Cell Division Research Articles

Bacteria divide to conquer antibiotics.

High-level resistance to methicillin requires a distinct form of cell division.

GPCR-like Protein ZmCOLD1 Regulate Plant Height in an ABA Manner.

G protein-coupled receptors (GPCRs) are sensors for the G protein complex to sense changes in environmental factors and molecular switches for G protein complex signal transduction. In this study, the homologous gene of GPCR-like proteins was identified from maize and named as ZmCOLD1. Subcellular analysis showed that the ZmCOLD1 protein is localized to the cell membrane and endoplasmic reticulum. A CRISPR/Cas9 knock-out line of ZmCOLD1 was further created and its plant height was significantly lower than the wild-type maize at both the seedling and adult stages. Histological analysis showed that the increased cell number but significantly smaller cell size may result in dwarfing of zmcold1, indicating that the ZmCOLD1 gene could regulate plant height development by affecting the cell division process. Additionally, ZmCOLD1 was verified to interact with the maize Gα subunit, ZmCT2, though the central hydrophilic loop domain by in vivo and in vitro methods. Abscisic acid (ABA) sensitivity analysis by seed germination assays exhibited that zmcold1 were hypersensitive to ABA, indicating its important roles in ABA signaling. Finally, transcriptome analysis was performed to investigate the transcriptional change in zmcold1 mutant. Overall, ZmCOLD1 functions as a GPCR-like protein and an important regulator to plant height.

Thymol as adjuvant in oncology: molecular mechanisms, therapeutic potentials, and prospects for integration in cancer management.

Cancer remains a global health challenge, prompting a search for effective treatments with fewer side effects. Thymol, a natural monoterpenoid phenol derived primarily from thyme (Thymus vulgaris) and other plants in the Lamiaceae family, is known for its diverse biological activities. It emerges as a promising candidate in cancer prevention and therapy. This study aims to consolidate current research on thymol's anticancer effects, elucidating its mechanisms and potential to enhance standard chemotherapy, and to identify gaps for future research. A comprehensive review was conducted using databases like PubMed/MedLine, Google Scholar, and ScienceDirect, focusing on studies from the last 6years. All cancer types were included, assessing thymol's impact in both cell-based (in vitro) and animal (in vivo) studies. Thymol has been shown to induce programmed cell death (apoptosis), halt the cell division cycle (cell cycle arrest), and inhibit cancer spread (metastasis) through modulation of critical signaling pathways, including phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), extracellular signal-regulated kinase (ERK), mechanistic target of rapamycin (mTOR), and Wnt/β-catenin. It also enhances the efficacy of 5-fluorouracil (5-FU) in colorectal cancer treatments. Thymol's broad-spectrum anticancer activities and non-toxic profile to normal cells underscore its potential as an adjunct in cancer therapy. Further clinical trials are essential to fully understand its therapeutic benefits and integration into existing treatment protocols.

CDC42 enhances phagocytic activity and exhibits antimicrobial functions through the recruitment of IQGAP1 in Apostichopus japonicus

Cell Division Cycle 42 protein (CDC42) is a pivotal member of the Rho family of small GTPases, integral to cell signaling and the regulation of cell polarity. CDC42 is extensively documented to participate in host immunity, particularly in processes such as phagocytosis and cell migration. Nevertheless, the precise mechanisms by which CDC42 affects host-bacteria interactions remain unclear in Echinoderms. In this study, we have isolated and characterized the CDC42 gene from Apostichopus japonicus, referred to as AjCDC42. The complete cDNA sequence of AjCDC42 extends 1282 base pairs, including a typical classical RHO domain, spanning amino acids from 6 to 179. Spatial expression analysis demonstrated that AjCDC42 is ubiquitously expressed across all examined tissues. Following infection with Vibrio splendidus and stimulation with LPS, the protein and mRNA levels of AjCDC42 were significantly upregulated in coelomocytes. Moreover, the knockdown of AjCDC42 significantly decreased V. splendidus-induced phagocytic activity in coelomocytes, leading to a reduced intracellular load of V. splendidus, as evidenced by flow cytometry and bacterial plate counting assays. Mechanistically, AjCDC42 was identified as an interacting partner of IQ Motif Containing GTPase Activating Protein 1 (AjIQGAP1) through the use of co-immunoprecipitation, immunofluorescence, and GST pull-down assays combined with mass spectrometry techniques. Additional functional analyses revealed that the knockdown of AjIQGAP1 similarly decreased the phagocytic activity and the intracellular load of V. splendidus in coelomocytes. Furthermore, we detected that decreased levels of AjCDC42 and AjIQGAP1 impaired cytoskeletal rearrangement and delayed lysosomal clearance of V. splendidus. In summary, our findings underscore the pivotal role of the AjCDC42-AjIQGAP1 axis in regulating coelomocytes phagocytosis in A. japonicus during V. splendidus infection.

