Kinesin-1 contains two identical heavy chains and therefore has two identical motor domains. In contrast, kinesin-5 contains four identical heavy chains. Electron microscopic analysis of metal-shadowed kinesins results in the images shown in the top panel. Pretreatment of these kinesins with an antibody that binds specifically to the kinesin motor domain results in the images shown in the lower panel. All four images are at the same approximate magnification. What can you deduce about the structure of kinesin-5 from these data?

b. To determine if kinesin-5 is a (+) or (-) end microtubule motor, polarity-marked microtubules are generated by assembling short microtubules from brightly fluorescent tubulin and then elongating those short, bright microtubules using less fluorescent tubulin. As a result, the microtubules are very fluorescent at one end and less fluorescent along most of their length. A perfusion chamber is then coated with purified kinesin-5, which becomes immobilized on the glass surface. The chamber is then perfused with the polarity-labeled microrubules and ATP, and microtubule gliding with respect to the immobilized kinesin-5 is observed. The following sequence of images is obtained. Which end of these microtubules, the bright or the less-bright end, is the (+) end? Do these microrubules glide on kinesin-5 with their (+) or (-) end leading? Based on these data, is kinesin-5 a (+) or (-) end microtubule motor?

c. Kinesin-5 can cross-bridge adjacent microtubules. Polarity-marked microtubules are assembled in which rubulin attached to a red fluorescent dye is assembled to form short red microrubules, which are then elongated with tubulin attached to a green fluorescent dye. The microtubules are mixed with kinesin-5 and observed by fluorescence microscopy as A TP is added. The following images show a time sequence of two overlapping and cross-bridged microtubules as A TP is added. The arrowhead is in a fixed position. Can you explain what happens when A TP is added to microtubules cross-bridged by kinesin-5?

d. Eg5 is a kinesin-5 family member in Xenopus. To understand Eg5 function in vivo, cells are transfected with RNAi directed against this motor. The following images are obtained of mitotic cells. What function might Eg5 play in cells?

Transcribed Image Text:

Kinesin-1 Kinesin-5 No antibody Decorated with kinesin motor domain antibody 26