Biology subjects

Purvis, J. E.

Publications and source records attributed to Purvis, J. E..

5 recordsLinked to original sources

bioRxiv2018

Precise detection of S phase onset reveals decoupled G1/S transition events

The eukaryotic cell division cycle is the process by which cells duplicate their genomes and proliferate. Transitions between sequential cell cycle phases are tightly orchestrated to ensure precise and efficient cell cycle progression. Interrogating molecular events at these transitions is important for understanding normal and pathological cell proliferation and mechanisms that ensure genome stability. A popular fluorescent reporter system known as \"FUCCI\" has been widely adopted for identifying cell cycle phases. Using time-lapse fluorescence microscopy, we quantitatively analyzed the dynamics of the FUCCI reporters relative to the transitions into and out of S phase. Although the original reporters reflect the E3 ubiquitin ligase activities for which they were designed, SCFSkp2 and APCCdh1, their dynamics are significantly and variably offset from actual S phase boundaries. To precisely mark these transitions, we generated and thoroughly validated a new reporter containing a PCNA-interacting protein degron whose oscillations are directly coupled to the process of DNA replication itself. We combined this reporter with the geminin-based APCCdh1 reporter to create \"PIP-FUCCI.\" PIP degron reporter dynamics closely correlate with S phase transitions irrespective of reporter expression levels. Using PIP-FUCCI, we made the unexpected observation that the apparent timing of APCCdh1 inactivation frequently varies relative to the onset of S phase. We demonstrate that APCCdh1 inactivation is not a strict pre-requisite for S phase entry, though delayed APCCdh1 inactivation correlates with longer S phase. Our results illustrate the benefits of precise delineation of cell cycle phase boundaries for uncovering the sequences of molecular events at critical cell cycle transitions.

cell biology↗

bioRxiv2018

Evidence that the cell cycle is a series of uncoupled, memoryless phases

The cell cycle is canonically described as a series of 4 phases: G1 (gap phase 1), S (DNA synthesis), G2 (gap phase 2), and M (mitosis). Various models have been proposed to describe the durations of each phase, including a two-state model with fixed S-G2-M duration and random G1 duration1,2; a \"stretched\" model in which phase durations are proportional3; and an inheritance model in which sister cells show correlated phase durations2,4. A fundamental challenge is to understand the quantitative laws that govern cell-cycle progression and to reconcile the evidence supporting these different models. Here, we used time-lapse fluorescence microscopy to quantify the durations of G1, S, G2, and M phases for thousands of individual cells from three human cell lines. We found no evidence of correlation between any pair of phase durations. Instead, each phase followed an Erlang distribution with a characteristic rate and number of steps. These observations suggest that each cell cycle phase is memoryless with respect to previous phase durations. We challenged this model by perturbing the durations of specific phases through oncogene activation, inhibition of DNA synthesis, reduced temperature, and DNA damage. Phase durations remained uncoupled in individual cells despite large changes in durations in cell populations. To explain this behavior, we propose a mathematical model in which the independence of cell-cycle phase durations arises from a large number of molecular factors that each exerts a minor influence on the rate of cell-cycle progression. The model predicts that it is possible to force correlations between phases by making large perturbations to a single factor that contributes to more than one phase duration, which we confirmed experimentally by inhibiting cyclin-dependent kinase 2 (CDK2). We further report that phases can show coupling under certain dysfunctional states such as in a transformed cell line with defective cell cycle checkpoints. This quantitative model of cell cycle progression explains the paradoxical observation that phase durations are both inherited and independent and suggests how cell cycle progression may be altered in disease states.

systems biology↗

bioRxiv2017

The Cell Cycle Browser: an interactive tool for visualizing, simulating, and perturbing cell cycle progression

SUMMARYThe cell cycle is driven by precise temporal coordination among many molecular activities. To understand and explore this process, we developed the Cell Cycle Browser (CCB), an interactive web interface based on real-time reporter data collected in proliferating human cells. This tool facilitates visualizing, simulating, and predicting the outcomes of perturbing cell cycle parameters. Time-series traces from individual cells can be combined to build a multi-layered timeline of molecular activities. Users can simulate the cell cycle using computational models that capture the dynamics of molecular activities and phase transitions. By adjusting individual expression levels and strengths of molecular relationships, users can predict effects on the cell cycle. Virtual assays, such as growth curves and flow cytometry, provide familiar outputs to compare cell cycle behaviors for data and simulations. The CCB serves to unify our understanding of cell cycle dynamics and provides a platform for generating hypotheses through virtual experiments.\n\nHIGHLIGHTSO_LIUsers can stack and align single-cell traces for different molecular reporters\nC_LIO_LIComputational models with adjustable parameters simulate cell cycle progression\nC_LIO_LIVirtual growth curves and flow cytometry assays predict cell cycle behaviors\nC_LI

systems biology↗

bioRxiv2017

Ratiometric Distribution Of OCT4 During Stem Cell Division Controls The Balance Between Self-Renewal And Differentiation

Clonal cells can make different fate decisions, but it is often unclear to what extent these decisions are autonomous or predetermined. Here, we introduce a live-cell reporter for the pluripotency factor OCT4 into human embryonic stem cells to understand how they choose between self-renewal and differentiation. By tracing the histories of individual cells over multiple generations, we found that cells whose offspring were destined to differentiate showed decreased expression of OCT4 long before exposure to the differentiation stimulus. OCT4 levels were lineage-dependent; however, during cell division, mother cells distributed OCT4 asymmetrically to daughters. The resulting ratio of OCT4 between sister cells--established within minutes of mitosis--was predictive of downstream fates: cells receiving a greater ratio of maternal OCT4 showed sustained OCT4 levels and a reduced capacity to differentiate. Our observations imply that the choice between two developmental fates is almost entirely predetermined at the time of cell birth through inheritance of a pluripotency factor.

systems biology↗

bioRxiv2017

DNA Damage Checkpoint Dynamics Drive Cell Cycle Phase Transitions

DNA damage checkpoints are cellular mechanisms that protect the integrity of the genome during cell cycle progression. In response to genotoxic stress, these checkpoints halt cell cycle progression until the damage is repaired, allowing cells enough time to recover from damage before resuming normal proliferation. Here, we investigate the temporal dynamics of DNA damage checkpoints in individual proliferating cells by observing cell cycle phase transitions following acute DNA damage. We find that in gap phases (G1 and G2), DNA damage triggers an abrupt halt to cell cycle progression in which the duration of arrest correlates with the severity of damage. However, cells that have already progressed beyond a proposed \"commitment point\" within a given cell cycle phase readily transition to the next phase, revealing a relaxation of checkpoint stringency during later stages of certain cell cycle phases. In contrast to G1 and G2, cell cycle progression in S phase is significantly less sensitive to DNA damage. Instead of exhibiting a complete halt, we find that increasing DNA damage doses leads to decreased rates of S-phase progression followed by arrest in the subsequent G2. Moreover, these phase-specific differences in DNA damage checkpoint dynamics are associated with corresponding differences in the proportions of irreversibly arrested cells. Thus, the precise timing of DNA damage determines the sensitivity, rate of cell cycle progression, and functional outcomes for damaged cells. These findings should inform our understanding of cell fate decisions after treatment with common cancer therapeutics such as genotoxins or spindle poisons, which often target cells in a specific cell cycle phase.

systems biology↗