Short Cell Cycle Research Articles

SPL13 controls a root apical meristem phase change by triggering oriented cell divisions.

Oriented cell divisions are crucial for determining the overall morphology and size of plants, but what controls the onset and duration of this process remains largely unknown. Here, we identified a small molecule that activates root apical meristem (RAM) expression of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE13 (SPL13) a known player in the shoot's juvenile-to-adult transition. This expression leads to oriented cell divisions in the RAM through SHORT ROOT (SHR) and cell cycle regulators. We further show that the RAM has distinct juvenile and adult phases typed by morphological and molecular characteristics and that SPL factors are crucially required for this transition in Arabidopsis and rice (Oryza sativa). In summary, we provide molecular insights into the age-dependent morphological changes occurring in the RAM during phase change.

Study of impacts of two types of cellular aging on the yeast bud morphogenesis.

Understanding the mechanisms of the cellular aging processes is crucial for attempting to extend organismal lifespan and for studying age-related degenerative diseases. Yeast cells divide through budding, providing a classical biological model for studying cellular aging. With their powerful genetics, relatively short cell cycle, and well-established signaling pathways also found in animals, yeast cells offer valuable insights into the aging process. Recent experiments suggested the existence of two aging modes in yeast characterized by nucleolar and mitochondrial declines, respectively. By analyzing experimental data, this study shows that cells evolving into those two aging modes behave differently when they are young. While buds grow linearly in both modes, cells that consistently generate spherical buds throughout their lifespan demonstrate greater efficacy in controlling bud size and growth rate at young ages. A three-dimensional multiscale chemical-mechanical model was developed and used to suggest and test hypothesized impacts of aging on bud morphogenesis. Experimentally calibrated model simulations showed that during the early stage of budding, tubular bud shape in one aging mode could be generated by locally inserting new materials at the bud tip, a process guided by the polarized Cdc42 signal. Furthermore, the aspect ratio of the tubular bud could be stabilized during the late stage as observed in experiments in this work. The model simulation results suggest that the localization of new cell surface material insertion, regulated by chemical signal polarization, could be weakened due to cellular aging in yeast and other cell types, leading to the change and stabilization of the bud aspect ratio.

Single cell expression and chromatin accessibility of the Toxoplasma gondii lytic cycle identifies AP2XII-8 as an essential ribosome regulon driver

Sequential lytic cycles driven by cascading transcriptional waves underlie pathogenesis in the apicomplexan parasite Toxoplasma gondii. This parasite’s unique division by internal budding, short cell cycle, and jumbled up classically defined cell cycle stages have restrained in-depth transcriptional program analysis. Here, unbiased transcriptome and chromatin accessibility maps throughout the lytic cell cycle are established at the single-cell level. Correlated pseudo-timeline assemblies of expression and chromatin profiles maps transcriptional versus chromatin level transition points promoting the cell division cycle. Sequential clustering analysis identifies functionally related gene groups promoting cell cycle progression. Promoter DNA motif mapping reveals patterns of combinatorial regulation. Pseudo-time trajectory analysis reveals transcriptional bursts at different cell cycle points. The dominant burst in G1 is driven largely by transcription factor AP2XII-8, which engages a conserved DNA motif, and promotes the expression of 44 ribosomal proteins encoding regulon. Overall, the study provides integrated, multi-level insights into apicomplexan transcriptional regulation.

The maternal-to-zygotic transition

Rapid cleavage divisions and the transition from maternal to zygotic control of gene expression are the hallmarks of early embryonic development in most species. Early development in insects, fish and amphibians is characterized by several short cell cycles with no gap phases, necessary for the rapid production of cells prior to patterning and morphogenesis. Maternal mRNAs and proteins loaded into the egg during oogenesis are essential to drive these rapid early divisions. Once the function of these maternal inputs is complete, the maternal-to-zygotic transition (MZT) marks the handover of developmental control to the gene products synthesized from the zygotic genome. The MZT requires three major events: the removal of a subset of maternal mRNAs, the initiation of zygotic transcription, and the remodeling of the cell cycle. In each species, the MZT occurs at a highly reproducible time during development due to a series of feedback mechanisms that tightly couple these three processes. Dissecting these feedback mechanisms and their spatiotemporal control will be essential to understanding the control of the MZT. In this primer, we outline the mechanisms that govern the major events of the MZT across species and highlight the role of feedback mechanisms that ensure the MZT is precisely timed and orchestrated.