TRBP2, a Major Component of the RNAi Machinery, Is Subjected to Cell Cycle-Dependent Regulation in Human Cancer Cells of Diverse Tissue Origin.

Transactivation Response Element RNA-binding Protein (TRBP2) is a double-stranded RNA-binding protein widely known for its critical contribution to RNA interference (RNAi), a conserved mechanism of gene-expression regulation mediated through small non-coding RNA moieties (ncRNAs). Nevertheless, TRBP2 has also proved to be involved in other molecular pathways and biological processes, such as cell growth, organism development, spermatogenesis, and stress response. Mutations or aberrant expression of TRBP2 have been previously associated with diverse human pathologies, including Alzheimer's disease, cardiomyopathy, and cancer, with TRBP2 playing an essential role(s) in proliferation, invasion, and metastasis of tumor cells. Hence, the present study aims to investigate, via employment of advanced flow cytometry, immunofluorescence, cell transgenesis and bioinformatics technologies, new, still elusive, functions and properties of TRBP2, particularly regarding its cell cycle-specific control during cancer cell division. We have identified a novel, mitosis-dependent regulation of TRBP2 protein expression, as clearly evidenced by the lack of its immunofluorescence-facilitated detection during mitotic phases, in several human cancer cell lines of different tissue origin. Notably, the obtained TRBP2-downregulation patterns seem to derive from molecular mechanisms that act independently of oncogenic activities (e.g., malignancy grade), metastatic capacities (e.g., low versus high), and mutational signatures (e.g., p53-/- or p53ΔΥ126) of cancer cells. Taken together, we herein propose that TRBP2 serves as a novel cell cycle-dependent regulator, likely exerting mitosis-suppression functions, and, thus, its mitosis-specific downregulation can hold strong promise to be exploited for the efficient and successful prognosis, diagnosis, and (radio-/chemo-)therapy of diverse human malignancies, in the clinic.

Lysate of bovine adipose-derived stem cells accelerates in-vitro development and increases cryotolerance through reduced content of lipid in the in vitro fertilized embryos

Mesenchymal stem cells such as adipose-derived stem cells (ADSCs) are known to secrete factors that stimulate cell division and promote regeneration in neighboring cells. While conditioned medium from stem cells has been used in blastocyst production, no studies have examined the use of cell lysates. In this study we investigated the effects of adding ADSC lysate to in vitro culture (IVC) medium. ADSCs and fibroblasts were isolated from bovine adipose tissue and auricular tissue, respectively, and their lysates were prepared by freeze-thaw disruption. ADSC lysate was added to synthetic oviductal fluid medium. The effects on cleavage, blastocyst development rates, cell numbers, cryotolerance, gene expression (POU5F1, BAX, IGF1R, IGF2R, SOD2), lipid content, and membrane integrity were evaluated according to the experimental design. In Expt. 1, the comparison involved adding ADSC or fibroblast lysate alongside the control group. The total blastocyst rate increased when ADSC lysate was introduced (ADSCs: 40.1%, fibroblasts: 33.1%, control: 27.3%). However, there were no significant differences in the number of trophoblast cells or in the inner cell mass. Experiment 2 confirmed that this increase in blastocyst development was not due to the solvent, PBS(-). In Expt. 3, addition of 10% fetal calf serum (FCS) or ADSC lysate increased the total blastocyst rate compared to the control (control, 26.2%; 10% FCS, 43.4%; 1% ADSC lysate, 34.2%; 10% ADSC lysate, 48.1%). After freezing and thawing, the survival and hatching rates of embryos with FCS were significantly lower than those of the control as well as those with added ADSC lysate. In Expt. 4, the addition of ADSC lysate or FCS had no significant effect on gene expression in blastocysts compared to control. However, the addition of FCS significantly increased the gray intensity, indicating higher lipid content compared to the control, with a significant increase in the number of dead cells in the blastocyst. These results indicate that the addition of ADSC lysate to the IVC medium accelerates bovine blastocyst development and that its 10% addition, corresponding to 1 x 105 cells/mL, is as effective as 10% FCS without a decrease in cryotolerance due to the increased lipid content.

