Abstract
The fundamental biophysics of gliding microtubule (MT) motility by surface-tethered kinesin-1 motor proteins has been widely studied, as well as applied to capture and transport analytes in bioanalytical microdevices. In these systems, phenomena such as molecular wear and fracture into shorter MTs have been reported due the mechanical forces applied on the MT during transport. In the present work, we show that MTs can be split longitudinally into protofilament bundles (PFBs) by the work performed by surface-bound kinesin motors. We examine the properties of these PFBs using several techniques (e.g., fluorescence microscopy, SEM, AFM), and show that the PFBs continue to be mobile on the surface and display very high curvature compared to MT. Further, higher surface density of kinesin motors and shorter kinesin-surface tethers promote PFB formation, whereas modifying MT with GMPCPP or higher paclitaxel concentrations did not affect PFB formation.
Highlights
The fundamental biophysics of gliding microtubule (MT) motility by surface-tethered kinesin-1 motor proteins has been widely studied, as well as applied to capture and transport analytes in bioanalytical microdevices
We describe a novel mechanism of MT damage in which MTs are sheared longitudinally as they are transported by kinesin motors in a gliding motility assay, leaving fragments consisting of curved protofilament bundles (PFB) reminiscent of the ram’s horn conformation
Using a GFP-kinesin-1 fusion protein adsorbed to a coverglass via an anti-GFP antibody, we observed splitting of MTs longitudinally into two fragments, which we propose are PFBs as shown in Fig. 1B
Summary
The fundamental biophysics of gliding microtubule (MT) motility by surface-tethered kinesin-1 motor proteins has been widely studied, as well as applied to capture and transport analytes in bioanalytical microdevices. In these systems, phenomena such as molecular wear and fracture into shorter MTs have been reported due the mechanical forces applied on the MT during transport. We describe a novel mechanism of MT damage in which MTs are sheared longitudinally as they are transported by kinesin motors in a gliding motility assay, leaving fragments consisting of curved protofilament bundles (PFB) reminiscent of the ram’s horn conformation. We further describe the dependence of this phenomenon on the surface density of the motor protein and the length and flexibility of the linker connecting the motor protein to the surface
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