Abstract
Rapid vesicle fusion with the plasma membrane for signaling or cargo release allows large and sudden signaling shifts by a cell. In readiness for repeating heavy requirements in signaling, bulk endocytosis fulfils this role, and is an area of study to understand how vesicle proteins are concentrated and the bulk endosomes partitioned into new vesicles. In this work, we examine the recovery of vesicles to maintain a ready supply for bovine chromaffin cells. Four general routes exist for vesicle recovery: Clathrin Mediated Endocytosis (CME), Clathrin Independent Endocytosis (CIE), Bulk Endocytosis (BE), and Kiss & Run. The prioritization of these mechanisms varies depending on the level of stimulation. This work examined the recycling of the plasma membrane into vesicles after stimulation within the BE pathway, as visualized by electron microscopy (EM). Negative stain EM allows a complete and detailed cross-sectional view of a cell, readily allowing before and after stimulation analysis for tallying a population of events. In this case, the distribution of recovered membrane for recycling into vesicles. A common problem with single layer sections can be determining when a bulk endocytotic event has completed fission from the plasma membrane. In this case, it is alleviated by using serial sections to determine the overall nature of bulk endocytotic structures as a cellular process for the recovery of vesicles.
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