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Membrane fusion in eukaryotic cells is thought to be mediated by a …


Biology Articles » Biophysics » Multiple intermediates in SNARE-induced membrane fusion

Abstract
- Multiple intermediates in SNARE-induced membrane fusion

Multiple intermediates in SNARE-induced membrane fusion
Tae-Young Yoon,* Burak Okumus, Fan Zhang, Yeon-Kyun Shin,§ and Taekjip Ha*§
*Howard Hughes Medical Institute,
Center for Biophysics and Computational Biology, and
Department of Physics, University of Illinois at Urbana–Champaign, Urbana, IL 61801; and
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011
§To whom correspondence may be addressed.
Edited by Thomas C. Südhof, University of Texas Southwestern Medical Center, Dallas, TX, and approved October 31, 2006
Author 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 paper.
 
Proc Natl Acad Sci U S A. 2006 December 26; 103(52): 19731–19736. Open Access Article.
 

Abstract

 
Membrane fusion in eukaryotic cells is thought to be mediated by a highly conserved family of proteins called SNAREs (soluble N-ethyl 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.
 
Keywords: FRET, single-molecule spectroscopy, lipid mixing
 
 
 
Single-liposome fluorescence imaging (15) is a powerful method for observing and dissecting the fusion dynamics of biological membranes (68). 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 (911). Recent studies provided evidence that SNARE-mediated fusion transits through hemifusion (1215) (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.

We 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 (14) 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.

 
 

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