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.

Two sequential gene expression programs bridged by cell division support long-distance collective cell migration.

The precise assembly of tissues and organs relies on spatiotemporal regulation of gene expression to coordinate the collective behavior of cells. In Drosophila embryos, the midgut musculature is formed through collective migration of caudal visceral mesoderm (CVM) cells, but how gene expression changes as cells migrate is not well understood. Here, we have focused on ten genes expressed in the CVM and the cis-regulatory sequences controlling their expression. Although some genes are continuously expressed, others are expressed only early or late during migration. Late expression relates to cell cycle progression, as driving string/Cdc25 causes earlier division of CVM cells and accelerates the transition to late gene expression. In particular, we found that the cell cycle effector transcription factor E2F1 is a required input for the late gene CG5080. Furthermore, whereas late genes are broadly expressed in all CVM cells, early gene transcripts are polarized to the anterior or posterior ends of the migrating collective. We show this polarization requires transcription factors Snail, Zfh1 and Dorsocross. Collectively, these results identify two sequential gene expression programs bridged by cell division that support long-distance directional migration of CVM cells.

Single-cell analysis reveals context-dependent, cell-level selection of mtDNA.

Heteroplasmy occurs when wild-type and mutant mitochondrial DNA (mtDNA) molecules co-exist in single cells1. Heteroplasmy levels change dynamically in development, disease and ageing2,3, but it is unclear whether these shifts are caused by selection or drift, and whether they occur at the level of cells or intracellularly. Here we investigate heteroplasmy dynamics in dividing cells by combining precise mtDNA base editing (DdCBE)4 with a new method, SCI-LITE (single-cell combinatorial indexing leveraged to interrogate targeted expression), which tracks single-cell heteroplasmy with ultra-high throughput. We engineered cells to have synonymous or nonsynonymous complex I mtDNA mutations and found that cell populations in standard culture conditions purge nonsynonymous mtDNA variants, whereas synonymous variants are maintained. This suggests that selection dominates over simple drift in shaping population heteroplasmy. We simultaneously tracked single-cell mtDNA heteroplasmy and ancestry, and found that, although the population heteroplasmy shifts, the heteroplasmy of individual cell lineages remains stable, arguing that selection acts at the level of cell fitness in dividing cells. Using these insights, we show that we can force cells to accumulate high levels of truncating complex I mtDNA heteroplasmy by placing them in environments where loss of biochemical complex I activity has been reported to benefit cell fitness. We conclude that in dividing cells, a given nonsynonymous mtDNA heteroplasmy can be harmful, neutral or even beneficial to cell fitness, but that the 'sign' of the effect is wholly dependent on the environment.

ARGONAUTE10 controls cell fate specification and formative cell divisions in the Arabidopsis root.

A key question in plant biology is how oriented cell divisions are integrated with patterning mechanisms to generate organs with adequate cell type allocation. In the root vasculature, a gradient of miRNA165/6 controls the abundance of HD-ZIP III transcription factors, which in turn control cell fate and spatially restrict vascular cell proliferation to specific cells. Here, we show that vascular development requires the presence of ARGONAUTE10, which is thought to sequester miRNA165/6 and protect HD-ZIP III transcripts from degradation. Our results suggest that the miR165/6-AGO10-HDZIP III module acts by buffering cytokinin responses and restricting xylem differentiation. Mutants of AGO10 show faster growth rates and strongly enhanced survival under severe drought conditions. However, this superior performance is offset by markedly increased variation and phenotypic plasticity in sub-optimal carbon supply conditions. Thus, AGO10 is required for the control of formative cell division and coordination of robust cell fate specification of the vasculature, while altering its expression provides a means to adjust phenotypic plasticity.

The association analysis of DNA methylation and transcriptomics identified BpCYCD3;2 as a participant in influencing cell division in autotetraploid birch (Betula pendula) leaves.

