Nanocones to Study Initial Steps of Endocytosis
Vesicle endocytosis at the plasma membrane is associated with a precise temporal choreography in the recruitment of cytosolic proteins that sense, generate, or stabilize locally curved membrane regions. To dissect the role of membrane curvature sensing from other co-occurring events during the initial steps of endocytosis, we developed a method to artificially induce nanoscale deformations of the PM in living cells that is based on cone-shaped nanostructures (i.e., Nanocones). When cultured on Nanocones, cells create stable inward plasma membrane deformations to which curvature-sensing proteins are recruited. Here, we provide a detailed protocol how to use Nanocones to study recruitment during the initial steps of endocytosis in cells by fluorescence and electron microscopy.
Key wordsNanocones Plasma membrane Membrane curvature Endocytosis Endocytic vesicles
S. J. acknowledges support from the Korea Foundation for Advanced Studies (KFAS) for graduate fellowship. M. G. was supported by fellowships from the Swiss National Science Foundation (No. PBBSP3-123159), Novartis Jubilaeumsstiftung, Stanford Deans Postdoctoral Fellowship and grants from the German Research Foundation (Cluster of Excellence EXC 1003, Cells in Motion, CiM, Münster, Germany).