Interzone microtubule behavior in late anaphase and telophase spindles

We have studied microtubule behavior in late anaphase and telophase spindles of PtK1 cells, using fluoresceinated tubulin (DTAF-tubulin), microinjection, and laser microbeam photobleaching. We present the results of two novel tests which add to the evidence that DTAF-tubulin closely mimics the behavior of native tubulin in vivo. (a) Microinjected DTAF-tubulin was as effective as injected native tubulin in promoting division of taxol-dependent mitotic mutant cells that had been deprived of taxol. (b) Microinjected colchicine-DTAF-tubulin complex was similar to injected colchicine-native tubulin complex in causing depolymerization of spindles. Immediately after microinjection of DTAF-tubulin into wild-type cells during late anaphase or telophase, fluorescence incorporation by microtubules was seen in chromosomal half-spindles and just behind the chromosomes, but there was no fluorescence incorporation near the middle of the interzone. Over the next few minutes, tubulin fluorescence accumulated at the center of the interzone (the equator), becoming progressively more intense. In other experiments, cells were microinjected with DTAF-tubulin at prophase and allowed to equilibrate for 30 min. Cells that had progressed to late anaphase were then photobleached to reduce the fluorescence in the central portion of the interzone. Over a period of several minutes, the only substantial redistribution of fluorescence was the appearance of a bright area at the equator of the interzone. Both the site of fluorescence incorporation and the photobleaching data suggest that tubulin adds to the elongating spindle interzone near the equator where the plus ends of the interdigitated microtubules are located. In further experiments, several dark lines were photobleached perpendicular to the pole-to-pole axis of fluorescent anaphase-telophase spindles. Time-dependent changes in the spacings between the lines indicated that the two halves of the interzone lying on opposite sides of the spindle equator moved away from one another. This shows that the interdigitated microtubules, which make up most of the interzone, can undergo antiparallel sliding. Our data support a model for anaphase B in which plus end elongation of interdigitated microtubules and antiparallel sliding contribute to chromosome separation.