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HIP1 exhibits an early recruitment and a late stage function in the maturation of coated pits

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Abstract

Huntingtin interacting protein 1 (HIP1) is an accessory protein of the clathrin-mediated endocytosis (CME) pathway, yet its precise role and the step at which it becomes involved are unclear. We employed live-cell imaging techniques to focus on the early steps of CME and characterize HIP1 dynamics. We show that HIP1 is highly colocalized with clathrin at the plasma membrane and shares similar dynamics with a subpopulation of clathrin assemblies. Employing transferrin receptor fused to pHluorin, we distinguished between open pits to which HIP1 localizes and newly internalized vesicles that are devoid of HIP1. Moreover, shRNA knockdown of clathrin compromised HIP1 membranal localization, unlike the reported behavior of Sla2p. HIP1 fragment, lacking its ANTH and Talin-like domains, inhibits internalization of transferrin, but retains colocalization with membranal clathrin assemblies. These data demonstrate HIP1’s role in pits maturation and formation of the coated vesicle, and its strong dependence on clathrin for membranal localization.

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Acknowledgments

We thank Prof. Erich Wanker and Dr. Stephanie Waelter for plasmids and discussions during the early stages of this project. We thank Reut Friedrich for her help with the statistical fittings and Ofer Yizhar for providing technical support for the tracking program. This work was supported by a grant from the Israel Science Foundation (grant no. 1211/07 to U.A.). I.G. was supported by the Clore Scholars Program.

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Authors and Affiliations

  1. Department of Neurobiology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, 69978, Tel Aviv, Israel

    Irit Gottfried & Uri Ashery

  2. Department of Cell Research and Immunology, Faculty of Life Sciences, Tel Aviv University, 69978, Tel Aviv, Israel

    Marcelo Ehrlich

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  1. Irit Gottfried
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Correspondence to Uri Ashery.

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18_2009_77_MOESM1_ESM.tif

HIP1218-604 localizes to small dynamic assemblies at the PM, colocalized with clathrin. A. An example of a short-lived (32 s) HIP1218-604 assembly (box). B. Filtered TIRF images of part of a cell coexpressing HIP1218-604-EGFP and tdTomato-CLC. Merged image demonstrates the high colocalization of the two proteins (HIP1218-604 in green, clathrin in red). (TIFF 651 kb)

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Gottfried, I., Ehrlich, M. & Ashery, U. HIP1 exhibits an early recruitment and a late stage function in the maturation of coated pits. Cell. Mol. Life Sci. 66, 2897–2911 (2009). https://doi.org/10.1007/s00018-009-0077-4

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