A decoy receptor derived from alternative splicing fine-tunes cytokinin signaling in Arabidopsis

Hormone perception and signaling pathways play a fundamental regulatory function in the physiological processes of plants. Cytokinins, plant hormones, regulate cell division and meristem maintenance. The cytokinin signaling pathway is well-established in model plant Arabidopsis. Several negative feedback mechanisms, tightly controlling the cytokinin signaling output, were described previously. Here, we identified a new feedback mechanism executed through an alternative splicing of the cytokinin receptor AHK4/CRE1. A novel splicing variant named CRE1int7 results from seventh intron retention, introducing a premature termination codon in the transcript. We show that CRE1int7 is translated in planta into a truncated receptor lacking the C-terminal receiver domain essential for signal transduction. The CRE1int7 can bind the cytokinin but cannot activate the downstream cascade. We present a novel negative feedback mechanism of the cytokinin signaling pathway facilitated by a decoy receptor, which can inactivate canonical cytokinin receptors via dimerization and compete with them for ligand binding. While a similar molecular mechanism is well-known in mammals, decoy receptors are rare in plants. Ensuring proper plant growth and development requires precise control of the cytokinin signaling pathway at several levels. The CRE1int7 represents a yet unknown mechanism for fine-tuning the cytokinin signaling pathway in Arabidopsis.

Two codependent routes lead to high-level MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA), in which acquisition of mecA [which encodes the cell wall peptidoglycan biosynthesis component penicillin-binding protein 2a (PBP2a)] confers resistance to β-lactam antibiotics, is of major clinical concern. We show that, in the presence of antibiotics, MRSA adopts an alternative mode of cell division and shows an altered peptidoglycan architecture at the division septum. PBP2a can replace the transpeptidase activity of the endogenous and essential PBP2 but not that of PBP1, which is responsible for the distinctive native septal peptidoglycan architecture. Successful division without PBP1 activity requires the alternative division mode and is enabled by several possible chromosomal potentiator (pot) mutations. MRSA resensitizing agents differentially interfere with the two codependent mechanisms required for high-level antibiotic resistance, which provides opportunities for new interventions.

CDC42 deficiency leads to endometrial stromal cell senescence in recurrent implantation failure.

