Individual Vesicle Fusion Events Mediated by Lipid-anchored DNA

TY - JOUR

T1 - Individual Vesicle Fusion Events Mediated by Lipid-anchored DNA

AU - van Lengerich, Bettina

AU - Rawle, Robert J.

AU - Bendix, Pól Martin

AU - Boxer, Steven G.

PY - 2013/7/16

Y1 - 2013/7/16

N2 - Membrane fusion consists of a complex rearrangement of lipids and proteins that results in the merger of two lipid bilayers. We have developed a model system that employs synthetic DNA-lipid conjugates as a surrogate for the membrane proteins involved in the biological fusion reaction. We previously showed that complementary DNA-lipids, inserted into small unilamellar vesicles, can mediate membrane fusion in bulk. Here, we use a model membrane architecture developed in our lab to directly observe single-vesicle fusion events using fluorescence microscopy. In this system, a planar tethered membrane patch serves as the target membrane for incoming vesicles. This allows us to quantify the kinetics and characteristics of individual fusion events from the perspective of the lipids or the DNA-lipids involved in the process. We find that the fusion pathways are heterogeneous, with an arrested hemi-fusion state predominating, and we quantitate the outcome and rate of fusion events to construct a mechanistic model of DNA-mediated vesicle fusion. The waiting times between docking and fusion are distributed exponentially, suggesting that fusion occurs in a single step. Our analysis indicates that when two lipid bilayers are brought into close proximity, fusion occurs spontaneously, with little or no dependence on the number of DNA hybrids formed.

AB - Membrane fusion consists of a complex rearrangement of lipids and proteins that results in the merger of two lipid bilayers. We have developed a model system that employs synthetic DNA-lipid conjugates as a surrogate for the membrane proteins involved in the biological fusion reaction. We previously showed that complementary DNA-lipids, inserted into small unilamellar vesicles, can mediate membrane fusion in bulk. Here, we use a model membrane architecture developed in our lab to directly observe single-vesicle fusion events using fluorescence microscopy. In this system, a planar tethered membrane patch serves as the target membrane for incoming vesicles. This allows us to quantify the kinetics and characteristics of individual fusion events from the perspective of the lipids or the DNA-lipids involved in the process. We find that the fusion pathways are heterogeneous, with an arrested hemi-fusion state predominating, and we quantitate the outcome and rate of fusion events to construct a mechanistic model of DNA-mediated vesicle fusion. The waiting times between docking and fusion are distributed exponentially, suggesting that fusion occurs in a single step. Our analysis indicates that when two lipid bilayers are brought into close proximity, fusion occurs spontaneously, with little or no dependence on the number of DNA hybrids formed.

U2 - 10.1016/j.bpj.2013.05.056

DO - 10.1016/j.bpj.2013.05.056

M3 - Journal article

C2 - 23870262

SN - 0006-3495

VL - 105

SP - 409

EP - 419

JO - Biophysical Journal (2011)

JF - Biophysical Journal (2011)

IS - 2

ER -