Chromosomes lacking direct connection to the pole are not lost during anaphase. (A–B) Behavior of a laser-severed K-fiber during anaphase in a PtK2 cell. (A) Overview of the cell. (A′) Selected frames from time-lapse recording. Notice the change in orientation of the stub and its subsequent poleward movement (A′). (A″) Kymogram demonstrates that the K-fiber stub remains motionless for ∼15 s and then moves poleward in a series of rapid but brief movements (marked by blue arrows). Notice that the length of the stub remains constant throughout the experiment. (B) Longer-term time-lapse recording of the cell in A. Notice that the chromosome with severed K-fiber (arrow) ultimately incorporates into the forming daughter nucleus. K, estimated positions of the kinetochore; Cut, position of laser cut and minus end of the K-fiber stub. (C and D) Distinct mechanisms drive segregation of kinetochores with direct versus indirect attachments to the pole. (C) Creation of a bleached fiduciary mark on a continuous K-fiber with a 488-nm continuous wave laser beam. (C′) Combined bleach/sever approach to create a K-fiber stub with fiduciary mark. Notice that cells in C and C′ are at a similar stage of anaphase. (D) Movement of kinetochores with continuous K-fibers to the fiduciary mark indicates depolymerization of MT plus ends at the kinetochore (i.e., Pacman). (D′) Depolymerization of MT plus ends is not observed in laser-severed K-fibers. Instead the entire stub is transported poleward. Red lines denote positions of kinetochores and blue lines show bleached segments of MTs. Time in seconds.