Polyploidization plays a crucial role in plant breeding and genetic improvement. Although the phenomenon of polyploidization affecting the area and number of plant epidermal pavement cells is well described, the underlying mechanism behind this phenomenon is still largely unknown. In this study, we found that the leaves of autotetraploid birch (Betula pendula) stopped cell division earlier and had a larger cell area. In addition, compared to diploids, tetraploids have a smaller stomatal density and fewer stomatal numbers. Genome-wide DNA methylation analysis revealed no significant difference in global DNA methylation levels between diploids and tetraploids. A total of 9154 differential methylation regions (DMRs) were identified between diploids and tetraploids, with CHH-type DMRs accounting for 91.73% of all types of DMRs. Further research has found that there are a total of 2105 differentially methylated genes (DMEGs) with CHH-type DMRs in birch. The GO functional enrichment results of DMEGs showed that differentially methylated genes were mainly involved in terms such as cellular process and metabolic process. The analysis of differentially methylated genes and differentially expressed genes suggests that hyper-methylation in the promoter region may inhibit the gene expression level of BpCYCD3;2 in tetraploids. To investigate the function of BpCYCD3;2 in birch, we obtained overexpression and repressed expression lines of BpCYCD3;2 through genetic transformation. The morphogenesis of both BpCYCD3;2-OE and BpCYCD3;2-RE lines was not affected. However, low expression of BpCYCD3;2 can lead to inhibition of cell division in leaves, and this inhibition of cell proliferation can be compensated for by an increase in cell size. Additionally, we found that the number and density of stomata in the BpCYCD3;2-RE lines were significantly reduced, consistent with the tetraploid. These data indicate that changes in cell division ability and stomatal changes in tetraploid birch can be partially attributed to low expression of the BpCYCD3;2 gene, which may be related to hyper-methylation in its promoter region. These results will provide new insights into the mechanism by which polyploidization affects plant development.

An epigenetic timer regulates the transition from cell division to cell expansion during Arabidopsis petal organogenesis.

A number of studies have demonstrated that epigenetic factors regulate plant developmental timing in response to environmental changes. However, we still have an incomplete view of how epigenetic factors can regulate developmental events such as organogenesis, and the transition from cell division to cell expansion, in plants. The small number of cell types and the relatively simple developmental progression required to form the Arabidopsis petal makes it a good model to investigate the molecular mechanisms driving plant organogenesis. In this study, we investigated how the RABBIT EARS (RBE) transcriptional repressor maintains the downregulation of its downstream direct target, TCP5, long after RBE expression dissipates. We showed that RBE recruits the Groucho/Tup1-like corepressor TOPLESS (TPL) to repress TCP5 transcription in petal primordia. This process involves multiple layers of changes such as remodeling of chromatin accessibility, alteration of RNA polymerase activity, and histone modifications, resulting in an epigenetic memory that is maintained through multiple cell divisions. This memory functions to maintain cell divisions during the early phase of petal development, and its attenuation in a cell division-dependent fashion later in development enables the transition from cell division to cell expansion. Overall, this study unveils a novel mechanism by which the memory of an epigenetic state, and its cell-cycle regulated decay, acts as a timer to precisely control organogenesis.

Cell division machinery drives cell-specific gene activation during differentiation in Bacillus subtilis.

When faced with starvation, the bacterium Bacillus subtilis transforms itself into a dormant cell type called a "spore". Sporulation initiates with an asymmetric division event, which requires the relocation of the core divisome components FtsA and FtsZ, after which the sigma factor σF is exclusively activated in the smaller daughter cell. Compartment-specific activation of σF requires the SpoIIE phosphatase, which displays a biased localization on one side of the asymmetric division septum and associates with the structural protein DivIVA, but the mechanism by which this preferential localization is achieved is unclear. Here, we isolated a variant of DivIVA that indiscriminately activates σF in both daughter cells due to promiscuous localization of SpoIIE, which was corrected by overproduction of FtsA and FtsZ. We propose that the core components of the redeployed cell division machinery drive the asymmetric localization of DivIVA and SpoIIE to trigger the initiation of the sporulation program.