Involvement of H2A variants in DNA damage response of zygotes

Phosphorylated H2AX, known as γH2AX, forms in response to genotoxic insults in somatic cells. Despite the high abundance of H2AX in zygotes, the level of irradiation-induced γH2AX is low at this stage. Another H2A variant, TH2A, is present at a high level in zygotes and can also be phosphorylated at its carboxyl end. We constructed H2AX- or TH2A-deleted mice using CRISPR Cas9 and investigated the role of these H2A variants in the DNA damage response (DDR) of zygotes exposed to γ-ray irradiation at the G2 phase. Our results showed that compared to irradiated wild-type zygotes, irradiation significantly reduced the developmental rates to the blastocyst stage in H2AX-deleted zygotes but not in TH2A-deleted ones. Furthermore, live cell imaging revealed that the G2 checkpoint was activated in H2AX-deleted zygotes, but the duration of arrest was significantly shorter than in wild-type and TH2A-deleted zygotes. The number of micronuclei was significantly higher in H2AX-deleted embryos after the first cleavage, possibly due to the shortened cell cycle arrest of damaged embryos and, consequently, the insufficient time for DNA repair. Notably, FRAP analysis suggested the involvement of H2AX in chromatin relaxation. Moreover, phosphorylated CHK2 foci were found in irradiated wild-type zygotes but not in H2AX-deleted ones, suggesting a critical role of these foci in maintaining cell cycle arrest for DNA repair. In conclusion, H2AX, but not TH2A, is involved in the DDR of zygotes, likely by creating a relaxed chromatin structure with enhanced accessibility for DNA repair proteins and by facilitating the formation of pCHK2 foci to prevent premature cleavage.

Short cell cycle duration is a phenotype of human epidermal stem cells

BackgroundA traditional view is that stem cells (SCs) divide slowly. Meanwhile, both embryonic and pluripotent SCs display a shorter cell cycle duration (CCD) in comparison to more committed progenitors (CPs).MethodsWe examined the in vitro proliferation and cycling behavior of somatic adult human cells using live cell imaging of passage zero keratinocytes and single-cell RNA sequencing.ResultsWe found two populations of keratinocytes: those with short CCD and protracted near exponential growth, and those with long CCD and terminal differentiation. Applying the ergodic principle, the comparative numbers of cycling cells in S phase in an enriched population of SCs confirmed a shorter CCD than CPs. Further, analysis of single-cell RNA sequencing of cycling adult human keratinocyte SCs and CPs indicated a shortening of both G1 and G2M phases in the SC.ConclusionsContrary to the pervasive paradigm, SCs progress through cell cycle more quickly than more differentiated dividing CPs. Thus, somatic human adult keratinocyte SCs may divide infrequently, but divide rapidly when they divide. Additionally, it was found that SC-like proliferation persisted in vitro.

Cellular Functions of High-Temperature Requirement Factor A4 in Placenta.

The expression of High-temperature requirement factor A4 (HtrA4) mRNA is significantly lower in the chorionic villi of patients with recurrent pregnancy loss (RPL) than in the control group. We conducted an investigation into the cellular functions of HtrA4 using the CRISPR/Cas9 system and shRNA-HtrA4 to create knockout BeWo cells and HtrA4 knockdown JEG3 cells. Our results indicated that the knockout BeWo cells exhibited reduced capacity for invasion and fusion, but increased levels of proliferation and migration, with a significantly shortened cell cycle compared to wild-type cells. Wild-type BeWo cells highly expressed cell invasion- and fusion-related factors, while knockout BeWo cells highly expressed migration-, proliferation-, and cell cycle-related factors. The shRNA-HtrA4 JEG3 cells showed a decreased capacity for invasion, but an increased capacity for migration, accompanied by a decrease in the expression of cell invasion-related factors and an increase in migration-related factors. Moreover, our ELISA results revealed that the serum HtrA4 level was lower in patients with RPL than in the controls. These findings suggest that HtrA4 depletion may be associated with placental dysfunction.

