RESUMO
During cytokinesis, a cleavage furrow generated by actomyosin ring contraction is restructured into the midbody, a platform for the assembly of the abscission machinery that controls the final separation of daughter cells. The polymerization state of F-actin is important during assembly, ingression, disassembly, and closure of the contractile ring and for the cytoskeletal remodeling that accompanies midbody formation and progression to abscission. Actin filaments must be cleared from the abscission sites before the final cut can take place. Although many conserved proteins interact with and influence the polymerization state of actin filaments, it is poorly understood how they regulate cytokinesis in higher eukaryotes. We report here that the actin capping protein (CP), a barbed end actin binding protein, participates in the control of actin polymerization during later stages of cytokinesis in human cells. Cells depleted of CP furrow and form early midbodies, but they fail cytokinesis. Appropriate recruitment of the ESCRT-III abscission machinery to the midbody is impaired, preventing the cell from progressing to the abscission stage. To generate actin filaments of optimal length, different actin nucleators, such as formins, balance CP's activity. Loss of actin capping activity leads to excessive accumulation of formin-based linear actin filaments. Depletion of the formin FHOD1 results in partial rescue of CP-induced cytokinesis failure, suggesting that it can antagonize CP activity during midbody maturation. Our work suggests that the actin cytoskeleton is remodeled in a stepwise manner during cytokinesis, with different regulators at different stages required for successful progression to abscission.
Assuntos
Proteínas de Capeamento de Actina/fisiologia , Citoesqueleto de Actina/fisiologia , Citocinese/fisiologia , Actinas , Membrana Celular , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Células Epiteliais/fisiologia , Epitélio Corneano/citologia , Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Forminas , Regulação da Expressão Gênica/fisiologia , Células HeLa , Humanos , Proteínas dos Microfilamentos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMO
Capping protein (CP) binds the fast growing barbed end of the actin filament and regulates actin assembly by blocking the addition and loss of actin subunits. Recent studies provide new insights into how CP and barbed-end capping are regulated. Filament elongation factors, such as formins and ENA/VASP (enabled/vasodilator-stimulated phosphoprotein), indirectly regulate CP by competing with CP for binding to the barbed end, whereas other molecules, including V-1 and phospholipids, directly bind to CP and sterically block its interaction with the filament. In addition, a diverse and unrelated group of proteins interact with CP through a conserved 'capping protein interaction' (CPI) motif. These proteins, including CARMIL (capping protein, ARP2/3 and myosin I linker), CD2AP (CD2-associated protein) and the WASH (WASP and SCAR homologue) complex subunit FAM21, recruit CP to specific subcellular locations and modulate its actin-capping activity via allosteric effects.
Assuntos
Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Capeamento de Actina/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/fisiologia , Proteínas de Ligação a DNA/fisiologia , Humanos , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/fisiologia , Modelos Moleculares , Fosfatos de Fosfatidilinositol/química , Ligação Proteica , Conformação ProteicaRESUMO
Epithelial cell-cell adhesion and morphogenesis require dynamic control of actin-driven membrane remodeling. The Rho guanosine triphosphatase (GTPase) Cdc42 regulates sequential molecular processes during cell-cell junction formation; hence, mechanisms must exist that inactivate Cdc42 in a temporally and spatially controlled manner. In this paper, we identify SH3BP1, a GTPase-activating protein for Cdc42 and Rac, as a regulator of junction assembly and epithelial morphogenesis using a functional small interfering ribonucleic acid screen. Depletion of SH3BP1 resulted in loss of spatial control of Cdc42 activity, stalled membrane remodeling, and enhanced growth of filopodia. SH3BP1 formed a complex with JACOP/paracingulin, a junctional adaptor, and CD2AP, a scaffolding protein; both were required for normal Cdc42 signaling and junction formation. The filamentous actin-capping protein CapZ also associated with the SH3BP1 complex and was required for control of actin remodeling. Epithelial junction formation and morphogenesis thus require a dual activity complex, containing SH3BP1 and CapZ, that is recruited to sites of active membrane remodeling to guide Cdc42 signaling and cytoskeletal dynamics.
