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1.
Cell Death Dis ; 15(4): 304, 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38693139

RESUMO

Abnormal intraneuronal accumulation of soluble and insoluble α-synuclein (α-Syn) is one of the main pathological hallmarks of synucleinopathies, such as Parkinson's disease (PD). It has been well documented that the reversible liquid-liquid phase separation of α-Syn can modulate synaptic vesicle condensates at the presynaptic terminals. However, α-Syn can also form liquid-like droplets that may convert into amyloid-enriched hydrogels or fibrillar polymorphs under stressful conditions. To advance our understanding on the mechanisms underlying α-Syn phase transition, we employed a series of unbiased proteomic analyses and found that actin and actin regulators are part of the α-Syn interactome. We focused on Neural Wiskott-Aldrich syndrome protein (N-WASP) because of its association with a rare early-onset familial form of PD. In cultured cells, we demonstrate that N-WASP undergoes phase separation and can be recruited to synapsin 1 liquid-like droplets, whereas it is excluded from α-Syn/synapsin 1 condensates. Consistently, we provide evidence that wsp-1/WASL loss of function alters the number and dynamics of α-Syn inclusions in the nematode Caenorhabditis elegans. Together, our findings indicate that N-WASP expression may create permissive conditions that promote α-Syn condensates and their potentially deleterious conversion into toxic species.


Assuntos
Caenorhabditis elegans , Proteína Neuronal da Síndrome de Wiskott-Aldrich , alfa-Sinucleína , alfa-Sinucleína/metabolismo , Animais , Humanos , Caenorhabditis elegans/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Actinas/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Sinapsinas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo
2.
Sci Adv ; 9(3): eadd6495, 2023 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-36662867

RESUMO

Regulation of the Arp2/3 complex is required for productive nucleation of branched actin networks. An emerging aspect of regulation is the incorporation of subunit isoforms into the Arp2/3 complex. Specifically, both ArpC5 subunit isoforms, ArpC5 and ArpC5L, have been reported to fine-tune nucleation activity and branch junction stability. We have combined reverse genetics and cellular structural biology to describe how ArpC5 and ArpC5L differentially affect cell migration. Both define the structural stability of ArpC1 in branch junctions and, in turn, by determining protrusion characteristics, affect protein dynamics and actin network ultrastructure. ArpC5 isoforms also affect the positioning of members of the Ena/Vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongators, which mediate ArpC5 isoform-specific effects on the actin assembly level. Our results suggest that ArpC5 and Ena/VASP proteins are part of a signaling pathway enhancing cell migration.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina , Actinas , Actinas/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/análise , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Isoformas de Proteínas/metabolismo
3.
J Cell Sci ; 135(15)2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35971979

RESUMO

Cell migration frequently involves the formation of lamellipodia induced by Rac GTPases activating WAVE regulatory complex (WRC) to drive Arp2/3 complex-dependent actin assembly. Previous genome editing studies in B16-F1 melanoma cells solidified the view of an essential, linear pathway employing the aforementioned components. Here, disruption of the WRC subunit Nap1 (encoded by Nckap1) and its paralog Hem1 (encoded by Nckap1l) followed by serum and growth factor stimulation, or active GTPase expression, revealed a pathway to formation of Arp2/3 complex-dependent lamellipodia-like structures (LLS) that requires both Rac and Cdc42 GTPases, but not WRC. These phenotypes were independent of the WRC subunit eliminated and coincided with the lack of recruitment of Ena/VASP family actin polymerases. Moreover, aside from Ena/VASP proteins, LLS contained all lamellipodial regulators tested, including cortactin (also known as CTTN), the Ena/VASP ligand lamellipodin (also known as RAPH1) and FMNL subfamily formins. Rac-dependent but WRC-independent actin remodeling could also be triggered in NIH 3T3 fibroblasts by growth factor (HGF) treatment or by gram-positive Listeria monocytogenes usurping HGF receptor signaling for host cell invasion. Taken together, our studies thus establish the existence of a signaling axis to Arp2/3 complex-dependent actin remodeling at the cell periphery that operates without WRC and Ena/VASP.


Assuntos
Actinas , Pseudópodes , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Movimento Celular/fisiologia , Pseudópodes/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo
4.
Curr Biol ; 31(10): 2051-2064.e8, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-33711252

RESUMO

Hematopoietic-specific protein 1 (Hem1) is an essential subunit of the WAVE regulatory complex (WRC) in immune cells. WRC is crucial for Arp2/3 complex activation and the protrusion of branched actin filament networks. Moreover, Hem1 loss of function in immune cells causes autoimmune diseases in humans. Here, we show that genetic removal of Hem1 in macrophages diminishes frequency and efficacy of phagocytosis as well as phagocytic cup formation in addition to defects in lamellipodial protrusion and migration. Moreover, Hem1-null macrophages displayed strong defects in cell adhesion despite unaltered podosome formation and concomitant extracellular matrix degradation. Specifically, dynamics of both adhesion and de-adhesion as well as concomitant phosphorylation of paxillin and focal adhesion kinase (FAK) were significantly compromised. Accordingly, disruption of WRC function in non-hematopoietic cells coincided with both defects in adhesion turnover and altered FAK and paxillin phosphorylation. Consistently, platelets exhibited reduced adhesion and diminished integrin αIIbß3 activation upon WRC removal. Interestingly, adhesion phenotypes, but not lamellipodia formation, were partially rescued by small molecule activation of FAK. A full rescue of the phenotype, including lamellipodia formation, required not only the presence of WRCs but also their binding to and activation by Rac. Collectively, our results uncover that WRC impacts on integrin-dependent processes in a FAK-dependent manner, controlling formation and dismantling of adhesions, relevant for properly grabbing onto extracellular surfaces and particles during cell edge expansion, like in migration or phagocytosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Adesão Celular , Movimento Celular , Integrinas/metabolismo , Macrófagos/metabolismo , Fagocitose , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Quinase 1 de Adesão Focal/metabolismo , Masculino , Camundongos , Paxilina/metabolismo , Fosforilação , Pseudópodes
5.
Small GTPases ; 12(2): 122-132, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-31451035

RESUMO

Cell migration frequently involves the formation of lamellipodial protrusions, the initiation of which requires Rac GTPases signalling to heteropentameric WAVE regulatory complex (WRC). While Rac-related RhoG and Cdc42 can potently stimulate lamellipodium formation, so far presumed to occur by upstream signalling to Rac activation, we show here that the latter can be bypassed by RhoG and Cdc42 given that WRC has been artificially activated. This evidence arises from generation of B16-F1 cells simultaneously lacking both Rac GTPases and WRC, followed by reconstitution of lamellipodia formation with specific Rho-GTPase and differentially active WRC variant combinations. We conclude that formation of canonical lamellipodia requires WRC activation through Rac, but can possibly be tuned, in addition, by WRC interactions with RhoG and Cdc42.


Assuntos
Pseudópodes
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