A Self-Growing 3D Porous Sn Protective Layer Enhanced Zn Anode

Aqueous zinc-ion batteries (ZIBs) have received much attention because of their high safety, low pollution, and satisfactory energy density (840 mAh g−1), which is important for the research of new energy storage devices. However, problems such as short cell cycle life and low coulombic efficiency (CE) of zinc (Zn) anodes due to disorderly growth of Zn dendrites and side reactions of hydrogen corrosion have delayed the practical application of ZIBs. In this work, a new “self-growth” method is proposed to build a robust and homogeneous three-dimensional (3D) nanoporous structure of tin (Sn)-coated Zn anodes (ZSN) in just 10 min by a simple and fast reaction, which can largely raise the surface area of the electrode plate. The ZSN not only provides abundant Zn nucleation sites, but also reduces the corrosion current, thus alleviating the self-corrosion of the electrolyte, reducing the occurrence of hydrogen precipitation side reactions, and effectively inhibiting the growth of Zn dendrites during cycling. Thus, a symmetric cell with a ZSN anode can be stabilized with very low voltage hysteresis (30 mV) for 480 h of stable plating/stripping cycles and can operate well for 200 h even at high current densities of 10 mA cm−2. Supercapacitors and button cells were assembled, respectively, to verify the performance of ZSN electrodes in different energy storage tools. The ZSN||AC supercapacitor exhibited superior capacity (75 mAh g−1) and high reversibility (98% coulombic efficiency) at a current density of 2 A g−1. With a MnVO (MVO) electrode as the cathode, the ZSN||MVO full cell presents excellent cycling stability with a capacity retention of 95.4% after 500 cycles at 2 A g−1, which far exceeds that of the bare Zn cell.

Dysregulated cholesterol metabolism, aberrant excitability and altered cell cycle of astrocytes in fragile X syndrome.

Fragile X syndrome (FXS), the most prevalent heritable form of intellectual disability, is caused by the transcriptional silencing of the FMR1 gene. While neuronal contribution to FXS has been extensively studied in both animal and human-based models of FXS, the roles of astrocytes, a type of glial cells in the brain, are largely unknown. Here, we generated a human-based FXS model via differentiation of astrocytes from human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) and characterized their development, function, and proteomic profiles. We identified shortened cell cycle, enhanced Ca2+ signaling, impaired sterol biosynthesis, and pervasive alterations in the proteome of FXS astrocytes. Our work identified astrocytic impairments that could contribute to the pathogenesis of FXS and highlight astrocytes as a novel therapeutic target for FXS treatment.

Space Microgravity Alters Neural Stem Cell Division: Implications for Brain Cancer Research on Earth and in Space.

Considering the imminence of long-term space travel, it is necessary to investigate the impact of space microgravity (SPC-µG) in order to determine if this environment has consequences on the astronauts' health, in particular, neural and cognitive functions. Neural stem cells (NSCs) are the basis for the regeneration of the central nervous system (CNS) cell populations and learning how weightlessness impacts NSCs in health and disease provides a critical tool for the potential mitigation of specific mechanisms leading to neurological disorders. In previous studies, we found that exposure to SPC-µG resulted in enhanced proliferation, a shortened cell cycle, and a larger cell diameter of NSCs compared to control cells. Here, we report the frequent occurrence of abnormal cell division (ACD) including incomplete cell division (ICD), where cytokinesis is not successfully completed, and multi-daughter cell division (MDCD) of NSCs following SPC-µG as well as secretome exposure compared to ground control (1G) NSCs. These findings provide new insights into the potential health implications of space travel and have far-reaching implications for understanding the mechanisms leading to the deleterious effects of long-term space travel as well as potential carcinogenic susceptibility. Knowledge of these mechanisms could help to develop preventive or corrective measures for successful long-term SPC-µG exposure.

