The nucleolus as a polarized coaxial cable in which the rDNA axis is surrounded by dynamic subunit-specific phases

Cell cycle arrest of S. cerevisiae in conjunction with labeling of the cell wall

“…The red bulk chromatin - which transits between mother and bud ( Palmer et al., 1989 ) - can be found on one or the other side of the bud neck or stretching across the bud neck. The nucleolus (labeled by Gar1-GFP) remains in the mother domain, as we have discussed elsewhere ( Rai et al., 2017 , Tartakoff et al., 2021 ). The same approach can be used to monitor the distribution of single proteins or of other organelles.…”

“… a.Transfer samples of microcultures with a multi-prong replicator device into a set of 50 μL microcultures with complete synthetic medium (i.e., including methionine) ( Amberg et al., 2005 ). Shake the cultures for 3–5 h and then observe them at 40× in an inverted brightfield microscope to see whether a preponderance of the cells have large buds whose size approximates that of the mother cell ( Tartakoff et al., 2021 ). Retain and expand clones if >50% of cells have large buds.…”

“…Shake the cultures for 3–5 h and then observe them at 40× in an inverted brightfield microscope to see whether a preponderance of the cells have large buds whose size approximates that of the mother cell ( Tartakoff et al., 2021 ).…”

“…We describe (1) how to achieve this arrest and verify it, (2) how to label cell surface glycans covalently to distinguish mother from bud, and (3) how to detect the nucleolus and learn that it remains in the mother domain upon arrest. For complete details on the use and execution of this protocol, please refer to Tartakoff et al. (2021) , Rai et al.…”

“…For complete details on the use and execution of this protocol, please refer to Tartakoff et al. (2021) , Rai et al.…”