Cargo and dynamin regulate clathrin-coated pit maturation

Total internal reflection fluorescence microscopy (TIR-FM) has become a powerful tool for studying clathrin-mediated endocytosis. However, due to difficulties in tracking and quantifying their heterogeneous dynamic behavior, detailed analyses have been restricted to a limited number of selected clat...

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Published in:PLoS biology 2009-03, Vol.7 (3), p.e57-e57
Main Authors: Loerke, Dinah, Mettlen, Marcel, Yarar, Defne, Jaqaman, Khuloud, Jaqaman, Henry, Danuser, Gaudenz, Schmid, Sandra L
Format: Article
Language:English
Subjects:
Algorithms
Animals
Biological Transport
Carrier Proteins - genetics
Carrier Proteins - metabolism
Cell Biology
Cell culture
Cell Line
Clathrin
Clathrin - genetics
Clathrin - metabolism
Coated Pits, Cell-Membrane - metabolism
Data processing
Dynamins - genetics
Dynamins - metabolism
Endocytosis
Enzymes
Fluorescence microscopy
Lifetime
Microscopy, Fluorescence - methods
Physiological aspects
Plasma
Proteins
Rats
Software
Statistical methods
Studies
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Summary:Total internal reflection fluorescence microscopy (TIR-FM) has become a powerful tool for studying clathrin-mediated endocytosis. However, due to difficulties in tracking and quantifying their heterogeneous dynamic behavior, detailed analyses have been restricted to a limited number of selected clathrin-coated pits (CCPs). To identify intermediates in the formation of clathrin-coated vesicles and factors that regulate progression through these stages, we used particle-tracking software and statistical methods to establish an unbiased and complete inventory of all visible CCP trajectories. We identified three dynamically distinct CCP subpopulations: two short-lived subpopulations corresponding to aborted intermediates, and one longer-lived productive subpopulation. In a manner dependent on AP2 adaptor complexes, increasing cargo concentration significantly enhances the maturation efficiency of productive CCPs, but has only minor effects on their lifetimes. In contrast, small interfering RNA (siRNA) depletion of dynamin-2 GTPase and reintroduction of wild-type or mutant dynamin-1 revealed dynamin's role in controlling the turnover of abortive intermediates and the rate of CCP maturation. From these data, we infer the existence of an endocytic restriction or checkpoint, responsive to cargo and regulated by dynamin.
ISSN:1545-7885
1544-9173
1545-7885
DOI:10.1371/journal.pbio.1000057