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 108

t-SNAREs. Ideally, the atomic coordinates of membrane-associated SNARE
complex using X-ray crystallography would help elucidate the chemistry of
SNARE-induced membrane fusion in cells. So far, such structural details at
the atomic level of membrane-associated t-/v-SNARE complex have not been
realized. This has been primarily due to solubility problems of membrane-
associated SNAREs, compounded with the fact that v-SNARE and t-SNAREs
need to reside in opposing membranes when they meet, to assemble in a
physiologically relevant SNARE complex. The remaining option has been
the use of nuclear magnetic resonance (NMR) spectroscopy. However, NMR
spectroscopy too has been of little help, since the size of t-/v-SNARE ring
complexes are beyond the maximum limit for NMR spectroscopy studies.
Regardless of these setbacks, AFM force spectroscopy has provided for the
irst time at nanometre to sub-nanometre resolution an understanding of the
structure, assembly and disassembly of membrane-associated t-/v-SNARE
complexes in physiological buffer solution. 20-27 A bilayer electrophysiological
setup allowed measurements of membrane conductance and capacitance
during fusion of v-SNARE-reconstituted liposomes with t-SNARE-
reconstituted membrane, and vice versa ( Fig. 5.5a,b ) . Results from these
studies demonstrated that t-SNAREs and v-SNARE when present in opposing
membrane interact and assemble in a circular array, and in the presence of
calcium, they form conducting channels. 20 The interaction of t-/v-SNARE
proteins to form such a conducting channel is strictly dependent on the
presence of t-SNAREs and v-SNARE in opposing bilayers. Addition of puriied
recombinant v-SNARE to a t-SNARE-reconstituted lipid membrane results
in non-physiological interactions and without inluence on the electrical
properties of the membrane.

However, in the presence of calcium, when
v-SNARE vesicles are added to t-SNARE-reconstituted membrane or vice
versa, SNAREs assemble in a ring conformation. The resultant increase in
membrane capacitance and conductance demonstrates the establishment
of continuity between the opposing t-SNARE- and v-SNARE-reconstituted
bilayers. These results conirm that t-SNARE and v-SNAREs are required to
reside in opposing membranes, as they exist in the physiological state in cells,
to allow appropriate t-/v-SNARE interactions that lead to membrane fusion
in the presence of calcium. Studies using SNARE-reconstituted liposomes
and bilayers 21,22 further demonstrate the following: (i) a low fusion rate
(

20

= 16 minutes) is obtained between t-SNARE- and v-SNARE-reconstituted
liposomes in the absence of Ca 2+ and (ii) exposure of t-/v-SNARE liposomes
to Ca 2+ drives vesicle fusion on a near-physiological relevant time-scale
(

τ

10 seconds), demonstrating Ca 2+ and SNAREs in combination to be the
minimal fusion machinery in cells.

τ

^

Native and synthetic vesicles exhibit
a signiicant negative surface charge primarily owing to the polar phosphate
head groups, generating a repulsive force that prevents the aggregation and

21,22