The global RNA-RNA interactome of Klebsiella pneumoniae unveils a small RNA regulator of cell division.

The ubiquitous RNA chaperone Hfq is involved in the regulation of key biological processes in many species across the bacterial kingdom. In the opportunistic human pathogen Klebsiella pneumoniae, deletion of the hfq gene affects the global transcriptome, virulence, and stress resistance; however, the ligands of the major RNA-binding protein in this species have remained elusive. In this study, we have combined transcriptomic, co-immunoprecipitation, and global RNA interactome analyses to compile an inventory of conserved and species-specific RNAs bound by Hfq and to monitor Hfq-mediated RNA-RNA interactions. In addition to dozens of RNA-RNA pairs, our study revealed an Hfq-dependent small regulatory RNA (sRNA), DinR, which is processed from the 3' terminal portion of dinI mRNA. Transcription of dinI is controlled by the master regulator of the SOS response, LexA. As DinR accumulates in K. pneumoniae in response to DNA damage, the sRNA represses translation of the ftsZ transcript by occupation of the ribosome binding site. Ectopic overexpression of DinR causes depletion of ftsZ mRNA and inhibition of cell division, while deletion of dinR antagonizes cell elongation in the presence of DNA damage. Collectively, our work highlights the important role of RNA-based gene regulation in K. pneumoniae and uncovers the central role of DinR in LexA-controlled division inhibition during the SOS response.

The bacterial division protein MinDE has an independent function in flagellation.

Before preparing for division, bacteria stop their motility. During the exponential growth phase in Escherichia coli, when the rate of bacterial division is highest, the expression of flagellar genes is repressed and bacterial adhesion is enhanced. Hence, it is evident that cell division and motility in bacteria are linked; however, the specific molecular mechanism by which these two processes are linked is not known. While observing E. coli, we found that compared to the WT, the E. coli (Δmin) cells show higher motility and flagellation. We demonstrated that the higher motility was due to the absence of the Min system and can be restored to normal in the presence of Min proteins, where Min system negatively regulates flagella formation. The Min system in E. coli is widely studied for its role in the inhibition of polar Z-ring formation through its pole-to-pole oscillation. However, its role in bacterial motility is not explored. MinD homologs, FlhG and FleN, are known to control flagellar expression through their interaction with FlrA and FleQ, respectively. AtoC, a part of the two-component system AtoSC complex, is homologous to FlrA/FleQ, and the complex is involved in E. coli flagellation via its interaction with the fliA promoter. We have shown that MinD interacts directly with the AtoS of AtoSC complex and controls the fliA expression. Our findings suggest that the Min system acts as a link between cell division and motility in E. coli.

DNA hypomethylation activates Cdk4/6 and Atr to induce DNA replication and cell cycle arrest to constrain liver outgrowth in zebrafish.

Coordinating epigenomic inheritance and cell cycle progression is essential for organogenesis. UHRF1 connects these functions during development by facilitating maintenance of DNA methylation and cell cycle progression. Here, we provide evidence resolving the paradoxical phenotype of uhrf1 mutant zebrafish embryos which have activation of pro-proliferative genes and increased number of hepatocytes in S-phase, but the liver fails to grow. We uncover decreased Cdkn2a/b and persistent Cdk4/6 activation as the mechanism driving uhrf1 mutant hepatocytes into S-phase. This induces replication stress, DNA damage and Atr activation. Palbociclib treatment of uhrf1 mutants prevented aberrant S-phase entry, reduced DNA damage, and rescued most cellular and developmental phenotypes, but it did not rescue DNA hypomethylation, transposon expression or the interferon response. Inhibiting Atr reduced DNA replication and increased liver size in uhrf1 mutants, suggesting that Atr activation leads to dormant origin firing and prevents hepatocyte proliferation. Cdkn2a/b was downregulated pro-proliferative genes were also induced in a Cdk4/6 dependent fashion in the liver of dnmt1 mutants, suggesting DNA hypomethylation as a mechanism of Cdk4/6 activation during development. This shows that the developmental defects caused by DNA hypomethylation are attributed to persistent Cdk4/6 activation, DNA replication stress, dormant origin firing and cell cycle inhibition.

