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Brr6 drives the Schizosaccharomyces pombe spindle pole body nuclear envelope insertion/extrusion cycle

The Journal of Cell Biology
The Rockefeller University Press
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Brr6 promotes spindle pole body insertion and extrusion • Tamm et al. T H E J O U R N A L O F C E L L B IO L O G Y S1 JCBSupplemental material Tamm et al., JCB S2 Figure S1.  Imaging wild-type specimens highlights the defects arising from compromised Brr6 function. (A and B) brr6+ cells were processed for anti- tubulin immunofluorescence (A) and high-pressure freezing fixation based electron microscopy (B) to provide a reference for the defects revealed by these approaches in other parts of the manuscript. (C) pcp1.RFP atb2.GFP brr6+ and pcp1.RFP atb2.GFP brr6.ts8 cells after the nitrogen starvation re-feed protocol to preferentially label the old SPB with actively fluorescent RFP. In the brr6+ image in panel C i, it is clear that the lower of the two SPBs that reside at either end of the green mitotic spindle is fluorescing, which confirms that this protocol results in fluorescence from the old SPB only. In the three examples of brr6.ts8 cells shown in C (ii–iv), it is clear that the green tubulin signal emerges from the single red dot, which indicates that it is the old, rather than the new, SPB that nucleates microtubules (MT) in brr6.ts8 cells at 36°C. NE, nuclear envelope. Bars: (A) 5 µm; (B) 200 nm; (C) 2 µm. Brr6 promotes spindle pole body insertion and extrusion • Tamm et al. S3 Figure S2.  Loss of Brr6 blocks spindle formation while the brr6.ts8 mutation is a recessive mutation. (A) The tetrad progeny of the diploid brr6+/brr6. was analyzed on YE5S plates. Only two spores from each tetrad gave rise to wild-type colonies. The other two spores arrested as single elongated cells. (B) Dilution spot tests of brr6+ leu1.32 pREP81brr6+ and brr6. leu1.32 pREP81brr6+ strains on medium with (right) or without (left) thiamine to repress the transcription of the nmt81 promoter. (C) Thiamine was added to a brr6. leu1.32 pREP81brr6+ culture that had been grown to a density of 5 × 105 in mi

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