Figure 8.

Effects of GST-KRP₁₈₀ dominant/negative protein microinjected into mitotic sea urchin (L. pictus) embryos. (A) Purified GST-KRP₁₈₀ stalk fusion proteins (Coil 1 and Coil 2) were used as dominant/negative proteins. The numbers below the linear map of the KRP₁₈₀ polypeptide correspond to the amino acids that comprise the fusion proteins. Molecular mass markers indicate the size of the glutathione–Sepharose-purified fusion proteins (lane Coil 1 and 2; Coomassie-stained 7.5% and lane GST; 12% SDS-PAGE). (B) Microinjection of GST-Coil 2 into mitotic embryos perturbs the maintenance of prometaphase spindle shape, whereas microinjection of GST-Coil 1 and control GST does not. Spindle pole-to-pole distances were measured over time for individual spindles in GST-, Coil 1–, and Coil 2–injected embryos. Time 0 represents NEBD and the final time point indicates the completion of cytokinesis. Anaphase B initiates ∼15 min post-NEBD in the three spindles shown. (C) Video frames of a representative GST-Coil 2 injected embryo are shown. The video frames (with time in minutes) show the effects of microinjection on first cleavage five minutes before NEBD (time −5.0) until near the completion of cytokinesis (time 34.0). This embryo was microinjected in interphase, shortly after fertilization. The center of each spindle pole is marked with a red dot to assist in spindle length measurements while the intracellular oil droplet (arrowhead) indicates a successful microinjection. This embryo divides asymmetrically due to mispositioning of the spindle after displaying a prometaphase spindle collapse.