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1.
J Cell Sci ; 136(6)2023 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-36994763

RESUMO

Looking back at two decades of research on SPIRE actin nucleator proteins, the first decade was clearly dominated by the discovery of SPIRE proteins as founding members of the novel WH2-domain-based actin nucleators, which initiate actin filament assembly through multiple WH2 actin-binding domains. Through complex formation with formins and class 5 myosins, SPIRE proteins coordinate actin filament assembly and myosin motor-dependent force generation. The discovery of SPIRE-regulated cytoplasmic actin filament meshworks in oocytes initiated the next phase of SPIRE research, which has found that SPIRE proteins are integrated in a diverse range of cell biological processes. In addition to regulating vesicle-based actin filament meshworks, SPIRE proteins function in the organisation of actin structures driving the inward movement of pronuclei of the mouse zygote. Localisation at cortical ring structures and the results of knockdown experiments indicate that SPIRE proteins function in the formation of meiotic cleavage sites in mammalian oocytes and the externalisation of von Willebrand factor from endothelial cells. Alternative splicing targets mammalian SPIRE1 towards mitochondria, where it has a role in fission. In this Review, we summarise the past two decades of SPIRE research by addressing the biochemical and cell biological functions of SPIRE proteins in mammalian reproduction, skin pigmentation and wound healing, as well as in mitochondrial dynamics and host-pathogen interactions.


Assuntos
Actinas , Proteínas dos Microfilamentos , Animais , Camundongos , Actinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Células Endoteliais/metabolismo , Citoesqueleto de Actina/metabolismo , Forminas/metabolismo , Mamíferos/metabolismo , Proteínas do Tecido Nervoso/metabolismo
2.
Cell Mol Life Sci ; 79(2): 96, 2022 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-35084586

RESUMO

Weibel-Palade bodies (WPB) are endothelial cell-specific storage granules that regulate vascular hemostasis by releasing the platelet adhesion receptor von Willebrand factor (VWF) following stimulation. Fusion of WPB with the plasma membrane is accompanied by the formation of actin rings or coats that support the expulsion of large multimeric VWF fibers. However, factor(s) organizing these actin ring structures have remained elusive. We now identify the actin-binding proteins Spire1 and Myosin Vc (MyoVc) as cytosolic factors that associate with WPB and are involved in actin ring formation at WPB-plasma membrane fusion sites. We show that both, Spire1 and MyoVc localize only to mature WPB and that upon Ca2+ evoked exocytosis of WPB, Spire1 and MyoVc together with F-actin concentrate in ring-like structures at the fusion sites. Depletion of Spire1 or MyoVc reduces the number of these actin rings and decreases the amount of VWF externalized to the cell surface after histamine stimulation.


Assuntos
Cálcio/metabolismo , Exocitose , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Miosina Tipo V/metabolismo , Proteínas Nucleares/metabolismo , Fator de von Willebrand/metabolismo , Western Blotting , Células Cultivadas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas dos Microfilamentos/genética , Microscopia Confocal , Modelos Biológicos , Miosina Tipo V/genética , Proteínas Nucleares/genética , Interferência de RNA , Corpos de Weibel-Palade/metabolismo
3.
J Cell Sci ; 130(20): 3427-3435, 2017 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-29032357

RESUMO

The actin cytoskeleton and associated motor proteins provide the driving forces for establishing the astonishing morphological diversity and dynamics of mammalian cells. Aside from functions in protruding and contracting cell membranes for motility, differentiation or cell division, the actin cytoskeleton provides forces to shape and move intracellular membranes of organelles and vesicles. To establish the many different actin assembly functions required in time and space, actin nucleators are targeted to specific subcellular compartments, thereby restricting the generation of specific actin filament structures to those sites. Recent research has revealed that targeting and activation of actin filament nucleators, elongators and myosin motors are tightly coordinated by conserved protein complexes to orchestrate force generation. In this Cell Science at a Glance article and the accompanying poster, we summarize and discuss the current knowledge on the corresponding protein complexes and their modes of action in actin nucleation, elongation and force generation.