Does the downregulation of cell division cycle 42 (CDC42) protein in endometrial stroma lead to endometrial senescence in patients with recurrent implantation failure (RIF), and what is the potential mechanism? CDC42 deficiency causes endometrial stromal senescence and decidualization defects, impairing uterine receptivity of RIF patients, via activation of Wnt signaling pathway. Uterine aging is unique due to the cyclic remodeling and decidualization of endometrial tissue. Several transcriptomic studies have reported increased senescence in the endometrium in young patients with RIF. Our previous transcriptomic sequencing study discovered that endometrium from women with RIF showed downregulation of CDC42, which is an essential molecule affected by various senescence-related diseases. The endometrial samples of a total of 71 fertile control patients and 37 RIF patients were collected to verify the association between CDC42 expression and endometrial senescence of RIF patients. Primary endometrial stromal cells (EnSCs) were isolated from endometrial biopsies taken from patients without any endometrial complications and planning to undergo IVF, then subjected to adenovirus-mediated CDC42 knockdown and decidualization induction to explore the detailed mechanism by which CDC42 governs stromal senescence and decidualization. Wnt inhibitor XAV-939 was used to correct the endometrial senescence and decidualization defect. Senescence was determined by cell cycle arrest markers (e.g. P16, P21, and P53), SASP molecules (e.g. IL6 and CXCL8), and SA-β-gal staining. Masson's staining and Sirius Red staining were used to detect the endometrial fibrosis. Decidualization was evaluated by the mRNA expression and protein secretion of PRL and IGFBP1, F-actin immunostaining, and the BeWo spheroids 'in vitro implantation' model. Methods used to assess cell function included adenovirus transduction, RNA-sequencing, bioinformatic analysis, western blotting, RT-qPCR, ELISA, and immunofluorescence. Here, we observed remarkably increased levels of stromal senescence and fibrosis, along with stromal CDC42 deficiency, in the endometrium of patients with RIF (P < 0.001). Knockdown of CDC42 effectively induced premature senescence in EnSCs, leading to aberrant accumulation of senescent EnSCs and collagen deposition during decidualization. CDC42 deficiency in EnSCs restrained the decidualization differentiation and receptivity to trophoblast cells. Transcriptomic analysis revealed Wnt signaling activation as a critical downstream alteration in CDC42-deficient EnSCs. Mechanistically, CDC42 interacted with AKT competitively to impede the binding of GSK3β to AKT. Knockdown of CDC42 increased AKT-mediated phosphorylation of GSK3β to inactivate the Axin-GSK3β destruction complex, leading to accumulation and nuclear translocation of β-catenin. Importantly, Wnt signaling inhibitors partially corrected the endometrial senescence caused by CDC42 deficiency, and improved both decidualization and trophoblast invasion. RNA-seq data sets generated in this study have been deposited at the NCBI database with BioProject accession number PRJNA1102745. The present study was based on in vitro cell cultures. Further studies involving CDC42-regulated endometrial senescence are needed in knockout mice model and human endometrial assembloids. In addition to uncovering endometrial senescence in RIF, our findings underscore the significance of CDC42 in modulating EnSC senescence to maintain the decidualization function, and suggest Wnt signaling inhibitors as potential therapeutic agents for alleviating endometrial senescence. This work was supported by the National Natural Science Foundation of China [82271698 (R.J.), 82030040 (H.S.), 82288102 (H.W.), and 82371680 (G.Y.)]; the Natural Science Foundation of Jiangsu Province [BK20231117 (R.J.)]; and the Medical Science and Technology Development Foundation of Nanjing Department of Health [YKK23097 (Y.Z.)]. The authors declare no potential conflicts of interest.

IL-21/IL-21R signaling renders acute myeloid leukemia stem cells more susceptible to cytarabine treatment and CAR Tcell therapy.

Self-renewal programs in leukemia stem cells (LSCs) predict poor prognosis in patients with acute myeloid leukemia (AML). We identify CD4+ Tcell-derived interleukin (IL)-21 as an important negative regulator of self-renewal of LSCs. IL-21/IL-21R signaling favors asymmetric cell division and differentiation in LSCs through the activation of p38-MAPK signaling, resulting in reduced LSC numbers and significantly prolonged survival in murine AML models. In human AML, serum IL-21 at diagnosis is identified as an independent positive prognostic biomarker for outcome and correlates with improved survival and higher complete remission rates in patients that underwent high-dose chemotherapy. IL-21 treatment inhibits primary LSC function and enhances the effect of cytarabine and CD70 CAR Tcell treatment on LSCs invitro. Low-dose IL-21 treatment prolongs the survival of AML mice in syngeneic and xenograft experiments. Therefore, promoting IL-21/IL-21R signaling on LSCs may be an approach to reduce stemness and increase differentiation in AML.

Establishment of a 3D organoid culture model for the investigation of adult slow-cycling putative intestinal stem cells.

The study of intestinal stem cells is a prerequisite for the development of therapies aimed at regenerating the gut. To enable investigation of adult slow-cycling H2B-GFP-retaining putative small intestinal (SI) stem cells in vitro, we have developed a three-dimensional (3D) SI organoid culture model based on the Tet-Op histone 2 B (H2B)-green fluorescent protein (GFP) fusion protein (Tet-Op-H2B-GFP) transgenic mouse. SI crypts were isolated from 6- to 12-week-old Tet-Op-H2B-GFP transgenic mice and cultured with appropriate growth factors and an animal-derived matrix (Matrigel). For in vitro transgene expression, doxycycline was added to the culture medium for 24h. By pulse-chase experiments, H2B-GFP expression and retention were assessed through direct GFP fluorescence observations, both by confocal and fluorescence microscopy and by immunohistochemistry. The percentages of H2B-GFP-retaining putative SI stem cells and H2B-GFP-retaining Paneth cells persisting in organoids were determined by scoring relevant GFP-positive cells. Our results indicate that 24 h exposure to doxycycline (pulse) induced ubiquitous expression of H2B-GFP in the SI organoids. During subsequent culture, in the absence of doxycycline (chase), there was a gradual loss (due to cell division) of H2B-GFP. At 6-day chase, slow-cycling H2B-GFP-retaining putative SI stem cells and H2B-GFP-retaining Paneth cells were detected in the SI organoids. The developed culture model allows detection of slow-cycling H2B-GFP-retaining putative SI stem cells and will enable the study of self-renewal and regeneration for further characterization of these cells.

