The spatial-temporal orchestration of Start in budding yeast

Cells must balance growth against division in order to achieve the appropriate size and ensure maximal fitness in changing environments. Deregulation of size control leads to loss of fitness in lower eukaryotes such as budding yeast, and diseases such as cancer, diabetes or cardiac hypertrophy in humans. Cell size in the single celled eukaryote, budding yeast, is determined at the transition from the G1 phase to the S phase of the cell cycle, called Start, driven by the expression of ~200 genes via molecular networks largely conserved from yeast to humans.

The fundamental molecular mechanisms underlying the spatial and temporal control of large transcriptional programs such as Start, have yet to be discovered. The experimental and modeling approaches in this project will conjointly reveal the mechanisms underlying the orchestration of the Start transition in time and space. Beyond this fundamental result, the PI's approach, once validated in yeast, will have wider applications for understanding the coordination of transcriptional programs responsible for cell state transitions such as differentiation or trans-formation in higher eukaryotes.

The project includes multiple levels of training and outreach, including high school, undergraduate, and graduate training. The PI participates in the Undergraduate Research Program at Rensselaer and teaches an undergraduate course in Methods in Biophysics. For several of these students, this in-class undergraduate experience is linked with undergraduate research in the PIs lab where they carry out advanced microscopy data acquisition and analysis.

Graduates trained in the context of the proposed work will learn both the experimental and modeling aspects of the cellular biophysics. The PI has been instrumental in initiating a pilot program for STEM summer internships for high school students in the Capitol District. This program, structured around graduate student mentoring of high school students, aims to minimize disparities in STEM learning, providing a positive and engaging STEM experience for students of all levels, with the goal of enhancing abilities, and leapfrogging, rather than tracking.

All of these activities are aimed at promoting equity in STEM education and research. In addition, as Director of the Biochemistry and Biophysics graduate program, the PI works to increase the diversity of the participating graduate students

Yeast cell size is an emergent property of the spatial-temporal activation of the G1/S regulon. To maximize fitness under different environmental conditions and in different genetic backgrounds, yeast adjust their size. How this size adjustment is made remains to be determined and is the goal of this project.

Start is regulated primarily by the SBF and MBF transcriptional factors (TFs), while a positive phosphorylation feedback loop renders the Start transition sharp and irreversible. The PI has shown using fluctuation microscopy, that in newborn cells, the absolute G1/S TF copy numbers are severely limiting with respect to their target G1/S promoters. They accumulate throughout G1 phase, titrating the promoters, thus defining G1/S TF copy number as a growth-dependent determinant of the timing of Start.

The PI also showed that the small cell size phenotype observed when cells are grown in poor nutrients likely arises from positive differential scaling of G1/S TF accumulation with respect to the growth rate under these conditions, providing the first plausible explanation for nutrient-dependent size adjustments. Moreover, using super-resolution PALM microscopy, it was discovered that the G1/S TFs form a few clusters in the nuclei of newborn cells that increase in number but not in size as cells accumulate the G1/S TFs and grow in G1 phase.

These results raise central questions concerning the mechanisms underlying the dynamic organization of the G1/S regulon and the timing and synchrony of Start. Firstly, are the G1/S promoters clustered? If so, is there a specificity or time-dependence to their clustering patterns.

Do the G1/S TFs randomly distribute into clusters or is there an order to the titration that impacts Start synchrony? The PI will approach these questions using super-resolution fluorescence imaging of the promoters of the G1/S regulon and their TFs. The PI will construct a quantitative, predictive mathematical model of size control in budding yeast, iteratively refined by our experimental results.

Knowledge of the mechanisms underlying Start in a model organism will provide testable hypotheses for how cell size may be controlled in human cells, with implications for therapeutic strategies against diseases such as cancer. Moreover, this approach and the resulting comprehensive description of the control of Start will establish a framework for understanding other important cell-state transitions such as cellular differentiation and transformation.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.