Continuous K-fiber connection to the spindle pole is not required for chromosome biorientation. (A–A″) Behavior of a laser-severed K-fiber during metaphase in a PtK2 cell. (A) Overview of the cell. (A′) Selected frames from time-lapse recording. Laser pulses aimed ∼1 µm in front of kinetochore (X) break the fiber into two fragments. The fragment attached to the spindle pole rapidly depolymerizes. In contrast, the stub attached to the kinetochore (yellow lines) changes orientation, appears to contact neighboring MTs, and subsequently moves slightly toward the spindle pole. (A″) Kymogram generated from the recording used for A′. Notice a sudden decrease of the distance between sister kinetochores followed by a rapid restoration of centromere stretching. Also notice a gradual increase in the length of K-fiber stub during the course of the experiment. K1 and K2, estimated positions of sister kinetochores; Cut, position of laser cut and minus end of the K-fiber stub. (B–D) Laser-severed K-fibers form contacts with adjacent MT bundles. (B) Overview of the cell before operation. (B′) Selected frames from time-lapse recording (similar to A). The cell was fixed soon after the stub initiated poleward movement. (B″) Selected focal plane from deconvolved 3D GFP fluorescence volume recorded after fixation. Notice numerous spindle MTs that are not detected in the live-cell recording. (C) Two consecutive 150-nm EM sections containing the kinetochore with severed K-fiber. Notice that the stub (orange dashed lines) crosses another MT bundle (blue dashed lines). (D) Tracing of MTs in three consecutive EM sections superimposed on the corresponding fluorescence planes. Yellow lines mark positions of the kinetochore and arrows point to the minus end of the stub. Time stamp in seconds from the time of laser irradiation.