P-258 Impact of Direct Unequal Cleavage (DUC) on embryo development, blastocyst formation and ploidy - artificial intelligence (AI) analysis

Abstract Study question Do DUCs significantly impact embryo development? In particular, morphokinetics, grading, and Pre-implantation Genetic Testing for Aneuploidy (PGT-A) outcome? Is this analysis corroborated by artificial intelligence? Summary answer DUC embryos develop slower, have lower rates of blastulation and lower CHLOE (Fairtility) scores for blastulation and implantation. However, occasionally euploid blastocysts form from DUCs. What is known already Time-lapse technology enables the identification of DUCs during embryo development. Previous research associates DUCs with poorer blastulation, implantation, and ploidy outcomes. However, DUCs are not routinely annotated in all clinics. Some algorithms, deselect embryos with short second cell cycles; hence, DUCs are rarely transferred. Whether DUC embryos should be automatically discarded or deprioritised is an ongoing debate which leads to inconsistency in clinical practices across fertility centres. AI image processing algorithms may assist embryologists in the identification of DUCs. Study design, size, duration A retrospective single-centre study of normally-fertilised embryos cultured in time-lapse incubators throughout 2019--2021. We reviewed 9284 time-lapse videos using an AI image processing tool (CHLOE, Fairtility), and assessed DUC embryo outcomes (ploidy, blastulation, and blastocyst quality). Additionally, we analysed pronuclei data searching for possible causes of DUCs. Participants/materials, setting, methods CHLOE (Fairtility) software analysed time-lapse videos identifying pronuclei, DUCs, and blastulation; recording all morphokinetic time points (tPNa,tPNf,t2,t3,t4,t5,t6,t7,t8,t9,tM, tSB,tB,tEB), morphological grades for the inner cell mass (ICM) and trophectoderm, blastocyst size at 116hpi; and assessing the likelihood of blastulation (at 30hpi) and implantation. We evaluated the statistical significance for all variables using t-tests (continuous variables) and chi-squared tests (categorical variables). We quantified the two pronuclei (2PN) detection efficacy using four metrics: accuracy, sensitivity, specificity, and informedness. Main results and the role of chance Of all the embryos analysed (n = 9284), 35% showed DUCs (n = 3269). Blastulation was significantly higher in non-DUC versus DUC embryos (76% and 49%, p < 0.0001). Of the embryos that blastulated, ICM quality (A,B,C,D: 24%,13%,19%,21% and 3%,4%,16%,47%, p < 0.001) and trophectoderm quality (20%,21%,15%,23% and 2%,7%,14%,52%, p < 0.0001) were significantly higher in non-DUC than in DUC embryos. As defined, DUC embryos were significantly quicker at reaching t3 than non-DUC [Mean(SD): 34(15) and 39(10), p < 0.0001], with the minimum times being 4hpi and 13hpi respectively. Interestingly, there was no significant difference in achieving t5 [52(21) and 51(12), NS]. For all other morphokinetic milestones, DUC embryos were 6 hours slower than non-DUC embryos. DUC embryos had an euploidy rate of 27.2% (12/44). Only one DUC embryo was transferred in a double embryo transfer cycle leading to a negative outcome. Implantation score [0.14(0.24) and 0.46(0.36), p < 0.0001] and blastulation score [0.4(0.46) and 0.75(0.4), p < 0.0001] were lower for DUC embryos than for non-DUC embryos. CHLOE automatic PN assessment agreed with human annotation in 92% of cases (TP = 388,TN=5,FP=29,FN=7). CHLOE Blastocyst prediction at 30hpi had an AUC of 0.89. The embryologist agreed on 97% of all 483 embryos that CHLOE classified as DUC. Discrepancies arose from CHLOE misclassifying fragments as blastomeres. Further studies warranted. Limitations, reasons for caution Differentiating between fragments and blastomeres within the 5 hours from the first division proves challenging for embryologists and, especially, AI algorithms. Hence, some embryos’ DUC status may be misclassified. Additionally, our sample sizes are limited and larger sizes are needed to corroborate our findings, especially those pertaining to ploidy status. Wider implications of the findings DUC embryos are associated with poorer outcomes and DUC status should be integrated into embryo classification frameworks. Nevertheless, some DUC embryos prove to be euploid. Hence, DUC embryos should not excluded from culture at cleavage stage and instead be allowed to reach blastocyst stage before assessing their suitability for transfer/vitrification/PGT-A. Trial registration number IRB-001C01-01-22

Transmembrane Protein-Based Risk Model and H3K4me3 Modification Characteristics in Lung Adenocarcinoma.

