Figure 4

Chemical-genetic inhibition of myosin-Vb halts myosin-Vb-decorated particles, including those being transported via microtubules. (A) Representative image of two HeLa cells expressing sensitized Y119G mutant eGFP-myosin-Vb before injection of the upper cell with PE-ADP. Bar, 15 μm. (B and C) Kymographs from the cells shown in panel A (y axes represent lines wx and yz from panel A). (D and E) Kymographs from additional negative control cells expressing wild-type myosin-Vb injected with PE-ADP and wild-type myosin-Vb injected with dextran respectively. (F and G) Histograms of instantaneous speeds of myosin-Vb-labeled vesicles before (black bars) and after (white bars) PE-ADP injection in cells expressing wild-type and Y119G mutant eGFP-myosin-Vb respectively; Speeds were measured for 1178 (before injection) and 621 particles (after) for panel F, and 717 and 551 respectively for panel G. (H) Instantaneous speeds of wild-type eGFP-myosin-Vb-labeled vesicles before (black bars) and after (white bars) depolymerization of actin by latrunculin A; the Y119G mutant gave indistinguishable results (data not shown). Speeds were measured for 707 (before) and 206 (after) particles. (I and J) Diagrams depicting additions to the dynamic tethering hypothesis to accommodate these data. Kinesin is represented with the letter "k" for the head domain.