Strengths and Weaknesses of Cell Synchronization Protocols Based on Inhibition of DNA Synthesis

Synchronous cell populations are commonly used for the analysis of various aspects of cellular metabolism at specific stages of the cell cycle. Cell synchronization at a chosen cell cycle stage is most frequently achieved by inhibition of specific metabolic pathway(s). In this respect, various protocols have been developed to synchronize cells in particular cell cycle stages. In this review, we provide an overview of the protocols for cell synchronization of mammalian cells based on the inhibition of synthesis of DNA building blocks—deoxynucleotides and/or inhibition of DNA synthesis. The mechanism of action, examples of their use, and advantages and disadvantages are described with the aim of providing a guide for the selection of suitable protocol for different studied situations.

A novel auxin-inducible degron system for rapid, cell cycle-specific targeted proteolysis

The OsTIR1/auxin-inducible degron (AID) system allows “on demand” selective and reversible protein degradation upon exposure to the phytohormone auxin. In the current format, this technology does not allow to study the effect of acute protein depletion selectively in one phase of the cell cycle, as auxin similarly affects all the treated cells irrespectively of their proliferation status. Therefore, the AID system requires coupling with cell synchronization techniques, which can alter the basal biological status of the studied cell population. Here, we introduce a new AID system to Regulate OsTIR1 Levels based on the Cell Cycle Status (ROLECCS system), which induces proteolysis of both exogenously transfected and endogenous gene-edited targets in specific phases of the cell cycle. This new tool paves the way to studying the differential roles that target proteins may have in specific phases of the cell cycle.

Systematic analysis of factors that improve homologous direct repair (HDR) efficiency in CRISPR/Cas9 technique

The CRISPR/Cas9 bacterial system has proven to be an powerful tool for genetic manipulation in several organisms, but the efficiency of sequence replacement by homologous direct repair (HDR) is substantially lower than random indel creation. Many studies focused on improving HDR efficiency using double sgRNA, cell synchronization cycle, and the delivery of single-stranded oligo DNA nucleotides (ssODN) with a rational design. In this study, we evaluate these three methods’ synergistic effects to improve HDR efficiency. For our tests, we have chosen the TNFα gene (NM_000594) for its crucial role in various biological processes and diseases. For the first time, our results showed how the use of two sgRNA with asymmetric donor design and triple transfection events dramatically increase the HDR efficiency from an undetectable HDR event to 39% of HDR efficiency and provide a new strategy to facilitate CRISPR/Cas9-mediated human genome editing. Besides, we demonstrated that the TNFα locus could be edited with CRISPR/Cas9 methodology, an opportunity to safely correct, in the future, the specific mutations of each patient.

CDK1 couples proliferation with protein synthesis

Cell proliferation exerts a high demand on protein synthesis, yet the mechanisms coupling the two processes are not fully understood. A kinase and phosphatase screen for activators of translation, based on the formation of stress granules in human cells, revealed cell cycle–associated kinases as major candidates. CDK1 was identified as a positive regulator of global translation, and cell synchronization experiments showed that this is an extramitotic function of CDK1. Different pathways including eIF2α, 4EBP, and S6K1 signaling contribute to controlling global translation downstream of CDK1. Moreover, Ribo-Seq analysis uncovered that CDK1 exerts a particularly strong effect on the translation of 5′TOP mRNAs, which includes mRNAs encoding ribosomal proteins and several translation factors. This effect requires the 5′TOP mRNA-binding protein LARP1, concurrent to our finding that LARP1 phosphorylation is strongly dependent on CDK1. Thus, CDK1 provides a direct means to couple cell proliferation with biosynthesis of the translation machinery and the rate of protein synthesis.

Systematic analysis of factors that improve HDR efficiency in CRISPR / Cas9 technique

The bacterial CRISPR/Cas9 system has a proven to be an efficient tool for genetic manipulation in various organisms, but the efficiency of sequence replacement by homologous direct repair (HDR) is substantially lower than random creation of indels. Many studies focused on improving the efficiency of HDR using double sgRNA, cell synchronization cycle and the delivery of ssODN with a rational design. In the present study, we tested and compared the combination of these three methods to improve HDR efficiency. To our tests, we chosen the TNFα gene (NM_000594) for its crucial role in a variety of biological processes and diseases. Our results showed a dramatically increases of HDR efficiency from undetectable HDR event to 39% of HDR efficiency and provide a new strategy to facilitate CRISPR/Cas9-mediated human genome targeting. Furthermore, we showed that TNFα gene could be edited with CRISPR/Cas9 methodology, an opportunity to safely correct, in the future, the specific mutations of each patient.

