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1. Deep learning-driven imaging of cell division and cell growth across an entire eukaryotic life cycle

   https://doi.org/10.1101/2024.04.25.591211  

   Ramakanth, Shreya; Kennedy, Taylor; Yalcinkaya, Berk; Neupane, Sandhya; Tadic, Nika; Buchler, Nicolas E; Argüello-Miranda, Orlando (April 2024, bioRxiv)

   Abstract The life cycle of biomedical and agriculturally relevant eukaryotic microorganisms involves complex transitions between proliferative and non-proliferative states such as dormancy, mating, meiosis, and cell division. New drugs, pesticides, and vaccines can be created by targeting specific life cycle stages of parasites and pathogens. However, defining the structure of a microbial life cycle often relies on partial observations that are theoretically assembled in an ideal life cycle path. To create a more quantitative approach to studying complete eukaryotic life cycles, we generated a deep learning-driven imaging framework to track microorganisms across sexually reproducing generations. Our approach combines microfluidic culturing, life cycle stage-specific segmentation of microscopy images using convolutional neural networks, and a novel cell tracking algorithm, FIEST, based on enhancing the overlap of single cell masks in consecutive images through deep learning video frame interpolation. As proof of principle, we used this approach to quantitatively image and compare cell growth and cell cycle regulation across the sexual life cycle ofSaccharomyces cerevisiae. We developed a fluorescent reporter system based on a fluorescently labeled Whi5 protein, the yeast analog of mammalian Rb, and a new High-Cdk1 activity sensor, LiCHI, designed to report during DNA replication, mitosis, meiotic homologous recombination, meiosis I, and meiosis II. We found that cell growth preceded the exit from non-proliferative states such as mitotic G1, pre-meiotic G1, and the G0 spore state during germination. A decrease in the total cell concentration of Whi5 characterized the exit from non-proliferative states, which is consistent with a Whi5 dilution model. The nuclear accumulation of Whi5 was developmentally regulated, being at its highest during meiotic exit and spore formation. The temporal coordination of cell division and growth was not significantly different across three sexually reproducing generations. Our framework could be used to quantitatively characterize other single-cell eukaryotic life cycles that remain incompletely described. An off-the-shelf user interfaceYeastvisionprovides free access to our image processing and single-cell tracking algorithms. 

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2. The Ribbon-Helix-Helix Domain Protein CdrS Regulates the Tubulin Homolog ftsZ2 To Control Cell Division in Archaea

   https://doi.org/10.1128/mBio.01007-20  

   Darnell, Cynthia L.; Zheng, Jenny; Wilson, Sean; Bertoli, Ryan M.; Bisson-Filho, Alexandre W.; Garner, Ethan C.; Schmid, Amy K. (August 2020, mBio)

   Søgaard-Andersen, Lotte (Ed.)

   ABSTRACT Precise control of the cell cycle is central to the physiology of all cells. In prior work we demonstrated that archaeal cells maintain a constant size; however, the regulatory mechanisms underlying the cell cycle remain unexplored in this domain of life. Here, we use genetics, functional genomics, and quantitative imaging to identify and characterize the novel CdrSL gene regulatory network in a model species of archaea. We demonstrate the central role of these ribbon-helix-helix family transcription factors in the regulation of cell division through specific transcriptional control of the gene encoding FtsZ2, a putative tubulin homolog. Using time-lapse fluorescence microscopy in live cells cultivated in microfluidics devices, we further demonstrate that FtsZ2 is required for cell division but not elongation. The cdrS-ftsZ2 locus is highly conserved throughout the archaeal domain, and the central function of CdrS in regulating cell division is conserved across hypersaline adapted archaea. We propose that the CdrSL-FtsZ2 transcriptional network coordinates cell division timing with cell growth in archaea. IMPORTANCE Healthy cell growth and division are critical for individual organism survival and species long-term viability. However, it remains unknown how cells of the domain Archaea maintain a healthy cell cycle. Understanding the archaeal cell cycle is of paramount evolutionary importance given that an archaeal cell was the host of the endosymbiotic event that gave rise to eukaryotes. Here, we identify and characterize novel molecular players needed for regulating cell division in archaea. These molecules dictate the timing of cell septation but are dispensable for growth between divisions. Timing is accomplished through transcriptional control of the cell division ring. Our results shed light on mechanisms underlying the archaeal cell cycle, which has thus far remained elusive. 

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3. Impact of variability in cell cycle periodicity on cell population dynamics

   https://doi.org/10.1371/journal.pcbi.1011080  

   Nowak, Chance M; Quarton, Tyler; Bleris, Leonidas (June 2023, PLOS Computational Biology)

   Csikász-Nagy, Attila (Ed.)

