Cytokinesis Defects Research Articles

In Search of a Mechanistic Link between Chlamydia trachomatis-Induced Cellular Pathophysiology and Oncogenesis.

Centrosome duplication and cell cycle progression are essential cellular processes that must be tightly controlled to ensure cellular integrity. Despite their complex regulatory mechanisms, microbial pathogens have evolved sophisticated strategies to co-opt these processes to promote infection. While misregulation of these processes can greatly benefit the pathogen, the consequences to the host cell can be devastating. During infection, the obligate intracellular pathogen Chlamydia trachomatis induces gross cellular abnormalities, including supernumerary centrosomes, multipolar spindles, and defects in cytokinesis. While these observations were made over 15 years ago, identification of the bacterial factors responsible has been elusive due to the genetic intractability of Chlamydia. Recent advances in techniques of genetic manipulation now allows for the direct linking of bacterial virulence factors to manipulation of centrosome duplication and cell cycle progression. In this review, we discuss the impact, both immediate and downstream, of C. trachomatis infection on the host cell cycle regulatory apparatus and centrosome replication. We highlight links between C. trachomatis infection and cervical and ovarian cancers and speculate whether perturbations of the cell cycle and centrosome are sufficient to initiate cellular transformation. We also explore the biological mechanisms employed by Inc proteins and other secreted effector proteins implicated in the perturbation of these host cell pathways. Future work is needed to better understand the nuances of each effector's mechanism and their collective impact on Chlamydia's ability to induce host cellular abnormalities.

Abstract PR018: Neuropeptide Y as a metastatic factor

Abstract Although neuropeptide Y (NPY) is known mainly as a sympathetic neurotransmitter, growing evidence indicates its role in tumor biology. Previous studies from our laboratory and other groups suggested associations of high expression of NPY and its Y5 receptor (Y5R) with invasive and metastatic phenotypes of various tumor types; yet the direct evidence for the metastatic activity was lacking. Our recent work began to fill this gap. Using Ewing sarcoma (ES) as a model of NPY-rich tumor, we identified the NPY/Y5R axis as a crucial pathway responsible for hypoxia-induced ES bone metastasis. This osseous dissemination was driven by hypoxia-dependent over-activation of the NPY/Y5R/RhoA pathway, which led to cytokinesis defects and formation of the aneuploid ES cell population with high propensity to bone metastasis. Here, we sought to determine the impact of the Y5R signaling on overall, hypoxia-independent metastatic capabilities of ES cells. To this end, a doxycycline-inducible CRIPSR/Cas9 system was used to knockout Y5R in ES orthotopic xenografts and test its impact on metastasis and cell migration assessed by a transwell assay. The frequency of the NPY5R gene modifications was determined by sequencing and confirmed by immunohistochemistry. FISH was used to identify NPY5R gene gains in ES xenografts. RhoA activity was measured by immunocytochemistry and pull-down assay. As expected, ES/Y5R-sgRNA primary tumors in doxycycline-treated mice consisted of a heterogeneous ES cell population with variable Y5R levels. In contrast, metastases developing from these xenografts were primarily initiated by ES clones with an intact NPY5R gene. The frequency of NPY5R gene modifications in metastases from the doxycycline-treated group was markedly reduced, as compared to the primary tumors. No metastases arising from ES clones lacking intact NPY5R gene were detected. Similarly, metastasis from wild type ES xenografts was associated with a selection of clones with the NPY5R gene gain that was present in a small percent of the parental cells. Subsequent in vitro assays implicated Y5R-dependent ES cell motility driven by the activation of a key cytoskeleton regulator, RhoA, as the mechanism underlying the metastatic effects of NPY. The above results, along with our previously published data, provide direct evidence for the crucial role of the NPY/Y5R axis in metastasis. We have shown that under basal conditions the NPY/Y5R/RhoA axis promotes tumor cell motility and overall cancer cell dissemination, while over-activation of this pathway under hypoxic conditions leads to genomic instability and bone metastasis. While our study focused on ES due to its high endogenous expression of NPY and its receptors, our findings may be relevant to other malignancies rich in NPY and Y5R, such as neuroblastoma, breast and prostate cancer. Given availability of the FDA approved Y5R antagonist, NPY/Y5R pathway may be a promising target for therapies preventing cancer progression. Yet, further research is required to establish effectiveness of such pharmacological interventions. Citation Format: Mina Adnani, Sung-Hyeok Hong, Susana Galli, Akanksha Mahajan, Nouran Abualsaud, Lindsay Caprio, Jason U. Tilan, Olga Rodriguez, Hingkun Wang, Christopher Albanese, Luciane R. Cavalli, Joanna Kitlinska. Neuropeptide Y as a metastatic factor [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR018.