SHR and SCR coordinate root patterning and growth early in the cell cycle.

Precise control of cell division is essential for proper patterning and growth during the development of multicellular organisms. Coordination of formative divisions that generate new tissue patterns with proliferative divisions that promote growth is poorly understood. SHORTROOT (SHR) and SCARECROW (SCR) are transcription factors that are required for formative divisions in the stem cell niche of Arabidopsis roots1,2. Here we show that levels of SHR and SCR early in the cell cycle determine the orientation of the division plane, resulting in either formative or proliferative cell division. We used 4D quantitative, long-term and frequent (every 15 min for up to 48 h) light sheet and confocal microscopy to probe the dynamics of SHR and SCR in tandem within single cells of living roots. Directly controlling their dynamics with an SHR induction system enabled us to challenge an existing bistable model3 of the SHR-SCR gene-regulatory network and to identify key features that are essential for rescue of formative divisions in shr mutants. SHR and SCR kinetics do not align with the expected behaviour of a bistable system, and only low transient levels, present early in the cell cycle, are required for formative divisions. These results reveal an uncharacterized mechanism by which developmental regulators directly coordinate patterning and growth.

Ploidy-specific transcriptomes shed light on the heterogeneous identity and metabolism of developing tomato pericarp cells.

Endoreduplication, during which cells increase their DNA content through successive rounds of full genome replication without cell division, is the major source of endopolyploidy in higher plants. Endoreduplication plays pivotal roles in plant growth and development and is associated with the activation of specific transcriptional programmes that are characteristic of each cell type, thereby defining their identity. In plants, endoreduplication is found in numerous organs and cell types, especially in agronomically valuable ones, such as the fleshy fruit (pericarp) of tomato presenting high ploidy levels. We used the tomato pericarp tissue as a model system to explore the transcriptomes associated with endoreduplication progression during fruit growth. We confirmed that expression globally scales with ploidy level and identified sets of differentially expressed genes presenting only developmental-specific, only ploidy-specific expression patterns or profiles resulting from an additive effect of ploidy and development. When comparing ploidy levels at a specific developmental stage, we found that non-endoreduplicated cells are defined by cell division state and cuticle synthesis while endoreduplicated cells are mainly defined by their metabolic activity changing rapidly over time. By combining this dataset with publicly available spatiotemporal pericarp expression data, we proposed a map describing the distribution of ploidy levels within the pericarp. These transcriptome-based predictions were validated by quantifying ploidy levels within the pericarp tissue. This in situ ploidy quantification revealed the dynamic progression of endoreduplication and its cell layer specificity during early fruit development. In summary, the study sheds light on the complex relationship between endoreduplication, cell differentiation and gene expression patterns in the tomato pericarp.

Balanced cell division is secured by two different regulatory sites in OxyS RNA.

The hydrogen peroxide-induced small RNA OxyS has been proposed to originate from the 3' UTR of a peroxide mRNA. Unexpectedly, phylogenetic OxyS targetome predictions indicate that most OxyS targets belong to the category of "cell cycle," including cell division and cell elongation. Previously, we reported that Escherichia coli OxyS inhibits cell division by repressing expression of the essential transcription termination factor nusG, thereby leading to the expression of the KilR protein, which interferes with the function of the major cell division protein, FtsZ. By interfering with cell division, OxyS brings about cell-cycle arrest, thus allowing DNA damage repair. Cell division and cell elongation are opposing functions to the extent that inhibition of cell division requires a parallel inhibition of cell elongation for the cells to survive. In this study, we report that in addition to cell division, OxyS inhibits mepS, which encodes an essential peptidoglycan endopeptidase that is responsible for cell elongation. Our study indicates that cell-cycle arrest and balancing between cell division and cell elongation are important and conserved functions of the oxidative stress-induced sRNA OxyS.

