functions for these actin-binding proteins. Analysis of the cables in these double-mutant cells supports a role for fimbrin in organizing cytoplasmic cables and for Mdm20p and tropomyosin in excluding cofilin from the cables.     

Aip1 and cofilin promote rapid turnover of yeast actin patches and cables: a coordinated mechanism for severing and capping filaments

Rapid turnover of actin structures is required for dynamic remodeling of the cytoskeleton and cell morphogenesis, but the mechanisms driving actin disassembly are poorly defined. Cofilin plays a central role in promoting actin turnover by severing/depolymerizing filaments. Here, we analyze the in vivo function of a ubiquitous actin-interacting protein, Aip1, suggested to work with cofilin. We provide the first demonstration that Aip1 promotes actin turnover in living cells. Further, we reveal an unanticipated role for Aip1 and cofilin in promoting rapid turnover of yeast actin cables, dynamic structures that are decorated and stabilized by tropomyosin. Through systematic mutagenesis of Aip1 surfaces, we identify two well-separated F-actin-binding sites, one of which contributes to actin filament binding and disassembly specifically in the presence of cofilin. We also observe a close correlation between mutations disrupting capping of severed filaments in vitro and reducing rates of actin turnover in vivo. We propose a model for balanced regulation of actin cable turnover, in which Aip1 and cofilin function together to "prune" tropomyosin-decorated cables along their lengths. Consistent with this model, deletion of AIP1 rescues the temperature-sensitive growth and loss of actin cable defects of tpm1Delta mutants.     

Disruption of the single tropomyosin gene in yeast results in the disappearance of actin cables from the cytoskeleton

The yeast tropomyosin gene, designated TPM1, is present in a single copy per haploid genome and encodes a protein with a predicted molecular weight of 23.5 kd. The protein sequence is homologous to higher cell tropomyosins, including the characteristic hydrophobic-hydrophilic pseudoheptapeptide repeats. Indirect immunofluorescence microscopy reveals that tropomyosin is localized with actin cables in wild-type cells. Disruption of TPM1 is not lethal, but results in a reduced growth rate and disappearance of actin cables. Strains carrying the conditional actin mutation act1-2 also lack actin cables; overexpression of tropomyosin in these strains partially restores actin cables. These results strongly suggest that tropomyosin interacts with F actin in vivo and may play an important role in assembling or stabilizing actin cables in yeast.     

Intrinsic capability of budding yeast cofilin to promote turnover of tropomyosin-bound actin filaments

The ability of actin filaments to function in cell morphogenesis and motility is closely coupled to their dynamic properties. Yeast cells contain two prominent actin structures, cables and patches, both of which are rapidly assembled and disassembled. Although genetic studies have shown that rapid actin turnover in patches and cables depends on cofilin, how cofilin might control cable disassembly remains unclear, because tropomyosin, a component of actin cables, is thought to protect actin filaments against the depolymerizing activity of ADF/cofilin. We have identified cofilin as a yeast tropomyosin (Tpm1) binding protein through Tpm1 affinity column and mass spectrometry. Using a variety of assays, we show that yeast cofilin can efficiently depolymerize and sever yeast actin filaments decorated with either Tpm1 or mouse tropomyosins TM1 and TM4. Our results suggest that yeast cofilin has the intrinsic ability to promote actin cable turnover, and that the severing activity may rely on its ability to bind Tpm1.     

Two general classes of cytoplasmic actin filaments in tissue culture cells: The role of tropomyosin

In the assembly of actin filaments that takes place during the spreading of a polulation of human lung cells, after trypsin detachment off the substratum and replating, tropomyosin exhibits a considrable lag in its association with the newly forming filament bundles; it begins to associate with them during the later stages of cell spreading as the actin filament bundles normally seen in interphase cells begin to organize. This lag is evident in a number of cell types that are spreading onto a substratum; it does not appear to be due to a selective degradation of this molecule during rounding up of the cells, since tropmyosin associates with the actin filament bundles after this lag even under conditions where the protein synthetic activity of the cell is inhibited to more than 95% by cycloheximide. The preferential binding of tropomyosin to fully assembled filament bundles but not to newly formed bundles of actin filaments suggests therefore the existence of two classes of action filaments: those that bind tropomyosin and those that do not. This selective localization of tropomyosin and those that do not. This selective localization of tropomyosin on actin filaments was further pursued by examining the localization of this molecule in membrane ruffles. The immunofluorescent results indicate that ruffling is an actin-filament-dependent, microtubule-independent phenomenon. Tropomyosin is absent from membrane ruffles under a variety of circumstances where ruffling is expressed and, more generally, from any other cellular activity where actin filaments are expected to be in a dynamic state of reorganization or are required to be in a flexible configuraion. It is concluded that in tissue culture cells tropomyosin binds preferentially to actin filaments involved in structural support to confer rigidity upon them as well as aid them in maintaining a stretched phenotype. The absence of tropomyosin from certain motile phenomena where actin filaments are involved indicates that these classes of actin filaments are regulated by cytoplasmic mechanisms distinct from that by which tropomyosin (and troponin) mediates contractility in skeletal mulscle; it opens the possibility that different types of actin filaments enagaged in different cellular motile phenomenon in tissue culture cells may be regulated by a host of coexisting regulatory mechanisms, some as yet undetermined.     

