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Spatial control of coated-pit dynamics in living cells

Abstract

Here we visualize new aspects of the dynamics of endocytotic clathrin-coated pits and vesicles in mammalian cells by using a fusion protein consisting of green fluorescent protein and clathrin light chain a. Clathrin-coated pits invaginating from the plasma membrane show definite, but highly limited, mobility within the membrane that is relaxed upon treatment with latrunculin B, an inhibitor of actin assembly, indicating that an actin-based framework may be involved in the mobility of these pits. Transient, motile coated vesicles that originate from coated pits can be detected, with multiple vesicles occasionally appearing to emanate from a single pit. Despite their seemingly random distribution, coated pits tend to form repeatedly at defined sites while excluding other regions. This spatial regulation of coated-pit assembly and function is attributable to the attachment of the coated pits to the membrane skeleton.

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Figure 1: A fusion protein consisting of GFP and clathrin light chain a is incorporated into structurally and functionally active clathrin trimers and coated pits in cells.
Figure 2: Dynamics of coated pits and vesicles containing GFP–clathrin in live cells.
Figure 3: Coated pits exhibit limited mobility on cell surfaces that is sensitive to treatment with latrunculin B.
Figure 4: Coated pits repeatedly form at discrete sites.
Figure 5: Coated pits are attached to an interconnected, detergent-resistant membrane skeleton.

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Acknowledgements

We thank M. Birnbaum and M. Marks for their critical review of the manuscript, and P. Hingorani for help with production of the figures. This work was supported by an NIH grant (to J.H.K.). Correspondence and requests for materials should be addressed to J.H.K.

Supplementary Information is available on Nature Cell Biology 's World-Wide Web site (ncb/suppl/ncb0599/ncb0599_001/).

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Correspondence to James H. Keen.

Supplementary information

Coated vesicle formation

Coated vesicle emerges from coated pit slightly left and above center of field. (MOV 185 kb)

Appearance of multiple coated vesicles

from region of coated pit slightly left and above center of field; coated pit eventually disappears. (MOV 204 kb)

Coated pits disappear and reappear at discrete sites

A larger field than that shown in the lower panel of Fig. 4a. (MOV 225 kb)

Coated pits move coordinately on detergent extraction

Image sequence is initiated approximately 10 sec before detergent addition. (MOV 12157 kb)

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Gaidarov, I., Santini, F., Warren, R. et al. Spatial control of coated-pit dynamics in living cells. Nat Cell Biol 1, 1–7 (1999). https://doi.org/10.1038/8971

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