Drive Cell Division Research Articles

Myosin II tension sensors visualize force generation within the actin cytoskeleton in living cells.

Nonmuscle myosin II generates cytoskeletal forces that drive cell division, embryogenesis, muscle contraction, and many other cellular functions. However, at present there is no method that can directly measure the forces generated by myosins in living cells. Here we describe a Förster resonance energy transfer (FRET)-based tension sensor that can detect myosin associated force along the filamentous actin network. Fluorescence lifetime imaging microscopy (FLIM)-FRET measurements indicate that the forces generated by NMIIB exhibit significant spatial and temporal heterogeneity as a function of donor lifetime and fluorophore energy exchange. These measurements provide a proxy for inferred forces that vary widely along the actin cytoskeleton. This initial report highlights the potential utility of myosin-based tension sensors in elucidating the roles of cytoskeletal contractility in a wide variety of contexts.

A de novo Mutation (p.Gln277X) of Cyclin D2 is Responsible for a Child with Megalencephaly-Polymicrogyria-Polydactyly-Hydrocephalus Syndrome.

Megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome (MPPH), a type of overgrowth syndrome, is characterized by progressive megalencephaly, cortical brain malformations, and distal limb anomalies. Previous studies have revealed that the overactivity of the phosphatidylinositol 3-kinase-Protein kinase Bpathway and the increased cyclin D2 (CCND2) expression were the main factors contributing to this disease. Here, we present the case of a patient who exhibited megalencephaly, polymicrogyria, abnormal neuronal migration, and developmental delay. Serum tandem mass spectrometry and chromosome examination did not detect any metabolic abnormalities or copy number variants. However, whole-exome sequencing and Sanger sequencing revealed a de novo nonsense mutation (NM_001759.3: c.829C>T; p.Gln277X) in the CCND2 gene of the patient. Bioinformatics analysis predicted that this mutation may disrupt the structure and surface charge of the CCND2 protein. This disruption could potentially prevent polyubiquitination of CCND2, leading to its resistance against degradation. Consequently, this could drive cell division and growth by altering the activity of key cell cycle regulatory nodes, ultimately contributing to the development of MPPH. This study not only presents a new case of MPPH and expands the mutation spectrum of CCND2 but also enhances our understanding of the mechanisms connecting CCND2 with overgrowth syndromes.

Protein Tyrosine Phosphatase regulation by Reactive Oxygen Species.

Protein Tyrosine Phosphatases (PTPs) help to maintain the balance of protein phosphorylation signals that drive cell division, proliferation, and differentiation. These enzymes are also well-suited to redox-dependent signaling and oxidative stress response due to their cysteine-based catalytic mechanism, which requires a deprotonated thiol group at the active site. This review focuses on PTP structural characteristics, active site chemical properties, and vulnerability to change by reactive oxygen species (ROS). PTPs can be oxidized and inactivated by H2O2 through three non-exclusive mechanisms. These pathways are dependent on the coordinated actions of other H2O2-sensitive proteins, such as peroxidases like Peroxiredoxins (Prx) and Thioredoxins (Trx). PTPs undergo reversible oxidation by converting their active site cysteine from thiol to sulfenic acid. This sulfenic acid can then react with adjacent cysteines to form disulfide bonds or with nearby amides to form sulfenyl-amide linkages. Further oxidation of the sulfenic acid form to the sulfonic or sulfinic acid forms causes irreversible deactivation. Understanding the structural changes involved in both reversible and irreversible PTP oxidation can help with their chemical manipulation for therapeutic intervention. Nonetheless, more information remains unidentified than is presently known about the precise dynamics of proteins participating in oxidation events, as well as the specific oxidation states that can be targeted for PTPs. This review summarizes current information on PTP-specific oxidation patterns and explains how ROS-mediated signal transmission interacts with phosphorylation-based signaling machinery controlled by growth factor receptors and PTPs.

