Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Thursday Speaker Abstracts

HOW AND HOW MUCH HAS IT FUSED? DETECTING FUSION INTERMEDIATES AND QUANTIFYING FUSION EFFICIENCY Rafael B. Lira 1 ; Tom Robinson 1 ; Karin A Riske 2 ; Rumiana Dimova 1 ; 1 Max Planck Institute of Colloids and Interfaces, Theory, Potsdam, Brandenburg, Germany 2 Federal University of Sao Paulo, Biophysics, Sao Paulo, Brazil Membrane fusion is a ubiquitous process in the cell. It transits through a number of fusion intermediates and involves the merging of two separated membranes, forming a compartment whose area is the sum of the two fusing bilayers; increase in area inevitably accompanies fusion. Due to the high complexity, membrane fusion is commonly studied with spectroscopic techniques and reconstituted bulk liposomal systems. They bear the disadvantage that intermediates are not explicitly detected, heterogeneity is hidden, efficiency is indirectly probed and increase in area is not measurable. We studied the fusion of cationic large unilamellar vesicles (LUVs) with giant unilamellar vesicles (GUVs) with increasing negative charge. We developed a method to detect fusion intermediates, quantify the fusion efficiency and the amount of membrane area gained via fusion on a single vesicle as assessed by observation of GUV morphology, intensity and lifetime FRET, content mixing and GUV electrodeformation. At low charge, LUVs dock and hemifuse to GUVs, increasing membrane tension that causes GUV rupture. At intermediate to high charge, fusion is very fast and efficient and the transferred lipids lead to GUV area gain, assessed from vesicle deformation in AC fields. The increase in area scales with GUV charge density. Initial LUV curvature leads to the formation of outward buds/tubules on the GUVs due to increased spontaneous curvature. Both hemifusion and full fusion are leakage-free. From FRET, we were able to retrieve the GUV final composition upon fusion. All of the above parameters can be measured in real-time on the single-vesicle level (Biophys. J. 116:79, 2019). The possibility to detect fusion intermediates, quantify fusion efficiency, retrieve membrane’s final composition and to manipulate membranes on a single- vesicle level opens up many possibilities not accessible with classical fusion systems.

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