Clathrin-coated vesicle preparation

The earliest electrophoretic techniques concentrated mainly on the preparation of mitochondria " and lysosomes, but further research has made possible the purification of secretory vesicles clathrin-coated vesicles " endoplasmic reticulum early, middle-, and late-stage endosomes peroxisomes " microsomes " and phagosomes." These preparatory techniques have functioned as the proof-of-principle for analytical separations of intact organelles using CE, by demonstrating that isolated organelles are amenable to eleetrophoretic separation techniques. 

This conclusion is based on immunogold localization of PrP in these organelles by electron microscopy inhibition of PrP internalization by incubation of cells in hypertonic sucrose, which disrupts clathrin lattices and detection of PrP in purified preparations of coated vesicles from brain. The N-terminal half of the PrP polypeptide chain is essential for efficient clathrin-mediated endocytosis, because... 

Fig. 7. Reversible trapping of synaptic vesicle membrane in the plasma membrane in reticulospinal synapses. (A) Electron micrograph of a lamprey reticulospinal synapse stimulated with action potentials at 20 Hz for 20 min and then incubated for 90 min in Ca " -free solution with 10 mM EGTA. Note the reduction in the number of synaptic vesicles and the presence of large membrane expansions compared to an unstimulated synapse (inset). (B) Activation of clathrin-mediated endocytosis in reticulospinal synapses by addition of Ca +-containing extracellular solution. Spinal cord preparations were stimulated at 20 Hz for 20 min, incubated for 90 min in Ca -free solution, and then incubated in Ca -containing solution (2.6 mM) for 120 s. Electron micrograph of a synapse shows the appearance of coated pits (arrows) lateral to the active zone. Designations as in Fig. 1. Scale bar, 0.2 p.m. Modified from Gad et al. (1998) Neuron 21 601-616, with permission copyright is held by Cell Press.

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