The role and mechanism of transmembrane proteins (TMEMs) in tumorigenesis remain unclear. Based on 4 independent cohorts containing 1,208 cases, we identified 3 TMEMs (TMEM273, TMEM164, and TMEM125), which were used to construct a risk model to predict the prognosis of LUAD. The two patterns based on the risk score exhibited a high degree of consistency with the characteristics of immune cell infiltration and epigenetic distribution. Patients with a low-risk score, characterized by an increased activation of immunity, H3K4me3 modification, tumor cell apoptosis, chemokine secretion, and TMB, had better disease-free survival (DFS) and overall survival (OS). Obvious immunosuppression, increased epithelial–mesenchymal transition, a low H3K4me3 level, shortened cell cycle, and accelerated cell division manifested in high-risk patients, with poorer DFS and OS. The model showed a better prognostic value than the tumor immune dysfunction and exclusion score. Correlation analysis told us that patients with high scores were suitable for treatment with CD276 inhibitors for their higher levels of CD276 expression. The risk score had a strong negative correlation with HAVCR2 and ICOS among patients with EGFR-WT, KRAS-WT, STK11-WT, or TP53-MUT, and patients with these mutation types with low scores were suitable for treatment with HAVCR2 or ICOS inhibitors. This work comprehensively analyzed the role and mechanism of TMEMs in LUAD and revealed the characteristics of histone methylation modification. The TMEM-based signature gave us deep insight into immune cell infiltration profiles and provided an individualized immunotherapy strategy.

Strategy for enhanced performance of silicon nanoparticle anodes for lithium-ion batteries.

The modification of silicon nanoparticles for lithium-ion battery anode materials has been a hot exploration subject in light of their excellent volume buffering performance. However, huge volume expansion leads to an unstable solid electrolyte interface (SEI) layer on the surface of the silicon anode material, resulting in short cell cycle life, which is an important factor limiting the application of silicon nanoparticles. Herein, a dual protection strategy to improve the cycling stability of commercial silicon nanoparticles is demonstrated. Specifically, the Si/s-C@TiO2 composite was produced by the hydrothermal method to achieve the embedding of commercial silicon nanoparticles in spherical carbon and the coating of the amorphous TiO2 shell on the outer surface. Buffering of silicon nanoparticle volume expansion by spherical carbon and also the stabilization of the TiO2 shell with high mechanical strength on the surface constructed a stable outer surface SEI layer of the new Si/s-C@TiO2 electrode during longer cycling. In addition, the spherical carbon and lithiated TiO2 further enhanced the electronic and ionic conductivity of the composite. Electrochemical measurements showed that the Si/s-C@TiO2 composite exhibited excellent lithium storage performance (780 mA h g−1 after 100 cycles at a current density of 0.2 A g−1 with a coulombic efficiency of 99%). Our strategy offers new ideas for the production of high stability and high-performance anode materials for lithium-ion batteries.

Developing Live Imaging Strategies to Analyze the Influence of Cell Cycle Rate on MEP Fate Decisions at the Single Cell Level

