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Cargo and Dynamin Regulate Clathrin-Coated Pit Maturation

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  • Dinah Loerke
  • Marcel Mettlen
  • Defne Yarar
  • Khuloud Jaqaman
  • Henry Jaqaman
  • Gaudenz Danuser
  • Sandra L Schmid

Abstract

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. : Clathrin-mediated endocytosis is the major pathway for the uptake of molecules into eukaryotic cells and is regulated by the GTPase dynamin. Adaptor proteins recruit clathrin to the plasma membrane, where clathrin-coated pits capture transmembrane cargo molecules, again via adaptors. The pits invaginate and pinch off to form clathrin-coated vesicles that carry the cargo into the cell. Live cell imaging has revealed striking heterogeneity in the dynamic behavior of clathrin-coated pits associated with the plasma membrane, yet the nature of this heterogeneity and its functional implications are unknown. We used particle-tracking software to establish an unbiased and complete inventory of the trajectories of clathrin-coated pits visible by total internal reflection fluorescence microscopy. Through statistical analyses, we identified three dynamically distinct subpopulations of coated pits: two short-lived subpopulations corresponding to aborted intermediates, and one longer-lived productive subpopulation. The proportion of each subpopulation and their lifetimes respond independently to molecular perturbations. As a result of systematic modulation of cargo concentration, adaptor levels, and analysis of dynamin mutants, we postulate the existence of an endocytic restriction or checkpoint that governs the rate of clathrin-mediated endocytosis by gating the maturation of clathrin-coated pits. Analysis of live-cell images reveals that clathrin-mediated endocytosis proceeds via distinct subpopulations of clathrin-coated pits, and suggests that an endocytosis check-point is regulated by dynamin and responsive to cargo load.

Suggested Citation

  • Dinah Loerke & Marcel Mettlen & Defne Yarar & Khuloud Jaqaman & Henry Jaqaman & Gaudenz Danuser & Sandra L Schmid, 2009. "Cargo and Dynamin Regulate Clathrin-Coated Pit Maturation," PLOS Biology, Public Library of Science, vol. 7(3), pages 1-12, March.
  • Handle: RePEc:plo:pbio00:1000057
    DOI: 10.1371/journal.pbio.1000057
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    References listed on IDEAS

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    1. Joëlle Bigay & Pierre Gounon & Sylviane Robineau & Bruno Antonny, 2003. "Lipid packing sensed by ArfGAP1 couples COPI coat disassembly to membrane bilayer curvature," Nature, Nature, vol. 426(6966), pages 563-566, December.
    2. Eva M. Schmid & Harvey T. McMahon, 2007. "Integrating molecular and network biology to decode endocytosis," Nature, Nature, vol. 448(7156), pages 883-888, August.
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    Cited by:

    1. Meiyan Jin & Cyna Shirazinejad & Bowen Wang & Amy Yan & Johannes Schöneberg & Srigokul Upadhyayula & Ke Xu & David G. Drubin, 2022. "Branched actin networks are organized for asymmetric force production during clathrin-mediated endocytosis in mammalian cells," Nature Communications, Nature, vol. 13(1), pages 1-12, December.

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