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PMID: 20587778 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Quantitative analysis of the mechanism of endocytic actin patch assembly and disassembly in fission yeast.

Molecular biology of the cell ·Vol. 21 ·No. 16 ·2010-08-15 ·Pages 2894-904

Sirotkin V, Berro J, Macmillan K, Zhao L, Pollard TD

Abstract

We used quantitative confocal microscopy to measure the numbers of 16 proteins tagged with fluorescent proteins during assembly and disassembly of endocytic actin patches in fission yeast. The peak numbers of each molecule that accumulate in patches varied <30-50% between individual patches. The pathway begins with accumulation of 30-40 clathrin molecules, sufficient to build a hemisphere at the tip of a plasma membrane invagination. Thereafter precisely timed waves of proteins reach characteristic peak numbers: endocytic adaptor proteins (approximately 120 End4p and approximately 230 Pan1p), activators of Arp2/3 complex (approximately 200 Wsp1p and approximately 340 Myo1p) and approximately 300 Arp2/3 complexes just ahead of a burst of actin assembly into short, capped and highly cross-linked filaments (approximately 7000 actins, approximately 200 capping proteins, and approximately 900 fimbrins). Coronin arrives last as all other components disperse upon patch internalization and movement over approximately 10 s. Patch internalization occurs without recruitment of dynamins. Mathematical modeling, described in the accompanying paper (Berro et al., 2010, MBoC 21: 2803-2813), shows that the dendritic nucleation hypothesis can account for the time course of actin assembly into a branched network of several hundred filaments 100-200 nm long and that patch disassembly requires actin filament fragmentation in addition to depolymerization from the ends.

MeSH Terms
Actin-Related Protein 2-3 Complex/genetics,metabolism Actins/genetics,metabolism Clathrin/genetics,metabolism Endocytosis Luminescent Proteins/genetics,metabolism Microfilament Proteins/genetics,metabolism Microscopy, Fluorescence/methods Models, Biological Protein Binding Schizosaccharomyces/cytology,genetics,metabolism Schizosaccharomyces pombe Proteins/genetics,metabolism Time Factors
Chemicals
Actin-Related Protein 2-3 Complex Actins Clathrin End4 protein, S pombe Luminescent Proteins Microfilament Proteins Schizosaccharomyces pombe Proteins
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Sirotkin Vladimir
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520-8103, USA.
Berro Julien
Macmillan Keely
Zhao Lindsey
Pollard Thomas D
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Article Info
Journal
Molecular biology of the cell
Abbr.
Mol Biol Cell
ISSN
1939-4586
DOI
10.1091/mbc.E10-02-0157
Published
2010-08-15
Epub
2010-00-29
Pages
2894-904
Language
English
Region
United States
NLM ID
9201390
PMCID
PMC2921122
Subset
IM
Grants
NIGMS NIH HHS · GM-026132 · United States
NIGMS NIH HHS · R37 GM026132 · United States
NIGMS NIH HHS · GM-0026338 · United States
NIGMS NIH HHS · R01 GM026338 · United States
NIGMS NIH HHS · R01 GM026132 · United States
External Links
PubMed source DOI: 10.1091/mbc.E10-02-0157 PMC Full Text View journal details
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