Elucidating the mechanisms underlying hematopoietic cell fate decision is critical to our understanding of physiological and pathological hematopoiesis. Robust response to physiological stress requires rapid commitment and expansion of mature blood cells derived from less committed progenitors. The processes dictating the directed differentiation of these progenitors are poorly understood.Preliminary data from our lab suggest that cell cycle rate influences commitment of the Megakaryocyte/Erythroid Progenitor (MEP) to the megakaryocytic and erythroid (Er) lineages. To test whether slower cycling MEPs tend to be more committed to the megakaryocytic lineage, we have developed a time-lapse microscopy imaging approach to measure the number and duration of cell cycles of single cells and their progeny as they make their cell fate decision determined by their fluorescent marker expression in real-time.CD34-selected mobilized peripheral blood cells were stained with desired antibodies and sorted using our recently published scheme: Lin-CD34+CD38midCD45RA-FLT3-MPL+CD36-CD41- (Sanada and Xavier-Ferrucio, et al. Blood 2016) that is highly enriched for bipotent MEPs than populations previously reported. MEPs were plated at low density (25 cells/15μL) in MegaCult collagen-based medium containing SCF, IL3, IL6, TPO, and EPO in the center of a MatTek plate under a coverslip to minimize focal range. After the MegaCult polymerized, 1.5mL of additional MegaCult and cytokines were added to the dish to support growth and expansion of megakaryocytes and erythroid cells for 12 days. Cells were imaged every 2 hours in DIC for the first three days, and every 20 minutes for 8 additional days. On the 10th day, fluorescently conjugated anti-CD41 (to identify Mks) and anti-CD235a (to identify Er cells) antibodies were diluted in IMDM and added to the imaged dish. The antibodies permeated the MegaCult and labeled the committed cells. Imaging continued every 20 minutes in DIC, FITC, and Cy5 channels for an additional day. Using the Baxter Algorithm to analyze time-lapse images, we traced the progeny back to the parent cell, and calculated the number of cell cycles, as well as the duration of each cycle leading to lineage commitment. We have validated this method by measuring cell cycle rates of bi-potent MEPs, Mk-restricted progenitors, and Er-restricted progenitors, and found Er-restricted progenitors to have a significantly shortened cell cycle rate compared to Mk-restricted progenitors.To further investigate the role of cell cycle rate in MEP commitment, we utilized our live imaging strategy to collect and analyze cell cycle rate data for transduced MEPs expressing shRNA against Myb. Myb is a known regulator of the G2/M transition by binding the promoter and altering expression of Cyclin B1 in human blood cells (Nakata et al., MCB, 2007). Here we show that MEPs knocked down for Myb have no difference in survival compared to scramble controls. Interestingly, the rate of cell cycle divisions is significantly longer in MEP progeny lacking Myb, and this correlates with an increase in the ratio of CFU-Mk to BFU-E and CFU-Mk/E. This data suggests that by lengthening the cell cycle rate, fate decision of the bipotent MEP can be swayed towards the Mk lineage, which has a longer cell cycle rate compared to the Er lineage. In the future, live imaging analysis of single cells can be utilized to answer a plethora of questions regarding fate decisions and lineage commitment of hematopoietic stem and progenitor cells. DisclosuresNo relevant conflicts of interest to declare.

Numerical simulation of skin formation: epidermal stem cells and undulation of the basal layer

The basal layer at the base of the epidermis produces new epidermal cells once a week and plays an important role in skin formation. Interestingly, the basal layer undulates, and the amplitude of this undulation is dependent on age or disease. However, the mechanisms driving this phenomenon have not been well studied. This work simulated the skin formation process using an original method to understand the mechanism of the undulation formation in the basal layer. The basal layer has cells with both short and long cell cycles, but only the former has been examined in previous studies. Accordingly, we more accurately simulated the skin formation process by including cells with a long cell cycle and observed undulation of the basal layer. Based on our findings, we propose that the basal layer undulates due to the interaction between two types of basal layer cells.

MiR-302 Regulates Glycolysis to Control Cell-Cycle during Neural Tube Closure.