Assuntos
Adesão Celular/fisiologia , Células Epiteliais/citologia , Proteínas Ativadoras de GTPase/fisiologia , Junções Intercelulares/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas de Capeamento de Actina/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células CACO-2 , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Feminino , Proteínas Ativadoras de GTPase/biossíntese , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Junções Intercelulares/metabolismo , Complexos Multiproteicos/fisiologia , RNA Interferente Pequeno/genética , Transdução de Sinais/fisiologiaRESUMO
We reconstructed cellular motility in vitro from individual proteins to investigate how actin filaments are organized at the leading edge. Using total internal reflection fluorescence microscopy of actin filaments, we tested how profilin, Arp2/3, and capping protein (CP) function together to propel thin glass nanofibers or beads coated with N-WASP WCA domains. Thin nanofibers produced wide comet tails that showed more structural variation in actin filament organization than did bead substrates. During sustained motility, physiological concentrations of Mg(2+) generated actin filament bundles that processively attached to the nanofiber. Reduction of total Mg(2+) abolished particle motility and actin attachment to the particle surface without affecting actin polymerization, Arp2/3 nucleation, or filament capping. Analysis of similar motility of microspheres showed that loss of filament bundling did not affect actin shell formation or symmetry breaking but eliminated sustained attachments between the comet tail and the particle surface. Addition of Mg(2+), Lys-Lys(2+), or fascin restored both comet tail attachment and sustained particle motility in low Mg(2+) buffers. TIRF microscopic analysis of filaments captured by WCA-coated beads in the absence of Arp2/3, profilin, and CP showed that filament bundling by polycation or fascin addition increased barbed end capture by WCA domains. We propose a model in which CP directs barbed ends toward the leading edge and polycation-induced filament bundling sustains processive barbed end attachment to the leading edge.
Assuntos
Citoesqueleto de Actina/fisiologia , Movimento Celular , Proteínas de Capeamento de Actina/fisiologia , Proteína 2 Relacionada a Actina/fisiologia , Animais , Humanos , Microscopia de Fluorescência , Profilinas/fisiologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/fisiologiaRESUMO
Erythrocyte tropomodulin (E-Tmod) is first isolated from human erythrocyte membrane as a TM-binding protein. Its N-terminus contains two TM-binding sites and one TM-dependent actin capping domain and C-terminus contains 5 leucine-rich repeats and a TM-independent actin capping domain. As the unique capping protein at the slow-growing end of F-actin, E-Tmod binds to N-terminus of TM and actin and decreases the TM-coated F-actin depolymerization. E-Tmod encoding gene is highly conserved and E-Tmod is distributed widely in erythrocytes and cardiomyocytes, etc. E-Tmod plays a pivotal role in organizing F-actin and cytoskeleton and maintaining the mechanical properties of the cells.
Assuntos
Proteínas de Capeamento de Actina/fisiologia , Citoesqueleto de Actina/fisiologia , Tropomodulina/fisiologia , Animais , Citoesqueleto/fisiologia , Humanos , Tropomiosina/fisiologiaRESUMO
Caenorhabditis elegans gelsolin-like protein-1 (GSNL-1) is a new member of the gelsolin family of actin regulatory proteins [Klaavuniemi, T., Yamashiro, S., and Ono, S. (2008) J. Biol. Chem. 283, 26071-26080]. It is an unconventional gelsolin-related protein with four gelsolin-like (G) domains (G1-G4), unlike typical gelsolin-related proteins with three or six G domains. GSNL-1 severs actin filaments and caps the barbed end in a calcium-dependent manner similar to that of gelsolin. In contrast, GSNL-1 has properties different from those of gelsolin in that it remains bound to F-actin and does not nucleate actin polymerization. To understand the mechanism by which GSNL-1 regulates actin dynamics, we investigated the domain-function relationship of GSNL-1 by analyzing activities of truncated forms of GSNL-1. G1 and the linker between G1 and G2 were sufficient for actin filament severing, whereas G1 and G2 were required for barbed end capping. The actin severing activity of GSNL-1 was inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), and a PIP2-sensitive domain was mapped to G1 and G2. At least two actin-binding sites were detected: a calcium-dependent G-actin-binding site in G1 and a calcium-independent G- and F-actin-binding site in G3 and G4. These results reveal both conserved and different utilization of G domains between C. elegans GSNL-1 and mammalian gelsolin for actin regulatory functions.
