Extending the Range of In Vivo Multimotor Force-Velocity Curves by Sizing Vesicles Below the Diffraction Limit

Abstract

In our previous work (Shtridelman et al. 2008, Cell Bioch. & Biophys), we presented three force-velocity curves corresponding to 1, 2, and 3 motors/vesicle. We constructed these curves via velocities obtained directly from vesicles (NT2 cells, 37°C) and via forces obtained indirectly from Stokes' Law using measured intracellular viscosity, and vesicle diameters and velocities as inputs. The range of these earlier curves was restricted by the diffraction limit. In our current work, we use the image intensity--obtained with a differential interference contrast (DIC) microscope--as a proxy for vesicle diameters smaller than the diffraction limit. This novel sizing method is surprisingly robust, allowing us to extend the range of our in vivo multimotor force velocity curves. As with our previous curves, our newly extended 1-motor in vivo curve is similar to in vitro single kinesin force-velocity curves obtained at 35°C and, qualitatively, to single dynein curves obtained at 25°C. However, the 2- and 3- motor curves have proportionally higher extrapolated stall forces and overall velocities. The figure accompanying this abstract shows the primary size-velocity data we used to generate our extended in vivo multimotor force-velocity curves.View Large Image | View Hi-Res Image | Download PowerPoint Slide