Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
1.
J Biol Chem ; 287(5): 3087-98, 2012 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-22094460

RESUMO

Formin proteins are actin assembly factors that accelerate filament nucleation then remain on the elongating barbed end and modulate filament elongation. The formin homology 2 (FH2) domain is central to these activities, but recent work has suggested that additional sequences enhance FH2 domain function. Here we show that the C-terminal 76 amino acids of the formin FMNL3 have a dramatic effect on the ability of the FH2 domain to accelerate actin assembly. This C-terminal region contains a WASp homology 2 (WH2)-like sequence that binds actin monomers in a manner that is competitive with other WH2 domains and with profilin. In addition, the C terminus binds filament barbed ends. As a monomer, the FMNL3 C terminus inhibits actin polymerization and slows barbed end elongation with moderate affinity. As a dimer, the C terminus accelerates actin polymerization from monomers and displays high affinity inhibition of barbed end elongation. These properties are not common to all formin C termini, as those of mDia1 and INF2 do not behave similarly. Interestingly, mutation of two aliphatic residues, which blocks high affinity actin binding by the WH2-like sequence, has no effect on the ability of the C terminus to enhance FH2-mediated polymerization. However, mutation of three successive basic residues at the C terminus of the WH2-like sequence compromises polymerization enhancement. These results illustrate that the C termini of formins are highly diverse in their interactions with actin.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Multimerização Proteica/fisiologia , Proteínas/metabolismo , Citoesqueleto de Actina/genética , Actinas/genética , Substituição de Aminoácidos , Animais , Forminas , Camundongos , Mutação de Sentido Incorreto , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/genética
2.
J Biol Chem ; 286(8): 6577-86, 2011 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-21148482

RESUMO

The maintenance of rapid and efficient actin dynamics in vivo requires coordination of filament assembly and disassembly. This regulation requires temporal and spatial integration of signaling pathways by protein complexes. However, it remains unclear how these complexes form and then regulate the actin cytoskeleton. Here, we identify a srGAP2 and formin-like 1 (FMNL1, also known as FRL1 or FRLα) complex whose assembly is regulated by Rac signaling. Our data suggest srGAP2 regulates FMNL1 in two ways; 1) Rac-mediated activation of FMNL1 leads to the recruitment of srGAP2, which contains a Rac-specific GAP domain; 2) the SH3 domain of srGAP2 binds the formin homology 1 domain of FMNL1 to inhibit FMNL1-mediated actin severing. Thus, srGAP2 can efficiently terminate the upstream activating Rac signal while also opposing an important functional output of FMNL1, namely actin severing. We also show that FMNL1 and srGAP2 localize to the actin-rich phagocytic cup of macrophage-derived cells, suggesting the complex may regulate this Rac- and actin-driven process in vivo. We propose that after Rac-dependent activation of FMNL1, srGAP2 mediates a potent mechanism to limit the duration of Rac action and inhibit formin activity during rapid actin dynamics.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Macrófagos/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Actinas , Proteínas do Citoesqueleto/genética , Forminas , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Células HeLa , Humanos , Complexos Multiproteicos/genética , Fagossomos/genética , Fagossomos/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Domínios de Homologia de src
3.
Sci Adv ; 7(38): eabg6908, 2021 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-34524844

RESUMO

Genomic instability is a hallmark of human cancer; yet the underlying mechanisms remain poorly understood. Here, we report that the cytoplasmic unconventional Myosin X (MYO10) regulates genome stability, through which it mediates inflammation in cancer. MYO10 is an unstable protein that undergoes ubiquitin-conjugating enzyme H7 (UbcH7)/ß-transducin repeat containing protein 1 (ß-TrCP1)­dependent degradation. MYO10 is upregulated in both human and mouse tumors and its expression level predisposes tumor progression and response to immune therapy. Overexpressing MYO10 increased genomic instability, elevated the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING)­dependent inflammatory response, and accelerated tumor growth in mice. Conversely, depletion of MYO10 ameliorated genomic instability and reduced the inflammation signaling. Further, inhibiting inflammation or disrupting Myo10 significantly suppressed the growth of both human and mouse breast tumors in mice. Our data suggest that MYO10 promotes tumor progression through inducing genomic instability, which, in turn, creates an immunogenic environment for immune checkpoint blockades.

4.
Elife ; 102021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34519272

RESUMO

Skeletal muscle fibers are multinucleated cellular giants formed by the fusion of mononuclear myoblasts. Several molecules involved in myoblast fusion have been discovered, and finger-like projections coincident with myoblast fusion have also been implicated in the fusion process. The role of these cellular projections in muscle cell fusion was investigated herein. We demonstrate that these projections are filopodia generated by class X myosin (Myo10), an unconventional myosin motor protein specialized for filopodia. We further show that Myo10 is highly expressed by differentiating myoblasts, and Myo10 ablation inhibits both filopodia formation and myoblast fusion in vitro. In vivo, Myo10 labels regenerating muscle fibers associated with Duchenne muscular dystrophy and acute muscle injury. In mice, conditional loss of Myo10 from muscle-resident stem cells, known as satellite cells, severely impairs postnatal muscle regeneration. Furthermore, the muscle fusion proteins Myomaker and Myomixer are detected in myoblast filopodia. These data demonstrate that Myo10-driven filopodia facilitate multinucleated mammalian muscle formation.


