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1.
Commun Biol ; 4(1): 763, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34155338

RESUMO

Mechanical forces control cell behavior, including cancer progression. Cells sense forces through actomyosin to activate YAP. However, the regulators of F-actin dynamics playing relevant roles during mechanostransduction in vitro and in vivo remain poorly characterized. Here we identify the Fascin1 F-actin bundling protein as a factor that sustains YAP activation in response to ECM mechanical cues. This is conserved in the mouse liver, where Fascin1 regulates YAP-dependent phenotypes, and in human cholangiocarcinoma cell lines. Moreover, this is relevant for liver tumorigenesis, because Fascin1 is required in the AKT/NICD cholangiocarcinogenesis model and it is sufficient, together with AKT, to induce cholangiocellular lesions in mice, recapitulating genetic YAP requirements. In support of these findings, Fascin1 expression in human intrahepatic cholangiocarcinomas strongly correlates with poor patient prognosis. We propose that Fascin1 represents a pro-oncogenic mechanism that can be exploited during intrahepatic cholangiocarcinoma development to overcome a mechanical tumor-suppressive environment.


Assuntos
Neoplasias dos Ductos Biliares/etiologia , Proteínas de Transporte/fisiologia , Proteínas de Ciclo Celular/fisiologia , Colangiocarcinoma/etiologia , Mecanotransdução Celular/fisiologia , Proteínas dos Microfilamentos/fisiologia , Fatores de Transcrição/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/fisiologia , Animais , Proteína de Capeamento de Actina CapZ/fisiologia , Moléculas de Adesão Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos , Fosfoproteínas/fisiologia
2.
Nat Cell Biol ; 17(9): 1094-6, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26316454

RESUMO

Compared with most intracellular vesicles, the autophagosome is formed by an unusual event of vesicle budding involving an elusive sequence of membrane expansions that ends with a double membrane vesicle. It is now shown that actin polymerization inside the forming autophagosome is a driving force for the expansion and assembly of a functional autophagosome.


Assuntos
Citoesqueleto de Actina/metabolismo , Autofagia , Proteína de Capeamento de Actina CapZ/fisiologia , Membranas Intracelulares/metabolismo , Fagossomos/metabolismo , Animais
3.
Nat Cell Biol ; 17(9): 1112-23, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26237647

RESUMO

A fundamental question regarding autophagosome formation is how the shape of the double-membrane autophagosomal vesicle is generated. Here we show that in mammalian cells assembly of an actin scaffold inside the isolation membrane (the autophagosomal precursor) is essential for autophagosomal membrane shaping. Actin filaments are depolymerized shortly after starvation and actin is assembled into a network within the isolation membrane. When formation of actin puncta is disrupted by an actin polymerization inhibitor or by knocking down the actin-capping protein CapZß, isolation membranes and omegasomes collapse into mixed-membrane bundles. Formation of actin puncta is PtdIns(3)P dependent, and inhibition of PtdIns(3)P formation by treating cells with the PI(3)K inhibitor 3-MA, or by knocking down Beclin-1, abolishes the formation of actin puncta. Binding of CapZ to PtdIns(3)P, which is enriched in omegasomes, stimulates actin polymerization. Our findings illuminate the mechanism underlying autophagosomal membrane shaping and provide key insights into how autophagosomes are formed.


Assuntos
Citoesqueleto de Actina/metabolismo , Autofagia , Proteína de Capeamento de Actina CapZ/fisiologia , Membranas Intracelulares/metabolismo , Fagossomos/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Células Cultivadas , Membranas Intracelulares/ultraestrutura , Fagossomos/ultraestrutura , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica , Multimerização Proteica , Transporte Proteico , Ratos
4.
Mol Biol Cell ; 25(14): 2152-60, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24829386

RESUMO

Capping protein (CP) binds to barbed ends of growing actin filaments and inhibits elongation. CP is essential for actin-based motility in cell-free systems and in Dictyostelium. Even though CP is believed to be critical for creating the lamellipodial actin structure necessary for protrusion and migration, CP's role in mammalian cell migration has not been directly tested. Moreover, recent studies have suggested that structures besides lamellipodia, including lamella and filopodia, may have unappreciated roles in cell migration. CP has been postulated to be absent from filopodia, and thus its role in filopodial activity has remained unexplored. We report that silencing CP in both cultured mammalian B16F10 cells and in neurons of developing neocortex impaired cell migration. Moreover, we unexpectedly observed that low levels of CP were detectable in the majority of filopodia. CP depletion decreased filopodial length, altered filopodial shape, and reduced filopodial dynamics. Our results support an expansion of the potential roles that CP plays in cell motility by implicating CP in filopodia as well as in lamellipodia, both of which are important for locomotion in many types of migrating cells.