Assuntos
Citoesqueleto de Actina/fisiologia , Pseudópodes/fisiologia , Citoesqueleto de Actina/ultraestrutura , Actinas/fisiologia , Actinas/ultraestrutura , Animais , Fenômenos Fisiológicos Celulares , Células Cultivadas , Humanos , Multimerização Proteica , Pseudópodes/ultraestrutura
4.
J Biol Chem ; 290(10): 6428-44, 2015 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-25564607

RESUMO

Spir and formin (FMN)-type actin nucleators initiate actin polymerization at vesicular membranes necessary for long range vesicular transport processes. Here we studied in detail the membrane binding properties and protein/protein interactions that govern the assembly of the membrane-associated Spir·FMN complex. Using biomimetic membrane models we show that binding of the C-terminal Spir-2 FYVE-type zinc finger involves both the presence of negatively charged lipids and hydrophobic contributions from the turret loop that intrudes the lipid bilayer. In solution, we uncovered a yet unknown intramolecular interaction between the Spir-2 FYVE-type domain and the N-terminal kinase non-catalytic C-lobe domain (KIND) that could not be detected in the membrane-bound state. Interestingly, we found that the intramolecular Spir-2 FYVE/KIND and the trans-regulatory Fmn-2-FSI/Spir-2-KIND interactions are competitive. We therefore characterized co-expressed Spir-2 and Fmn-2 fluorescent protein fusions in living cells by fluorescence cross-correlation spectroscopy. The data corroborate a model according to which Spir-2 exists in two different states, a cytosolic monomeric conformation and a membrane-bound state in which the KIND domain is released and accessible for subsequent Fmn-2 recruitment. This sequence of interactions mechanistically couples membrane binding of Spir to the recruitment of FMN, a pivotal step for initiating actin nucleation at vesicular membranes.


Assuntos
Actinas/metabolismo , Proteínas dos Microfilamentos/química , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/química , Actinas/química , Sequência de Aminoácidos , Forminas , Células HEK293 , Humanos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteínas Nucleares/metabolismo , Mapas de Interação de Proteínas/genética
5.
Biol Chem ; 394(12): 1649-60, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23863697

RESUMO

The diversity of cellular actin functions is attained by the activation of actin nucleator complexes, which initiate the polymerization of actin monomers into a helical double-stranded filament at defined subcellular compartments. Next to actin functions at the cell membrane, including different forms of membrane protrusions and invaginations, actin dynamics at intracellular membranes has recently become a research focus. Experiments addressing the vesicle-associated Spir WH2 domain containing actin nucleators have provided novel mechanistic insights into the function of actin dynamics at intracellular membranes. Spir proteins are targeted by a modified FYVE zinc finger motif toward endosomal and vesicle membranes, where they interact and cooperate with the distinct nucleators of the FMN subfamily of formins in the nucleation of actin filaments. The function of the Spir/formin actin nucleator complex is closely related to the Rab11 small G protein, which is a key regulator of recycling and exocytic transport processes. Together with the actin motor protein and Rab11 effector myosin Vb, Spir/formin nucleated actin filaments mediate actin-dependent vesicle transport processes. Drosophila and mouse genetic studies as well as cell biology experiments point toward an important role of the Spir/formin complex in oocyte maturation and in the structure and signaling of the nervous system.


Assuntos
Actinas/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Fetais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Animais , Proteínas de Drosophila/genética , Forminas , Humanos , Proteínas dos Microfilamentos/genética , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso , Oócitos/fisiologia , Estrutura Terciária de Proteína , Transdução de Sinais , Vesículas Transportadoras/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
6.
J Biol Chem ; 286(35): 30732-30739, 2011 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-21705804

