SINGLE KINESIN MOLECULES CROSSBRIDGE MICROTUBULES IN-VITRO

Kinesin is a cytoplasmic motor protein that moves along microtubules a nd can induce microtubule bundling and sliding in vitro. To determine how kinesin mediates microtubule interactions, we determined the shape s and mass distributions of squid brain kinesin, taxol-stabilized micr otubules (squid and bovine), and adenosine 5'-[beta,gamma-imido]tripho sphate-stabilized kinesin-microtubule complexes by high-resolution met al replication and by low-temperature, low-dose dark-field scanning tr ansmission electron microscopy of unfixed, directly frozen preparation s. Mass mapping by electron microscopy revealed kinesins loosely attac hed to the carbon support as asymmetrical dumbbell-shaped molecules, 4 0-52 nm long, with a mass of 379 +/- 15 kDa. The mass distribution and shape of these molecules suggest that these images represent kinesin in a shortened conformation. Kinesin-microtubule complexes were organi zed as bundles of linearly arrayed microtubules, stitched together at irregular intervals by crossbridges typically less-than-or-equal-to 25 nm long. The crossbridges had a mass of 360 +/- 15 kDa, consistent wi th one kinesin per crossbridge. These results suggest that kinesin has a second microtubule binding site in addition to the known site on th e motor domain of the heavy chain; this second site may be located nea r the C terminus of the heavy chains or on the associated light chains . Thus, kinesin could play a role in either crosslinking or sliding mi crotubules.