Gibberellins as a novel mutagen for inducing 2n gametes in plants.

The plant hormone gibberellin (GA) regulates many physiological processes, such as cell differentiation, cell elongation, seed germination, and the response to abiotic stress. Here, we found that injecting male flower buds with exogenous gibberellic acid (GA3) caused defects in meiotic cytokinesis by interfering with radial microtubule array formation resulting in meiotic restitution and 2n pollen production in Populus. A protocol for inducing 2n pollen in Populus with GA3 was established by investigating the effects of the dominant meiotic stage, GA3 concentration, and injection time. The dominant meiotic stage (F = 41.882, P < 0.001) and GA3 injection time (F = 172.466, P < 0.001) had significant effects on the frequency of induced 2n pollen. However, the GA3 concentration (F = 1.391, P = 0.253) did not have a significant effect on the frequency of induced 2n pollen. The highest frequency of GA3-induced 2n pollen (21.37%) was observed when the dominant meiotic stage of the pollen mother cells was prophase II and seven injections of 10 μM GA3 were given. Eighteen triploids were generated from GA3-induced 2n pollen. Thus, GA3 can be exploited as a novel mutagen to induce flowering plants to generate diploid male gametes. Our findings provide some new insight into the function of GAs in plants.

STYK1/NOK affects cell cycle late mitosis and directly interacts with anaphase-promoting complex activator CDH1

The novel oncogene STYK1/NOK plays critical roles in cancer development. However, its regulation during cell division is less defined. In this paper, we show that over-expression of STYK1/NOK caused mitotic arrest and cytokinesis defects. The protein level of STYK/NOK fluctuated during the cell cycle, with a peak at mitosis and a quick reduction upon mitotic exit. The cell cycle-related expression pattern of STYK1/NOK resembled the one of aurora kinases and polo-like kinase 1. Depletion of APC3 led to accumulation of STYK1/NOK and to the G2/M arrest. Co-immunoprecipitation experiment demonstrated the direct interaction of STYK1/NOK with CDH1. Overexpression of CDH1 shortened the half-life of STYK1/NOK. The kinase domain, but not the five D boxes, of STYK1/NOK was responsible for the interaction with CDH1. Altogether, our data demonstrated for the first time that STYK1/NOK could affect cell division, probably by directly targeting key components of APC/C such as CDH1 at late mitosis. Current study may provide a vital mechanistic clue for understanding the roles of STYK1/NOK in mitosis and cytokinesis during STYK1NOK mediated genomic instability and oncogenesis.

CLK1/CLK2-driven signalling at the Leishmania kinetochore is captured by spatially referenced proximity phosphoproteomics

Kinetochores in the parasite Leishmania and related kinetoplastids appear to be unique amongst eukaryotes and contain protein kinases as core components. Using the kinetochore kinases KKT2, KKT3 and CLK2 as baits, we developed a BirA* proximity biotinylation methodology optimised for sensitivity, XL-BioID, to investigate the composition and function of the Leishmania kinetochore. We could detect many of the predicted components and also discovered two novel kinetochore proteins, KKT24 and KKT26. Using KKT3 tagged with a fast-acting promiscuous biotin ligase variant, we took proximity biotinylation snapshots of the kinetochore in synchronised parasites. To quantify proximal phosphosites at the kinetochore as the parasite progressed through the cell cycle, we further developed a spatially referenced proximity phosphoproteomics approach. This revealed a group of phosphosites at the kinetochore that were highly dynamic during kinetochore assembly. We show that the kinase inhibitor AB1 targets CLK1/CLK2 (KKT10/KKT19) in Leishmania leading to defective cytokinesis. Using AB1 to uncover CLK1/CLK2 driven signalling pathways important for kinetochore function at G2/M, we found a set of 16 inhibitor responsive kinetochore-proximal phosphosites. Our results exploit new proximity labelling approaches to provide a direct analysis of the Leishmania kinetochore, which is emerging as a promising drug target.