RESUMO

The distinct actin nucleation factors of the Spir and formin subgroup families cooperate in actin nucleation. The Spir/formin cooperativity has been identified to direct two essential steps in mammalian oocyte maturation, the asymmetric spindle positioning and polar body extrusion during meiosis. Understanding the nature and regulation of the Spir/Fmn cooperation is an important requirement to comprehend mammalian reproduction. Recently we dissected the structural elements of the Spir and Fmn family proteins, which physically link the two actin nucleation factors. The trans-regulatory interaction is mediated by the Spir kinase non-catalytic C-lobe domain (KIND) and the C-terminal formin Spir interaction motif (FSI). The interaction inhibits formin nucleation activity and enhances the Spir activity. To get insights into the molecular mechanism of the Spir/Fmn interaction, we determined the crystal structure of the KIND domain alone and in complex with the C-terminal Fmn-2 FSI peptide. Together they confirm the proposed structural homology of the KIND domain to the protein kinase fold and reveal the basis of the Spir/formin interaction. The complex structure showed a large interface with conserved and positively charged residues of the Fmn FSI peptide mediating major contacts to an acidic groove on the surface of KIND. Protein interaction studies verified the electrostatic nature of the interaction. The data presented here provide the molecular basis of the Spir/formin interaction and give a first structural view into the mechanisms of actin nucleation factor cooperativity.


Assuntos
Actinas/química , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Catálise , Cristalização , Cristalografia por Raios X/métodos , Forminas , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Oócitos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Propriedades de Superfície
7.
J Cell Biol ; 179(1): 117-28, 2007 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-17923532

RESUMO

Spire and Cappuccino are actin nucleation factors that are required to establish the polarity of Drosophila melanogaster oocytes. Their mutant phenotypes are nearly identical, and the proteins interact biochemically. We find that the interaction between Spire and Cappuccino family proteins is conserved across metazoan phyla and is mediated by binding of the formin homology 2 (FH2) domain from Cappuccino (or its mammalian homologue formin-2) to the kinase noncatalytic C-lobe domain (KIND) from Spire. In vitro, the KIND domain is a monomeric folded domain. Two KIND monomers bind each FH2 dimer with nanomolar affinity and strongly inhibit actin nucleation by the FH2 domain. In contrast, formation of the Spire-Cappuccino complex enhances actin nucleation by Spire. In Drosophila oocytes, Spire localizes to the cortex early in oogenesis and disappears around stage 10b, coincident with the onset of cytoplasmic streaming.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas dos Microfilamentos/metabolismo , Actinas/metabolismo , Animais , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Microtúbulos/metabolismo , Oogênese , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas
8.
Trends Cell Biol ; 16(9): 477-83, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16901698

RESUMO

The initiation of actin polymerization from free monomers requires actin-nucleation factors. Spir proteins nucleate actin polymerization by a novel mechanism that is distinct from actin nucleation by the Arp2/3 complex or by formins. In vitro actin polymerization assays and electron microscopic data show that Spire nucleates actin polymerization by binding four actin monomers to a cluster of four Wiskott-Aldrich syndrome protein-homology domain 2 (WH2) domains in the central region of the proteins. Although the exact cell biological function and regulation of Spir proteins is still unknown, data from genetic studies in Drosophila, cell biological studies and protein interaction experiments have provided insight into the biology of these interesting and novel actin-nucleation factors and suggest a role in vesicle transport processes and in the coordination of cortical microtubule and actin filaments. Phosphorylation by mitogen-activated protein kinases and interaction with Rho GTPases have been proposed as regulatory mechanisms.


Assuntos
Actinas/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas dos Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/química , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Proteínas dos Microfilamentos/química , Microtúbulos/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich
9.
Nature ; 433(7024): 382-8, 2005 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-15674283

RESUMO

The actin cytoskeleton is essential for many cellular functions including shape determination, intracellular transport and locomotion. Previous work has identified two factors--the Arp2/3 complex and the formin family of proteins--that nucleate new actin filaments via different mechanisms. Here we show that the Drosophila protein Spire represents a third class of actin nucleation factor. In vitro, Spire nucleates new filaments at a rate that is similar to that of the formin family of proteins but slower than in the activated Arp2/3 complex, and it remains associated with the slow-growing pointed end of the new filament. Spire contains a cluster of four WASP homology 2 (WH2) domains, each of which binds an actin monomer. Maximal nucleation activity requires all four WH2 domains along with an additional actin-binding motif, conserved among Spire proteins. Spire itself is conserved among metazoans and, together with the formin Cappuccino, is required for axis specification in oocytes and embryos, suggesting that multiple actin nucleation factors collaborate to construct essential cytoskeletal structures.