Cell Division: More Help in Forensic Medicine

Cell Division is a extremely regulated process. Cell mechanism and its Division is a very important step in solving human health problem’s. dismember the mechanisms of cell Division used classical genetics approaches to identify genes involved in mitosis and deployed biochemical approaches to isolate and identify proteins critical for cell Division. If it is my intent in this brief review to discuss the strategies in this genetics approaches of the new conclusions that have come to light

Genotoxic and Cytotoxic Activities of Cornhusk Extract of Zea mays and Leaf Extract of Sacharum officinarum

Zea mays husk and Saccharum officinarum have been used for years in ethnomedicine for their antimalarial, anti-inflammatory, antipyretic, antidiabetic, and antiphlogistic activities. The cytotoxic and genotoxic effects of Zea mays husk and Saccharum officinarum leaf extracts on the root meristem cells of Allium cepa were investigated. Onion bulbs were exposed to 2.5 mg/ml, 5mg/ml, and 10 mg/ml concentrations of the extracts for macroscopic and microscopic analysis. Tap water was used as a negative control and Methotrexate (0.1 mg/ml) was used as a positive control. There was statistically significant (p &lt; 0.05) inhibition of root growth depending on concentration by the extracts when compared with the negative control group. All the tested extracts were observed to have cytotoxic effects on cell division in A. cepa. The extract induced chromosomal aberrations and micronuclei (MNC) formations in A. cepa root tip cells were significant (p&lt;0.05) when compared with the control group. The extracts treatment further induced cell death, ghost cells, cells membrane damage, and binucleated cells. The Zea mays husk extract was found to exhibit higher cytotoxic and genotoxic potential than Saccharum officinarum leaf extract. These results suggest that Zea mays husk and Saccharum officinarum leaf extracts possess cytotoxic and genotoxic effects on A. cepa.

Research on the Mechanisms of Phytohormone Signaling in Regulating Root Development.

Phytohormones are organic compounds produced in trace amounts within plants that regulate their physiological processes. Their physiological effects are highly complex and diverse. They influence processes ranging from cell division, elongation, and differentiation to plant germination and rooting. Therefore, phytohormones play a crucial regulatory role in plant growth and development. Recently, various studies have highlighted the role of PHs, such as auxin, cytokinin (CK), and abscisic acid (ABA), and newer classes of PHs, such as brassinosteroid (BR) and peptide hormone, in the plant responses toward environmental stresses. These hormones not only have distinct roles at different stages of plant growth but also interact to promote or inhibit each other, thus effectively regulating plant development. Roots are the primary organs for water and mineral absorption in plants. During seed germination, the radicle breaks through the seed coat and grows downward to form the primary root. This occurs because the root needs to quickly penetrate the soil to absorb water and nutrients, providing essential support for the plant's subsequent growth. Root development is a highly complex and precisely regulated process influenced by various signals. Changes in root architecture can affect the plant's ability to absorb nutrients and water, which in turn impacts crop yield. Thus, studying the regulation of root development is of great significance. Numerous studies have reported on the role of phytohormones, particularly auxins, in root regulation. This paper reviews recent studies on the regulation of root development by various phytohormones, both individually and in combination, providing a reference for researchers in this field and offering perspectives on future research directions for improving crop yields.

Proteomics analysis of round and wrinkled pea (Pisum sativum L.) seeds during different development periods.

