Titlebook: Clathrin-Mediated Endocytosis; Methods and Protocol Laura E.‘Swan Book 2018 Springer Science+Business Media, LLC, part of Springer Nature 2

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Preparation of Synaptosomes from Mammalian Brain by Subcellular Fractionation and Gradient Centrifuommunication. Among other techniques, the isolation of nerve terminals [or synaptosomes (Whittaker et al. Biochem J, 90(2):293–303, 1964)] has been fundamental to study the biochemistry and the physiology of the nervous system. This chapter describes the isolation and purification of intact synaptos

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Probing Endocytosis During the Cell Cycle with Minimal Experimental Perturbation, during infection. Endocytosis activity is known to vary during the cell cycle, in particular during mitosis. Importantly, different experimental conditions can lead to opposite results and conclusions, thereby emphasizing the need for a careful design of protocols. For example, experiments using se

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Assaying the Contribution of Membrane Tension to Clathrin-Mediated Endocytosis, Since the implementation of this methodology to the field of clathrin-mediated endocytosis (CME), this approach has revolutionized our molecular understanding of clathrin-driven cellular uptake. Conventional live cell microscopy approaches are used to determine the precise functions of specific pro

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Identifying Small-Molecule Inhibitors of the Clathrin Terminal Domain,ncluding receptors for nutrients and signaling molecules, as well as synaptic vesicle reformation. Multiple genetic and chemical approaches have been developed to interfere with this process. However, many of these tools do not selectively block CME, for example by targeting components shared with c

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Reconstitution of Clathrin Coat Disassembly for Fluorescence Microscopy and Single-Molecule Analysi recruitment of auxilin and Hsc70, an ATP-driven molecular clamp. Here, we describe the preparation of reagents and the single-particle fluorescence microscopy imaging assay in which we visualize directly the Hsc70-driven uncoating of synthetic clathrin coats or clathrin-coated vesicles.

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Spatial and Temporal Aspects of Phosphoinositides in Endocytosis Studied in the Isolated Plasma Memembrane phospholipids have key regulatory functions in endocytosis and membrane traffic. I have previously described an in vitro assay based on the isolated, substrate-attached plasma membrane to study the spatial distribution and levels of phosphoinositides, in particular phosphatidylinositol-4,5-b