Assuntos
Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas dos Microfilamentos/metabolismo , Actinas/ultraestrutura , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Drosophila/química , Proteínas de Drosophila/ultraestrutura , Drosophila melanogaster/citologia , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/ultraestrutura , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína
10.
J Biol Chem ; 284(37): 25324-33, 2009 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-19605360

RESUMO

The actin nucleation factors Spire and Cappuccino interact with each other and regulate essential cellular events during Drosophila oogenesis in a cooperative fashion. The interaction blocks formin actin nucleation activity and enhances the Spire activity. Analogous to Spire and Cappuccino, the mammalian homologs Spir-1 and formin-2 show a regulatory interaction. To get an understanding of the nature of the Spir-formin cooperation, we have analyzed the interaction biochemically and biophysically. Our data shows that the association of Spir-1 and formin-2 is not significantly mediated by binding of the Spir-1-KIND domain to the formin FH2 core domain. Instead, a short sequence motif C-terminal adjacent to the formin-2-FH2 domain could be characterized that mediates the interaction and is conserved among the members of the Fmn subgroup of formins. In line with this, we found that both mammalian Spir proteins, Spir-1 and Spir-2, interact with mammalian Fmn subgroup proteins formin-1 and formin-2.


Assuntos
Proteínas Fetais/química , Proteínas dos Microfilamentos/química , Proteínas do Tecido Nervoso/química , Proteínas Nucleares/química , Sequência de Aminoácidos , Animais , Anisotropia , Proliferação de Células , Forminas , Células HeLa , Humanos , Microscopia de Fluorescência/métodos , Modelos Biológicos , Dados de Sequência Molecular , Oogênese , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
11.
Nat Commun ; 11(1): 3495, 2020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32661310

RESUMO

Cell biologists generally consider that microtubules and actin play complementary roles in long- and short-distance transport in animal cells. On the contrary, using melanosomes of melanocytes as a model, we recently discovered that the motor protein myosin-Va works with dynamic actin tracks to drive long-range organelle dispersion in opposition to microtubules. This suggests that in animals, as in yeast and plants, myosin/actin can drive long-range transport. Here, we show that the SPIRE-type actin nucleators (predominantly SPIRE1) are Rab27a effectors that co-operate with formin-1 to generate actin tracks required for myosin-Va-dependent transport in melanocytes. Thus, in addition to melanophilin/myosin-Va, Rab27a can recruit SPIREs to melanosomes, thereby integrating motor and track assembly activity at the organelle membrane. Based on this, we suggest a model in which organelles and force generators (motors and track assemblers) are linked, forming an organelle-based, cell-wide network that allows their collective activity to rapidly disperse the population of organelles long-distance throughout the cytoplasm.


Assuntos
Actinas/metabolismo , Proteínas rab27 de Ligação ao GTP/metabolismo , Biologia Celular , Citoesqueleto/metabolismo , Células HEK293 , Humanos , Microtúbulos/metabolismo , Organelas , Filogenia , Proteínas rab27 de Ligação ao GTP/genética
12.
Small GTPases ; 10(2): 111-121, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-28394692

RESUMO

Spir actin nucleators and myosin V motor proteins were recently discovered to coexist in a protein complex. The direct interaction allows the coordinated activation of actin motor proteins and actin filament track generation at vesicle membranes. By now the cooperation of myosin V (MyoV) motors and Spir actin nucleation function has only been shown in the exocytic transport of Rab11 vesicles in metaphase mouse oocytes. Next to Rab11, myosin V motors however interact with a variety of Rab GTPases including Rab3, Rab8 and Rab10. As a common theme most of the MyoV interacting Rab GTPases function at different steps along the exocytic transport routes. We here summarize the different transport functions of class V myosins and provide as proof of principle data showing a colocalization of Spir actin nucleators and MyoVa at Rab8a vesicles. This suggests that besides Rab11/MyoV transport also the Rab8/MyoV and possibly other MyoV transport processes recruit Spir actin filament nucleation function.