The divergent early divisome: is there a functional core?

The bacterial divisome is a complex nanomachine that drives cell division and separation. The essentiality of these processes leads to the assumption that proteins with core roles will be strictly conserved across all bacterial genomes. However, recent studies in diverse proteobacteria have revealed considerable variation in the early divisome compared with Escherichia coli. While some proteins are highly conserved, their specific functions and interacting partners vary. Meanwhile, different subphyla use clade-specific proteins with analogous functions. Thus, instead of focusing on gene conservation, we must also explore how key functions are maintained during early division by diverging protein networks. An enhanced awareness of these complex genetic networks will clarify the physical and evolutionary constraints of bacterial division.

The ClpX chaperone and a hypermorphic FtsA variant with impaired self-interaction are mutually compensatory for coordinating Staphylococcus aureus cell division.

Bacterial cell division requires the coordinated assembly and disassembly of a large protein complex called the divisome; however, the exact role of molecular chaperones in this critical process remains unclear. We here provide genetic evidence that ClpX unfoldase activity is a determinant for proper coordination of bacterial cell division by showing the growth defect of a Staphylococcus aureus clpX mutant is rescued by a spontaneously acquired G325V substitution in the ATP-binding domain of the essential FtsA cell division protein. The polymerization state of FtsA is thought to control initiation of bacterial septum synthesis and, while restoring the aberrant FtsA dynamics in clpX cells, the FtsAG325V variant displayed reduced ability to interact with itself and other cell division proteins. In wild-type cells, the ftsAG325V allele shared phenotypes with Escherichia coli superfission ftsA mutants and accelerated the cell cycle, increased the risk of daughter cell lysis, and conferred sensitivity to heat and antibiotics inhibiting cell wall synthesis. Strikingly, lethality was mitigated by spontaneous mutations that inactivate ClpX. Taken together, our results suggest that ClpX promotes septum synthesis by antagonizing FtsA interactions and illuminates the critical role of a protein unfoldase in coordinating bacterial cell division.

Overexpression of a TaATL1 encoding RING-type E3 ligase negatively regulates cell division and flowering time in Arabidopsis thaliana.

The transition of food crops from the vegetative to reproductive stages is an important process that affects the final yield. Despite extensive characterization of E3 ligases in model plants, their roles in wheat development remain unknown. In this study, we elucidated the molecular function of wheat TaATL1 (Arabidopsis thaliana Toxicos EN Levadura), which acts as a negative regulator of flowering time and cell division. TaATL1 amino acid residues contain a RING domain and exist mainly in a beta-turn form. The expression level of TaATL1 was highly reduced during the transition from vegetative to reproductive stages. TaATL1 is localized in the nucleus and exhibits E3 ligase activity. Transgenic Arabidopsis plants, in which the TaATL1 gene is constitutively overexpressed under the control of the cauliflower mosaic virus 35 S promoter, exhibited regulation of cell numbers, thereby influencing both leaf and root growth. Moreover, TaATL1 overexpression plants showed a late-flowering phenotype compared to wild-type (WT) plants. Following transcriptome analysis, it was discovered that 1661 and 901 differentially expressed genes were down- or up- regulated, respectively, in seedling stages between WT and TaATL1 overexpression. TaATL1 transcripts are involved in cell division, flowering, and signaling. Overall, our findings demonstrated that the regulatory mechanism of wheat TaATL1 gene plays a significant role in cell division-mediated flowering in Arabidopsis.

Integration of cell wall synthesis and chromosome segregation during cell division in Caulobacter.

