Figure 5.

Cells lacking Mia1p do not sustain normal PAA. (A) Mia1p-GFP localizes to the eMTOC at the division site in live cells. (B) Immunoelectron micrograph image of cell expressing Mia1p-GFP using 10-nm gold particles, where Mia1p-GFP is found on both sides of the division site (indicated by stars). Bar, 300 nm. (C) Time-lapse sequence of the dividing α-tubulin-GFP–expressing wild-type cell. Once established, the microtubule-organizing structures are maintained and eventually constrict together with the actomyosin ring. The medial microtubule foci are marked with stars in time point 1′30”. Tracking of these structures from their first appearance to ring constriction suggests that they are relatively persistent (as shown in C′). (D) In contrast, time-lapse sequence of the dividing α-tubulin-GFP–expressing mia1Δ cell suggests that although microtubules can be nucleated from the eMTOC, there are no persistent nucleation sites (compare D′ with C′). Different colors in C′ and D′ represent independent nucleation sites. Ejection event is marked by arrow in time point 1′30”. Shown are single maximum intensity reconstructions of z-stacks. Numbers refer to the time, in minutes and seconds. Bar, 1 μm. (E) Proteins associated with the γ-tubulin complex, Alp4p-GFP, Mto1p-GFP, and Rsp1p-GFP localize to the equatorial ring in the absence of Mia1p.