Seed development is complex, influenced by genetic and environmental factors. Understanding proteome profiles at different seed developmental stages is key to improving seed composition and quality. We used label-free quantitative proteomics to analyze round and wrinkled pea seeds at five growth stages: 4, 7, 12, 15, and days after anthesis (DAA), and at maturity. Wrinkled peas had lower starch content (30%) compared to round peas (47%-55%). Proteomic analysis identified 3659 protein groups, with 21%-24% shared across growth stages. More proteins were identified during early seed development than at maturity. Statistical analysis found 735 significantly different proteins between wrinkled and round seeds, regardless of the growth stage. The detected proteins were categorized into 31 functional classes, including metabolic enzymes, proteins involved in protein biosynthesis and homeostasis, carbohydrate metabolism, and cell division. Cell division-related proteins were more abundant in early stages, while storage proteins were more abundant later in seed development. Wrinkled seeds had lower levels of the starch-branching enzyme (SBEI), which is essential for amylopectin biosynthesis. Seed storage proteins like legumin and albumin (PA2) were more abundant in round peas, whereas vicilin was more prevalent in wrinkled peas. This study enhances our understanding of seed development in round and wrinkled peas. The study highlighted the seed growth patterns and protein profiles in round and wrinkled peas during seed development. It showed how protein accumulation changed, particularly focusing on proteins implicated in cell division, seed reserve metabolism, as well as storage proteins and protease inhibitors. These findings underscore the crucial role of these proteins in seed development. By linking the proteins identified to Cameor-based pea reference genome, our research can open avenues for deeper investigations into individual proteins, facilitate their practical application in crop improvement, and advance our knowledge of seed development.

Shugoshin 1 expression in various cancers: a potential target for therapy.

Shugoshin 1 (SGO1) is one of the Shugoshin (guardian spirit) family proteins, which is reported to be majorly involved in the protection of centromeres and proper segregation of chromosomes during cell division. Recent studies found that the altered expression of SGO1 is associated with various cancers and genetic disorders, and suggested as a target for therapy. In the present study, we have reviewed the available literature on SGO1 gene and protein expression in various cancer-cell lines, animal models and cancer patients, and targeting SGO1 with siRNA/shRNA. A significant increase in the expression of SGO1 mRNA and protein levels were observed in prostate, renal, lung, breast, neuroblastoma, leukemia, hepatocellular, and colon cancer-cell lines and the levels were associated with increased cellular proliferation, invasion, and metastasis. The altered SGO1 levels were observed in SGO1 knockout/haploinsufficient mice compared to wild type and the levels were associated with increased chromosome instability and tumorigenesis. Consistent with cell lines, higher SGO1 expression was also observed in tumor tissues of cancer patients compared to adjacent normal tissue and the levels were positively correlated with tumor stage, grade, size, and hormonal status. Higher SGO1 expression was related to resistance to chemotherapeutic agents and the knockdown of SGO1 increased sensitivity to those agents. Furthermore, targeting SGO1 with siRNA/shRNA reduced the expression of SGO1 and proliferation, and induced apoptosis of cancer cells. Overall, the SGO1 expression levels were significantly higher in various cancers, and targeting SGO1 with siRNA and shRNA reduced the levels of SGO1, proliferation and metastasis of cancers.

PTPN1/2 inhibition promotes muscle stem cell differentiation in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal disease caused by mutations in the DMD gene that encodes dystrophin. Dystrophin deficiency also impacts muscle stem cells (MuSCs), resulting in impaired asymmetric stem cell division and myogenic commitment. Using MuSCs from DMD patients and the DMD mouse model mdx, we found that PTPN1 phosphatase expression is up-regulated and STAT3 phosphorylation is concomitantly down-regulated in DMD MuSCs. To restore STAT3-mediated myogenic signaling, we examined the effect of K884, a novel PTPN1/2 inhibitor, on DMD MuSCs. Treatment with K884 enhanced STAT3 phosphorylation and promoted myogenic differentiation of DMD patient-derived MuSCs. In MuSCs from mdx mice, K884 treatment increased the number of asymmetric cell divisions, correlating with enhanced myogenic differentiation. Interestingly, the pro-myogenic effect of K884 is specific to human and murine DMD MuSCs and is absent from control MuSCs. Moreover, PTPN1/2 loss-of-function experiments indicate that the pro-myogenic impact of K884 is mediated mainly through PTPN1. We propose that PTPN1/2 inhibition may serve as a therapeutic strategy to restore the myogenic function of MuSCs in DMD.

