A Novel Pathway that Coordinates Mitotic Exit with Spindle Position -- Nelson and Cooper 18 (9): 3440 Data Supplement - Supplemental Materials -- Molecular Biology of the Cell

A Novel Pathway that Coordinates Mitotic Exit with Spindle Position
Mol. Biol. Cell Nelson and Cooper 18: 3440

Supplemental Materials

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Movie 1 - Premature spindle breakdown in an msl1Δ arp1Δ mutant. A representative cell with a late-anaphase spindle in the mother exits mitosis with a cytoplasmic microtubule through the mother-bud neck. Cytokinesis cleaves the cytoplasmic microtubule after the spindle breaks down. Frames were collected 10 s apart, and they play at a rate of 8 per s. Pseudocolor is based on the "Fire" lookup table in ImageJ. Each image is 8.7 µm wide.
Figure S1 - snRNP components have no role in the SPC. Cells lacking the U2 snRNP component Lea1 or the U5 snRNP component Mud1 have no significant checkpoint phenotype (p = 0.42 and 0.24, respectively). n≥27.
Figure S2 - Timing of mitosis, from early anaphase to mitotic exit. Cells with short spindles were identified and followed by movie analysis until spindle breakdown. Cells where the spindle entered the neck and then broke down are grey, and cells where the spindle remained in the mother until spindle breakdown are in black. For all the mutants, the time of mitosis is 20 to 30 min regardless of whether the spindle enters the neck or remains in the mother. In the otherwise wt strain, mitosis in the mother is prolonged. The strain background was arp1 with Tub1-GFP. No mutant shows is no significant difference in the timing of mitotic exit in any mutants except cla4, which is delayed by an average of 5 minutes (p=0.04). The values are the mean number of cells, and the error bar is one standard error.
Figure S3 - Timing of the delay in mitotic exit in the mother cell. In this analysis, cells in late anaphase, with long spindles in the mother, were identified and followed by time-lapse movies, as was done for Figures 2 and 4. Time until spindle breakdown was measured and is plotted here as histograms. The strain background was arp1 with Tub1-GFP. For otherwise wild-type cells, the upper left panel shows data for cells in which the spindle remains in the mother. Here, the spindle breaks down after a variable delay. The upper right panel shows that when the spindle enters the neck, mitotic exit occurs at a normal time. For the mutants, in the lower panels, only cells with spindles in the mother were followed. In the mutants, the spindles in most cells broke down after 20-30 min, and some cells showed substantial delay.
Figure S4 - Localization of Atc1-GFP and MSL1-GFP. Atc1-GFP is localized throughout the cell. Msl1-GFP is diffuse in the cytoplasm, with partial concentration in the nucleus and exclusion from the vacuole. For comparison, cells not expressing GFP, middle right, are barely visible at the same settings; their locations are revealed by the transmitted light image from the spinning-disk confocal, lower right. Plain GFP under control of the GAL1-promoter, middle left, shows bright fluorescence in the cytoplasm and nucleus, with exclusion from the vacuole.
Figure S5 - Microtubule-cortex interactions in kip2 and bik1 mutants. Microtubules in kip2 and bik1 mutants contact the cell cortex at positions away from Bud6-GFP foci with the same frequency as they do in wild type cells, despite being shorter.
Figure S6 - Specificity of checkpoint defect in bud6 mutants. A) DNA damage checkpoint assay. 10-fold serial dilutions of liquid cultures on a plate containing hydroxyurea. Wild type and bud6Δ, but not rad50Δ, rad51Δ, or rad52Δ cells grew in hydroxyurea-containing media. B) Morphogenesis checkpoint. Cells were arrested in G0 with alpha factor, and then released into Latrunculin A. Samples were collected at the indicated time points, fixed, and stained with DAPI. The percentages of unbudded cells that underwent nuclear division are shown. Neither wild type nor bud6Δ mutant cells underwent nuclear division in unbudded cells, unlike the checkpoint-defective swe1Δ mutant.
Figure S7 - Bud site selection, formins, and actin cables have no role in the SPC. Cells with the indicated mutations were assayed as in Figure 2A. A) Mutants lacking Bud1 or Bud5 have normal SPC integrity. B) Mutants defective for one or both formins (Bni1 and Bnr1), or structural components of actin cables (Fimbrin / Sac6 or Tropomyosin / Tpm1) have normal SPC integrity.
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