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.

SETD2 Regulates Nuclear Division to Achieve Polyploidization in Megakaryocyte

Megakaryocytes (MKs) undergo a fascinating endomitosis process to become naturally large multilobulated, polyploid nuclei cells that give rise to platelets. Two key steps involving in this process are the controlled failure in cytokinesis (cytoplasm division) and karyokinesis (nuclear division). The cytokinesis defect has been reported to be regulated by factors related to abnormal actomyosin ring and cleavage furrow formation such as RhoA, MYH9 and Rac1. In contrast, it remains largely unknown how nuclear division defect is regulated during MK endomitosis. Using a mouse model and an in vitro differentiation system, we found SETD2 expression markedly declined when MKs underwent polyploidization. Knockdown of SETD2 increased the high ploidy of primary bone marrow (BM) murine MKs, as well as MKs derived from cultured human cord-blood CD34+ cells and MEG-01 cells. Furthermore, the higher ploidy of MKs was accompanied by the increase of platelet formation both in the mouse model and the in vitro differentiation system. With forced overexpression of SETD2, the proportion of high ploidy MKs (≥8N) was remarkably decreased. These data suggest that SETD2, an α-tubulin methyltransferase, negatively regulates polyploidization during megakaryopoiesis. To investigate how SETD2 regulates endomitosis, we employed live-cell imaging to monitor the whole process of endomitosis at the single-cell level by observing the spindle geometry. We found SETD2 deficient MKs were capable of completing the endomitotic cycle, allowing cells to reach a higher ploidy in a significantly shorter time compared to control cells (2.9 hours vs. 4.9 hours). In comparison, overexpression of SETD2 significantly increased endomitosis cycle time compared to control conditions (5.2 hours vs. 4.3 hours). These results show that SETD2 deficiency accelerates the endomitotic cell cycle. Moreover, loss of SETD2 increased the percentage of cells within the presence of nucleoplasmic bridges (NPBs), a thin DNA link referred as a characteristic structure for karyokinesis defect. These results suggest that SETD2 may regulate endomitosis through facilitating the defect of karyokinesis. During karyokinesis, microtubules localized near spindle poles are need to be depolymerized as force-generating apparatus to separate nuclei. Thus, we measured the amount of polymerized and depolymerized microtubule forms in the presence and absence of SETD2. We found that loss of SETD2 decreased the proportion of the depolymerized form of tubulin in spindles, while SETD2 overexpression increased depolymerized microtubule forms. These data suggest that loss of SETD2 promotes the defect of karyokinesis by reducing depolymerized microtubule state. Mechanistically, as SETD2 could trimethylate α-tubulin at lysine 40 (α-tubK40me3), we found that the levels of α-TubK40me3 near spindle poles were inversely correlated with the degree of karyokinesis defect and MK polyploidization. Moreover, SETD2-deficiency resulted in an absence of α-TubK40me3 with an increase of the MK ploidy. And this ploidy increase was largely reversed by overexpressing a mutant tubulin protein that mimics the α-tubK40me3. Together, these data indicate that SETD2-mediated α-TubK40me3 regulates MK polyploidization by influencing microtubule dynamics. In summary, our study reveals that SETD2 is an important, novel regulator of MK endomitosis by regulating α-TubK40me3 to influence microtubule dynamics. These findings help fill current knowledge gaps in the mechanism of the karyokinesis defect during MK endomitosis. Targeting SETD2 may improve high ploidy MKs and platelet production that could promote the regeneration of platelets in vitro and help treat megakaryocytic dysplasia diseases with abnormal polyploidization in the future.

Rice microtubule-associated protein OsMAP65-3.1, but not OsMAP65-3.2, plays a critical role in phragmoplast microtubule organization in cytokinesis.

