460 The Journal of Cell Biology | Volume 156, Number 3, 2002 Figure 9. Spc42p assembly is compro- mised in the mps1-1 mutant background. Asynchronously growing MPS1 GAL-SPC42-myc (ACY122-1c) and mps1-1 GAL-SPC42-myc (ACY123-10a) strains were arrested in G1 using -factor and then released at both 25 and 30 C into galactose-containing media to induce overexpression of SPC42-myc (under control of GAL1,10 promoter). Samples were collected for indirect immunofluorescence 3 h after release into inducing media (A, C, and E); DNA is stained with DAPI (blue), and antibodies were used to detect -tubulin (red) and the myc epitope of Spc42-myc (green). A similar experiment was performed to generate samples for EM (B, D, and F). The only modification was that the strains were released from -factor into inducing media at 25 and 34 C. Samples were high pressure frozen and processed for EM (as described in Materials and methods). Formation of the organized “super plaque” composed of Spc42-myc protein is observed in mps1-1 (two cells are shown) and MPS1 (unpublished data) cells grown at 25 C (A) and in MPS1 cells grown at 30 C (two cells shown) (C). (E) An apparently smaller less organized Spc42-myc protein structure forms in mps1-1 cells grown at the mps1-1 restrictive (30 C; four cells shown) temperature. (F) Serial EM sections (i, ii, iii) reveal that this Spc42-myc structure forms at the half-bridge of the existing SPB and lacks the symmetry reported previously for the “super plaque” (Donaldson and Kilmartin, 1996). The “super plaque” observed in mps1-1 cells grown at the mps1-1 permissive (25 C) temperature and in MPS1 cells grown at 34 C (B and D) have a morphology similar to that reported previously for the “super plaque” (Donaldson and Kilmartin, 1996). Bars: (A, C, and D) 1.0 m. when we treated extract from the MSP1 strain with calf alka- line phosphatase before two-dimensional analysis, the same five spots of Spc42p-myc were absent (Fig. 8 F). The phos- phatase-treated sample is missing an additional form of Spc42p-myc detected in the untreated MPS1 extract and shows accumulation of two new forms of Spc42p-myc (Fig. 8 F). Loss of this additional form of Spc42p in the phos- phatase-treated sample suggests that another kinase may contribute to Spc42p phosphorylation. The new forms of Spc42p migrate faster and in a more basic position than forms present in the untreated MPS1 sample, suggesting that they are less phosphorylated or unphosphorylated forms of Spc42p (Fig. 8 F, arrow). Interestingly, we do not detect these new forms of Spc42p-myc in the mps1-1 strain. Failure of the phosphatase treatment to produce a single form of Spc42p-myc could be due to incomplete dephosphorylation of Spc42p-myc or to additional posttranslational modifica- tion on Spc42p. Phosphorylation of Spc42p by Mps1p in vitro and dependence on Mps1p in vivo suggests that Mps1p may regulate Spc42p during SPB duplication. Misassembly of Spc42p in MPS1 mutant cells Overexpression of Spc42p-myc from an inducible promoter results in the lateral expansion of the central layer of the SPB so that organized layers of Spc42p extend from the SPB in all directions and appear to rest on the nuclear envelope (Donaldson and Kilmartin, 1996; Bullitt et al., 1997; O’Toole et al., 1999). This “super plaque” structure appears by EM to be organized into a two-dimensional crystalline lattice identical to what is observed for Spc42p in the central plaque of a normal SPB (Bullitt et al., 1997). Spc42p iso- lated from both of these structures is phosphorylated; how- ever, it is not yet clear what role phosphorylation of Spc42p might play in their assembly (Donaldson and Kilmartin, 1996). Here, we analyze formation of the “super plaque” upon SPC42 overexpression in MPS1 mutants. The assem-