Assuntos
Proteína de Capeamento de Actina CapZ/fisiologia , Movimento Celular , Pseudópodes/ultraestrutura , Actinas/metabolismo , Animais , Linhagem Celular Tumoral , Forma Celular , Técnicas de Silenciamento de Genes , Camundongos , Pseudópodes/metabolismo
5.
Nat Rev Mol Cell Biol ; 14(2): 113-9, 2013 02.
Artigo em Inglês | MEDLINE | ID: mdl-23299957

RESUMO

Correct specification of myofilament length is essential for efficient skeletal muscle contraction. The length of thin actin filaments can be explained by a novel 'two-segment' model, wherein the thin filaments consist of two concatenated segments, which are of either constant or variable length. This is in contrast to the classic 'nebulin ruler' model, which postulates that thin filaments are uniform structures, the lengths of which are dictated by nebulin. The two-segment model implicates position-specific microregulation of actin dynamics as a general principle underlying actin filament length and stability.


Assuntos
Citoesqueleto de Actina/química , Citoesqueleto de Actina/fisiologia , Modelos Biológicos , Músculo Esquelético/ultraestrutura , Animais , Proteína de Capeamento de Actina CapZ/metabolismo , Proteína de Capeamento de Actina CapZ/fisiologia , Humanos , Contração Muscular/fisiologia , Proteínas Musculares/metabolismo , Proteínas Musculares/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Miofibrilas/química , Miofibrilas/metabolismo , Miofibrilas/fisiologia , Miofibrilas/ultraestrutura , Miopatias da Nemalina/genética , Miopatias da Nemalina/metabolismo , Miopatias da Nemalina/patologia , Miopatias da Nemalina/fisiopatologia , Sarcômeros/metabolismo , Sarcômeros/fisiologia , Tropomiosina/metabolismo , Tropomiosina/fisiologia
6.
Cell Mol Life Sci ; 68(19): 3261-74, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21290163

RESUMO

DNA aneuploidy has been identified as a prognostic factor for epithelial malignancies. Further understanding of the translation of DNA aneuploidy into protein expression will help to define novel biomarkers to improve therapies and prognosis. DNA ploidy was assessed by image cytometry. Comparison of gel-electrophoresis-based protein expression patterns of three diploid and four aneuploid colorectal cancer cell lines detected 64 ploidy-associated proteins. Proteins were identified by mass spectrometry and subjected to Ingenuity Pathway Analysis resulting in two overlapping high-ranked networks maintaining Cellular Assembly and Organization, Cell Cycle, and Cellular Growth and Proliferation. CAPZA1, TXNL1, and HDAC2 were significantly validated by Western blotting in cell lines and the latter two showed expression differences also in clinical samples using a tissue microarray of normal mucosa (n=19), diploid (n=31), and aneuploid (n=47) carcinomas. The results suggest that distinct protein expression patterns, affecting TXNL1 and HDAC2, distinguish aneuploid with poor prognosis from diploid colorectal cancers.


Assuntos
Aneuploidia , Carcinoma/genética , Neoplasias Colorretais/genética , Diploide , Histona Desacetilase 2/genética , Tiorredoxinas/genética , Western Blotting , Proteína de Capeamento de Actina CapZ/genética , Proteína de Capeamento de Actina CapZ/metabolismo , Proteína de Capeamento de Actina CapZ/fisiologia , Carcinoma/diagnóstico , Carcinoma/patologia , Linhagem Celular Tumoral , Estudos de Coortes , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/patologia , DNA de Neoplasias/química , Instabilidade Genômica , Histona Desacetilase 2/metabolismo , Histona Desacetilase 2/fisiologia , Humanos , Prognóstico , Tiorredoxinas/metabolismo , Tiorredoxinas/fisiologia
7.
PLoS Biol ; 7(10): e1000208, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19806181

RESUMO

Capping protein (CP) is a heterodimer that regulates actin assembly by binding to the barbed end of F-actin. In cultured nonneuronal cells, each CP subunit plays a critical role in the organization and dynamics of lamellipodia and filopodia. Mutations in either alpha or beta CP subunit result in retinal degeneration in Drosophila. However, the function of CP subunits in mammalian neurons remains unclear. Here, we investigate the role of the beta CP subunit expressed in the brain, Capzb2, in growth cone morphology and neurite outgrowth. We found that silencing Capzb2 in hippocampal neurons resulted in short neurites and misshapen growth cones in which microtubules overgrew into the periphery and completely overlapped with F-actin. In searching for the mechanisms underlying these cytoskeletal abnormalities, we identified beta-tubulin as a novel binding partner of Capzb2 and demonstrated that Capzb2 decreases the rate and the extent of tubulin polymerization in vitro. We mapped the region of Capzb2 that was required for the subunit to interact with beta-tubulin and inhibit microtubule polymerization. A mutant Capzb2 lacking this region was able to bind F-actin and form a CP heterodimer with alpha2-subunit. However, this mutant was unable to rescue the growth cone and neurite outgrowth phenotypes caused by Capzb2 knockdown. Together, these data suggest that Capzb2 plays an important role in growth cone formation and neurite outgrowth and that the underlying mechanism may involve direct interaction between Capzb2 and microtubules.


