Myosin 1E coordinates actin assembly and cargo trafficking during clathrin-mediated endocytosis

Myosin 1E (Myo1E) is recruited to sites of clathrin-mediated endocytosis coincident with a burst of actin assembly. The recruitment dynamics and lifetime of Myo1E are similar to those of tagged actin polymerization regulatory proteins. Like inhibition of actin assembly, depletion of Myo1E causes reduced transferrin endocytosis and a significant delay in transferrin trafficking to perinuclear compartments, demonstrating an integral role for Myo1E in these actin-mediated steps. Mistargeting of GFP-Myo1E or its src-homology 3 domain to mitochondria results in appearance of WIP, WIRE, N-WASP, and actin filaments at the mitochondria, providing evidence for Myo1E's role in actin assembly regulation. These results suggest for mammalian cells, similar to budding yeast, interdependence in the recruitment of type I myosins, WIP/WIRE, and N-WASP to endocytic sites for Arp2/3 complex activation to assemble F-actin as endocytic vesicles are being formed.

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Switch-like Arp2/3 activation upon WASP and WIP recruitment to an apparent threshold level by multivalent linker proteins in vivo

eLife ◽

10.7554/elife.29140 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 28

Author(s):

Yidi Sun ◽

Nicole T Leong ◽

Tommy Jiang ◽

Astou Tangara ◽

Xavier Darzacq ◽

...

Keyword(s):

Quantitative Imaging ◽

Threshold Level ◽

Actin Assembly ◽

Cellular Processes ◽

Src Homology 3 ◽

Filament Assembly ◽

Tightly Coupled ◽

Src Homology 3 Domain ◽

Src Homology

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.

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Saccharomyces cerevisiae Bzz1p Is Implicated with Type I Myosins in Actin Patch Polarization and Is Able To Recruit Actin-Polymerizing Machinery In Vitro

Molecular and Cellular Biology ◽

10.1128/mcb.22.22.7889-7906.2002 ◽

2002 ◽

Vol 22 (22) ◽

pp. 7889-7906 ◽

Cited By ~ 70

Author(s):

Alexandre Soulard ◽

Terry Lechler ◽

Vladislav Spiridonov ◽

Andrej Shevchenko ◽

Anna Shevchenko ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Actin Polymerization ◽

Nacl Stress ◽

Cell Cortex ◽

Type I ◽

Cortical Actin ◽

Src Homology 3 ◽

Src Homology ◽

Actin Patch

ABSTRACT In Saccharomyces cerevisiae, the WASP (Wiskott-Aldrich syndrome protein) homologue Las17p (also called Bee1p) is an important component of cortical actin patches. Las17p is part of a high-molecular-weight protein complex that regulates Arp2/3 complex-dependent actin polymerization at the cell cortex and that includes the type I myosins Myo3p and Myo5p and verprolin (Vrp1p). To identify other factors implicated with this complex in actin regulation, we isolated proteins that bind to Las17p by two-hybrid screening and affinity chromatography. Here, we report the characterization of Lsb7/Bzz1p (for Las seventeen binding protein 7), an Src homology 3 (SH3) domain protein that interacts directly with Las17p via a polyproline-SH3 interaction. Bzz1p coimmunoprecipitates in a complex with Las17p, Vrp1p, Myo3/5p, Bbc1p, Hsp70p, and actin. It colocalizes with cortical actin patches and with Las17p. This localization is dependent on Las17p, but not on F-actin. Bzz1p interacts physically and genetically with type I myosins. While deletion of BZZ1 shows no obvious phenotype, simultaneous deletion of the BZZ1, MYO3, and MYO5 genes is lethal. Overexpression of Bzz1p inhibits cell growth, and a bzz1Δ myo5Δ double mutant is unable to restore actin polarity after NaCl stress. Finally, Bzz1p in vitro is able to recruit a functional actin polymerization machinery through its SH3 domains. Its interactions with Las17p, Vrp1p, and the type I myosins are essential for this process. This suggests that Bzz1p could be implicated in the regulation of actin polymerization.

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