Neural tube closure is a critical early step in central nervous system development that requires precise control of metabolism to ensure proper cellular proliferation and differentiation. Dysregulation of glucose metabolism during pregnancy has been associated with neural tube closure defects (NTDs) in humans suggesting that the developing neuroepithelium is particularly sensitive to metabolic changes. However, it remains unclear how metabolic pathways are regulated during neurulation. Here, we used single-cell mRNA-sequencing to analyze expression of genes involved in metabolism of carbon, fats, vitamins, and antioxidants during neurulation in mice and identify a coupling of glycolysis and cellular proliferation to ensure proper neural tube closure. Using loss of miR-302 as a genetic model of cranial NTD, we identify misregulated metabolic pathways and find a significant upregulation of glycolysis genes in embryos with NTD. These findings were validated using mass spectrometry-based metabolite profiling, which identified increased glycolytic and decreased lipid metabolites, consistent with a rewiring of central carbon traffic following loss of miR-302. Predicted miR-302 targets Pfkp, Pfkfb3, and Hk1 are significantly upregulated upon NTD resulting in increased glycolytic flux, a shortened cell cycle, and increased proliferation. Our findings establish a critical role for miR-302 in coordinating the metabolic landscape of neural tube closure.

Intramitochondrial Src kinase links mitochondrial dysfunctions and aggressiveness of breast cancer cells

High levels and activity of Src kinase are common among breast cancer subtypes, and several inhibitors of the kinase are currently tested in clinical trials. Alterations in mitochondrial activity is also observed among the different types of breast cancer. Src kinase is localized in several subcellular compartments, including mitochondria where it targets several proteins to modulate the activity of the organelle. Although the subcellular localization of other oncogenes modulates the potency of known treatments, nothing is known about the specific role of intra-mitochondrial Src (mtSrc) in breast cancer. The aim of this work was to determine whether mtSrc kinase has specific impact on breast cancer cells. We first observed that activity of mtSrc is higher in breast cancer cells of the triple negative subtype. Over-expression of Src specifically targeted to mitochondria reduced mtDNA levels, mitochondrial membrane potential and cellular respiration. These alterations of mitochondrial functions led to lower cellular viability, shorter cell cycle and increased invasive capacity. Proteomic analyses revealed that mtSrc targets the mitochondrial single-stranded DNA-binding protein, a regulator of mtDNA replication. Our findings suggest that mtSrc promotes aggressiveness of breast cancer cells via phosphorylation of mitochondrial single-stranded DNA-binding protein leading to reduced mtDNA levels and mitochondrial activity. This study highlights the importance of considering the subcellular localization of Src kinase in the development of potent therapy for breast cancer.

Developing Single Cell Live Imaging Strategies to Determine MEP Fate and Predict Potential

The molecular mechanisms underlying lineage commitment of stem and progenitor cells have implications for deriving specific cell types in vitro for regenerative medicine purposes and elucidating the aberrant pathways responsible for pathological conditions. We investigated Megakaryocytic-Erythroid Progenitors (MEP) commitment to the megakaryocytic (Mk) and erythroid (E) lineages as a model of cell fate decisions. Colony forming unit (CFU) assays are used to test the functional output, or lineage potential, of progenitor cell populations. As single progenitor cells proliferate, their progeny remain near each other to form a colony. This potential is deduced from the mature cell types comprising the colony. However, this assay has several limitations: 1) there is an assumption that each colony arose from a single progenitor cell based on the low probability that individual progenitors would form overlapping colonies when plated sparsely; 2) there is a surfeit of kinetic data about the colony forming cells that is not collected with a single end-point; and 3) the outcome may be misleading if there is selective loss of specific cell types of early committed progeny prior to the endpoint. To overcome these limitations, we developed a time-lapse microscopy and lineage tracing approach to visualize single sorted MEP, as well as committed Megakaryocytic progenitors (MkP) and Erythroid progenitors (ErP), as they proliferate, specify and progress down either the Mk or E lineages. We plated primary adult human MEP (Sanada and Xavier-Ferrucio, et al. Blood, 2016) at low density in semisolid media supplemented with cytokines in a MatTek plate under a coverslip to minimize focal range and support colony formation for up to 14 days. The plated cells were imaged with an Olympus VivaView. Fluorescently conjugated anti-CD41 (a marker of Mk) and anti-CD235a (a marker of E) were added to the culture dishes at the end to confirm the lineage of each cell in the colonies. The average time from plating to the first division was 38 h, and not significantly different between MEP, ErP, and MkP. We optimized the image acquisition settings to permit accurate tracking of the plated cells and their progeny for up to 11 days. Average colony forming efficiency was 75%, which was equal to unimaged CFU assays, indicating minimal phototoxicity. Acquired images were stacked into time-lapse videos and automatically tracked with the Baxter Algorithm (Magnusson et al. IEEE Trans Med Imaging, 2015). After manual segmentation and track correction, lineage trees were generated for each colony (Figure 1). We exported quantifiable features of the cells including the time between cell divisions and velocity of each cell from the algorithm. We defined cells that give rise to only CD41-labeled cells as committed MkP, cells that give rise to only CD235-labeled cells as ErP, and cells that give rise to at least one progeny of both as MEP. However, these progenitor definitions are based on an inaccurate assumption that fate reflects potential, since a bipotent cell may stochastically give rise to progeny that are committed to a single lineage. To convert fate to potential, we first need to identify the precise period of commitment to more accurately define MEP, MkP, and ErP in our culture system. To achieve this, we are developing a mathematical model that can predict the potential of cells based on quantifiable features that are measured form the time-lapse images and are significantly different between MEP, MkP, and ErP. Utilizing statistical tools, we have identified that the duration of the cell cycle and the velocity of single progenitor cells along with their lineage history, permit predictions of progenitor potential and provide several novel insights into the process of lineage commitment. We found that ErP have a significantly shortened cell cycle duration and slower velocity compared to MkP, while MEP have the longest cell cycle and an intermediate velocity until undergoing their fate decisions (Figures 2 and 3). With this time-lapse imaging approach, we hope to better investigate molecular mechanisms that direct fate decisions of multipotent progenitor cells. Disclosures No relevant conflicts of interest to declare.

