In vivo Multimotor Force–Velocity Curves by Tracking and Sizing Sub-Diffraction Limited Vesicles
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Determining in vivo force–velocity relationships of motor proteins is a critical step toward clarifying how they accomplish intracellular transport. We show that in vivo force–velocity curves corresponding to an estimated 1, 2, and 3 motors-per-vesicle can be constructed by tracking and sizing transported vesicles. The force range for these curves would normally be constrained by diffraction limited diameter measurements. However, we present a new method that uses the image intensity obtained with differential interference contrast microscopy as a proxy for vesicle diameters smaller than the diffraction limit. We calibrate this novel sizing method in vitro with polystyrene microsphere standards and apply it in vivo to vesicles. The resulting diameter vs. velocity data for large, small, and sub-diffraction limited vesicles is used to construct force–velocity curves that extend the force range of our previous curves. These extended 1-, 2-, and 3-motor in vivo curves qualitatively agree with a simple model of load sharing for motors that jointly transport a single vesicle.
KeywordsProcessive molecular motors Cooperative fast vesicle transport Anterograde retrograde traffic Intracellular motion DIC microscopy Kinesin Dyenin Myosin Particle tracking
We thank Keith Bonin for helpful discussions for input at various stages. This work was supported by a start-up grant by Wake Forest University to JCM, an NIH grant (NS-053493) to GMH and by an NIH grant (AG-020996) to DAD.
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