What is Roadblock gliding assays stepping assays kinesin Road block microtubule When road blocks are in front of motor protein Motro proteins moment wait in front of the roadblock detach from the microtubule Perform a sidestep and bypass the roadblock. micorotubles moment dependency of transport reduction on roadblock concentration of streptavidin and nanoparticles.

explore the interdependence of regulative properties of roadblocks Roadblocks in this reseach explore the interdependence of regulative properties of roadblocks Geometrical property => density or binding position Nanomechanical propety => size of roadblocks Electrochemical properties => netcharge of roadblocks

MT decorated with tau proteins Gray symbols indicate gliding velocities before tau addition the line symbols are drawn to guide the eye. Error bars are SD Decrease of microtubule gliding velocities with increasing tau density. a charge dependency for the roadblock effect of tau was not evident (by higher ionic strength situation)

MT with (strept)avidin

Roadblock( Mw vs Net charge ) To estimate the relative contributions of these properties , they performed a linear regression analysis. The roadblock density and molecular weight correlate notably with the gliding velocity ((Pearson’s r ∼ −0.9 (p < 0.001) for biotin ratio and r ∼ −0.2 (p < 0.06) for molecular weight)

Roadblock avidin + DNA The attachment of DNA the roadblock 53 kDato +69 kDa (ssDNA) + 83 kDa(dsDNA). double stranded DNA to microtubules decorated with st-avidins did not further decrease microtubule gliding velocities

relative sizes of the kinesin heads with roadblocks (strept)avidins at pH 6.9: (a) rhizavidin, (b) bradavidin core I, (c) streptavidin, and (d) avidin. Negative charges of amino acid residues are colored in red, neutral residues in white, and positively charged residues in blue. Kinesin is depicted in light green. The green arrow indicates kinesin’s moving direction. Lysines (to which biotin is coupled) on the microtubule surface are colored in yellow

Mechabisim of road block (head 1, ADP state) and the other head is tightly attached to the microtubule (head 2, ATP state). (b) While kinesin’s leading head (head 1) undergoes a biased diffusional search for its next binding site on the microtubule, roadblock properties like size, charge or flexibility might influence how kinesin by passes the roadblock. (c) Kinesin might overcome the roadblock by various mechanisms: By performing a side step, by pushing the roadblock partially aside, By detaching and rebinding behind the roadblock if pulled across by synergetically working other kinesins.

Mechabisim of road block If only a few kinesins push the microtubule forward ,microtubules detach from kinsin surfaces This indicates that kinesin probably stops in front of a roadblock and thendetaches.

Mechanism of road block Roadblock of　negatively charged (strept)avidins →roadblock density and roadblock size　reduce the velocity of gliding moments roadblock size by the addition of flexible molecules like DNA →not further decrease the gliding velocities. the design of nanodevices based on motor protein transport → cargo linkers and cargo molecules are best　tailored for specific applications. Smaller and negatively charged molecules like rhizavidin or bradavidin might be appropriate to link cargo to microtubules