Fig. 3. Ssm4p and the dynein complex interact. (A) cyr1 sxa2 ssm4-HA and cyr1 sxa2 dhc1-myc fission yeast cells were grown in the absence (–P) and in the presence (+P) of pheromone for 6 hours. Boiled cell extracts were prepared as described and western blots were probed with anti-HA or anti-MYC antibodies. (B) cyr1 sxa2 dhc1-myc ssm4-HA cells were induced with pheromone for 6 hours. Native cell extracts were immunoprecipitated with anti-HA and anti-MYC antibodies. Western blots of immunoprecipitates (IPs), supernatants and total cell extracts were carried out as described and probed with HA and MYC antibodies (right panel). Control IPs with anti-HA of cyr1 sxa2 dhc1-myc did not precipitate any Dhc1p-myc (middle panel) and IPs with anti-MYC of cyr1 sxa2 ssm4-HA did not precipitate any Ssm4p-HA (left panel). (C) Two-hybrid interaction of Ssm4p with Dlc1p. ß-galactosidase activity was assayed in Saccharomyces cerevisiae cells expressing proteins as indicated. The combination of Ras and Raf was used as a positive control. (D) Localisation of Ssm4p-GFP in meiotic cells. Homothallic haploid cells that carried the ssm4-GFP fusion gene and the CFP-atb2 fusion gene encoding CFP-tubulin were subjected to nitrogen starvation to induce mating and subsequent meiosis in zygotes. They were monitored by fluorescence microscopy for localisation of Ssm4p and microtubules. Nuclear DNA was stained with Hoechst 33342. Merged images are shown in the right panels. Green, GFP; red, CFP; blue, DNA. The arrowheads and the asterisks indicate intense GFP fluorescence at the leading edge of the nucleus and at the microtubular tip contacting the cell cortex, respectively. Bar, 10 µm.