Novel optical trapping methodology reveals cooperative function of kinesin motors on cellular organelles

Abstract

Optical trap force measurements give a precise readout of how motor proteins function against a calibrated load. To understand how motors work inside cells, optical trapping must be extended to motors transporting real cellular organelles. However, the size of organelles in a cell is variable and unknown. To date, it has not been possible to precisely calculate the force exerted by an optical trap on single cellular organelles. We have reconstituted robust kinesin-driven in vitro motility of lipid droplets in a cell extract of mammalian liver. A new method of optical trapping is then used to calibrate the optical trap precisely for every single lipid droplet, irrespective of its size. We have measured and analyzed the cooperative function of multiple endogenously assembled kinesins against a calibrated load-force with precision and spatio-temporal resolution comparable to kinesin-coated beads. Our method of force measurement makes accessible for experimental scrutiny a whole new range of physiologically important questions: How much force do motors exert on cellular cargoes? How are motors assembled on the cargo membrane? What is the emergent function of such motor-assemblies? Do multiple motors compete or cooperate? How do motor-associated regulatory proteins fine tune this process? How does emergent function of kinesins differ from dyneins? Our work will answer some of these questions. Finally, our method of force measurement provides for the first time an ability to biophysically interrogate how motors may regulate storage and lipolysis of cytosolic fat in mammals. This may further our understanding of diseases related to (mis)management of fat.