Myo1C and Endophilin Promote Kinesin-Driven Tubulation at Engineered Cytoskeletal Intersections

Abstract

In cells, kinesin-driven membrane tubulation along microtubules (MT) typically initiates in regions with a locally high actin filament (AF) density, suggesting that membrane deformations may result from the cooperative activities of kinesin and myosin motors at cytoskeletal intersections. While kinesin-1 motors are likely the primary drivers of tubule extension, membrane-binding myosin-I motors have also been proposed to participate in tubulation and membrane deformation in vivo. We hypothesize that Myo1c anchors membrane vesicles at AF/MT intersections, providing a resistive force that enables effective kinesin-1-driven tubulation. We tested this proposal by attaching kinesin-1 and Myo1c motors to giant unilamellar vesicles (GUVs); a truncated, His-tagged kinesin-1 construct (K560) was bound to NTA(Ni)-functionalized lipids and full length Myo1c was bound to PtdIns(4,5)P2 incorporated into the membrane. GUVs were introduced into chambers with engineered cytoskeletal arrays of sparse MTs bridging stripes densely-coated with short AFs. In the absence of Myo1c, kinesin-driven vesicles paused periodically at AF/MT intersections, but vesicle deformation was not observed. In contrast, GUVs with bound kinesin-1 and Myo1c motors paused at AF/MT intersections and deformed readily along MTs. However, extended tubules of diffraction-limited diameter were rarely observed. Next, we tested whether the addition of a BAR domain protein would facilitate vesicle tubulation by adding the membrane-binding domain of endophilin-A1, which is necessary for many endocytic trafficking tubulation events in vivo. We noted a significant, concentration-dependent, increase in the frequency and length of GUV tubulation events in the presence of endophilin. Importantly, this increased tubulation frequency required both kinesin-1 and Myo1c. These data provide insights into the interplay between cytoskeletal motors and BAR domain proteins in facilitating membrane tubulation. This work was supported by NIH grants GM087253 to E.M.O. and E.L.F.H. and T32-AR053461 to B.B.M.