   The cell cycle consists of a series of orchestrated events controlled by molecular sensing and feedback networks that ultimately drive the duplication of total DNA and the subsequent division of a single parent cell into two daughter cells. The ability to block the cell cycle and synchronize cells within the same phase has helped understand factors that control cell cycle progression and the properties of each individual phase. Intriguingly, when cells are released from a synchronized state, they do not maintain synchronized cell division and rapidly become asynchronous. The rate and factors that control cellular desynchronization remain largely unknown. In this study, using a combination of experiments and simulations, we investigate the desynchronization properties in cervical cancer cells (HeLa) starting from the G₁/S boundary following double-thymidine block. Propidium iodide (PI) DNA staining was used to perform flow cytometry cell cycle analysis at regular 8 hour intervals, and a custom auto-similarity function to assess the desynchronization and quantify the convergence to an asynchronous state. In parallel, we developed a single-cell phenomenological model the returns the DNA amount across the cell cycle stages and fitted the parameters using experimental data. Simulations of population of cells reveal that the cell cycle desynchronization rate is primarily sensitive to the variability of cell cycle duration within a population. To validate the model prediction, we introduced lipopolysaccharide (LPS) to increase cell cycle noise. Indeed, we observed an increase in cell cycle variability under LPS stimulation in HeLa cells, accompanied with an enhanced rate of cell cycle desynchronization. Our results show that the desynchronization rate of artificially synchronized in-phase cell populations can be used a proxy of the degree of variance in cell cycle periodicity, an underexplored axis in cell cycle research. 

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4. Non-genetic inheritance restraint of cell-to-cell variation

   https://doi.org/10.7554/eLife.64779  

   Vashistha, Harsh; Kohram, Maryam; Salman, Hanna (February 2021, eLife)

   null (Ed.)

   All the different forms of life on our planet – including animals, plants, fungi and bacteria – tend to grow, multiply and expand. This happens through a process called cell division, where one cell becomes two; two cells become four; four cells become eight; and so on. Each dividing cell passes on the same set of genetic instructions to its two daughter cells in the form of DNA. Its remaining contents, made up of a mixture of proteins, RNA and other chemicals, also get divided up equally between the two new cells. This division of cellular assets establishes a form of 'cellular memory', where daughter cells retain very similar properties to their ancestors, which helps them remain stable over time. Yet this memory can fade, and small changes in how a cell looks or acts can appear over many generations of cell division. This happens even when the exact same set of DNA-based genetic instructions have been passed down to daughter cells, confirming that other factors aside from DNA do influence cellular properties and can act to maintain them or introduce variation over time. Here, Vashistha, Kohram and Salman set out to understand how long cellular memory could be maintained in dividing E. coli bacteria. To do this, they created a technique to track cellular memory as it passed down from a single mother cell to two daughter cells over dozens of generations. Using this technique, Vashistha, Kohram and Salman found that some inherited elements, including cell size and the time cells took to divide, were maintained between mother and daughter cells for almost 10 generations. Other elements, such as the density of proteins inside each cell, started changing almost immediately after daughter cells were formed, and only remained similar for about two generations. These findings suggest that cellular memory may be long, but is not infinite, and that inheritance of non-genetic elements can help maintain cellular memory and reduce variation among new-born cells for considerable number of generations. Building on this research to achieve a better understanding of cellular memory may allow researchers to harness these insights to direct the evolution of different cellular properties over time. This could have a wide range of potential applications, such as designing new infection control measures for viruses or bacteria; enhancing our ability to grow working organs for tissue transplant; or improving the texture and consistency of cultured, lab-grown meat. 

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5. Using conditional independence tests to elucidate causal links in cell cycle regulation in Escherichia coli

   https://doi.org/10.1073/pnas.2214796120  

   Kar, Prathitha; Tiruvadi-Krishnan, Sriram; Männik, Jaana; Männik, Jaan; Amir, Ariel (March 2023, Proceedings of the National Academy of Sciences)

   How cells regulate their cell cycles is a central question for cell biology. Models of cell size homeostasis have been proposed for bacteria, archaea, yeast, plant, and mammalian cells. New experiments bring forth high volumes of data suitable for testing existing models of cell size regulation and proposing new mechanisms. In this paper, we use conditional independence tests in conjunction with data of cell size at key cell cycle events (birth, initiation of DNA replication, and constriction) in the model bacterium Escherichia coli to select between the competing cell cycle models. We find that in all growth conditions that we study, the division event is controlled by the onset of constriction at midcell. In slow growth, we corroborate a model where replication-related processes control the onset of constriction at midcell. In faster growth, we find that the onset of constriction is affected by additional cues beyond DNA replication. Finally, we also find evidence for the presence of additional cues triggering initiations of DNA replication apart from the conventional notion where the mother cells solely determine the initiation event in the daughter cells via an adder per origin model. The use of conditional independence tests is a different approach in the context of understanding cell cycle regulation and it can be used in future studies to further explore the causal links between cell events. 

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