Deciphering start dynamics in budding yeast using scanning number and brightness

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Goswami, Pooja
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Electronic thesis
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Cell size homeostasis reflects the coordination between cell growth and division and is vital to all living organisms. This coordination is achieved primarily at the G1/S phase transition, termed Start in budding yeast. The major G1/S factors and their epistatic relationships are known. Cells pass Start upon activation of kinase Cdc28 by the G1 cyclin, Cln3, which phosphorylates the repressor Whi5, thereby activating expression of the downstream G1 cyclins CLN1/2 and ~200 other genes in the G1/S regulon by the SBF/MBF transcription factor complexes. It has been reported that dilution of Whi5 with cell growth controls the timing of Start while Swi4 concentration remains constant. Our previous results showed that the concentration of Whi5 remains constant with increasing size in G1 cells, while Swi4 is upregulated. In Chapter 4 of this thesis, single-cell imaging was used to quantify Whi5-GFP intensity as a function of time, rather than size, under experimental conditions that largely eliminate confounding effects of photo-bleaching. The results showed no significant time-dependent change in Whi5-GFP intensity in G1 cells. Measurements in a heterozygous WHI5 deletion diploid strain further validated the conclusion that Whi5 dosage is not a critical determinant of Start. In 2018, our group demonstrated that SBF/MBF subunit copy numbers, in particular Swi4, are sub-saturating with respect to their target promoters in small cells, but increase to a near 1:1 ratio as cells reach the Start threshold. That work also revealed that these factors, and consequently Cln1/2 levels, are upregulated in poor nutrients, conditions under which Start occurs at a smaller cell size. Chapter 5 of this thesis presents results describing a novel feedback loop for the expression of Swi4, the DNA-binding component of the dominant SBF transcription complex. It is likely that this additional regulatory mechanism contributes to the fine tuning of the Start transition. Finally, although it is known that the process of ribosome biogenesis is involved in regulating the cell size threshold, and moreover, is highly sensitive to external nutrient and stress conditions, the relationship between this pathway and the Start network remains to be determined. Preliminary results in Chapter 6 suggest the existence of a G1/S bypass mechanism that may come into play under conditions of decreased ribosome production. Future research will seek to further investigate this potential link between these growth and Start networks.
December 2021
School of Science
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Rensselaer Polytechnic Institute, Troy, NY
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