One SPB fails to insert in cut12.1 mutant cells at 36°C. (A–D) cut12.1 cells were grown to mid-log phase in minimal medium at 25°C and shifted to 36°C after synchronization by centrifugal elutriation. 100 min after the shift, when the condensed chromosome index was 30%, cells were fixed by high pressure freezing and processed for transmission EM. The panels show different magnifications of the same section of a mutant mitotic nucleus. The enlargements in C and D correspond to the regions delimited by the dotted boxes in A. Although the cell is clearly in mitosis because the right SPB (SPB2) is nucleating microtubules (MT, arrows), the left SPB (SPB1) has not inserted into the nuclear envelope (NE) and retains the region of differentiated nuclear envelope that is typical of interphase SPBs (indicated by the bracket in C), which presents an electron-dense material. Only the pole that is inserted is nucleating mitotic microtubules. It is noteworthy that the connection between the mitochondria and the SPB reported in previous EM analyses of mitotic cells is maintained in the defective mitoses of cut12.1 cells (McCully and Robinow, 1971; Kanbe et al., 1989). Bars, 500 nm.