Regulation of Membrane Fusion

Membrane fusion by SNAREs is thermodynamically favored, and is therefore constitutively “on.”

Regulatory factors are needed, especially in tightly controlled processes such as neurotransmission and hormone release, to keep fusion off when it is not needed and to activate it when it is.

We mainly utilize ‘flipped SNAREs’ expressed on the surface of transfected cells – triggering cell-cell fusion – as a platform to establish the mechanism of action of regulatory proteins.

SNARE-Mediated Fusion at the Single Event Level: the "flipped" SNARE Cell-Fusion Assay

Cells expressing Flipped SNAREs (SNAREs ectopically expressed to face outside the cell), could fuse when grown next to cells with the cognate Flipped SNARE.

We demonstrated that cells expressing Flipped SNAREs (SNAREs ectopically expressed to face outside the cell), could fuse when grown next to cells with the cognate Flipped SNARE (above). Thus, SNAREs are sufficient even when the fusing membranes are relatively flat and chemically complex.

This assay also provided the first opportunity to visualize individual SNARE-mediated fusion events and to record them in real time.

Membrane Fusion Intermediates

Stalk model of membrane fusion

The identification of intermediate structures composed of both lipid and protein which may have physiologic lifetimes on the millisecond time scale is a daunting challenge.  Our best understanding of how membranes merge comes from a large body of work on viral membrane fusion processes and is summed up in the stalk model of membrane fusion (roughly sketched on the right).

We are attempting to isolate intermediates on this pathway by manipulating the protein, lipid, and experimental conditions to trap membrane fusion at a particular step. 

For example, we have recently shown, with our flipped-SNARE cell-fusion assay (below), that when the transmembrane domain of a SNARE is replaced with a GPI anchor, membrane fusion is halted at a hemifusion stage, in which only the outer leaflets of the interacting cellular membranes mix. 

Confocal microscopy of flipped t-SNARE expressing cells (yellow with red PM) hemifused to flipped v-SNARE cell (red PM only, due to lipid transfer from t-cell)

Whether we have isolated an on-pathway intermediate, or forced the membrane fusion reaction down a non-productive alternate pathway, is a question we continue to explore.