contributions: T.-Y.Y., B.O., and F.Z. contributed equally to this
work; Y.-K.S. and T.H. designed research; T.-Y.Y., B.O., and F.Z.
performed research; Y.-K.S. and T.H. contributed new reagents/analytic
tools; T.-Y.Y. analyzed data; and T.-Y.Y., Y.-K.S., and T.H. wrote the
maleimide sensitive-factor attachment protein receptors). The
vesicle-associated v-SNARE engages with its partner t-SNAREs on the
target membrane to form a coiled coil that bridges two membranes and
facilitates fusion. As demonstrated by recent findings on the
hemifusion state, identifying intermediates of membrane fusion can help
unveil the underlying fusion mechanism. Observation of SNARE-driven
fusion at the single-liposome level has the potential to dissect and
characterize fusion intermediates most directly. Here, we report on the
real-time observation of lipid-mixing dynamics in a single fusion event
between a pair of SNARE-reconstituted liposomes. The assay reveals
multiple intermediate states characterized by discrete values of FRET
between membrane-bound fluorophores. Hemifusion, flickering of fusion
pores, and kinetic transitions between intermediates, which would be
very difficult to detect in ensemble assays, are now identified. The
ability to monitor the time course of fusion events between two
proteoliposomes should be useful for addressing many important issues
in SNARE-mediated membrane fusion.
Because it can follow directly the time course of a single reaction
without the need for synchronization, the single-liposome approach has
the potential to clarify important issues that spatiotemporal averaging
in ensemble measurements cannot.
Soluble N-ethyl maleimide
sensitive-factor attachment protein receptor (SNARE) proteins are
involved in membrane fusion during exocytosis and vesicular trafficking
(9–11). Recent studies provided evidence that SNARE-mediated fusion transits through hemifusion (12–15) (however, see also ref. 16), similar to the fusion pathway proposed for type I (17, 18) and II viruses (19, 20) and lipidic membrane fusion (1, 21). Hemifusion is a metastable membrane structure in which the outer leaflets are merged but the inner leaflets remain intact (22).
It has been shown that the rate of inner leaflet mixing is slower than
that of outer leaflet mixing in a SNARE-reconstituted fusion reaction (15). The lipid mixing was also shown to occur earlier than aqueous content mixing in fusion between native vacuoles (14). These results are in favor of the mechanism through hemifusion (22).
However, an alternative mechanistic model in which hemifusion is
designated as an off-pathway state that can be reversed by liposomes
detachment can explain the results equally well (15).
This issue of hemifusion along with many other compelling questions
surrounding the topic of SNARE-induced membrane fusion may be clearly
addressed by observing a fusion event at the level of single liposomes.
have established a fluorescence-based single liposome fusion assay that
enables us to monitor lipid mixing between two proteoliposomes in real
time. Previous approaches have been focused on fusion of single
liposomes with larger-scale membranes such as supported (1–4) or plasma membranes (5).
The fluorophores contained in single liposomes then inevitably diffuse
away subsequent to fusion. In contrast, the fusing objects in our
assay, two proteoliposomes, constitute a small closed system. The
number of fluorophores therefore is preserved throughout the fusion
process such that each fusion intermediate with a certain degree of
lipid mixing corresponds to a discrete FRET value. The dwell time in
each intermediate can also be precisely determined. As a result, the
complete lipid-mixing dynamics of SNARE-mediated fusion, that is, from
docking to full fusion, has been monitored and dissected in detail.