Actin filament oxidation by MICAL1 suppresses protections from cofilin‐induced disassembly

Proteins of the ADF/cofilin family play a central role in the disassembly of actin filaments, and their activity must be tightly regulated in cells. Recently, the oxidation of actin filaments by the enzyme MICAL1 was found to amplify the severing action of cofilin through unclear mechanisms. Using single filament experiments in vitro, we found that actin filament oxidation by MICAL1 increases, by several orders of magnitude, both cofilin binding and severing rates, explaining the dramatic synergy between oxidation and cofilin for filament disassembly. Remarkably, we found that actin oxidation bypasses the need for cofilin activation by dephosphorylation. Indeed, non‐activated, phosphomimetic S3D‐cofilin binds and severs oxidized actin filaments rapidly, in conditions where non‐oxidized filaments are unaffected. Finally, tropomyosin Tpm1.8 loses its ability to protect filaments from cofilin severing activity when actin is oxidized by MICAL1. Together, our results show that MICAL1‐induced oxidation of actin filaments suppresses their physiological protection from the action of cofilin. We propose that, in cells, direct post‐translational modification of actin filaments by oxidation is a way to trigger their disassembly.     

Filamin A regulates cell spreading and survival via beta1 integrins

Cell spreading and exploration of topographically complex substrates require tightly-regulated interactions between extracellular matrix receptors and the cytoskeleton, but the molecular determinants of these interactions are not defined. We examined whether the actin-binding proteins cortactin, vinculin and filamin A are involved in the formation of the earliest extensions of cells spreading over collagen or poly-L-lysine-coated smooth and beaded substrates. Spreading of human gingival fibroblasts was substantially reduced on beaded or poly-L-lysine-coated substrates. Filamin A, vinculin and cortactin were found in cell extensions on smooth collagen. HEK-293 cells also spread rapidly on smooth collagen and formed numerous cell extensions enriched with filamin A. Knockdown of filamin A in HEK-293 cells by short hairpin RNA reduced spreading and the number of cell extensions. Blocking beta1 integrin function significantly reduced cell spreading and localization of filamin A to cell extensions. Conversely, filamin A-knockdown reduced beta1 integrin-collagen binding as measured by 12G10 antibody, suggesting co-dependence between filamin A and beta1 integrin functions. TUNEL staining showed higher percentages of apoptosis after filamin A-knockdown in spreading cells. Chelation of [Ca2+]i with BAPTA/AM reduced spreading of wild-type and filamin A-knockdown cells, however wild-type cells showed recruitment of filamin A to the subcortex, indicating independent roles of filamin A and [Ca2+]i in cell spreading. We conclude that filamin A integrates with beta1 integrins to mediate cell spreading and prevent apoptosis.     

Cofilin, a protein in porcine brain that binds to actin filaments and inhibits their interactions with myosin and tropomyosin

Cofilin, a 21 000 molecular weight protein of porcine brain, reacts stoichiometrically with actin in a 1:1 molar ratio. Upon binding of cofilin, the fluorescence of pyrene-labeled actin under polymerizing conditions is changed into the monomer form, irrespective of whether cofilin is added to actin before or after polymerization. Cofilin decreases the viscosity of actin filaments but increases the light-scattering intensity of the filaments. The centrifugation assay and the DNase I inhibition assay demonstrate that cofilin binds to actin filaments in a 1:1 molar ratio of cofilin to actin monomer in the filament and that cofilin increases the monomeric actin to a limited extent (up to 1.1-1.5 microM monomer) in the presence of physiological concentrations of Mg2+ and KCl. Cofilin is also able to bind to monomeric actin, as demonstrated by gel filtration. Electron microscopy showed that actin filaments are shortened and slightly thickened in the presence of cofilin. No bundle formation was observed in the presence of various concentrations of cofilin. The gel point assay using an actin cross-linking protein and the nucleation assay also suggested that cofilin shortens the actin filaments and hence increases the filament number. Cofilin blocks the binding of tropomyosin to actin filaments. Tropomyosin is dissociated from actin filaments by the binding of cofilin to actin filaments. Cofilin was found to inhibit the superprecipitation of actin-myosin mixtures as well as the actin-activated myosin ATPase. All these results suggest that cofilin is a new type of actin-associated protein.     