A noncanonical GTPase signaling mechanism controls exit from mitosis in budding yeast

In the budding yeast Saccharomyces cerevisiae, exit from mitosis is coupled to spindle position to ensure successful genome partitioning between mother and daughter cells. This coupling occurs through a GTPase signaling cascade known as the mitotic exit network (MEN). The MEN senses spindle position via a Ras-like GTPase Tem1 which localizes to the spindle pole bodies (SPBs, yeast equivalent of centrosomes) during anaphase and signals to its effector protein kinase Cdc15. How Tem1 couples the status of spindle position to MEN activation is not fully understood. Here, we show that Cdc15 has a relatively weak preference for Tem1GTP and Tem1's nucleotide state does not change upon MEN activation. Instead, we find that Tem1's nucleotide cycle establishes a localization-based concentration difference in the cell where only Tem1GTP is recruited to the SPB, and spindle position regulates the MEN by controlling Tem1 localization to the SPB. SPB localization of Tem1 primarily functions to promote Tem1-Cdc15 interaction for MEN activation by increasing the effective concentration of Tem1. Consistent with this model, we demonstrate that artificially tethering Tem1 to the SPB or concentrating Tem1 in the cytoplasm with genetically encoded multimeric nanoparticles could bypass the requirement of Tem1GTP and correct spindle position for MEN activation. This localization/concentration-based GTPase signaling mechanism for Tem1 differs from the canonical Ras-like GTPase signaling paradigm and is likely relevant to other localization-based signaling scenarios.

Clonal dynamics after allogeneic haematopoietic cell transplantation.

Allogeneic haematopoietic cell transplantation (HCT) replaces the stem cells responsible for blood production with those from a donor1,2. Here, to quantify dynamics of long-term stem cell engraftment, we sequenced genomes from 2,824 single-cell-derived haematopoietic colonies of ten donor-recipient pairs taken 9-31 years after HLA-matched sibling HCT3. With younger donors (18-47 years at transplant), 5,000-30,000 stem cells had engrafted and were still contributing to haematopoiesis at the time of sampling; estimates were tenfold lower with older donors (50-66 years). Engrafted cells made multilineage contributions to myeloid, B lymphoid and T lymphoid populations, although individual clones often showed biases towards one or other mature cell type. Recipients had lower clonal diversity than matched donors, equivalent to around 10-15 years of additional ageing, arising from up to 25-fold greater expansion of stem cell clones. A transplant-related population bottleneck could not explain these differences; instead, phylogenetic trees evinced two distinct modes of HCT-specific selection. In pruning selection, cell divisions underpinning recipient-enriched clonal expansions had occurred in the donor, preceding transplant-their selective advantage derived from preferential mobilization, collection, survival ex vivo or initial homing. In growth selection, cell divisions underpinning clonal expansion occurred in the recipient's marrow after engraftment, most pronounced in clones with multiple driver mutations. Uprooting stem cells from their native environment and transplanting them to foreign soil exaggerates selective pressures, distorting and accelerating the loss of clonal diversity compared to the unperturbed haematopoiesis of donors.

On the possibility of yet a third kinetochore system in the protist phylum Euglenozoa.

A macromolecular assembly called the kinetochore is essential for the segregation of genetic material during eukaryotic cell division. Therefore, characterization of kinetochores across species is essential for understanding the mechanisms involved in this key process across the eukaryotic tree of life. In particular, little is known about kinetochores in divergent protists such as Euglenozoa, a group of unicellular flagellates that includes kinetoplastids, euglenids, and diplonemids, the latter being a highly diverse and abundant component of marine plankton. While kinetoplastids have an unconventional kinetochore system and euglenids have a canonical one similar to traditional model eukaryotes, preliminary searches detected neither unconventional nor canonical kinetochore components in diplonemids. Here, we employed state-of-the-art deep homology detection protocols but still could not detect orthologs for the bulk of kinetoplastid-specific nor canonical kinetochore proteins in diplonemids except for a putative centromere-specific histone H3 variant. Our results suggest that diplonemids evolved kinetochores that do not resemble previously known ones.