In plants, MAP65 preferentially cross-links the anti-parallel microtubules (MTs) and plays an important role for cytokinesis. However, the functions of MAP65 isoforms in rice (Oryza sativa. L) are largely unknown. Here, we identified two MAP65-3 homologs in rice, OsMAP65-3.1 and OsMAP65-3.2. We found that both OsMAP65-3.1 and OsMAP65-3.2 were similar in dimerization and location to AtMAP65-3, and the expression of either rice genes driven by the AtMAP65-3 promoter suppressed the cytokinesis failure and growth defect of atmap65-3. However, OsMAP65-3.1 with native promoter also recovered the atmap65-3, but OsMAP65-3.2 with its own promoter had no effects. OsMAP65-3.1 but not OsMAP65-3.2 was actively expressed in tissues enriched with dividing cells. R1R2R3-Myb (MYB3R) transcription factors directly bound to the OsMAP65-3.1 promoter but not that of OsMAP65-3.2. Furthermore, osmap65-3.2 had no obvious phenotype, while either osmap65-3.1 or osmap65-3.1(+/-) was lethal. The eminent MTs around the daughter nuclei and cytokinesis defects were frequently observed in OsMAP65-3.1-defective plants. Taken together, our findings suggest that OsMAP65-3.1, rather than OsMAP65-3.2, plays essential roles in rice cytokinesis resulting from their differential expression which were passably directly regulated by OsMYB3Rs.

Rapamycin and Torin2 inhibit Candida auris TOR: Insights through growth profiling, docking, and MD simulations

The fungus Candida auris is a pathogen of utmost concern due to its rapid emergence across the globe, acquired antifungal drug tolerance, thermotolerance, and ability to survive in hospital settings and preserved foods. Recent incidences of comorbidity of corona patients with its infection in hospital settings highlighted the importance of understanding the pathobiology and drug tolerance of this fungus on priority. The Target of rapamycin (TOR) is a central regulator of growth across eukaryotes with an illustrated role in fungal pathology. The role of the TOR signalling pathway in the growth of C. auris is yet to be described. In-silico, analysis revealed the presence of highly conserved Tor kinase, components of TORC, and key downstream components in C. auris. Rapamycin and Torin2, the specific inhibitors of Tor reduce the growth of C. auris. An inhibition of Tor leads to cell cycle arrest at the G1 phase with a defect in cytokinesis. Interestingly, with an insignificant difference in growth at 30 and 37 °C, a sharp decline in growth is seen with Torin2 at 37 °C. The heterogeneous response emphasizes the importance of physiology-based differential cellular response at different temperatures. In addition, the inhibition of Tor suppresses the biofilm formation. In silico studies through docking and simulations showed rapamycin and torin2 as specific inhibitors of C. auris Tor kinase (CauTor kinase) and hence can be exploited for a thorough understanding of the TOR signalling pathway in pathobiology and drug tolerance of C. auris. HIGHLIGHTS Conservation of TOR signalling pathway in Candida auris Rapamycin and torin2 are specific inhibitors of Cau Tor Understanding of the role of TOR signalling pathway through the use of inhibitors rapamycin and torin2. Heterogenous response of C. auris to torin2 at different physiological conditions. Communicated by Ramaswamy H. Sarma

Delivery of ceramide phosphoethanolamine lipids to the cleavage furrow through the endocytic pathway is essential for male meiotic cytokinesis.

Cell division, wherein 1 cell divides into 2 daughter cells, is fundamental to all living organisms. Cytokinesis, the final step in cell division, begins with the formation of an actomyosin contractile ring, positioned midway between the segregated chromosomes. Constriction of the ring with concomitant membrane deposition in a specified spatiotemporal manner generates a cleavage furrow that physically separates the cytoplasm. Unique lipids with specific biophysical properties have been shown to localize to intercellular bridges (also called midbody) connecting the 2 dividing cells; however, their biological roles and delivery mechanisms remain largely unknown. In this study, we show that ceramide phosphoethanolamine (CPE), the structural analog of sphingomyelin, has unique acyl chain anchors in Drosophila spermatocytes and is essential for meiotic cytokinesis. The head group of CPE is also important for spermatogenesis. We find that aberrant central spindle and contractile ring behavior but not mislocalization of phosphatidylinositol phosphates (PIPs) at the plasma membrane is responsible for the male meiotic cytokinesis defect in CPE-deficient animals. Further, we demonstrate the enrichment of CPE in multivesicular bodies marked by Rab7, which in turn localize to cleavage furrow. Volume electron microscopy analysis using correlative light and focused ion beam scanning electron microscopy shows that CPE-enriched Rab7 positive endosomes are juxtaposed on contractile ring material. Correlative light and transmission electron microscopy reveal Rab7 positive endosomes as a multivesicular body-like organelle that releases its intraluminal vesicles in the vicinity of ingressing furrows. Genetic ablation of Rab7 or Rab35 or expression of dominant negative Rab11 results in significant meiotic cytokinesis defects. Further, we show that Rab11 function is required for localization of CPE positive endosomes to the cleavage furrow. Our results imply that endosomal delivery of CPE to ingressing membranes is crucial for meiotic cytokinesis.