Muscle arm development in Caenorhabditis elegans

In several types of animals, muscle cells use membrane extensions to contact motor axons during development. To better understand the process of membrane extension in muscle cells, we investigated the development of Caenorhabditis elegans muscle arms, which extend to motor axons and form the postsynaptic element of the neuromuscular junction. We found that muscle arm development is a highly regulated process: the number of muscle arms extended by each muscle, the shape of the muscle arms and the path taken by the muscle arms to reach the motor axons are largely stereotypical. We also investigated the role of several cytoskeletal components and regulators during arm development, and found that tropomyosin (LEV-11), the actin depolymerizing activity of ADF/cofilin (UNC-60B) and, surprisingly, myosin heavy chain B (UNC-54) are each required for muscle arm extension. This is the first evidence that UNC-54, which is found in thick filaments of sarcomeres, can also play a role in membrane extension. The muscle arm phenotypes produced when these genes are mutated support a 'two-phase' model that distinguishes passive muscle arm development in embryogenesis from active muscle arm extension during larval development.     

Attachment of HeLa cells during early G1 phase

Both growth factor directed and integrin dependent signal transduction were shown to take place directly after completion of mitosis. The local activation of these signal transduction cascades was investigated in early G1 cells. Interestingly, various key signal transduction proteins were found in blebs at the cell membrane within 30 min after mitosis. These membrane blebs appeared in round, mitotic-like cells and disappeared rapidly during spreading of the cells in G1 phase. In addition to tyrosine-phosphorylated proteins, the blebs contained also phosphorylated FAK and phosphorylated MAP kinase. The formation of membrane blebs in round, mitotic cells before cell spreading is not specific for mitotic cells, because similar features were observed in trypsinized cells. Just before cell spreading also these cells exhibited membrane blebs containing active signal transduction proteins. Inhibition of signal transduction did not affect membrane bleb formation, suggesting that the membrane blebs were formed independent of signal transduction.     

Cofilin phosphorylation and actin cytoskeletal dynamics regulated by rho- and Cdc42-activated LIM-kinase 2

The rapid turnover of actin filaments and the tertiary meshwork formation are regulated by a variety of actin-binding proteins. Protein phosphorylation of cofilin, an actin-binding protein that depolymerizes actin filaments, suppresses its function. Thus, cofilin is a terminal effector of signaling cascades that evokes actin cytoskeletal rearrangement. When wild-type LIMK2 and kinase-dead LIMK2 (LIMK2/KD) were respectively expressed in cells, LIMK2, but not LIMK2/KD, phosphorylated cofilin and induced formation of stress fibers and focal complexes. LIMK2 activity toward cofilin phosphorylation was stimulated by coexpression of activated Rho and Cdc42, but not Rac. Importantly, expression of activated Rho and Cdc42, respectively, induced stress fibers and filopodia, whereas both Rho- induced stress fibers and Cdc42-induced filopodia were abrogated by the coexpression of LIMK2/KD. In contrast, the coexpression of LIMK2/KD with the activated Rac did not affect Rac-induced lamellipodia formation. These results indicate that LIMK2 plays a crucial role both in Rho- and Cdc42-induced actin cytoskeletal reorganization, at least in part by inhibiting the functions of cofilin. Together with recent findings that LIMK1 participates in Rac-induced lamellipodia formation, LIMK1 and LIMK2 function under control of distinct Rho subfamily GTPases and are essential regulators in the Rho subfamilies-induced actin cytoskeletal reorganization.     

Beta1 integrin-mediated T cell adhesion and cell spreading are regulated by calpain

To investigate the function of calpain in T cells, we sought to determine the role of this protease in cellular events mediated by beta1 integrins. T cell receptor cross-linked or phorbol ester-stimulated T cells binding to immobilized fibronectin induce the translocation of calpain to the cytoskeletal/membrane fraction of these cells. Such translocation of calpain is associated with proteolytic modification of protein tyrosine phosphatase 1B, increased cellular adhesion, and dramatic alterations in cellular morphology. However, affinity-related increases in T cell adhesion induced by the anti-beta1 integrin antibody 8A2 occur in a calpain-independent manner and in the absence of morphological shape changes. Furthermore, calpain undergoes activation in response to either alpha4beta1 or alpha5beta1 integrin binding to fibronectin in appropriately stimulated T cells, and calpain II as well as protein tyrosine phosphatase 1B accumulates at sites of focal contact formation. Inhibition of calpain activity not only inhibits the proteolytic modification of protein tyrosine phosphatase 1B, but also decreases the ability of T cells to adhere to and spread on immobilized fibronectin. Thus, we describe a potential regulatory role for calpain in beta1 integrin-mediated signaling events associated with T cell adhesion and cell spreading on fibronectin.     

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.