Assuntos
Actinas/metabolismo , Miosina Tipo V/metabolismo , Transporte Biológico , Humanos , Proteínas rho de Ligação ao GTP/metabolismo
13.
Brain Pathol ; 28(5): 695-709, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29222813

RESUMO

Glioblastoma (GBM) represents the most common and most malignant type of primary brain tumor and significantly contributes to cancer morbidity and mortality. Invasion into the healthy brain parenchyma is a major feature of glioblastoma aggressiveness. Reelin (RELN) is a large secreted extracellular matrix glycoprotein that regulates neuronal migration and positioning in the developing brain and sustains functionality in the adult brain. We here show that both RELN and its main downstream effector DAB1 are silenced in glioblastoma as compared to non-neoplastic tissue and mRNA expression is inversely correlated with malignancy grade. Furthermore, RELN expression is positively correlated with patient survival in two large, independent clinically annotated datasets. RELN silencing occurs via promoter hypermethylation as shown by both database mining and bisulfite sequencing of the RELN promoter. Consequently, treatment with 5'-Azacytidine and trichostatin A induced RELN expression in vitro. On the functional level, we found RELN to regulate glioblastoma cell migration both in a DAB1 (tyrosine phosphorylation)-dependent and -independent fashion, depending on the substrate provided. Moreover, stimulation of RELN signaling strongly reduced proliferation in glioblastoma cells. This phenotype depends on DAB1 stimulation by RELN, as a mutant that lacks all RELN induced tyrosine phosphorylation sites (DAB1-5F) failed to induce a growth arrest. Proteomic analyzes revealed that these effects are mediated by a reduction in E2F targets and dephosphorylation of ERK1/2. Taken together, our data establish a relevance of RELN signaling in glioblastoma pathology and thereby might unearth novel, yet unrecognized treatment options.


Assuntos
Neoplasias Encefálicas/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Movimento Celular/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Glioblastoma/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais/metabolismo , Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Simulação por Computador , Proteínas da Matriz Extracelular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Masculino , Camundongos , Proteínas do Tecido Nervoso/genética , Proteoma , RNA Mensageiro/metabolismo , Proteína Reelina , Serina Endopeptidases/genética , Transdução de Sinais , Adulto Jovem
14.
PLoS One ; 11(9): e0161965, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27627128

RESUMO

Salmonella Typhimurium (S. Tm) is a leading cause of diarrhea. The disease is triggered by pathogen invasion into the gut epithelium. Invasion is attributed to the SPI-1 type 3 secretion system (T1). T1 injects effector proteins into epithelial cells and thereby elicits rearrangements of the host cellular actin cytoskeleton and pathogen invasion. The T1 effector proteins SopE, SopB, SopE2 and SipA are contributing to this. However, the host cell factors contributing to invasion are still not completely understood. To address this question comprehensively, we used Hela tissue culture cells, a genome-wide siRNA library, a modified gentamicin protection assay and S. TmSipA, a sopBsopE2sopE mutant which strongly relies on the T1 effector protein SipA to invade host cells. We found that S. TmSipA invasion does not elicit membrane ruffles, nor promote the entry of non-invasive bacteria "in trans". However, SipA-mediated infection involved the SPIRE family of actin nucleators, besides well-established host cell factors (WRC, ARP2/3, RhoGTPases, COPI). Stage-specific follow-up assays and knockout fibroblasts indicated that SPIRE1 and SPIRE2 are involved in different steps of the S. Tm infection process. Whereas SPIRE1 interferes with bacterial binding, SPIRE2 influences intracellular replication of S. Tm. Hence, these two proteins might fulfill non-redundant functions in the pathogen-host interaction. The lack of co-localization hints to a short, direct interaction between S. Tm and SPIRE proteins or to an indirect effect.


