UNDERSTANDING CELL SECRETION AND MEMBRANE FUSION PROCESSES ON THE NANOSCALE USING THE ATOMIC FORCE MICROSCOPE - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 105

The immunoisolated porosome complex has also been both structurally and
functionally reconstituted into liposomes and lipid bilayer membranes. 9,11-13
Transmission electron micrographs of pancreatic porosomes reconstituted
into liposomes exhibit a 150-200 nm cup-shaped basket-like morphology,
similar to what is observed in its native state when co-isolated with ZGs. To
test the functionality of the isolated porosome complex, puriied porosomes
obtained from exocrine pancreas or neurons were subjected to reconstitution
in lipid membrane of the electrophysiological setup (EPC9) and challenged
with isolated ZGs or synaptic vesicles. Electrical activity of the reconstituted
membrane as well as the transport of vesicular contents from the

cis to the

trans compartments of the bilayer chambers was monitored. Results from

these experiments demonstrate that the lipid membrane-reconstituted
porosomes are indeed functional, 9,11 since in the presence of calcium,
isolated secretory vesicles dock and fuse to transfer intravesicular contents
from the

compartment of the bilayer chamber. ZGs fused
with the porosome-reconstituted bilayer as demonstrated by an increase
in capacitance and conductance and a time-dependent transport of the ZG
enzyme amylase from

cis to the trans

compartment of the bilayer chamber.
Amylase is detected using immunoblot analysis of the buffer in the

cis to the trans

cis and

trans chambers. As observed in immunoblot assays of isolated porosomes,

chloride channel activity is present in the reconstituted porosome complex. 11
Furthermore, the chloride channel inhibitor DIDS was found to inhibit
current activity through the porosome-reconstituted bilayer, demonstrating
a requirement of the porosome-associated chloride channel activity in
porosome function. Similarly, the structure and biochemical composition
of the neuronal porosome, and the docking and fusion of synaptic vesicles
at the neuronal porosome complex, have also been elucidated. In summary,
these studies demonstrate. Porosomes to be permanent supramolecular
lipoprotein structures at the cell plasma membrane, where membrane-bound
secretory vesicles transiently dock and fuse to release intravesicular contents
to the outside. Porosomes have therefore been designated as universal
secretory machinery in cells. 29,30

5.3 AFM: ELUCIDATING SNAREINDUCED MEMBRANE
FUSION IN CELLS

As outlined in the preceding section, in live cells, membrane fusion is
mediated via a specialized set of proteins present in opposing bilayers. 15-27
Target membrane proteins, SNAP-25 and syntaxin (t-SNAREs) and secretory
vesicle-associated protein (v-SNARE), are part of the conserved protein