Assuntos
Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Mioblastos Esqueléticos/metabolismo , Miosinas/metabolismo , Pseudópodes/metabolismo , Animais , Diferenciação Celular , Fusão Celular , Linhagem Celular , Proliferação de Células , Modelos Animais de Doenças , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Knockout , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Mioblastos Esqueléticos/patologia , Miosinas/genética , Pseudópodes/genética , Regeneração , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/patologia , Fatores de Tempo
5.
Genetics ; 216(4): 905-930, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33067325

RESUMO

The laboratory mouse is the most widely used animal model for biomedical research, due in part to its well-annotated genome, wealth of genetic resources, and the ability to precisely manipulate its genome. Despite the importance of genetics for mouse research, genetic quality control (QC) is not standardized, in part due to the lack of cost-effective, informative, and robust platforms. Genotyping arrays are standard tools for mouse research and remain an attractive alternative even in the era of high-throughput whole-genome sequencing. Here, we describe the content and performance of a new iteration of the Mouse Universal Genotyping Array (MUGA), MiniMUGA, an array-based genetic QC platform with over 11,000 probes. In addition to robust discrimination between most classical and wild-derived laboratory strains, MiniMUGA was designed to contain features not available in other platforms: (1) chromosomal sex determination, (2) discrimination between substrains from multiple commercial vendors, (3) diagnostic SNPs for popular laboratory strains, (4) detection of constructs used in genetically engineered mice, and (5) an easy-to-interpret report summarizing these results. In-depth annotation of all probes should facilitate custom analyses by individual researchers. To determine the performance of MiniMUGA, we genotyped 6899 samples from a wide variety of genetic backgrounds. The performance of MiniMUGA compares favorably with three previous iterations of the MUGA family of arrays, both in discrimination capabilities and robustness. We have generated publicly available consensus genotypes for 241 inbred strains including classical, wild-derived, and recombinant inbred lines. Here, we also report the detection of a substantial number of XO and XXY individuals across a variety of sample types, new markers that expand the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. We provide preliminary evidence that the array can be used to identify both partial sex chromosome duplication and mosaicism, and that diagnostic SNPs can be used to determine how long inbred mice have been bred independently from the relevant main stock. We conclude that MiniMUGA is a valuable platform for genetic QC, and an important new tool to increase the rigor and reproducibility of mouse research.


Assuntos
Estudo de Associação Genômica Ampla/métodos , Técnicas de Genotipagem/métodos , Camundongos/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Feminino , Estudo de Associação Genômica Ampla/normas , Genótipo , Técnicas de Genotipagem/normas , Masculino , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos/normas , Polimorfismo Genético , Reprodutibilidade dos Testes , Processos de Determinação Sexual
6.
Sci Rep ; 7(1): 17354, 2017 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-29229982

RESUMO

Myosin-X (Myo10) is an unconventional myosin best known for its striking localization to the tips of filopodia. Despite the broad expression of Myo10 in vertebrate tissues, its functions at the organismal level remain largely unknown. We report here the generation of KO-first (Myo10 tm1a/tm1a ), floxed (Myo10 tm1c/tm1c ), and KO mice (Myo10 tm1d/tm1d ). Complete knockout of Myo10 is semi-lethal, with over half of homozygous KO embryos exhibiting exencephaly, a severe defect in neural tube closure. All Myo10 KO mice that survive birth exhibit a white belly spot, all have persistent fetal vasculature in the eye, and ~50% have webbed digits. Myo10 KO mice that survive birth can breed and produce litters of KO embryos, demonstrating that Myo10 is not absolutely essential for mitosis, meiosis, adult survival, or fertility. KO-first mice and an independent spontaneous deletion (Myo10 m1J/m1J ) exhibit the same core phenotypes. During retinal angiogenesis, KO mice exhibit a ~50% decrease in endothelial filopodia, demonstrating that Myo10 is required to form normal numbers of filopodia in vivo. The Myo10 mice generated here demonstrate that Myo10 has important functions in mammalian development and provide key tools for defining the functions of Myo10 in vivo.


Assuntos
Miosinas/fisiologia , Neovascularização Patológica , Tubo Neural/fisiopatologia , Artéria Oftálmica/fisiopatologia , Pigmentação , Pseudópodes/patologia , Corpo Vítreo/patologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Artéria Oftálmica/metabolismo , Pseudópodes/metabolismo , Corpo Vítreo/irrigação sanguínea , Corpo Vítreo/metabolismo
7.
Curr Eye Res ; 31(12): 1073-80, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17169846

RESUMO

Human retinal pigmented epithelial cells (ARPE-19) grown in euglycemic media (5.5 mM) had lower cell number, significantly different cell morphology, and lower levels of vascular endothelial growth factor (VEGF) in the culture media than those grown in hyperglycemic media (18 mM) customarily used for culturing ARPE-19 cells. Although it has been shown that within a 24-hour period, all-trans retinoic acid significantly reduces VEGF secretion by retinal pigmented epithelial cells (grown in 18 mM glucose), such an inhibitory effect was not observed in cells grown in 5.5 mM glucose. Our results suggest that ARPE-19 cells grown in euglycemic media exhibit distinctly different cell growth, cell differentiation, and cell functions in comparison to cells grown in hyperglycemic media. Because euglycemic conditions provide a physiological glucose environment, this glucose concentration must be included in all future investigations of the mechanism of diabetic retinopathy when studying VEGF secretion by ARPE-19 cells.