Chromosome segregation failure and cytokinesis defect producing unreduced pollen in the diploid rubber tree (Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg.)

Chromosome segregation failure and cytokinesis defect producing unreduced pollen in the diploid rubber tree (Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg.)

Cell cycle expression of FLJ25439, a cytokinesis-associated protein, is mediated by D-box recognition and APC/C-Cdc20 regulated degradation

The midbody is a transient structure forming out of the central spindle at late telophase. Both the midbody and central spindle have important functions ensuring completion of cytokinesis and defects in this process may lead to genetic diseases, including cancer. Thus, understanding the mechanisms that control cytokinesis during mitosis can reveal the key components taking part in some of the processes that promote accurate cell division. Our previous study showed that overexpression of FLJ25439 causes cytokinesis defect with midbody arrest and induces tetraploids with prolonged cell growth/cell cycle progression (Pan et al., 2015). Here, we extend our investigation with regard to the expression profile/regulation and cellular localization/function of FLJ25439 during mitosis/cytokinesis. Using a monoclonal antibody 2A4 we found that FLJ25439 expression is cell cycle-dependent and subjected to APC/C complex regulation. Furthermore, it is a novel substrate for the APC/C-Cdc20 complex and its degradation is proteasome-dependent through D-box recognition during mitotic exit. Immunofluorescence microscopy showed it is distributed at the central spindle and midbody, two structures considered important for completion of cell division, in telophase and cytokinesis, respectively, during cell cycle progression. Depletion of FLJ25439 expression revealed defects in chromosome alignment/segregation and delayed mitosis/cytokinesis progression. We thus conclude that FLJ25439 is a hitherto undiscovered factor involved in cytokinesis regulation.

Stabilization of F-Actin Cytoskeleton by Paclitaxel Improves the Blastocyst Developmental Competence through P38 MAPK Activity in Porcine Embryos.

Changes in F-actin distribution and cortical F-actin morphology are important for blastocyst developmental competence during embryogenesis. However, the effect of paclitaxel as a microtubule stabilizer on embryonic development in pigs remains unclear. We investigated the role of F-actin cytoskeleton stabilization via P38 MAPK activation using paclitaxel to improve the developmental potential of blastocysts in pigs. In this study, F-actin enrichment and adducin expression based on blastomere fragment rate and cytokinesis defects were investigated in cleaved embryos after in vitro fertilization (IVF). Adducin and adhesive junction F-actin fluorescence intensity were significantly reduced with increasing blastomere fragment rate in porcine embryos. In addition, porcine embryos were cultured with 10 and 100 nM paclitaxel for two days after IVF. Adhesive junction F-actin stabilization and p-P38 MAPK activity in embryos exposed to 10 nM paclitaxel increased significantly with blastocyst development competence. However, increased F-actin aggregation, cytokinesis defects, and over-expression of p-P38 MAPK protein by 100 nM paclitaxel exposure disrupted blastocyst development in porcine embryos. In addition, exposure to 100 nM paclitaxel increased the misaligned α-tubulin of spindle assembly and adhesive junction F-actin aggregation at the blastocyst stage, which might be caused by p-P38 protein over-expression-derived apoptosis in porcine embryos.

