KINESIN-LIKE MOLECULES INVOLVED IN SPINDLE FORMATION

To study the possible involvement of kinesin-like molecules in mitosis a polyclonal antibody against the head domain of Drosophila kinesin h eavy chain (HD antibody) was microinjected into PtK1 cells at the prop hase-prometaphase transition. Progress of the cell through mitosis was recorded for subsequent detailed analysis. Cells injected with pre-im mune IgG progressed through mitosis at rates similar to those for noni njected cells. After HD antibody injections, chromosomes failed to con gress to an equatorial plane and cells failed to form a bipolar spindl e. Rather, the spindle poles came together, resulting in a monopolar-l ike configuration with chromosomes arranged about the poles in a roset te. Sometimes the monopolar array moved to the margin of the cell in a way similar to anaphase B movement in normal cells. Antibody-injected cells progressed into the next cell cycle as evidenced by chromosome decondensation and nuclear envelope reformation. Anti-tubulin immunofl uorescence confirmed the presence of a radial monopolar array of micro tubules in injected cells. HD antibody stained in a punctate pattern i n interphase and the spindle region in mitotic PtK1 cells. The antibod y also reacted with spindle fibers of isolated mitotic CHO spindles an d with kinetochores of isolated CHO chromosomes. Immunoblotting indica ted that the major component recognized by the antibody is the 120 kDa kinesin heavy chain. At higher protein loads the antibody recognized also a 34 kDa polypeptide in PtK1 cell extracts, a 135 kDa polypeptide in a preparation of CHO spindles and a 300 kDa polypeptide in a prepa ration of CHO mitotic chromosomes. We conclude that a kinesinlike mole cule is important for the formation and/or maintenance of the structur e of mitotic spindle.