Nucleolar asymmetry and the importance of septin integrity upon cell cycle arrest

Abstract

Cell cycle arrest can be imposed by inactivating the anaphase promoting complex (APC). In S. cerevisiae this arrest has been reported to stabilize a metaphase-like intermediate in which the nuclear envelope spans the bud neck, while chromatin repeatedly translocates between the mother and bud domains. The present investigation was undertaken to learn how other features of nuclear organization are affected upon depletion of the APC activator, Cdc20. We observe that the spindle pole bodies and the spindle repeatedly translocate across the narrow orifice at the level of the neck. Nevertheless, we find that the nucleolus (organized around rDNA repeats on the long right arm of chromosome XII) remains in the mother domain, marking the polarity of the nucleus. Accordingly, chromosome XII is polarized: TelXIIR remains in the mother domain and its centromere is predominantly located in the bud domain. In order to learn why the nucleolus remains in the mother domain, we studied the impact of inhibiting rRNA synthesis in arrested cells. We observed that this fragments the nucleolus and that these fragments entered the bud domain. Taken together with earlier observations, the restriction of the nucleolus to the mother domain therefore can be attributed to its massive structure. We also observed that inactivation of septins allowed arrested cells to complete the cell cycle, that the alternative APC activator, Cdh1, was required for completion of the cell cycle and that induction of Cdh1 itself caused arrested cells to progress to the end of the cell cycle.

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

“…[22][23][24] Nucleolar markers remain in the mother domain in arrested cells (Figure 1D). 10 Moreover, especially when chromatin (Htb2-mRFP) is in the bud, rDNA and Hmo1 form an elongated filament in the mother domain (Figure 1E), whose length appears comparable to the length of rDNA in cycling cells. 25 Histones (Htb2-mRFP) can be detected along the gnarled Hmo1-GFPpositive filament and occasionally highlights the end of the filament, presumably near the right telomere of chromosome XII (Figures 1A and 1F).…”

“…Correspondingly, cotranscriptional events endow particles with surfaces that are densely covered with AFs. As post-transcriptional steps in subunit genesis entail 10 For all panels, B, bud; M, mother. (E) Projected image of arrested cells that express the histone, Htb2-mRFP, and Hmo1-GFP.…”

Protein-protein interaction analysis highlights the role of septins in membrane enclosed lumen and mRNA processing

“…Thus, in these nuclear relationships in the PPI networks of septins, we highlighted links between chromatin partners and septins, coupled with links to splicing molecules. Chromatin has already been described as being link to splicing regulation (de Almeida and Carmo-Fonseca, 2014) and the importance of septin integrity at cell cycle arrest has recently been connected to nucleolar asymmetry (Rai et al, 2017). In this network, we showed that through their partner relationships, septins could participate in the interplay between the spliceosome and chromatin in the nuclear part of cells.…”

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.…”

“…For complete details on the use and execution of this protocol, please refer to Tartakoff et al. (2021) , Rai et al. (2017) , and Zapanta Rinonos et al.…”