Abstract 5660: Anti-proliferation effect of polo-like kinase-4 inhibitor in ovarian cancer via AKT/mTOR/p70S6K pathway

Abstract Background: Chromosomal instability is a hallmark in epithelial ovarian cancer. Polo-like kinases (PLK) play a vital role in cell cycle regulation. In particular, PLK4 is crucial in centriole duplication. PLK4 overexpression causes defect in cytokinesis, which in turn leads to aneuploidy and mitosis catastrophe. Previous studies showed that PLK4 inhibitors are effective against cell growth in different cancers like breast cancer and lung cancer. We hypothesize that the AKT/mTOR and p70S6K pathway is involved in this mechanism. Methods: The importance of cell proliferation of PLK4 was assessed by its inhibition in ovarian cancer cell lines and patient-derived organoids by CFI-400945. RNA sequencing was performed. The effects of PLK4 inhibition on the AKT/mTOR and p70S6K pathway was demonstrated by qPCR and western blot. Correlation of the mRNA expression level with the IC50 of CFI-400945 in ovarian cancer cell lines. Results: PLK4 inhibition led to reduced cell proliferation in ovarian cancer cells based on XTT and colony formation assays. The anti-proliferative effect was further confirmed in 12 patient-derived organoids using ATP bioluminescent assay. RNA sequencing showed that PLK4 inhibition led to downregulation of the AKT/mTOR/p70S6K pathway. It was found that the most sensitive cell line had the highest mRNA expression of mTOR and p70S6K, while those less sensitive had lower mTOR and p70S6K mRNA expression. Treatment with CFI-400945 showed a reduction of total and phosphorylated forms of AKT, mTOR and p70S6K in a dose-dependent level. Combined treatment with metformin, a drug that had mTOR inhibition effect, showed a synergistic effect in one ovarian cancer cell line. Conclusion: PLK4 inhibition caused reduced cell proliferation in in vitro and ex vivo assays, and this was accompanied by a downregulation of the AKT/mTOR/p70S6K pathway. This suggested that this pathway might be one of the downstream effector pathways of PLK4. Citation Format: Ka Yu Tse, Wai Sun Chan, Moxil Xuetang Mo, Horace Hok Yeung Lee, Philip Pun Ching Ip, Tak Wah Mak. Anti-proliferation effect of polo-like kinase-4 inhibitor in ovarian cancer via AKT/mTOR/p70S6K pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5660.

Exogenous 6-benzylaminopurine inhibits tip growth and cytokinesis via regulating actin dynamics in the moss Physcomitrium patens.

Exogenous BAP but not 2iP disrupts actin structures and induces tip-growth retardation and cytokinesis failure in the moss Physcomitrium patens. Synthetic cytokinins have been widely used to address hormonal responses during plant development. However, exogenous cytokinins can cause a variety of cellular effects. A detailed characterization of such effects has not been well studied. Here, using Physcomitrium patens as a model, we show that the aromatic cytokinin 6-benzylaminopurine (BAP) inhibits tip growth at concentrations above 0.2µM. At higher concentrations (0.6-1µM), BAP can additionally block mitotic entry and induce cytokinesis defects and cell death. These effects are associated with altered actin dynamics and structures. By contrast, 2-isopentenyladenine (2iP) does not cause marked defects at various concentrations up to 10µM, while t-zeatin (tZ) can moderately inhibit moss growth. Our results provide mechanistic insight into the inhibitory effects of BAP on cell growth and cell division and call for attention to the use of synthetic cytokinins for bioassays.

Sporophytic control of pollen meiotic progression is mediated by tapetum expression of ABORTED MICROSPORES.

Pollen development is dependent on the tapetum, a sporophytic anther cell layer surrounding the microspores that functions in pollen wall formation but is also essential for meiosis-associated development. There is clear evidence of crosstalk and co-regulation between the tapetum and microspores, but how this is achieved is currently not characterized. ABORTED MICROSPORES (AMS), a tapetum transcription factor, is important for pollen wall formation, but also has an undefined role in early pollen development. We conducted a detailed investigation of chromosome behaviour, cytokinesis, radial microtubule array (RMA) organization, and callose formation in the ams mutant. Early meiosis initiates normally in ams, shows delayed progression after the pachytene stage, and then fails during late meiosis, with disorganized RMA, defective cytokinesis, abnormal callose formation, and microspore degeneration, alongside abnormal tapetum development. Here, we show that selected meiosis-associated genes are directly repressed by AMS, and that AMS is essential for late meiosis progression. Our findings indicate that AMS has a dual function in tapetum-meiocyte crosstalk by playing an important regulatory role during late meiosis, in addition to its previously characterized role in pollen wall formation. AMS is critical for RMA organization, callose deposition, and therefore cytokinesis, and is involved in the crosstalk between the gametophyte and sporophytic tissues, which enables synchronous development of tapetum and microspores.

