Mammalian actin binding protein 1 is essential for endocytosis but not lamellipodia formation: functional analysis by RNA interference

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Abstract

Mammalian actin binding protein 1 (mAbp1, also called SH3P7/Hip55) is structurally and functionally related to yeast Abp1 and to cortactin, both of which have been implicated in endocytotic processes. mAbp1 associates through its SH3 domain with dynamin, a large GTPase essential for vesicle fission. To clarify the function of mAbp1, we specifically knocked down its expression in human embryonic kidney 293T cells, using RNA interference (RNAi). Co-transfection of a short interfering RNA (siRNA) together with a plasmid coding for a surface marker, followed by purification of transfected cells, enabled us to obtain a cell population having up to 90% inhibition of mAbp1 expression. In mAbp1-knocked down cells, transferrin (Tf) receptor endocytosis was significantly inhibited and intracellular distribution of the early endosomal compartment was modified. In contrast, in these cells actin and microtubule filaments appeared normal, and formation of lamellipodia induced by active Rac was not inhibited. This study provides definitive evidence that mAbp1 is indispensable for receptor-mediated endocytosis.

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Materials and methods

Antibodies and other reagents. The polyclonal anti-mAbp1antibody was produced by immunizing rabbit with a glutathione S-transferase (GST) fusion protein encompassing amino acids 146–326 of human mAbp1. For immunofluorescence, the antibody was purified using CNBr-activated Sepharose immobilized with the immunogen. Anti-Erk and anti-CrkL (Santa Cruz Biotechnology, CA) were rabbit polyclonal antibodies and anti-Akt (Santa Cruz Biotechnology) was goat polyclonal. Mouse monoclonal antibodies

Results and discussion

To knock down mAbp1 protein expression, a synthetic double-stranded siRNA directed to the gene sequence of human mAbp1 was transfected into 293T cells together with EGFP-expression plasmid utilized to monitor cells receiving the siRNA. As a negative control, cells were transfected with siRNA to the Dok2 gene [16], which is not expressed in 293T cells (data not shown). Forty-eight hours after transfection, mAbp1 was visualized by immunofluorescence using an affinity-purified anti-mAbp1

Acknowledgements

We thank Drs. F. Michel and T. Tuschl for help with some experiments and suggestions. We also thank Drs. G. Langsley, S. Pellegrini, and F. Michel for critical reading of the manuscript. S.M.-O. was a recipient of an Association pour la Recherche sur le Cancer fellowship. This work was supported by grants from the Institut Pasteur and the Centre National pour la Recherche Scientifique.

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Abbreviations: mAbp1, mammalian actin binding protein 1; siRNA, short interfering RNA; Tf, transferrin; RNAi, RNA interference, EGFP, enhanced green fluorescent protein.

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