A functional GFP fusion for imaging clathrin-mediated endocytosis.
The ability to localize proteins of interest in live cells through imaging inherently fluorescent protein tags has provided an unprecedented level of information on cellular organization. However, there are numerous cases where fluorescent tags alter the localization and/or function of the proteins to which they are appended. Clathrin-mediated endocytosis from the plasma membrane is a physiologically important process evolutionarily conserved from yeast to humans. Some proteins that are associated with the machinery of clathrin-mediated endocytosis have been tagged with fluorescent proteins. However, it has not yet been possible to study this process through a protein marker that is specific to this step and still fully functional when linked to a fluorescent protein. In this study, we present the first demonstration that one of these proteins, in this case a green fluorescent protein (GFP) fusion to alpha-adaptin, a marker of the adaptor protein-2 complex, functionally complements knockdown of endogenous protein through small interfering RNA silencing. GFP-alpha-adaptin, as well as the techniques used to test the fusion protein, represents an important contribution to the cell biologist's toolbox, which will permit a greater understanding of vesicle trafficking in live cells.
Rappoport
Joshua Z
JZ
Laboratory of Cellular Biophysics, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA. j.rappoport@bham.ac.uk
Simon
Sanford M
SM
D033965:Adaptor Protein Complex alpha Subunits / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002966:Clathrin / Q000737:chemistry*; D004705:Endocytosis; D020868:Gene Silencing; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D007107:Immune System; D051379:Mice; D008954:Models, Biological; D034741:RNA, Small Interfering / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry*
D033965:Adaptor Protein Complex alpha Subunits; D002966:Clathrin; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins