Cell cycle control is highly conserved through the eukaryotic kingdom, and the budding yeast model system has been the source of major insights applicable to issues in human development and disease. This proposal continues systems-level analysis of the eukaryotic cell cycle using this model system. The cell cycle 'clock'functions with high reliability and low noise, even though individual components and circuits making up the clock are frequently known to be highly variable. For example, gene expression is known to be highly variable between individual cells, and yet cell-cycle-regulated gene expression can be highly reliable with respect to timing and amplitude. Threshold responses to rising cyclin-Cdk activity levels can provide switch-like behavior, but such switches can frequently come at the cost of highly variable onset time; the overall cell cycle control circuitry avoids this variability. We are pursuing an emerging concept of multiple independent oscillators contributing to cell cycle control;while uncoupled oscillators result in highly variable and irregular sequences of cell cycle events, we propose that coupling ('phase-locking') of otherwise independent oscillators to the central cyclin-Cdk oscillator can yield a robust and accurate overall system. This proposal continues our innovative use of quantitative time-lapse fluorescence microscopy, over multi-cell cycle timescales, combined with semi-automated image analysis and in-depth genetic and quantitative analysis to drive systems-level understanding of cell cycle control. We are developing new methods of mathematical modeling. There is a pressing need in biology for simple but experimentally constrained models that can reveal basic control principles. The challenge is to find the most illuminating balance between the detail required for a connection to biological reality, and model simplicity required for transparency and insight. We are exploring methods to use geometrical, low-dimensionality representations of the cell cycle control network that can still be experimentally constrained, and that will yield testable predictions. In a new direction to provide evolutionary contrast from a critical but underexplored branch of the eukaryotic kingdom, we will carry out a genetic screen aiming at saturated detection of cell cycle control elements in the green alga, Chlamydomonas reinhardtii. We have devised robotic methods for the microbiology of mutant isolation, which combined with deep sequencing, allows a massive speedup of this project compared to traditional means.

Public Health Relevance

Cell division is a central process in growth and development of all organisms, which must be tightly regulated to provide new cells only at the right time and place. While we understand a lot about individual sub-mechanisms of cell cycle control, we do not understand how these potentially noise-ridden components work together to yield reliable control. We study this question in the experimentally tractable budding yeast. For broader evolutionary perspective, we have also initiated a search for cell cycle control elements in green algae. Green algae diverged from animal and yeast lineages long before they diverged from each other, so learning about all three provides triangulation on basic eukaryotic principles;at the same time, paradoxically, green algae have greater conservation of some critical animal cell cycle control proteins than do yeast. Thus green algae provide the sole system for microbial genetic exploration of functions of the proteins, such as the retinoblastoma protein and cyclin A.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Cellular Signaling and Regulatory Systems Study Section (CSRS)
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Hamlet, Michelle R
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Rockefeller University
Other Domestic Higher Education
New York
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Onishi, Masayuki; Pecani, Kresti; Jones 4th, Taylor et al. (2018) F-actin homeostasis through transcriptional regulation and proteasome-mediated proteolysis. Proc Natl Acad Sci U S A 115:E6487-E6496
Rahi, Sahand Jamal; Larsch, Johannes; Pecani, Kresti et al. (2017) Oscillatory stimuli differentiate adapting circuit topologies. Nat Methods 14:1010-1016
Rahi, Sahand Jamal; Pecani, Kresti; Ondracka, Andrej et al. (2016) The CDK-APC/C Oscillator Predominantly Entrains Periodic Cell-Cycle Transcription. Cell 165:475-87
Breker, Michal; Lieberman, Kristi; Tulin, Frej et al. (2016) High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii. J Vis Exp :
Pecani, Kresti; Cross, Frederick R (2016) Degradation of the Mitotic Cyclin Clb3 Is not Required for Mitotic Exit but Is Necessary for G1 Cyclin Control of the Succeeding Cell Cycle. Genetics 204:1479-1494
Tulin, Frej; Cross, Frederick R (2016) Patching Holes in the Chlamydomonas Genome. G3 (Bethesda) 6:1899-910
Onishi, Masayuki; Pringle, John R; Cross, Frederick R (2016) Evidence That an Unconventional Actin Can Provide Essential F-Actin Function and That a Surveillance System Monitors F-Actin Integrity in Chlamydomonas. Genetics 202:977-96
Cross, Frederick R; Umen, James G (2015) The Chlamydomonas cell cycle. Plant J 82:370-92
Cross, Frederick R (2015) Tying Down Loose Ends in the Chlamydomonas Genome: Functional Significance of Abundant Upstream Open Reading Frames. G3 (Bethesda) 6:435-46
Tulin, Frej; Cross, Frederick R (2015) Cyclin-Dependent Kinase Regulation of Diurnal Transcription in Chlamydomonas. Plant Cell 27:2727-42

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