Biology subjects

Limas, J. C.

Publications and source records attributed to Limas, J. C..

4 recordsLinked to original sources

bioRxiv2022

The molecular architecture of cell cycle arrest

The cellular decision governing the transition between proliferative and arrested states is crucial to the development and function of every tissue. While the molecular mechanisms that regulate the proliferative cell cycle are well established, we know comparatively little about what happens to cells as they diverge into cell cycle arrest. We performed hyperplexed imaging of 49 cell cycle effectors to obtain a map of the molecular architecture that governs cell cycle exit and progression into reversible ("quiescent") and irreversible ("senescent") arrest states. Using this map, we found multiple points of divergence from the proliferative cell cycle; identified stress-specific states of arrest; and resolved the molecular mechanisms governing these fate decisions, which we validated by single-cell, time-lapse imaging. Notably, we found that cells can exit into senescence from either G1 or G2; however, both subpopulations converge onto a single senescent state with a G1-like molecular signature. Cells can escape from this "irreversible" arrest state through the upregulation of G1 cyclins. This comprehensive map provides a first glimpse of the overall organization of cell proliferation and arrest.

cell biology↗

bioRxiv2021

Quantitative Profiling of Adaptation to Cyclin E Overproduction

Cyclin E/CDK2 drives cell cycle progression from G1 to S phase. Cyclin E overproduction is toxic to mammalian cells, although the gene encoding cyclin E (CCNE1) is overexpressed in some cancers. To gain insight into how cancer cells tolerate high cyclin E, we extensively characterized non-transformed epithelial cells throughout a time course of chronic cyclin E overproduction. Cells overproducing human cyclin E, but not cyclin D or cyclin A, briefly experienced truncated G1 phases, then endured a transient period of DNA replication origin underlicensing, replication stress, and severely impaired proliferation. Individual cells displayed substantial intercellular heterogeneity in cell cycle dynamics and CDK activity. Each phenotype improved rapidly despite maintaining high cyclin E-associated activity. Transcriptome analysis revealed that adapted cells downregulated a cohort of G1-regulated genes. Withdrawing cyclin E induction only partially reversed the intermediate licensing phenotype of adapted cells indicating that adaptation is at least partly independent of mutations. This study provides evidence that mammalian cyclin E/CDK inhibits origin licensing by an indirect mechanism through premature S phase onset and provides further insight into the relationship between CDK activity and licensing in mammals. It serves as an example of specific oncogene adaptation that may recapitulate molecular changes during tumorigenesis.

cell biology↗

bioRxiv2021

The structure of the human cell cycle

The human cell cycle is conventionally depicted as a five-phase model consisting of four proliferative phases (G1, S, G2, M) and a single state of arrest (G0). However, recent studies show that individual cells can take different paths through the cell cycle and exit into distinct arrest states, thus necessitating an update to the canonical model. We combined time lapse microscopy, highly multiplexed single cell imaging and manifold learning to determine the underlying "structure" of the human cell cycle under multiple growth and arrest conditions. By visualizing the cell cycle as a complete biological process, we identified multiple points of divergence from the proliferative cell cycle into distinct states of arrest, revealing multiple mechanisms of cell cycle exit and re-entry and the molecular routes to senescence, endoreduplication and polyploidy. These findings enable the visualization and comparison of alternative cell cycles in development and disease. One-sentence summaryA systems-level view of single-cell states reveals the underlying architecture of the human cell cycle

cell biology↗

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↗