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Elife ; 82019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31829937


Conserved proteins drive clathrin-mediated endocytosis (CME), which from yeast to humans involves a burst of actin assembly. To gain mechanistic insights into this process, we performed a side-by-side quantitative comparison of CME in two distantly related yeast species. Though endocytic protein abundance in S. pombe and S. cerevisiae is more similar than previously thought, membrane invagination speed and depth are two-fold greater in fission yeast. In both yeasts, accumulation of ~70 WASp molecules activates the Arp2/3 complex to drive membrane invagination. In contrast to budding yeast, WASp-mediated actin nucleation plays an essential role in fission yeast endocytosis. Genetics and live-cell imaging revealed core CME spatiodynamic similarities between the two yeasts, although the assembly of two zones of actin filaments is specific for fission yeast and not essential for CME. These studies identified conserved CME mechanisms and species-specific adaptations with broad implications that are expected to extend from yeast to humans.

Elife ; 62017 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-28813247


Actin-related protein 2/3 (Arp2/3) complex activation by nucleation promoting factors (NPFs) such as WASP, plays an important role in many actin-mediated cellular processes. In yeast, Arp2/3-mediated actin filament assembly drives endocytic membrane invagination and vesicle scission. Here we used genetics and quantitative live-cell imaging to probe the mechanisms that concentrate NPFs at endocytic sites, and to investigate how NPFs regulate actin assembly onset. Our results demonstrate that SH3 (Src homology 3) domain-PRM (proline-rich motif) interactions involving multivalent linker proteins play central roles in concentrating NPFs at endocytic sites. Quantitative imaging suggested that productive actin assembly initiation is tightly coupled to accumulation of threshold levels of WASP and WIP, but not to recruitment kinetics or release of autoinhibition. These studies provide evidence that WASP and WIP play central roles in establishment of a robust multivalent SH3 domain-PRM network in vivo, giving actin assembly onset at endocytic sites a switch-like behavior.

Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Microscopia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteína da Síndrome de Wiskott-Aldrich/genética