Assuntos
Proteína de Capeamento de Actina CapZ/fisiologia , Cones de Crescimento/ultraestrutura , Tubulina (Proteína)/fisiologia , Actinas/metabolismo , Animais , Sítios de Ligação , Proteína de Capeamento de Actina CapZ/genética , Proteína de Capeamento de Actina CapZ/metabolismo , Dimerização , Cones de Crescimento/fisiologia , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Camundongos , Microtúbulos/metabolismo , Mutação , Regeneração Nervosa , Neuritos/ultraestrutura , Interferência de RNA , Tubulina (Proteína)/metabolismo
8.
J Mol Cell Cardiol ; 41(3): 537-43, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16870209

RESUMO

Actin capping protein (CapZ) anchors the barbed ends of sarcomeric actin to the Z-disc. Myofilaments from transgenic mice (TG-CapZ) expressing a reduced amount of CapZ demonstrate altered function and protein kinase C (PKC) signaling [Pyle WG, Hart MC, Cooper JA, Sumandea MP, de Tombe PP, and Solaro RJ., Circ. Res. 90 (2002) 1299-306]. The aims of the current study were to determine the direct effects of CapZ on myofilament function and on PKC signaling to the myofilaments. Our studies compared mechanical properties of single myocytes from TG-CapZ mouse hearts to wild-type myocytes from which CapZ was extracted using PIP(2). We found that myofilaments from CapZ-deficient transgenic myocardium exhibited increased Ca(2+) sensitivity and maximum isometric tension. The extraction of CapZ from wild-type myofilaments replicated the increase in maximum isometric tension, but had no effect on myofilament Ca(2+) sensitivity. Immunoblot analysis revealed that the extraction of CapZ was associated with a reduction in myofilament-associated PKC-beta(II) and that CapZ-deficient transgenic myofilaments also lacked PKC-beta(II). Treatment of wild-type myofilaments with recombinant PKC-beta(II) reduced myofilament Ca(2+) sensitivity, whereas this effect was attenuated in myofilaments from TG-CapZ mice. Our results indicate that cardiac CapZ directly controls maximum isometric tension generation, and establish CapZ as an important component in anchoring PKC-beta(II) at the myofilaments, and for mediating the effects of PKC-beta(II) on myofilament function.


Assuntos
Proteína de Capeamento de Actina CapZ/fisiologia , Coração/fisiologia , Contração Miocárdica , Miocárdio/metabolismo , Proteína Quinase C/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Relação Dose-Resposta a Droga , Camundongos , Camundongos Transgênicos , Proteína Quinase C beta , Sensibilidade e Especificidade , Transdução de Sinais
9.
Methods Enzymol ; 406: 190-214, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16472659

RESUMO

Formins are members of a conserved family of proteins, present in all eukaryotes, that regulate actin dynamics. Mammals have 15 distinct formin genes. From studies to date, surprising variability between these isoforms has been uncovered. All formins examined have several common effects on actin dynamics in that they: (1) accelerate nucleation rate; (2) alter filament barbed end elongation/depolymerization rates; and (3) antagonize capping protein. However, the potency of each effect can vary greatly between formins. In addition, a subset of formins binds tightly to filament sides and bundle filaments. Even isoforms that are closely related phylogenetically can display marked differences in their effects on actin. This chapter discusses several methods for examining formin function in vitro. We also discuss pitfalls associated with these assays. As one example, the effect of profilin on formin function is difficult to interpret by "pyrene-actin" polymerization assays commonly used in the field and requires assays that can distinguish between filament nucleation and filament elongation. The regulatory mechanisms for formins are not clear and certainly vary between isoforms. A subset of formins is regulated by Rho GTPases, and the assays described in this chapter have been used for characterization of this regulation.


Assuntos
Actinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas dos Microfilamentos/fisiologia , Actinas/química , Actinas/ultraestrutura , Animais , Proteína de Capeamento de Actina CapZ/fisiologia , Proteínas de Transporte/fisiologia , Forminas , Profilinas/fisiologia , Pirenos/química , Espectrometria de Fluorescência
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