To divide, bacteria must synthesize their peptidoglycan (PG) cell wall, a protective meshwork that maintains cell shape. FtsZ, a tubulin homolog, dynamically assembles into a midcell band, recruiting division proteins, including the PG synthases FtsW and FtsI. FtsWI are activated to synthesize PG and drive constriction at the appropriate time and place. However, their activation pathway remains unresolved. In Caulobacter crescentus, FtsWI activity requires FzlA, an essential FtsZ-binding protein. Through time-lapse imaging and single-molecule tracking of Caulobacter FtsW and FzlA, we demonstrate that FzlA is a limiting constriction activation factor that signals to promote conversion of inactive FtsW to an active, slow-moving state. We find that FzlA interacts with the DNA translocase FtsK and place FtsK genetically in a pathway with FzlA and FtsWI. Misregulation of the FzlA-FtsK-FtsWI pathway leads to heightened DNA damage and cell death. We propose that FzlA integrates the FtsZ ring, chromosome segregation, and PG synthesis to ensure robust and timely constriction during Caulobacter division.

Global and local functions of the Fused kinase ortholog CdaH in intracellular patterning in Tetrahymena.

Ciliates assemble numerous microtubular structures into complex cortical patterns. During ciliate division, the pattern is duplicated by intracellular segmentation that produces a tandem of daughter cells. In Tetrahymena thermophila, the induction and positioning of the division boundary involves two mutually antagonistic factors: posterior CdaA (cyclin E) and anterior CdaI (Hippo kinase). Here, we characterized the related cdaH-1 allele, which confers a pleiotropic patterning phenotype including an absence of the division boundary and an anterior-posterior mispositioning of the new oral apparatus. CdaH is a Fused or Stk36 kinase ortholog that localizes to multiple sites that correlate with the effects of its loss, including the division boundary and the new oral apparatus. CdaH acts downstream of CdaA to induce the division boundary and drives asymmetric cytokinesis at the tip of the posterior daughter. CdaH both maintains the anterior-posterior position of the new oral apparatus and interacts with CdaI to pattern ciliary rows within the oral apparatus. Thus, CdaH acts at multiple scales, from induction and positioning of structures on the cell-wide polarity axis to local organelle-level patterning.

LinRace: cell division history reconstruction of single cells using paired lineage barcode and gene expression data.

Lineage tracing technology using CRISPR/Cas9 genome editing has enabled simultaneous readouts of gene expressions and lineage barcodes in single cells, which allows for inference of cell lineage and cell types at the whole organism level. While most state-of-the-art methods for lineage reconstruction utilize only the lineage barcode data, methods that incorporate gene expressions are emerging. Effectively incorporating the gene expression data requires a reasonable model of how gene expression data changes along generations of divisions. Here, we present LinRace (Lineage Reconstruction with asymmetric cell division model), which integrates lineage barcode and gene expression data using asymmetric cell division model and infers cell lineages and ancestral cell states using Neighbor-Joining and maximum-likelihood heuristics. On both simulated and real data, LinRace outputs more accurate cell division trees than existing methods. With inferred ancestral states, LinRace can also show how a progenitor cell generates a large population of cells with various functionalities.

Biased placement of Mitochondria fission facilitates asymmetric inheritance of protein aggregates during yeast cell division.

Mitochondria are essential and dynamic eukaryotic organelles that must be inherited during cell division. In yeast, mitochondria are inherited asymmetrically based on quality, which is thought to be vital for maintaining a rejuvenated cell population; however, the mechanisms underlying mitochondrial remodeling and segregation during this process are not understood. We used high spatiotemporal imaging to quantify the key aspects of mitochondrial dynamics, including motility, fission, and fusion characteristics, upon aggregation of misfolded proteins in the mitochondrial matrix. Using these measured parameters, we developed an agent-based stochastic model of dynamics of mitochondrial inheritance. Our model predicts that biased mitochondrial fission near the protein aggregates facilitates the clustering of protein aggregates in the mitochondrial matrix, and this process underlies asymmetric mitochondria inheritance. These predictions are supported by live-cell imaging experiments where mitochondrial fission was perturbed. Our findings therefore uncover an unexpected role of mitochondrial dynamics in asymmetric mitochondrial inheritance.