Assuntos
Proteínas de Bactérias/fisiologia , Estudo de Associação Genômica Ampla/métodos , Interações Hospedeiro-Patógeno/fisiologia , Proteínas dos Microfilamentos/fisiologia , Proteínas Nucleares/fisiologia , Salmonella typhimurium/patogenicidade , Animais , Linhagem Celular , Imunofluorescência , Células HeLa/metabolismo , Células HeLa/microbiologia , Humanos , Camundongos , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Salmonella typhimurium/fisiologia
15.
Elife ; 52016 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-27623148

RESUMO

There is growing evidence for a coupling of actin assembly and myosin motor activity in cells. However, mechanisms for recruitment of actin nucleators and motors on specific membrane compartments remain unclear. Here we report how Spir actin nucleators and myosin V motors coordinate their specific membrane recruitment. The myosin V globular tail domain (MyoV-GTD) interacts directly with an evolutionarily conserved Spir sequence motif. We determined crystal structures of MyoVa-GTD bound either to the Spir-2 motif or to Rab11 and show that a Spir-2:MyoVa:Rab11 complex can form. The ternary complex architecture explains how Rab11 vesicles support coordinated F-actin nucleation and myosin force generation for vesicle transport and tethering. New insights are also provided into how myosin activation can be coupled with the generation of actin tracks. Since MyoV binds several Rab GTPases, synchronized nucleator and motor targeting could provide a common mechanism to control force generation and motility in different cellular processes.


Assuntos
Vesículas Citoplasmáticas/metabolismo , Membranas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Miosina Tipo V/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Cristalografia por Raios X , Camundongos , Proteínas dos Microfilamentos/química , Modelos Moleculares , Miosina Tipo V/química , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas rab de Ligação ao GTP/química
16.
Oncogene ; 21(22): 3651-8, 2002 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-12032867

RESUMO

Low level expression of an active Raf kinase results in a transformed phenotype; however, high intensity Raf signals block cell cycle progression. Phospholipase D (PLD) has been implicated in regulating cell cycle progression and PLD activity is elevated in Raf transformed cells. We report here that high intensity Raf signals reduce PLD activity and that elevated expression of either PLD1 or PLD2 prevents cell cycle arrest induced by high intensity Raf signals. Overexpression of either PLD1 or PLD2 also reversed increases in p21(Cip1) and protein kinase C delta (PKC delta) cleavage seen with high intensity Raf signals. These data indicate that PLD signaling provides a novel survival signal that overcomes cell cycle arrest induced by high intensity Raf signaling.


Assuntos
Fosfolipase D/fisiologia , Proteínas Proto-Oncogênicas c-raf/fisiologia , Transdução de Sinais , Células 3T3 , Animais , Ciclo Celular , Divisão Celular , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Isoenzimas/metabolismo , Cinética , Camundongos , Fosfolipase D/metabolismo , Proteína Quinase C/metabolismo , Proteína Quinase C-delta , Proteínas Proto-Oncogênicas c-raf/genética , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão/metabolismo
17.
Biochim Biophys Acta ; 1589(2): 151-9, 2002 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-12007790

RESUMO

Activated Raf kinases and Rac GTPases were shown to cooperate in the oncogenic transformation of fibroblasts, which is characterised by the disassembly of the cellular actin cytoskeleton, a nearly complete loss of focal adhesion complexes and deregulated cell proliferation. This is surprising since the Rac GTPase induces actin structures and the adhesion of suspended cells to extracellular matrix proteins. NIH 3T3 cells expressing a hydroxytamoxifen-inducible oncogenic c-Raf-1-oestrogen receptor fusion protein (c-Raf-1-BxB-ER, N-BxB-ER cells) undergo morphological transformation upon stimulation of the Raf kinase. We show that treatment with the Rac, Rho and Cdc42 activating Escherichia coli toxin CNF1 or coexpression of an activated RacV12 mutant partially inhibits and reverses the disassembly of cellular actin structures and focal adhesion complexes by oncogenic Raf. Activation of the Rac GTPase restores actin structures and focal adhesion complexes at the cellular boundary, leading to spreading of the otherwise spindle-shaped Raf-transformed cells. Actin stress fibres, however, which are regulated by the function of the Rho GTPase, are disassembled by oncogenic Raf even in the presence of activated Rac and Rho. With respect to the RacV12-mediated spreading of Raf-transformed cells, we postulate an anti-oncogenic function of the activated Rac. Another feature of cell transformation is the deregulation of cell cycle control. NIH 3T3 cells expressing high levels of the c-Raf-1-BxB-ER protein undergo a cell cycle arrest upon stimulation of the oncogenic Raf kinase. Our results show that in N-BxB-ER-RacV12 cells the expression of the activated RacV12 mediates cell proliferation in the presence of high-intensity Raf signals and high levels of the Cdk inhibitor p21(Cip1). These results indicate a pro-oncogenic function of the Rac GTPase with respect to the deregulation of cell cycle control.