Assuntos
Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Sensoras de Cálcio Intracelular/química , Proteínas Sensoras de Cálcio Intracelular/metabolismo , Fosfatidilinositóis/metabolismo , Proteínas de Capeamento de Actina/química , Proteínas de Capeamento de Actina/fisiologia , Fatores de Despolimerização de Actina/química , Fatores de Despolimerização de Actina/genética , Fatores de Despolimerização de Actina/metabolismo , Fatores de Despolimerização de Actina/fisiologia , Actinas/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiologia , Gelsolina/química , Gelsolina/metabolismo , Gelsolina/fisiologia , Proteínas Sensoras de Cálcio Intracelular/genética , Proteínas Sensoras de Cálcio Intracelular/fisiologia , Modelos Biológicos , Peso Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica/fisiologia , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína/fisiologiaRESUMO
Actin cytoskeleton undergoes rapid reorganization in response to internal and external cues. How the dynamics of actin cytoskeleton are regulated, and how its dynamics relate to its function are fundamental questions in plant cell biology. The pollen tube is a well characterized actin-based cell morphogenesis in plants. One of the striking features of actin cytoskeleton characterized in the pollen tube is its surprisingly low level of actin polymer. This special phenomenon might relate to the function of actin cytoskeleton in pollen tubes. Understanding the molecular mechanism underlying this special phenomenon requires careful analysis of actin-binding proteins that modulate actin dynamics directly. Recent biochemical and biophysical analyses of several highly conserved plant actin-binding proteins reveal unusual and unexpected properties, which emphasizes the importance of carefully analyzing their action mechanism and cellular activity. In this review, we highlight an actin monomer sequestering protein, a barbed end capping protein and an F-actin severing and dynamizing protein in plant. We propose that these proteins function in harmony to regulate actin dynamics and maintain the low level of actin polymer in pollen tubes.
Assuntos
Actinas/metabolismo , Tubo Polínico/metabolismo , Proteínas de Capeamento de Actina/química , Proteínas de Capeamento de Actina/genética , Proteínas de Capeamento de Actina/metabolismo , Proteínas de Capeamento de Actina/fisiologia , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/fisiologia , Modelos Biológicos , Modelos Moleculares , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiologiaRESUMO
Summary Successful malaria transmission from the mosquito vector to the mammalian host depends crucially on active sporozoite motility. Sporozoite locomotion and host cell invasion are driven by the parasite's own actin/myosin motor. A unique feature of this motor machinery is the presence of very short subpellicular actin filaments. Therefore, F-actin stabilizing proteins likely play a central role in parasite locomotion. Here, we investigated the role of the Plasmodium berghei actin capping protein (PbCP), an orthologue of the heterodimeric regulator of filament barbed end growth, by reverse genetics. Parasites containing a deletion of the CP beta-subunit developed normally during the pathogenic erythrocytic cycle. However, due to reduced ookinete motility, mutant parasites form fewer oocysts and sporozoites in the Anopheles vector. These sporozoites display a vital deficiency in forward gliding motility and fail to colonize the mosquito salivary glands, resulting in complete attenuation of life cycle progression. Together, our results show that the CP beta-subunit exerts an essential role in the insect vector before malaria transmission to the mammalian host. The vital role is restricted to fast locomotion, as displayed by Plasmodium sporozoites.
Assuntos
Proteínas de Capeamento de Actina/fisiologia , Anopheles/parasitologia , Locomoção , Plasmodium berghei/fisiologia , Proteínas de Protozoários/fisiologia , Esporozoítos/fisiologia , Proteínas de Capeamento de Actina/genética , Actinas/metabolismo , Estruturas Animais/parasitologia , Animais , Deleção de Genes , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Proteínas de Protozoários/genética , Glândulas Salivares/parasitologiaRESUMO
During development, cells craft an impressive array of actin-based structures, mediating events as diverse as cytokinesis, apical constriction, and cell migration. One challenge is to determine how cells regulate actin assembly and disassembly to carry out these cell behaviors. During Drosophila oogenesis diverse cell behaviors are seen in the soma and germline. We used oogenesis to explore developmental roles of two important actin regulators: Enabled/VASP proteins and Capping protein. We found that Enabled plays an important role in cortical integrity of nurse cells, formation of robust bundled actin filaments in late nurse cells that facilitate nurse cell dumping, and migration of somatic border cells. During nurse cell dumping, Enabled localizes to barbed ends of the nurse cell actin filaments, suggesting its mechanism of action. We further pursued this mechanism using mutant Enabled proteins, each affecting one of its protein domains. These data suggest critical roles for the EVH2 domain and its tetramerization subdomain, while the EVH1 domain appears less critical. Enabled appears to be negatively regulated during oogenesis by Abelson kinase. We also explored the function of Capping protein. This revealed important roles in oocyte determination, nurse cell cortical integrity and nurse cell dumping, and support the idea that Capping protein and Enabled act antagonistically during dumping. Together these data reveal places that these actin regulators shape oogenesis.
