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Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. I. Discharge of vesicular contents across the planar membrane

Rockefeller University Press
Journal of General Physiology (JGP)
Authors:
Joshua Zimmerberg at National Institutes of Health
Joshua Zimmerberg
F S Cohen
F S Cohen
F S Cohen
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A Finkelstein
A Finkelstein
A Finkelstein
  • This person is not on ResearchGate, or hasn't claimed this research yet.
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Abstract

Multilamellar phospholipid vesicles are introduced into the cis compartment on one side of a planar phospholipid bilayer membrane. The vesicles contain a water-soluble fluorescent dye trapped in the aqueous phases between the lamellae. If a vesicle containing n lamellae fuses with a planar membrane, an n-1 lamellar vesicle should be discharged into the opposite trans compartment, where it would appear as a discernible fluorescent particle. Thus, fusion events can be assayed by counting the number of fluorescent particles appearing in the trans compartment. In the absence of divalent cation, fusion does not occur, even after vesicles have been in the cis compartment for 40 min. When CaCl2 is introduced into the cis compartment to a concentration of greater than or equal to 20 mM, fusion occurs within the next 20 min; it generally ceases thereafter because of vesicle aggregation in the cis compartment. With approximately 3 x 10(8) vesicles/cm3 in the cis compartment, about 25-50 fusion events occur following CaCl2 addition. The discharge of vesicular contents across the planar membrane is the most convincing evidence of vesicle-membrane fusion and serves as a model for that ubiquitous biological phenomenon--exocytosis.
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... The very first studies providing convincing evidence that artificial vesicles could fuse with an artificial planar membrane appeared in back-to-back articles in JGP in 1980 by Alan Finkelstein, Fred Cohen, and Josh Zimmerberg (Cohen et al., 1980;Zimmerberg et al., 1980a). There are remarkable aspects of these studies that merit a more detailed retelling. ...
... The vesicles themselves were multilammelar and housed a fluorescent soluble dye within all lipid compartments. Based on this design, the criterion used to score fusion was the appearance of fluorescein marker on the side of the planar membrane opposite (trans) to the side on which vesicles were added (Zimmerberg et al., 1980a;Fig. 2 A). The use of the soluble marker distinguished this study from others assaying membrane continuity, which were confounded by the possible nonfusion exchange of markers including diffusion of membrane intercalating dyes from one compartment to the other. ...
... High background signal, membrane breakage, and malfunctions of a fluorescence-activated cell sorter all conspired to make investigations cumbersome and inconvenient. Indeed, the discussion offers the trenchant observation that the "small number [of successful experiments] reflects not a lack of diligence and zeal on the part of the authors, but rather their finite capacity for heroics" (Zimmerberg et al., 1980a). Thus, in a companion article, the authors describe how an additional membrane reporter could be used to complement the dye-based reporter described above, or to be used in its stead (Cohen et al., 1980). ...
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Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. II. Incorporation of a vesicular membrane marker into the planar membrane
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Article
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