Tuesday, January 28, 2014

Controlled Particle Collision Leads to Direct Observation of Docking and Fusion of Lipid Droplets in an Optical Trap

Chiran Ghimire , Deepak Koirala , Malcom B. Mathis , Edgar Eduard Kooijman , and Hanbin Mao

As an intracellular organelle, phospholipid coated lipid droplets have shown increasing importance due to their expanding biological functions other than the lipid storage. The growing biological significance necessitates a close scrutiny on lipid droplets, which have been proposed to mature in a cell through processes such as fusions. Unlike phospholipid vesicles that are well known to fuse through docking and hemifusion steps, little is known on the fusion of lipid droplets. Herein, we used laser tweezers to capture two micrometer-sized 1,2,3-trioleoylglycerol (triolein) droplets coated with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) that closely resemble intracellular lipid droplets. We started the fusion processes by a well-controlled collision between the two lipid droplets in phosphate buffers at pH 7.4. By monitoring the change in the pathway of a trapping laser that captures the collided lipid droplets, docking and fusion events were clearly distinguished for the first time and their lifetimes were determined with a resolution of 10 microseconds after post-synchronization analyses. Our method revealed that the rate limiting docking process is affected by anions according to a Hofmeister series, which sheds light on the important role of interfacial water shedding during the process. During the actual fusion, the kinetics between bare triolein droplets is faster than lipid droplets, suggesting that breaking of phospholipid coating is involved in the process. This scenario was further supported by direct observation of a short-lived hemifusion state with ~ 46 milliseconds lifetime in POPC coated lipid droplets, but not in bare triolein droplets.

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