Assuntos
Linhagem Celular Transformada/efeitos dos fármacos , Proteínas de Escherichia coli , Proteínas Proto-Oncogênicas c-raf , Proteínas rac de Ligação ao GTP/farmacologia , Células 3T3 , Animais , Toxinas Bacterianas/farmacologia , Resinas de Troca de Cátion , Ciclo Celular/efeitos dos fármacos , Citotoxinas/farmacologia , Lipídeos , Camundongos , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Transfecção
18.
Gene Expr Patterns ; 6(1): 79-85, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16099729

RESUMO

The kinase non-catalytic c-lobe domain (KIND) evolved from the catalytic protein kinase fold into a potential protein interaction module for signalling proteins. Spir family actin organizers and the non-receptor phosphatase type 13 (PTP type 13) encode a KIND domain in the very N-terminal parts of the proteins. Here we report the characterization and cloning of a third member of the KIND protein family, which we have named very-KIND (VKIND) because of its two KIND domains. Like the other members of the protein family, VKIND has a KIND domain at the N-terminus. A second KIND domain is located in the central part of the protein. The C-terminal half encodes a guanine nucleotide exchange factor motif for Ras-like GTPases (RasGEF) and a RasGEF N-terminal module (RasGEFN). There is only one VKIND gene in the mammalian genomes and up to now we have found the gene only in vertebrates. During mouse embryogenesis the VKIND gene was specifically expressed in the developing nervous system. In adult mice Northern hybridizations revealed high expression only in brain. Low expression could be detected in ovary. In situ hybridizations showed a specific expression of VKIND in neuronal cells of the granular and Purkinje cell layers of the cerebellum.


Assuntos
Encéfalo/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Camundongos/embriologia , Proteínas do Tecido Nervoso/genética , Sistema Nervoso/embriologia , Sequência de Aminoácidos , Animais , Encéfalo/embriologia , Clonagem Molecular , Embrião de Mamíferos/enzimologia , Olho/embriologia , Olho/enzimologia , Feminino , Fatores de Troca do Nucleotídeo Guanina/análise , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Camundongos/genética , Camundongos/metabolismo , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/metabolismo , Sistema Nervoso/enzimologia , Ovário/enzimologia , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Proteínas ras/metabolismo
19.
Gene Expr Patterns ; 4(3): 249-55, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15053972

RESUMO

The Wiskott-Aldrich homology domain 2 (WH2) family protein Spir and the formin Cappuccino belong to two distinct classes of actin organizers. Despite their functional classification as actin organizers, a major defect of Drosophila spire and cappuccino mutant oocytes is a failure in the orientation of microtubule plus ends towards the posterior pole. Mammalian homologues of spire are the spir-1 and spir-2 genes. The mouse and human formin-1 and formin-2 genes have high similarity to the cappuccino gene. The mouse formin-2 gene has been found to be expressed in the developing nervous system and in neuronal cells of the adult brain. By analyzing the expression of the spir-1 gene we show that spir-1 and formin-2 have a nearly identical expression pattern during mouse embryogenesis and in the adult brain. In mouse embryos both genes are expressed in the developing nervous system. In the adult brain high expression of the genes was found in the Purkinje cells of the cerebellum and in neuronal cells of the hippocampus and dentate gyrus.


Assuntos
Encéfalo/metabolismo , Camundongos/embriologia , Proteínas dos Microfilamentos/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Sistema Nervoso/embriologia , Actinas/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas do Citoesqueleto , Proteínas de Drosophila/genética , Hibridização In Situ , Camundongos/genética , Camundongos/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/genética , Sistema Nervoso/metabolismo , Oligorribonucleotídeos Antissenso/genética , Estrutura Terciária de Proteína , Células de Purkinje/metabolismo
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