Nucleoid-mediated positioning and transport in bacteria.

Precise management of the spatiotemporal position of subcellular components is critical to a number of essential processes in the bacterial cell. The bacterial nucleoid is a highly structured yet dynamic object that undergoes significant reorganization during the relatively short cell cycle, e.g. during gene expression, chromosome replication, and segregation. Although the nucleoid takes up a large fraction of the volume of the cell, the mobility of macromolecules within these dense regions is relatively high and recent results suggest that the nucleoid plays an integral role of dynamic localization in a host of seemingly disparate cellular processes. Here, we review a number of recent reports of nucleoid-mediated positioning and transport in the model bacteria Escherichia coli. These results viewed as a whole suggest that the dynamic, cellular-scale structure of the nucleoid may be a key driver of positioning and transport within the cell. This model of a global, default positioning and transport system may help resolve many unanswered questions about the mechanisms of partitioning and segregation in bacteria.

Multiple Negative Regulators Restrict Recruitment of the SWI/SNF Chromatin Remodeler to the HO Promoter in Saccharomyces cerevisiae.

Activation of the Saccharomyces cerevisiae HO promoter is highly regulated, requiring the ordered recruitment of activators and coactivators and allowing production of only a few transcripts in mother cells within a short cell cycle window. We conducted genetic screens to identify the negative regulators of HO expression necessary to limit HO transcription. Known repressors of HO (Ash1 and Rpd3) were identified, as well as several additional chromatin-associated factors including the Hda1 histone deacetylase, the Isw2 chromatin remodeler, and the corepressor Tup1 We also identified clusters of HO promoter mutations that suggested roles for the Dot6/Tod6 (PAC site) and Ume6 repression pathways. We used ChIP assays with synchronized cells to validate the involvement of these factors and map the association of Ash1, Dot6, and Ume6 with the HO promoter to a brief window in the cell cycle between binding of the initial activating transcription factor and initiation of transcription. We found that Ash1 and Ume6 each recruit the Rpd3 histone deacetylase to HO, and their effects are additive. In contrast, Rpd3 was not recruited significantly to the PAC site, suggesting this site has a distinct mechanism for repression. Increases in HO expression and SWI/SNF recruitment were all additive upon loss of Ash1, Ume6, and PAC site factors, indicating the convergence of independent pathways for repression. Our results demonstrate that multiple protein complexes are important for limiting the spread of SWI/SNF-mediated nucleosome eviction across the HO promoter, suggesting that regulation requires a delicate balance of activities that promote and repress transcription.