Dr. Bhanu Jena
Discovery of the Molecular Mechinery & Mechanism of Cell Secretion

Secretion is a fundamental cellular process responsible for numerous physiological functions in living organisms, such as neurotransmission and the secretion of hormones and digestive enzymes. Hence, secretory defects in cells are responsible for a host of debilitating diseases. Therefore, this field has been the subject of intense investigation for over half a century. My studies almost 30 years ago which began as an inquiry in to how cells secrete, has provided only in the last decade an understanding of the process at the molecular level. Our studies provide for the first time an understanding of the universal molecular machinery involved, and the mechanism of cell secretion.

Where at the cell plasma membrane do secretory vesicles dock and fuse to release their contents?

A major breakthrough in our understanding of cell secretion came with the discovery of a supramolecular structure at the cell plasma membrane, the 'porosome', and its determination as the universal secretory machinery. Secretory vesicles dock and fuse at the porosome to release intravesicular contents to the outside. We determined the structure and dynamics of the porosome in live cells, at nm resolution and in real time, using atomic force microscopy. In both exocrine pancreas and in neuroendocrine cells, porosomes measured 100-150 nm in diameter, however at the nerve terminal they were an order of magnitude smaller, measuring 8-12 nm in diameter. The porosome structure was further confirmed by electron microscopy in whole cells, and in isolated porosome preparations. Subsequently, the isolated porosome was both structurally and functionally reconstituted in lipid membrane, and its composition determined. The discovery of the porosome as a new cellular structure, and an understanding of its structure, function, composition, and reconstitution, opens a new chapter in cell biology; nano cell biology.

How do the membrane-encapsulated secretory vesicles establish continuity with the porosome membrane?

Two sets of proteins, one set present at the base of porosomes and composed of two proteins called t-SNAREs, and the other protein at the secretory vesicle membrane called v-SNARE, have been implicated in the establishment of continuity between secretory vesicle membrane and the cell plasma membrane.

Our studies demonstrate that in the presence of calcium, t- and v-SNAREs in opposing membranes interact in a circular array to form conducting channels, thus establishing continuity between the vesicle interior and the porosome opening to outside of the cell.

How are intravesicular contents expelled from the cell during secretion?

Our studies demonstrate that the release of secretory products to outside of the cell via the SNARE channel and the porosome opening, occurs as a result of swelling of secretory vesicles. The extent of secretory vesicle swelling is directly proportional to the amount of intravesicular contents expelled.

Hence, the cell is able to precisely regulate the amount of vesicle content secreted. Further, the molecular mechanism of secretory vesicle swelling has been determined from our studies.

These findings provide a fundamental understanding of the molecular machinery and the mechanism of cell secretion. These new discoveries have brought about a clear understanding of cell secretion. Old questions such as, why do partially empty vesicles appear following cell secretion, or why is there no

loss in vesicle number following secretion, are answered. We now understand that membrane-bound secretory vesicles transiently dock at the porosome, establish continuity with the porosome membrane via SNAREs, and expel intravesicular contents through the porosome opening, following vesicle swelling which provides the energy for such expulsion.