|
|
|
|
|
|
|
||
The Journal of General Physiology, Vol 107, 329-351, Copyright © 1996 by The Rockefeller University Press
ARTICLES |
WD Niles, JR Silvius and FS Cohen
Department of Molecular Biophysics and Physiology, Rush Medical College, Chicago, Illinois 60612, USA. wniles@uic.edu
Membrane fusion of a phospholipid vesicle with a planar lipid bilayer is preceded by an initial prefusion stage in which a region of the vesicle membrane adheres to the planar membrane. A resonance energy transfer (RET) imaging microscope, with measured spectral transfer functions and a pair of radiometrically calibrated video cameras, was used to determine both the area of the contact region and the distances between the membranes within this zone. Large vesicles (5-20 microns diam) were labeled with the donor fluorophore coumarin- phosphatidylethanolamine (PE), while the planar membrane was labeled with the acceptor rhodamine-PE. The donor was excited with 390 nm light, and separate images of donor and acceptor emission were formed by the microscope. Distances between the membranes at each location in the image were determined from the RET rate constant (kt) computed from the acceptor:donor emission intensity ratio. In the absence of an osmotic gradient, the vesicles stably adhered to the planar membrane, and the dyes did not migrate between membranes. The region of contact was detected as an area of planar membrane, coincident with the vesicle image, over which rhodamine fluorescence was sensitized by RET. The total area of the contact region depended biphasically on the Ca2+ concentration, but the distance between the bilayers in this zone decreased with increasing [Ca2+]. The changes in area and separation were probably related to divalent cation effects on electrostatic screening and binding to charged membranes. At each [Ca2+], the intermembrane separation varied between 1 and 6 nm within each contact region, indicating membrane undulation prior to adhesion. Intermembrane separation distances < or = 2 nm were localized to discrete sites that formed in an ordered arrangement throughout the contact region. The area of the contact region occupied by these punctate attachment sites was increased at high [Ca2+]. Membrane fusion may be initiated at these sites of closest membrane apposition.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  R. Parthasarathy and J. T. Groves From the Cover: Protein patterns at lipid bilayer junctions PNAS, August 31, 2004; 101(35): 12798 - 12803. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. P. Wong and J. T. Groves Molecular topography imaging by intermembrane fluorescence resonance energy transfer PNAS, October 29, 2002; 99(22): 14147 - 14152. [Abstract] [Full Text] [PDF]
|
|
|
|