Assuntos
Glucose/farmacologia , Epitélio Pigmentado Ocular/citologia , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultura , Humanos , Epitélio Pigmentado Ocular/efeitos dos fármacos , Epitélio Pigmentado Ocular/metabolismo , Tretinoína/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
8.
Mol Biol Cell ; 26(25): 4646-59, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26446836

RESUMO

Filopodia are finger-like protrusions from the plasma membrane and are of fundamental importance to cellular physiology, but the mechanisms governing their assembly are still in question. One model, called convergent elongation, proposes that filopodia arise from Arp2/3 complex-nucleated dendritic actin networks, with factors such as formins elongating these filaments into filopodia. We test this model using constitutively active constructs of two formins, FMNL3 and mDia2. Surprisingly, filopodial assembly requirements differ between suspension and adherent cells. In suspension cells, Arp2/3 complex is required for filopodial assembly through either formin. In contrast, a subset of filopodia remains after Arp2/3 complex inhibition in adherent cells. In adherent cells only, mDia1 and VASP also contribute to filopodial assembly, and filopodia are disproportionately associated with focal adhesions. We propose an extension of the existing models for filopodial assembly in which any cluster of actin filament barbed ends in proximity to the plasma membrane, either Arp2/3 complex dependent or independent, can initiate filopodial assembly by specific formins.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Proteínas de Transporte/genética , Proteínas/genética , Pseudópodes/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Dendritos/genética , Dendritos/metabolismo , Forminas , Humanos , Células Jurkat , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Pseudópodes/metabolismo
9.
Nat Struct Mol Biol ; 20(1): 111-8, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23222643

RESUMO

Formins are actin-assembly factors that act in a variety of actin-based processes. The conserved formin homology 2 (FH2) domain promotes filament nucleation and influences elongation through interaction with the barbed end. FMNL3 is a formin that induces assembly of filopodia but whose FH2 domain is a poor nucleator. The 3.4-Å structure of a mouse FMNL3 FH2 dimer in complex with tetramethylrhodamine-actin uncovers details of formin-regulated actin elongation. We observe distinct FH2 actin-binding regions; interactions in the knob and coiled-coil subdomains are necessary for actin binding, whereas those in the lasso-post interface are important for the stepping mechanism. Biochemical and cellular experiments test the importance of individual residues for function. This structure provides details for FH2-mediated filament elongation by processive capping and supports a model in which C-terminal non-FH2 residues of FMNL3 are required to stabilize the filament nucleus.


Assuntos
Actinas/química , Actinas/metabolismo , Proteínas/química , Proteínas/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Forminas , Humanos , Células Jurkat , Camundongos , Modelos Moleculares , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Terciária de Proteína , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , Rodaminas/metabolismo
10.
Cytoskeleton (Hoboken) ; 67(12): 755-72, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20862687

RESUMO

Actin-dependent finger-like protrusions such as filopodia and microvilli are widespread in eukaryotes, but their assembly mechanisms are poorly understood. Filopodia assembly requires at least three biochemical activities on actin: actin filament nucleation, prolonged actin filament elongation, and actin filament bundling. These activities are shared by several mammalian formin proteins, including mDia2, FRL1 (also called FMNL1), and FRL2 (FMNL3). In this paper, we compare the abilities of constructs from these three formins to induce filopodia. FH1-FH2 constructs of both FRL2 and mDia2 stimulate potent filopodia assembly in multiple cell types, and enrich strongly at filopodia tips. In contrast, FRL1 FH1-FH2 lacks this activity, despite possessing similar biochemical activities and being highly homologous to FRL2. Chimeric FH1-FH2 experiments between FRL1 and FRL2 show that, while both an FH1 and an FH2 are needed, either FH1 domain supports filopodia assembly but only FRL2's FH2 domain allows this activity. A mutation that compromises FRL2's barbed end binding ability abolishes filopodia assembly. FRL2's ability to stimulate filopodia assembly is not altered by additional domains (GBD, DID, DAD), but is significantly reduced in the full-length construct, suggesting that FRL2 is subject to inhibitory regulation. The data suggest that the FH2 domain of FRL2 possesses properties not shared by FRL1 that allow it to generate filopodia.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas/metabolismo , Pseudópodes/metabolismo , Citoesqueleto de Actina/química , Actinas/química , Actinas/metabolismo , Animais , Forminas , Células HeLa , Humanos , Células Jurkat , Camundongos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Proteínas/química , Células Swiss 3T3 , Transfecção
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA