Low-frequency ERK and Akt activity dynamics are predictive of stochastic cell division events.

Understanding the dynamics of intracellular signaling pathways, such as ERK1/2 (ERK) and Akt1/2 (Akt), in the context of cell fate decisions is important for advancing our knowledge of cellular processes and diseases, particularly cancer. While previous studies have established associations between ERK and Akt activities and proliferative cell fate, the heterogeneity of single-cell responses adds complexity to this understanding. This study employed a data-driven approach to address this challenge, developing machine learning models trained on a dataset of growth factor-induced ERK and Akt activity time courses in single cells, to predict cell division events. The most predictive models were developed by applying discrete wavelet transforms (DWTs) to extract low-frequency features from the time courses, followed by using Ensemble Integration, a data integration and predictive modeling framework. The results demonstrated that these models effectively predicted cell division events in MCF10A cells (F-measure=0.524, AUC=0.726). ERK dynamics were found to be more predictive than Akt, but the combination of both measurements further enhanced predictive performance. The ERK model`s performance also generalized to predicting division events in RPE cells, indicating the potential applicability of these models and our data-driven methodology for predicting cell division across different biological contexts. Interpretation of these models suggested that ERK dynamics throughout the cell cycle, rather than immediately after growth factor stimulation, were associated with the likelihood of cell division. Overall, this work contributes insights into the predictive power of intra-cellular signaling dynamics for cell fate decisions, and highlights the potential of machine learning approaches in unraveling complex cellular behaviors.

Opposing polarity domains provide direction and play a role in cell division in plant growth.

Polar localization of proteins is important for plant growth and development. Identifying the interactors of polarized proteins provides spatial information and cell-type functions. In this issue of Developmental Cell, Wallner et al. (2024) utilize opposing polarity domain proteins to identify interactors and their functions during cell division in Arabidopsis stomata.

Escherichia coli self-organizes developmental rosettes.

Rosettes are self-organizing, circular multicellular communities that initiate developmental processes, like organogenesis and embryogenesis, in complex organisms. Their formation results from the active repositioning of adhered sister cells and is thought to distinguish multicellular organisms from unicellular ones. Though common in eukaryotes, this multicellular behavior has not been reported in bacteria. In this study, we found that Escherichia coli forms rosettes by active sister-cell repositioning. After division, sister cells "fold" to actively align at the 2- and 4-cell stages of clonal division, thereby producing rosettes with characteristic quatrefoil configuration. Analysis revealed that folding follows an angular random walk, composed of ~1 µm strokes and directional randomization. We further showed that this motion was produced by the flagellum, the extracellular tail whose rotation generates swimming motility. Rosette formation was found to require de novo flagella synthesis suggesting it must balance the opposing forces of Ag43 adhesion and flagellar propulsion. We went on to show that proper rosette formation was required for subsequent morphogenesis of multicellular chains, rpoS gene expression, and formation of hydrostatic clonal-chain biofilms. Moreover, we found self-folding rosette-like communities in the standard motility assay, indicating that this behavior may be a general response to hydrostatic environments in E. coli. These findings establish self-organization of clonal rosettes by a prokaryote and have implications for evolutionary biology, synthetic biology, and medical microbiology.

Differential growth regulates asymmetric size partitioning in Caulobacter crescentus.

Cell size regulation has been extensively studied in symmetrically dividing cells, but the mechanisms underlying the control of size asymmetry in asymmetrically dividing bacteria remain elusive. Here, we examine the control of asymmetric division in Caulobacter crescentus, a bacterium that produces daughter cells with distinct fates and morphologies upon division. Through comprehensive analysis of multi-generational growth and shape data, we uncover a tightly regulated cell size partitioning mechanism. We find that errors in division site positioning are promptly corrected early in the division cycle through differential growth. Our analysis reveals a negative feedback between the size of daughter cell compartments and their growth rates, wherein the larger compartment grows slower to achieve a homeostatic size partitioning ratio at division. To explain these observations, we propose a mechanistic model of differential growth, in which equal amounts of growth regulators are partitioned into daughter cell compartments of unequal sizes and maintained over time via size-independent synthesis.

The first two blastomeres contribute unequally to the human embryo.

Retrospective lineage reconstruction of humans predicts that dramatic clonal imbalances in the body can be traced to the 2-cell stage embryo. However, whether and how such clonal asymmetries arise in the embryo is unclear. Here, we performed prospective lineage tracing of human embryos using live imaging, non-invasive cell labeling, and computational predictions to determine the contribution of each 2-cell stage blastomere to the epiblast (body), hypoblast (yolk sac), and trophectoderm (placenta). We show that the majority of epiblast cells originate from only one blastomere of the 2-cell stage embryo. We observe that only one to three cells become internalized at the 8-to-16-cell stage transition. Moreover, these internalized cells are more frequently derived from the first cell to divide at the 2-cell stage. We propose that cell division dynamics and a cell internalization bottleneck in the early embryo establish asymmetry in the clonal composition of the future human body.

Structure and activity of the septal peptidoglycan hydrolysis machinery crucial for bacterial cell division.

The peptidoglycan (PG) layer is a critical component of the bacterial cell wall and serves as an important target for antibiotics in both gram-negative and gram-positive bacteria. The hydrolysis of septal PG (sPG) is a crucial step of bacterial cell division, facilitated by FtsEX through an amidase activation system. In this study, we present the cryo-EM structures of Escherichia coli FtsEX and FtsEX-EnvC in the ATP-bound state at resolutions of 3.05 Å and 3.11 Å, respectively. Our PG degradation assays in E. coli reveal that the ATP-bound conformation of FtsEX activates sPG hydrolysis of EnvC-AmiB, whereas EnvC-AmiB alone exhibits autoinhibition. Structural analyses indicate that ATP binding induces conformational changes in FtsEX-EnvC, leading to significant differences from the apo state. Furthermore, PG degradation assays of AmiB mutants confirm that the regulation of AmiB by FtsEX-EnvC is achieved through the interaction between EnvC-AmiB. These findings not only provide structural insight into the mechanism of sPG hydrolysis and bacterial cell division, but also have implications for the development of novel therapeutics targeting drug-resistant bacteria.

Defensins regulate cell cycle: Insights of defensins on cellular proliferation and division.

Defensins are a class of small antimicrobial peptides that play a crucial role against pathogens. However, recent research has highlighted defensins exhibit the ability to influence cell cycle checkpoints, promoting or inhibiting specific phases such as G1 arrest or S/M transition. By regulating the cell cycle, defensins impact the proliferation of normal and cancerous cells, with implications for cancer development and progression. Dysregulation of defensin expression can disrupt the delicate balance of cell cycle regulation, leading to uncontrolled cell growth and an increased risk of tumor formation. Defensins contribute to the resolution of inflammation, stimulate angiogenesis, and enhance the migration and proliferation of cells involved in tissue repair. Furthermore, The ability of defensins to respond to microenvironmental changes further demonstrates the significance of these peptides in host defense mechanisms and immune function. By adjusting their expression, defensins continue to combat pathogens effectively and maintain homeostasis within the body. This review highlights the multifaceted role of defensins in regulating the cell cycle and their broader implications in cancer progression, tissue repair, and microenvironmental response.

Feedback between stochastic gene networks and population dynamics enables cellular decision-making.

Phenotypic selection occurs when genetically identical cells are subject to different reproductive abilities due to cellular noise. Such noise arises from fluctuations in reactions synthesizing proteins and plays a crucial role in how cells make decisions and respond to stress or drugs. We propose a general stochastic agent-based model for growing populations capturing the feedback between gene expression and cell division dynamics. We devise a finite state projection approach to analyze gene expression and division distributions and infer selection from single-cell data in mother machines and lineage trees. We use the theory to quantify selection in multi-stable gene expression networks and elucidate that the trade-off between phenotypic switching and selection enables robust decision-making essential for synthetic circuits and developmental lineage decisions. Using live-cell data, we demonstrate that combining theory and inference provides quantitative insights into bet-hedging-like response to DNA damage and adaptation during antibiotic exposure in Escherichia coli.

Mechanisms of cell size regulation in slow-growing Escherichia coli cells: discriminating models beyond the adder.

Under ideal conditions, Escherichia coli cells divide after adding a fixed cell size, a strategy known as the adder. This concept applies to various microbes and is often explained as the division that occurs after a certain number of stages, associated with the accumulation of precursor proteins at a rate proportional to cell size. However, under poor media conditions, E. coli cells exhibit a different size regulation. They are smaller and follow a sizer-like division strategy where the added size is inversely proportional to the size at birth. We explore three potential causes for this deviation: degradation of the precursor protein and two models where the propensity for accumulation depends on the cell size: a nonlinear accumulation rate, and accumulation starting at a threshold size termed the commitment size. These models fit the mean trends but predict different distributions given the birth size. To quantify the precision of the models to explain the data, we used the Akaike information criterion and compared them to open datasets of slow-growing E. coli cells in different media. We found that none of the models alone can consistently explain the data. However, the degradation model better explains the division strategy when cells are larger, whereas size-related models (power-law and commitment size) account for smaller cells. Our methodology proposes a data-based method in which different mechanisms can be tested systematically.

Queuosine biosynthetic enzyme, QueE moonlights as a cell division regulator.

In many organisms, stress responses to adverse environments can trigger secondary functions of certain proteins by altering protein levels, localization, activity, or interaction partners. Escherichia coli cells respond to the presence of specific cationic antimicrobial peptides by strongly activating the PhoQ/PhoP two-component signaling system, which regulates genes important for growth under this stress. As part of this pathway, a biosynthetic enzyme called QueE, which catalyzes a step in the formation of queuosine (Q) tRNA modification is upregulated. When cellular QueE levels are high, it co-localizes with the central cell division protein FtsZ at the septal site, blocking division and resulting in filamentous growth. Here we show that QueE affects cell size in a dose-dependent manner. Using alanine scanning mutagenesis of amino acids in the catalytic active site, we pinpoint residues in QueE that contribute distinctly to each of its functions-Q biosynthesis or regulation of cell division, establishing QueE as a moonlighting protein. We further show that QueE orthologs from enterobacteria like Salmonella typhimurium and Klebsiella pneumoniae also cause filamentation in these organisms, but the more distant counterparts from Pseudomonas aeruginosa and Bacillus subtilis lack this ability. By comparative analysis of E. coli QueE with distant orthologs, we elucidate a unique region in this protein that is responsible for QueE's secondary function as a cell division regulator. A dual-function protein like QueE is an exception to the conventional model of "one gene, one enzyme, one function", which has divergent roles across a range of fundamental cellular processes including RNA modification and translation to cell division and stress response.

GTPase activity regulates FtsZ ring positioning in Caulobacter crescentus.

Bacterial cell division is crucial for replication and requires careful coordination via proteins collectively called the divisome. The tubulin-like GTPase FtsZ is the master regulator of this process and serves to recruit downstream divisome proteins and regulate their activities. Upon assembling at mid-cell, FtsZ exhibits treadmilling motion driven by GTP binding and hydrolysis. Treadmilling is proposed to play roles in Z-ring condensation and in distribution and regulation of peptidoglycan (PG) cell wall enzymes. FtsZ polymer superstructure and dynamics are central to its function, yet their regulation is incompletely understood. We addressed these gaps in knowledge by evaluating the contribution of GTPase activity to FtsZ's function in vitro and in Caulobacter crescentus cells. We observed that a lethal mutation that abrogates FtsZ GTP hydrolysis impacts FtsZ dynamics and Z-ring positioning, but not constriction. Aberrant Z-ring positioning was due to insensitivity to the FtsZ regulator MipZ when GTPase activity is reduced. Z-ring mislocalization resulted in DNA damage, likely due to constriction over the nucleoid. Collectively, our results indicate that GTP hydrolysis serves primarily to position the Z-ring at mid-cell in Caulobacter. Proper Z-ring localization is required for effective coordination with chromosome segregation to prevent DNA damage and ensure successful cell division.

Arabidopsis AN3 and OLIGOCELLULA genes link telomere maintenance mechanisms with cell division and expansion control.

Telomeres are conserved chromosomal structures necessary for continued cell division and proliferation. In addition to the classical telomerase pathway, multiple other genes including those involved in ribosome metabolism and chromatin modification contribute to telomere length maintenance. We previously reported that Arabidopsis thaliana ribosome biogenesis genes OLI2/NOP2A, OLI5/RPL5A and OLI7/RPL5B have critical roles in telomere length regulation. These three OLIGOCELLULA genes were also shown to function in cell proliferation and expansion control and to genetically interact with the transcriptional co-activator ANGUSTIFOLIA3 (AN3). Here we show that AN3-deficient plants progressively lose telomeric DNA in early homozygous mutant generations, but ultimately establish a new shorter telomere length setpoint by the fifth mutant generation with a telomere length similar to oli2/nop2a -deficient plants. Analysis of double an3 oli2 mutants indicates that the two genes are epistatic for telomere length control. Telomere shortening in an3 and oli mutants is not caused by telomerase inhibition; wild type levels of telomerase activity are detected in all analyzed mutants in vitro. Late generations of an3 and oli mutants are prone to stem cell damage in the root apical meristem, implying that genes regulating telomere length may have conserved functional roles in stem cell maintenance mechanisms. Multiple instances of anaphase fusions in late generations of oli5 and oli7 mutants were observed, highlighting an unexpected effect of ribosome biogenesis factors on chromosome integrity. Overall, our data implicate AN3 transcription coactivator and OLIGOCELLULA proteins in the establishment of telomere length set point in plants and further suggest that multiple regulators with pleiotropic functions can connect telomere biology with cell proliferation and cell expansion pathways.

Germ fate determinants protect germ precursor cell division by reducing septin and anillin levels at the cell division plane.

Animal cell cytokinesis, or the physical division of one cell into two, is thought to be driven by constriction of an actomyosin contractile ring at the division plane. The mechanisms underlying cell type-specific differences in cytokinesis remain unknown. Germ cells are totipotent cells that pass genetic information to the next generation. Previously, using formincyk-1(ts) mutant Caenorhabditis elegans 4-cell embryos, we found that the P2 germ precursor cell is protected from cytokinesis failure and can divide with greatly reduced F-actin levels at the cell division plane. Here, we identified two canonical germ fate determinants required for P2-specific cytokinetic protection: PIE-1 and POS-1. Neither has been implicated previously in cytokinesis. These germ fate determinants protect P2 cytokinesis by reducing the accumulation of septinUNC-59 and anillinANI-1 at the division plane, which here act as negative regulators of cytokinesis. These findings may provide insight into the regulation of cytokinesis in other cell types, especially in stem cells with high potency.

Deciphering molecular heterogeneity and dynamics of human hippocampal neural stem cells at different ages and injury states.

While accumulated publications support the existence of neurogenesis in the adult human hippocampus, the homeostasis and developmental potentials of neural stem cells (NSCs) under different contexts remain unclear. Based on our generated single-nucleus atlas of the human hippocampus across neonatal, adult, aging, and injury, we dissected the molecular heterogeneity and transcriptional dynamics of human hippocampal NSCs under different contexts. We further identified new specific neurogenic lineage markers that overcome the lack of specificity found in some well-known markers. Based on developmental trajectory and molecular signatures, we found that a subset of NSCs exhibit quiescent properties after birth, and most NSCs become deep quiescence during aging. Furthermore, certain deep quiescent NSCs are reactivated following stroke injury. Together, our findings provide valuable insights into the development, aging, and reactivation of the human hippocampal NSCs, and help to explain why adult hippocampal neurogenesis is infrequently observed in humans.

Cambium Reactivation Is Closely Related to the Cell-Cycle Gene Configuration in Larix kaempferi.

Dormancy release and reactivation in temperate trees are mainly controlled by temperature and are affected by age, but the underlying molecular mechanisms are still unclear. In this study, we explored the effects of low temperatures in winter and warm temperatures in spring on dormancy release and reactivation in Larix kaempferi. Further, we established the relationships between cell-cycle genes and cambium cell division. The results showed that chilling accelerated L. kaempferi bud break overall, and the longer the duration of chilling is, the shorter the bud break time is. After dormancy release, warm temperatures induced cell-cycle gene expression; when the configuration value of the cell-cycle genes reached 4.97, the cambium cells divided and L. kaempferi reactivated. This study helps to predict the impact of climate change on wood production and provides technical support for seedling cultivation in greenhouses.

Development and validation of a nomogram to predict cancer-specific survival of elderly patients with unresected gastric cancer who received chemotherapy.

This investigation aimed to explore the prognostic factors in elderly patients with unresected gastric cancer (GC) who have received chemotherapy and to develop a nomogram for predicting their cancer-specific survival (CSS). Elderly gastric cancer patients who have received chemotherapy but no surgery in the Surveillance, Epidemiology, and End Results Database between 2004 and 2015 were included in this study. Cox analyses were conducted to identify prognostic factors, leading to the formulation of a nomogram. The nomogram was validated using receiver operating characteristic (ROC) and calibration curves. The findings elucidated six prognostic factors encompassing grade, histology, M stage, radiotherapy, tumor size, and T stage, culminating in the development of a nomogram. The ROC curve indicated that the area under curve of the nomogram used to predict CSS for 3, 4, and 5 years in the training queue as 0.689, 0.708, and 0.731, and in the validation queue, as 0.666, 0.693, and 0.708. The calibration curve indicated a high degree of consistency between actual and predicted CSS for 3, 4, and 5 years. This nomogram created to predict the CSS of elderly patients with unresected GC who have received chemotherapy could significantly enhance treatment accuracy.

Mitochondria complex I deficiency in Candida albicans arrests the cell cycle at S phase through suppressive TOR and PKA pathways.

How mutations in mitochondrial electron transport chain (ETC) proteins impact the cell cycle of Candida albicans was investigated in this study. Using genetic null mutants targeting ETC complexes I (CI), III (CIII), and IV (CIV), the cell cycle stages (G0/G1, S phase, and G2/M) were analyzed via fluorescence-activated cell sorting (FACS). Four CI null mutants exhibited distinct alterations, including extended S phase, shortened G2/M population, and a reduction in cells size exceeding 10 µM. Conversely, CIII mutants showed an increased population in G1/G0 phase. Among four CI mutants, ndh51Δ/Δ and goa1Δ/Δ displayed aberrant cell cycle patterns correlated with previously reported cAMP/PKA downregulation. Specifically, nuo1Δ/Δ and nuo2Δ/Δ mutants exhibited increased transcription of RIM15, a central hub linking cell cycle with nutrient-dependent TOR1 and cAMP/PKA pathways and Snf1 aging pathway. These findings suggest that suppression of TOR1 and cAMP/PKA pathways or enhanced Snf1 disrupts cell cycle progression, influencing cell longevity and growth among CI mutants. Overall, our study highlights the intricate interplay between mitochondrial ETC, cell cycle, and signaling pathways.

Pyrrole-based inhibitors of RND-type efflux pumps reverse antibiotic resistance and display anti-virulence potential.

Efflux pumps of the resistance-nodulation-cell division (RND) superfamily, particularly the AcrAB-TolC, and MexAB-OprM, besides mediating intrinsic and acquired resistance, also intervene in bacterial pathogenicity. Inhibitors of such pumps could restore the activities of antibiotics and curb bacterial virulence. Here, we identify pyrrole-based compounds that boost antibiotic activity in Escherichia coli and Pseudomonas aeruginosa by inhibiting their archetype RND transporters. Molecular docking and biophysical studies revealed that the EPIs bind to AcrB. The identified efflux pump inhibitors (EPIs) inhibit the efflux of fluorescent probes, attenuate persister formation, extend post-antibiotic effect, and diminish resistant mutant development. The bacterial membranes remained intact upon exposure to the EPIs. EPIs also possess an anti-pathogenic potential and attenuate P. aeruginosa virulence in vivo. The intracellular invasion of E. coli and P. aeruginosa inside the macrophages was hampered upon treatment with the lead EPI. The excellent efficacy of the EPI-antibiotic combination was evidenced in animal lung infection and sepsis protection models. These findings indicate that EPIs discovered herein with negligible toxicity are potential antibiotic adjuvants to address life-threatening Gram-negative bacterial infections.

Compound 225# inhibits the proliferation of human colorectal cancer cells by promoting cell cycle arrest and apoptosis induction.

Colorectal cancer (CRC) ranks as the second leading cause of cancer­related death worldwide due to its aggressive nature. After surgical resection, >50% of patients with CRC require adjuvant therapy. As a result, eradicating cancer cells with medications is a promising method to treat patients with CRC. In the present study, a novel compound was synthesized, which was termed compound 225#. The inhibitory activity of compound 225# against CRC was determined by MTT assay, EdU fluorescence labeling and colony formation assay; the effects of compound 225# on the cell cycle progression and apoptosis of CRC cells were detected by flow cytometry and western blotting; and the changes in autophagic flux after the administration of compound 225# were detected using the double fluorescence fusion protein mCherry­GFP­LC3B and western blotting. The results demonstrated that compound 225# exhibited antiproliferative properties, inhibiting the proliferation and expansion of CRC cell lines in a time­ and dose­dependent manner. Furthermore, compound 225# triggered G2/M cell cycle arrest by influencing the expression of cell cycle regulators, such as CDK1, cyclin A1 and cyclin B1, which is also closely related to the activation of DNA damage pathways. The cleavage of PARP and increased protein expression levels of PUMA suggested that apoptosis was triggered after treatment with compound 225#. Moreover, the increase in LC3­II expression and stimulation of autophagic flux indicated the activation of an autophagy pathway. Notably, compound 225# induced autophagy, which was associated with endoplasmic reticulum (ER) stress. In accordance with the in vitro findings, the in vivo results demonstrated that compound 225# effectively inhibited the growth of HCT116 tumors in mice without causing any changes in their body weight. Collectively, the present results demonstrated that compound 225# not only inhibited proliferation and promoted G2/M­phase cell cycle arrest and apoptosis, but also initiated cytoprotective autophagy in CRC cells by activating ER stress pathways. Taken together, these findings provide an experimental basis for the evaluation of compound 225# as a novel potential medication for CRC treatment.

Determinants of minor satellite RNA function in chromosome segregation in mouse embryonic stem cells.

The centromere is a fundamental higher-order structure in chromosomes ensuring their faithful segregation upon cell division. Centromeric transcripts have been described in several species and suggested to participate in centromere function. However, low sequence conservation of centromeric repeats appears inconsistent with a role in recruiting highly conserved centromeric proteins. Here, we hypothesized that centromeric transcripts may function through a secondary structure rather than sequence conservation. Using mouse embryonic stem cells (ESCs), we show that an imbalance in the levels of forward or reverse minor satellite (MinSat) transcripts leads to severe chromosome segregation defects. We further show that MinSat RNA adopts a stem-loop secondary structure, which is conserved in human α-satellite transcripts. We identify an RNA binding region in CENPC and demonstrate that MinSat transcripts function through the structured region of the RNA. Importantly, mutants that disrupt MinSat secondary structure do not cause segregation defects. We propose that the conserved role of centromeric transcripts relies on their secondary RNA structure.