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Proc Natl Acad Sci U S A. 2013 Nov 26;110(48):E4591-600. doi: 10.1073/pnas.1315202110. Epub 2013 Nov 11.

Quantifying the dynamic interactions between a clathrin-coated pit and cargo molecules.

Author information

1
School of Biomedical Engineering and Departments of Biomedical Sciences, Biochemistry and Molecular Biology, and Electrical and Computer Engineering, Colorado State University, Fort Collins, CO 80523.

Abstract

Clathrin-mediated endocytosis takes place through the recruitment of cargo molecules into a growing clathrin-coated pit (CCP). Despite the importance of this process to all mammalian cells, little is yet known about the interaction dynamics between cargo and CCPs. These interactions are difficult to study because CCPs display a large degree of lifetime heterogeneity and the interactions with cargo molecules are time dependent. We use single-molecule total internal reflection fluorescence microscopy, in combination with automatic detection and tracking algorithms, to directly visualize the recruitment of individual voltage-gated potassium channels into forming CCPs in living cells. We observe association and dissociation of individual channels with a CCP and, occasionally, their internalization. Contrary to widespread ideas, cargo often escapes from a pit before abortive CCP termination or endocytic vesicle production. Thus, the binding times of cargo molecules associating to CCPs are much shorter than the overall endocytic process. By measuring tens of thousands of capturing events, we build the distribution of capture times and the times that cargo remains confined to a CCP. An analytical stochastic model is developed and compared with the measured distributions. Due to the dynamic nature of the pit, the model is non-Markovian and it displays long-tail power law statistics. The measured distributions and model predictions are in excellent agreement over more than five orders of magnitude. Our findings identify one source of the large heterogeneities in CCP dynamics and provide a mechanism for the anomalous diffusion of proteins in the plasma membrane.

KEYWORDS:

Levy statistics; TIRF; live-cell imaging; single-molecule tracking

PMID:
24218552
PMCID:
PMC3845133
DOI:
10.1073/pnas.1315202110
[Indexed for MEDLINE]
Free PMC Article

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