Distinct mechanisms of PTEN inactivation in dogs and humans highlight convergent molecular events that drive cell division in the pathogenesis of osteosarcoma

A hallmark of osteosarcoma in both human and canine tumors is somatic fragmentation and rearrangement of chromosome structure which leads to recurrent increases and decreases in DNA copy number. The PTEN gene has been implicated as an important tumor suppressor in osteosarcoma via forward genetic screens. Here, we analyzed copy number changes, promoter methylation and transcriptomes to better understand the role of PTEN in canine and human osteosarcoma. Reduction in PTEN copy number was observed in 23 of 95 (25%) of the canine tumors examined leading to corresponding decreases in PTEN transcript levels from RNA-Seq samples. Unexpectedly, canine tumors with an intact PTEN locus had higher levels of PTEN transcripts than human tumors. This variation in transcript abundance was used to evaluate the role of PTEN in osteosarcoma biology. Decreased PTEN copy number and transcript level was observed in - and likely an important driver of - increases in cell cycle transcripts in four independent canine transcriptional datasets. In human osteosarcoma, homozygous copy number loss was not observed, instead increased methylation of the PTEN promoter was associated with increased cell cycle transcripts. Somatic modification of PTEN, either by homozygous deletion in dogs or by promoter methylation in humans, is clinically relevant to osteosarcoma, because the cell cycle related transcripts are associated with patient outcomes. The PTEN gene is part of a syntenic rearrangement unique to the canine genome, making it susceptible to somatic loss of both copies of distal chromosome 26 which also includes the FAS death receptor. Significance StatementPTEN function is abrogated by different mechanisms in canine and human osteosarcoma tumors leading to uncontrolled cell cycling. Somatic loss of this canine specific syntenic region may help explain why the canine genome appears to be uniquely susceptible to osteosarcoma. Syntenic arrangement, in the context of copy number change, may lead to synergistic interactions that in turn modify species specific cancer risk. Comparative models of tumorigenesis may utilize different driver mechanisms.

Mutant K-Ras in Pancreatic Cancer: An Insight on the Role of Wild-Type N-Ras and K-Ras-Dependent Cell Cycle Regulation.

The development of K-Ras independence may explain the failure of targeted therapy for pancreatic cancer (PC). In this paper, active N as well as K-Ras was shown in all human cell lines tested. In a cell line dependent on mutant K-Ras, it was shown that depleting K-Ras reduced total Ras activity, while cell lines described as independent had no significant decline in total Ras activity. The knockdown of N-Ras showed it had an important role in controlling the relative level of oxidative metabolism, but only K-Ras depletion caused a decrease in G2 cyclins. Proteasome inhibition reversed this, and other targets of APC/c were also decreased by K-Ras depletion. K-Ras depletion did not cause an increase in ubiquitinated G2 cyclins but instead caused exit from the G2 phase to slow relative to completion of the S-phase, suggesting that the mutant K-Ras may inhibit APC/c prior to anaphase and stabilise G2 cyclins independently of this. We propose that, during tumorigenesis, cancer cells expressing wild-type N-Ras protein are selected because the protein protects cancer cells from the deleterious effects of the cell cycle-independent induction of cyclins by mutant K-Ras. Mutation independence results when N-Ras activity becomes adequate to drive cell division, even in cells where K-Ras is inhibited.

Insights on the Role of PGRMC1 in Mitotic and Meiotic Cell Division.

During mitosis, chromosome missegregation and cytokinesis defects have been recognized as hallmarks of cancer cells. Cytoskeletal elements composing the spindle and the contractile ring and their associated proteins play crucial roles in the faithful progression of mitotic cell division. The hypothesis that PGRMC1, most likely as a part of a yet-to-be-defined complex, is involved in the regulation of spindle function and, more broadly, the cytoskeletal machinery driving cell division is particularly appealing. Nevertheless, more than ten years after the preliminary observation that PGRMC1 changes its localization dynamically during meiotic and mitotic cell division, this field of research has remained a niche and needs to be fully explored. To encourage research in this fascinating field, in this review, we will recap the current knowledge on PGRMC1 function during mitotic and meiotic cell division, critically highlighting the strengths and limitations of the experimental approaches used so far. We will focus on known interacting partners as well as new putative associated proteins that have recently arisen in the literature and that might support current as well as new hypotheses of a role for PGRMC1 in specific spindle subcompartments, such as the centrosome, kinetochores, and the midzone/midbody.

Relating individual cell division events to single-cell ERK and Akt activity time courses

Biochemical correlates of stochastic single-cell fates have been elusive, even for the well-studied mammalian cell cycle. We monitored single-cell dynamics of the ERK and Akt pathways, critical cell cycle progression hubs and anti-cancer drug targets, and paired them to division events in the same single cells using the non-transformed MCF10A epithelial line. Following growth factor treatment, in cells that divide both ERK and Akt activities are significantly higher within the S-G2 time window (~ 8.5–40 h). Such differences were much smaller in the pre-S-phase, restriction point window which is traditionally associated with ERK and Akt activity dependence, suggesting unappreciated roles for ERK and Akt in S through G2. Simple metrics of central tendency in this time window are associated with subsequent cell division fates. ERK activity was more strongly associated with division fates than Akt activity, suggesting Akt activity dynamics may contribute less to the decision driving cell division in this context. We also find that ERK and Akt activities are less correlated with each other in cells that divide. Network reconstruction experiments demonstrated that this correlation behavior was likely not due to crosstalk, as ERK and Akt do not interact in this context, in contrast to other transformed cell types. Overall, our findings support roles for ERK and Akt activity throughout the cell cycle as opposed to just before the restriction point, and suggest ERK activity dynamics may be more important than Akt activity dynamics for driving cell division in this non-transformed context.

The archaeal division protein CdvB1 assembles into polymers that are depolymerized by CdvC.

The Cdv proteins constitute the cell division system of the Crenarchaea, a machinery closely related to the ESCRT system of eukaryotes. Using a combination of TEM imaging and biochemical assays, we here present an in vitro study of Metallosphaera sedula CdvB1, the Cdv protein that is believed to play a major role in the constricting ring that drives cell division in the Crenarchaea. We show that CdvB1 self‐assembles into filaments that are depolymerized by the Vps4‐homolog ATPase CdvC. Furthermore, we find that CdvB1 binds to negatively charged lipid membranes and can be detached from the membrane by the action of CdvC. Our findings provide novel insight into one of the main components of the archaeal cell division machinery.

Folic Acid, Folinic Acid, 5 Methyl TetraHydroFolate Supplementation for Mutations That Affect Epigenesis through the Folate and One-Carbon Cycles.

Methylation is an essential biochemical mechanism that is central to the transmission of life, and crucially responsible for regulating gametogenesis and continued embryo development. The methylation of DNA and histones drives cell division and regulation of gene expression through epigenesis and imprinting. Brain development and its maturation also depend on correct lipid methylation, and continued neuronal function depends on biogenic amines that require methylation for their synthesis. All methylation processes are carried out via a methyltransferase enzyme and its unique co-factor S-adenosylmethionine (SAM); the transfer of a methyl group to a target molecule results in the release of SAH (SA homocysteine), and then homocysteine (Hcy). Both of these molecules are toxic, inhibiting methylation in a variety of ways, and Hcy recycling to methionine is imperative; this is achieved via the one carbon cycle, supported by the folates cycle. Folate deficiency causes hyperhomocysteinaemia, with several associated diseases; during early pregnancy, deficiency interferes with closure of the neural tube at the fourth week of gestation, and nutraceutical supplementation has been routinely prescribed to prevent neural tube defects, mainly involving B vitamins, Zn and folates. The two metabolic pathways are subject to single nucleotide polymorphisms that alter their activity/capacity, often severely, impairing specific physiological functions including fertility, brain and cardiac function. The impact of three types of nutraceutical supplements, folic acid (FA), folinic acid (FLA) and 5 Methyl THF (MTHF), will be discussed here, with their positive effects alongside potentially hazardous secondary effects. The issue surrounding FA and its association with UMFA (unmetabolized folic acid) syndrome is now a matter of concern, as UMFA is currently found in the umbilical cord of the fetus, and even in infants’ blood. We will discuss its putative role in influencing the acquisition of epigenetic marks in the germline, acquired during embryogenesis, as well as the role of FA in the management of cancerous disease.

Structural and Functional Insights into the Microtubule Organizing Centers of Toxoplasma gondii and Plasmodium spp.

Microtubule organizing centers (MTOCs) perform critical cellular tasks by nucleating, stabilizing, and anchoring microtubule’s minus ends. These capacities impact tremendously a wide array of cellular functions ranging from ascribing cell shape to orchestrating cell division and generating motile structures, among others. The phylum Apicomplexa comprises over 6000 single-celled obligate intracellular parasitic species. Many of the apicomplexan are well known pathogens such as Toxoplasma gondii and the Plasmodium species, causative agents of toxoplasmosis and malaria, respectively. Microtubule organization in these parasites is critical for organizing the cortical cytoskeleton, enabling host cell penetration and the positioning of large organelles, driving cell division and directing the formation of flagella in sexual life stages. Apicomplexans are a prime example of MTOC diversity displaying multiple functional and structural MTOCs combinations within a single species. This diversity can only be fully understood in light of each organism’s specific MT nucleation requirements and their evolutionary history. Insight into apicomplexan MTOCs had traditionally been limited to classical ultrastructural work by transmission electron microscopy. However, in the past few years, a large body of molecular insight has emerged. In this work we describe the latest insights into nuclear MTOC biology in two major human and animal disease causing Apicomplexans: Toxoplasma gondii and Plasmodium spp.

The anti-cancer drug dabrafenib is not cardiotoxic and inhibits cardiac remodelling and fibrosis in a murine model of hypertension.

Raf kinases signal via extracellular signal-regulated kinases 1/2 (ERK1/2) to drive cell division. Since activating mutations in BRAF (B-Raf proto-oncogene, serine/threonine kinase) are highly oncogenic, BRAF inhibitors including dabrafenib have been developed for cancer. Inhibitors of ERK1/2 signalling used for cancer are cardiotoxic in some patients, raising the question of whether dabrafenib is cardiotoxic. In the heart, ERK1/2 signalling promotes not only cardiomyocyte hypertrophy and is cardioprotective but also promotes fibrosis. Our hypothesis is that ERK1/2 signalling is not required in a non-stressed heart but is required for cardiac remodelling. Thus, dabrafenib may affect the heart in the context of, for example, hypertension. In experiments with cardiomyocytes, cardiac fibroblasts and perfused rat hearts, dabrafenib inhibited ERK1/2 signalling. We assessed the effects of dabrafenib (3 mg/kg/d) on male C57BL/6J mouse hearts in vivo. Dabrafenib alone had no overt effects on cardiac function/dimensions (assessed by echocardiography) or cardiac architecture. In mice treated with 0.8 mg/kg/d angiotensin II (AngII) to induce hypertension, dabrafenib inhibited ERK1/2 signalling and suppressed cardiac hypertrophy in both acute (up to 7 d) and chronic (28 d) settings, preserving ejection fraction. At the cellular level, dabrafenib inhibited AngII-induced cardiomyocyte hypertrophy, reduced expression of hypertrophic gene markers and almost completely eliminated the increase in cardiac fibrosis both in interstitial and perivascular regions. Dabrafenib is not overtly cardiotoxic. Moreover, it inhibits maladaptive hypertrophy resulting from AngII-induced hypertension. Thus, Raf is a potential therapeutic target for hypertensive heart disease and drugs such as dabrafenib, developed for cancer, may be used for this purpose.

The nature of cell division forces in epithelial monolayers.

Epithelial cells undergo striking morphological changes during division to ensure proper segregation of genetic and cytoplasmic materials. These morphological changes occur despite dividing cells being mechanically restricted by neighboring cells, indicating the need for extracellular force generation. Beyond driving cell division itself, forces associated with division have been implicated in tissue-scale processes, including development, tissue growth, migration, and epidermal stratification. While forces generated by mitotic rounding are well understood, forces generated after rounding remain unknown. Here, we identify two distinct stages of division force generation that follow rounding: (1) Protrusive forces along the division axis that drive division elongation, and (2) outward forces that facilitate postdivision spreading. Cytokinetic ring contraction of the dividing cell, but not activity of neighboring cells, generates extracellular forces that propel division elongation and contribute to chromosome segregation. Forces from division elongation are observed in epithelia across many model organisms. Thus, division elongation forces represent a universal mechanism that powers cell division in confining epithelia.

Cellular Pushing Forces during Mitosis Drive Mitotic Elongation in Collagen Gels.

Cell elongation along the division axis, or mitotic elongation, mediates proper segregation of chromosomes and other intracellular materials, and is required for completion of cell division. In three‐dimensionally confining extracellular matrices, such as dense collagen gels, dividing cells must generate space to allow mitotic elongation to occur. In principle, cells can generate space for mitotic elongation during cell spreading, prior to mitosis, or via extracellular force generation or matrix degradation during mitosis. However, the processes by which cells drive mitotic elongation in collagen‐rich extracellular matrices remains unclear. Here, it is shown that single cancer cells generate substantial pushing forces on the surrounding collagen extracellular matrix to drive cell division in confining collagen gels and allow mitotic elongation to proceed. Neither cell spreading, prior to mitosis, nor matrix degradation, during spreading or mitotic elongation, are found to be required for mitotic elongation. Mechanistically, laser ablation studies, pharmacological inhibition studies, and computational modeling establish that pushing forces generated during mitosis in collagen gels arise from a combination of interpolar spindle elongation and cytokinetic ring contraction. These results reveal a fundamental mechanism mediating cell division in confining extracellular matrices, providing insight into how tumor cells are able to proliferate in dense collagen‐rich tissues.

Modelling the spatial crosstalk between two biochemical signals explains wood formation dynamics and tree-ring structure.

In conifers, xylogenesis during a growing season produces a very characteristic tree-ring structure: large, thin-walled earlywood cells followed by narrow, thick-walled latewood cells. Although many factors influence the dynamics of differentiation and the final dimensions of xylem cells, the associated patterns of variation remain very stable from one year to the next. While radial growth is characterized by an S-shaped curve, the widths of xylem differentiation zones exhibit characteristic skewed bell-shaped curves. These elements suggest a strong internal control of xylogenesis. It has long been hypothesized that much of this regulation relies on a morphogenetic gradient of auxin. However, recent modelling studies have shown that while this hypothesis could account for the dynamics of stem radial growth and the zonation of the developing xylem, it failed to reproduce the characteristic tree-ring structure. Here, we investigated the hypothesis of regulation by a crosstalk between auxin and a second biochemical signal, by using computational morphodynamics. We found that, in conifers, such a crosstalk is sufficient to simulate the characteristic features of wood formation dynamics, as well as the resulting tree-ring structure. In this model, auxin controls cell enlargement rates while another signal (e.g. cytokinin, tracheary element differentiation inhibitory factor) drives cell division and auxin polar transport.

Genome-wide CRISPR screen identifies noncanonical NF-κB signaling as a regulator of density-dependent proliferation.

Epithelial cells possess intrinsic mechanisms to maintain an appropriate cell density for normal tissue morphogenesis and homeostasis. Defects in such mechanisms likely contribute to hyperplasia and cancer initiation. To identify genes that regulate the density-dependent proliferation of murine mammary epithelial cells, we developed a fluorescence-activated cell sorting assay based on fluorescence ubiquitination cell cycle indicator, which marks different stages of the cell cycle with distinct fluorophores. Using this powerful assay, we performed a genome-wide CRISPR/Cas9 knockout screen, selecting for cells that proliferate normally at low density but continue to divide at high density. Unexpectedly, one top hit was Traf3, a negative regulator of NF-κB signaling that has never previously been linked to density-dependent proliferation. We demonstrate that loss of Traf3 specifically activates noncanonical NF-κB signaling. This in turn triggers an innate immune response and drives cell division independently of known density-dependent proliferation mechanisms, including YAP/TAZ signaling and cyclin-dependent kinase inhibitors, by blocking entry into quiescence.

Mitochondrial Potentiation Ameliorates Age-Related Heterogeneity in Hematopoietic Stem Cell Function.

Aging is associated with reduced fitness and increased myeloid bias of the hematopoietic stem cell (HSC) compartment, causing increased risk of immune compromise, anemia, and malignancy. We show that mitochondrial membrane potential (MMP) can be used to prospectively isolate chronologically old HSCs with transcriptional features and functional attributes characteristic of young HSCs, including a high rate of transcription and balanced lineage-affiliated programs. Strikingly, MMP is a stronger determinant of the quantitative and qualitative transcriptional state of HSCs than chronological age, and transcriptional consequences of manipulation of MMP in HSCs within their native niche suggest a causal relationship. Accordingly, we show that pharmacological enhancement of MMP in old HSCs invivo increases engraftment potential upon transplantation and reverses myeloid-biased peripheral blood output at steady state. Our results demonstrate that MMP is a source of heterogeneity in old HSCs, and its pharmacological manipulation can alter transcriptional programs with beneficial consequences for function.

Cell growth dilutes the cell cycle inhibitor Rb to trigger cell division.

Cell size is fundamental to cell physiology. For example, cell size determines the spatial scale of organelles and intracellular transport and thereby affects biosynthesis. Although some genes that affect mammalian cell size have been identified, the molecular mechanisms through which cell growth drives cell division have remained elusive. We show that cell growth during the G1 phase of the cell division cycle dilutes the cell cycle inhibitor Retinoblastoma protein (Rb) to trigger division in human cells. RB overexpression increased cell size and G1 duration, whereas RB deletion decreased cell size and removed the inverse correlation between cell size at birth and the duration of the G1 phase. Thus, Rb dilution through cell growth in G1 provides one of the long-sought molecular mechanisms that promotes cell size homeostasis.

Cdk1 Controls Global Epigenetic Landscape in Embryonic Stem Cells

The cyclin-dependent kinase 1 (Cdk1) drives cell division. To uncover additional functions of Cdk1, we generated knockin mice expressing an analog-sensitive version of Cdk1 in place of wild-type Cdk1. In our study, we focused on embryonic stem cells (ESCs), because this cell type displays particularly high Cdk1 activity. We found that in ESCs, a large fraction of Cdk1 substrates is localized on chromatin. Cdk1 phosphorylates many proteins involved in epigenetic regulation, including writers and erasers of all major histone marks. Consistent with these findings, inhibition of Cdk1 altered histone-modification status of ESCs. High levels of Cdk1 in ESCs phosphorylate and partially inactivate Dot1l, the H3K79 methyltransferase responsible for placing activating marks on gene bodies. Decrease of Cdk1 activity during ESC differentiation de-represses Dot1l, thereby allowing coordinated expression of differentiation genes. These analyses indicate that Cdk1 functions to maintain the epigenetic identity of ESCs.

The cell cycle in stem cell proliferation, pluripotency and differentiation.

Cyclins, cyclin-dependent kinases and other components of the core cell cycle machinery drive cell division. Growing evidence indicates that this machinery operates in a distinct fashion in some mammalian stem cell types, such as pluripotent embryonic stem cells. In this Review, we discuss our current knowledge of how cell cycle proteins mechanistically link cell proliferation, pluripotency and cell fate specification. We focus on embryonic stem cells, induced pluripotent stem cells and embryonic neural stem/progenitor cells.

ALDH1A3 Regulations of Matricellular Proteins Promote Vascular Smooth Muscle Cell Proliferation.

SummaryVascular smooth muscle cell (VSMC) proliferation promotes intimal hyperplasia (IH) in occluding vascular diseases. Here we identified a positive role of ALDH1A3 (an aldehyde dehydrogenase) in this pro-IH process. The expression of ALDH1A3, but not that of 18 other isoforms of the ALDH family, was substantially increased in cytokine-stimulated VSMCs. PDGF(BB) stimulated VSMC total ALDH activity and proliferation, whereas ALDH1A3 silencing abolished this effect. ALDH1A3 silencing also diminished the expression of two matricellular proteins (TNC1 and ESM1), revealing a previously unrecognized ALDH1A3 function. Loss-of-function experiments demonstrated that TNC1 and ESM1 mediated ALDH1A3's pro-proliferative function via activation of AKT/mTOR and/or MEK/ERK pathways. Furthermore, ALDH inhibition with disulfiram blocked VSMC proliferation/migration in vitro and decreased TNC1 and ESM1 and IH in angioplasty-injured rat carotid arteries. Thus, ALDH1A3 promotes VSMC proliferation at least partially through TNC1/ESM1 upregulation; dampening excessive ALDH1A3 activity represents a potential approach to IH mitigation.