CDK1 couples proliferation with protein synthesis

ABSTRACTCell proliferation exerts a high demand on protein synthesis, yet the mechanisms coupling the two processes are not fully understood. A kinase and phosphatase screen for activators of translation, based on the formation of stress granules in human cells, revealed cell cycle-associated kinases as major candidates. CDK1 was identified as a positive regulator of global translation, and cell synchronization experiments showed that this is an extra-mitotic function of CDK1. Dephosphorylation of eIF2α and S6K1 signaling were found to act downstream of CDK1. Moreover, Ribo-Seq analysis uncovered that CDK1 exerts a particularly strong effect on the translation of 5’TOP mRNAs, which includes mRNAs encoding for ribosomal proteins and several translation factors. This effect requires the 5’TOP mRNA-binding protein LARP1, concurrent to our finding that LARP1 phosphorylation is strongly dependent on CDK1. Taken together, our results show that CDK1 provides a direct means to couple cell proliferation with biosynthesis of the translation machinery and the rate of protein synthesis.

Cell cycle analysis of MDA-MB-157 and APC knockdown cells

Background and Hypothesis: A majority of sporadic breast cancers include deficits in the expression of Adenomatous Polyposis Coli (APC), a tumor suppressor. Deficits in APC are more common in patients with TNBC (triple negative breast cancer), which is also a cancer prone to chemotherapy resistance. The Prosperi lab previously found that APC knockdown cells (APCKD) were resistant to Paclitaxel (PTX). We hypothesize that APCKD cells are resistant to PTX treatment through avoidance of G2/M arrest. This summer, my goal was to investigate the mechanism by which PTX works to arrest the cell cycle at G2/M. Experimental Design or Project Methods: Cell Synchronization: To synchronize MDA-MB-157 and APCKD cells, we first tried serum starvation for 24-72 hours. Second, we tried synchronizing the cells using a double thymidine block as described (Bostock, 1971). In either protocol, cells were then stained with Propidium Iodide (PI) and flow cytometry was performed. Paclitaxel (PTX) treatment and cell cycle analysis: MDA-MB-157 and APCKD cells were grown to confluency and then treated with 0.078µM PTX for 12, 24, and 48 hours. Cells were stained with PI and flow cytometry was performed. Results: Cell synchronization: APCKD cells cells have an increased cell population in the G2/M phase than the parental cells after serum starvation. Importantly, APC knockdown cells are not impacted by serum starvation up to 72 hours. Additionally, a double thymidine block is insufficient to synchronize MDA-MB-157 and APCKD cells. A double thymidine block did shorten the S phase and move MDA-MB-157 and APCKD cells closer to G0-G1 arrest, but did not synchronize. Paclitaxel (PTX) treatment and cell cycle analysis: MDA-MB-157 and APCKD cells treated for longer intervals experienced more cell death and were further arrested in G2/M. Conclusion and Potential Impact: We learned that MDA-MB-157 and APCKD cannot be easily synchronized using serum starving or a double thymidine block. Future investigations will require alternative methods of synchronization or will proceed without synchronization. Furthermore, APCKD cells do not avoid G2/M arrest when treated with Paclitaxel, indicating a different mechanism of PTX resistance.

Architecture of a multi-cellular polygenic network governing immune homeostasis

SummaryComplex physiological functionality is often the outcome of multiple interacting cell-types, yet mechanistically how a large number of trait-associated genes yield a single multi-cellular network governing the phenotype has not been well defined. Individuals’ immune-cellular profiles at homeostasis show high heritability and inter-individual variation with functional and clinical implications. We profiled immune cellular variation by mass-cytometry in 55 genetically diverse mouse strains. We identify 788 genes associated with cellular homeostasis, supporting a polygenic model where 52% of genes correspond to core homeostatic functions whose genetic variants suffice to predict phenotype. Trait genes form a multi-cellular network architecture showing increased functional complexity over evolutionary timescales for shared regulation to all cells, specialized cell-specific programs, and between-cell synchronization. Contrasting to human studies suggests the regulatory network expands with environmental exposure history. Our findings shed light on the origin of immune-cellular variation and regulatory architectures that may generalize to other environmentally sensitive systems.