Secretory vesicles in live cells are not free-floating but tethered to filamentous structures: a study using photonic force microscopy.

It is well established that actin and microtubule cytoskeletal systems are involved in organelle transport and membrane trafficking in cells. This is also true for the transport of secretory vesicles in neuroendocrine cells and neurons. It was however unclear whether secretory vesicles remain free-floating, only to associate with such cytoskeletal systems when needing transport. This hypothesis was tested using live pancreatic acinar cells in physiological buffer solutions, using the photonic force microscope (PFM). When membrane-bound secretory vesicles (0.2-1.2 microm in diameter) in live pancreatic acinar cells were trapped at the laser focus of the PFM and pulled, they were all found tethered to filamentous structures. Mild exposure of cells to nocodazole and cytochalasin B, disrupts the tether. Immunoblot analysis of isolated secretory vesicles, further demonstrated the association of actin, myosin V, and kinesin. These studies demonstrate for the first time that secretory vesicles in live pancreatic acinar cells are tethered and not free-floating, suggesting that following vesicle biogenesis, they are placed on their own railroad track, ready to be transported to their final destination within the cell when required. This makes sense, since precision and regulation are the hallmarks of all cellular process, and therefore would hold true for the transport and localization of subcellular organelles such as secretory vesicles.

[1]  M. Mooseker,et al.  Vesicle-associated brain myosin-V can be activated to catalyze actin-based transport. , 1998, Journal of cell science.

[2]  G. Rutter,et al.  Myosin Va Transports Dense Core Secretory Vesicles in Pancreatic MIN6 β-Cells , 2005 .

[3]  B. Jena,et al.  Protein tyrosine phosphatase stimulates Ca(2+)-dependent amylase secretion from pancreatic acini. , 1991, The Journal of biological chemistry.

[4]  B. Jena,et al.  Gi regulation of secretory vesicle swelling examined by atomic force microscopy. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[5]  J. Lippincott-Schwartz,et al.  Kinetic Analysis of Secretory Protein Traffic and Characterization of Golgi to Plasma Membrane Transport Intermediates in Living Cells , 1998, The Journal of cell biology.

[6]  D. Ogden,et al.  Interaction of the actin cytoskeleton with microtubules regulates secretory organelle movement near the plasma membrane in human endothelial cells , 2003, Journal of Cell Science.

[7]  H. Gerdes,et al.  Dynamics of immature secretory granules: role of cytoskeletal elements during transport, cortical restriction, and F-actin-dependent tethering. , 2001, Molecular biology of the cell.

[8]  H. Gerdes,et al.  Myosin Va facilitates the distribution of secretory granules in the F-actin rich cortex of PC12 cells , 2003, Journal of Cell Science.

[9]  Ernst H. K. Stelzer,et al.  Local viscosity probed by photonic force microscopy , 1998 .

[10]  B. Jena,et al.  Vesicle swelling regulates content expulsion during secretion , 2004, Cell biology international.

[11]  Dieter G. Weiss,et al.  Actin-dependent organelle movement in squid axoplasm , 1992, Nature.

[12]  M. Sheetz,et al.  Functions of microtubule-based motors. , 1991, Annual review of physiology.

[13]  E. Stelzer,et al.  Three‐dimensional high‐resolution particle tracking for optical tweezers by forward scattered light , 1999, Microscopy research and technique.

[14]  P. Forscher,et al.  Brain myosin-V is a two-headed unconventional myosin with motor activity , 1993, Cell.

[15]  Samara L. Reck-Peterson,et al.  Class V myosins. , 2000, Biochimica et biophysica acta.

[16]  B. Jena,et al.  Structure, isolation, composition and reconstitution of the neuronal fusion pore , 2004, Cell biology international.