Assuntos
Proteínas de Capeamento de Actina/fisiologia , Citoesqueleto de Actina/fisiologia , Proteínas de Ligação a DNA/fisiologia , Animais , Movimento Celular/fisiologia , Forma Celular/fisiologia , Drosophila , Feminino , Oogênese/fisiologiaRESUMO
Tissue patterning must be translated into morphogenesis through cell shape changes mediated by remodeling of the actin cytoskeleton. We have found that Capping protein alpha (Cpa) and Capping protein beta (Cpb), which prevent extension of the barbed ends of actin filaments, are specifically required in the wing blade primordium of the Drosophila wing disc. cpa or cpb mutant cells in this region, but not in the remainder of the wing disc, are extruded from the epithelium and undergo apoptosis. Excessive actin filament polymerization is not sufficient to explain this phenotype, as loss of Cofilin or Cyclase-associated protein does not cause cell extrusion or death. Misexpression of Vestigial, the transcription factor that specifies the wing blade, both increases cpa transcription and makes cells dependent on cpa for their maintenance in the epithelium. Our results suggest that Vestigial specifies the cytoskeletal changes that lead to morphogenesis of the adult wing.
Assuntos
Proteínas de Capeamento de Actina/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/crescimento & desenvolvimento , Asas de Animais/crescimento & desenvolvimento , Proteínas de Capeamento de Actina/genética , Junções Aderentes/fisiologia , Animais , Apoptose/fisiologia , Padronização Corporal , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Imuno-Histoquímica , Hibridização In Situ , Morfogênese , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Fenótipo , Transdução de Sinais , Asas de Animais/citologiaRESUMO
Actin-capping protein (CP) is a heterodimeric protein which is expressed in various eukaryotic cells. CP binds to the barbed end of the actin filaments in vitro and inhibits both the association and dissociation of actin monomers at this end. However, the cellular role of CP has not been uncovered. Here we investigated the function of CP in fission yeast cells. The fission yeast CP is composed of Acp1 and Acp2. It was found that Acp2 accumulated as cortical dots at the cell ends during interphase and the mid-region of mitotic cells, which disappeared in the absence of Acp1 or F-actin. Acp1 and Acp2, when co-over-expressed, decreased F-actin structures in cells, and cytokinesis was often interrupted in these cells. On the other hand, disruption of one of the CP genes affected the distribution of F-actin patches at cell ends and decreased the rate of actin depolymerization in vivo. Moreover, genetic analysis showed that CP controls actin dynamics together with ADF/cofilin and profilin. In addition, CP is likely involved in assembling the F-actin contractile ring and F-actin patch with F-actin-crosslinking proteins.
Assuntos
Proteínas de Capeamento de Actina/fisiologia , Fatores de Despolimerização de Actina/fisiologia , Proteínas de Transporte/fisiologia , Citoesqueleto/metabolismo , Proteínas dos Microfilamentos/fisiologia , Profilinas/fisiologia , Schizosaccharomyces/fisiologia , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Interfase/fisiologia , Modelos Biológicos , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Polímeros/metabolismo , Subunidades Proteicas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Working in concert, multiple actin-binding proteins regulate the dynamic turnover of actin networks. Here, we define a novel function for the conserved actin-binding protein twinfilin, which until now was thought to function primarily as a monomer-sequestering protein. We show that purified budding yeast twinfilin (Twf1) binds to and severs actin filaments in vitro at pH below 6.0 in bulk kinetic and fluorescence microscopy assays. Further, we use total internal reflection fluorescence (TIRF) microscopy to demonstrate that Twf1 severs individual actin filaments in real time. It has been shown that capping protein directly binds to Twf1 and is required for Twf1 localization to cortical actin patches in vivo. We demonstrate that capping protein directly inhibits the severing activity of Twf1, the first biochemical function ascribed to this interaction. In addition, phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] inhibits Twf1 filament-severing activity. Consistent with these biochemical activities, a twf1Delta mutation causes reduced rates of cortical actin patch turnover in living cells. Together, our data suggest that twinfilin coordinates filament severing and monomer sequestering at sites of rapid actin turnover and is controlled by multiple regulatory inputs.