Flickering fusion pores comparable with initial exocytotic pores occur in protein-free phospholipid bilayers

Abstract

For the act of membrane fusion, there are two competing, mutually exclusive molecular models that differ in the structure of the initial pore, the pathway for ionic continuity between formerly separated volumes. Because biological ''fusion pores'' can be as small as ionic channels or gap junctions, one model posits a proteinaceous initial fusion pore. Because biological fusion pore conductance varies widely, another model proposes a lipidic initial pore. We have found pore opening and f lickering during the fusion of protein-free phospholipid vesicles with planar phospholipid bilayers. Fusion pore formation appears to follow the coalescence of contacting monolayers to create a zone of hemifusion where continuity between the two adherent membranes is lipidic, but not aqueous. Hypotonic stress, causing tension in the vesicle membrane, promotes complete fusion. Pores closed soon after opening (f lickering), and the distribution of fusion pore conductance appears similar to the distribution of initial fusion pores in biological fusion. Because small f lickering pores can form in the absence of protein, the existence of small pores in biological fusion cannot be an argument in support of models based on proteinaceous pores. Rather, these results support the model of a lipidic fusion pore developing within a hemifused contact site.

Cholesterol facilitates the native mechanism of Ca2+-triggered membrane fusion

“…Although perhaps most consistent with the stalk-pore hypothesis of fusion pore formation (Kozlov and Markin, 1983;Chernomordik and Zimmerberg, 1995;Chernomordik et al, 1995a;Chernomordik et al, 1995b;Chanturiya et al, 1997;Kozlovsky et al, 2002), alternate local effects of cholesterol including the direct facilitation of protein-based pores can not be excluded. Nonetheless, these studies clearly differentiate the dual role of cholesterol in the process of Ca 2+ -triggered membrane fusion: as a prefusion organizer contributing to the efficiency of fusion (e.g.…”

“…The simplest interpretation is that cholesterol contributes negative curvature to the membrane, promoting the formation of transient lipidic fusion intermediates (Kozlov and Markin, 1983;Chernomordik and Zimmerberg, 1995;Chernomordik et al, 1995a;Chernomordik et al, 1995b;Chanturiya et al, 1997;Kozlovsky et al, 2002). Of increasing complexity, in a proteinaceous fusion pore model, cholesterol could contribute to the mixing and coalescence of apposed membranes via expansion of a proteinaceous fusion pore (Lindau and Almers, 1995;Peters and Mayer, 1998;Bayer et al, 2003).…”

A Suppressor of fab1 Challenges Hypotheses on the Role of Thylakoid Unsaturation in Photosynthetic Function

“…Thus, one possibility is that changes in the shape of MGD molecules compensate for disruptive changes in the shape of PG molecules induced by the fab1 mutation, for example by altering the packing relationships between the thylakoid lipids and membrane proteins of the photosynthetic complexes (Gounaris and Barber, 1983;Simidjiev et al, 2000). Changes in the shape of lipid molecules is known to be important for a number of membrane functions including protein trafficking and membrane fusion (Rietveld et al, 1995;Chanturiya et al, 1997;Bruce, 1998). Certainly, the decreased unsaturation of lipids in fab1 fad5-2 plants relative to fab1 means that it is reasonable to consider molecular shape rather than membrane fluidity or phase changes as the basis of the suppressor phenotype.…”

Single-particle kinetics of influenza virus membrane fusion

“…The hemifusion stalk (in which only the proximal leaflets of the two bilayers have merged) may proceed directly toward nascent fusion pores or toward a longer-lived hemifusion diaphragm (11). Formation of a committed, expanding pore from a hemifusion structure appears to be an additional, kinetically disfavored step (12).…”