Abstract 463: The Smooth-Muscle-Cell-Angiotensin II-Sensitive LncRNA Controls Cell Division Fidelity And Mitochondrial Organization

Reactivation of the cell cycle and increase in proliferation rate (hyperplasia) is a common response of vascular smooth muscle cells (SMC) to modifications of their environment during remodeling. Although SMC hyperplasia is a predominant feature of many vascular diseases, SMC can also increase their mass within the remodeled vessel wall by enlarging their size and becoming hypertrophic. Hypertrophy is usually accompanied by cell cycle defects, cell polyploidy and binucleation, and senescence. However, the molecular mechanisms favoring SMC hypertrophy and their repercussion on SMC phenotype are not fully understood. Long-non-coding-RNAs (LncRNAs) are epigenetic regulators of gene expression, and they have been identified as modulators of cell division. We recently discovered a novel lncRNA, SAS (SMC-Angiotensin II-Sensitive), whose expression was markedly decreased in multiple models of SMC dedifferentiation, suggesting that SAS could regulate SMC phenotype and function. Publicly available transcriptional datasets revealed that SAS is preferentially expressed in SMC-rich tissues, including the aorta, in humans and mice. Yet, the functional relevance of SAS in SMC has never been investigated. Knockdown of SAS reduces proliferation, cell arrest and migration in aortic and renal artery-derived SMC treated with Platelet Derived Growth Factor. SAS knockdown was also associated with distinct SMC morphological changes including increase in cell size and binucleation demonstrating hypertrophy. Together, these observations suggest that decrease in SAS causes SMC hypertrophy due to defects in cell cycle completion and cytokinesis. Interestingly, SAS expression is decreased in response to Angiotensin-II in cultured VSMC and in the aorta of hypertensive mice (2 Kidney-1 Clip model), suggesting a role in mediating hypertension induced SMC hypertrophy. Similarly, to Angiotensin-II treatment, SAS knockdown promoted senescence. Furthermore, SAS deficient cells present mitochondria hyperfusion and increased oxygen consumption that correlates with the observed exacerbated senescence. Altogether, our results indicate that SAS is a potent regulator of VSMC morphology and is required for proper cell division and mitochondria organization.

Palmitic acid-induced defects in cell cycle progression and cytokinesis in Neuro-2a cells

ABSTRACT Obesity is associated with elevated levels of free fatty acids (FFAs). Excessive saturated fatty acids (SFAs) exhibit significant deleterious cytotoxic effects in many types of cells. However, the effects of palmitic acid (PA), the most common circulating SFA, on cell cycle progression in neuronal cells have not been well-examined. The aim of this study was to examine whether PA affects the proliferation and cell cycle progression in mouse neuroblastoma Neuro-2a (N2a) cells. Our studies found that 200 µM PA significantly decreased DNA synthesis and mitotic index in N2a cells as early as 4 h following treatment. 24 h treatment with 200 µM PA significantly decreased the percentage of diploid (2 N) cells while dramatically increasing the percentage of tetraploid (4 N) cells as compared to the BSA control. Moreover, our studies found that 24 h treatment with 200 µM PA increased the percentage of binucleate cells as compared to the BSA control. Our studies also found that unsaturated fatty acids (UFAs), including linoleic acid, oleic acid, α-linolenic acid, and docosahexaenoic acid, were able to abolish PA-induced decrease of 2 N cells, increase of 4 N cells, and accumulation of binucleate cells. Taken together, these results suggest that PA may affect multiple aspects of the cell cycle progression in N2a cells, including decreased DNA synthesis, G2/M arrest, and cytokinetic failure, which could be abolished by UFAs. Abbreviations: 4-PBA, 4-Phenylbutyric Acid; ALA, α-linolenic acid; BrdU, 5-bromo-2’-deoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole; ER, endoplasmic reticulum; FFA, free fatty acids; FITC, fluorescein isothiocyanate; LA, linoleic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; N2a, Neuro-2a; NAC, N-acetyl cysteine; OA, oleic acid; PA, palmitic acid; pHH3, Phosphorylation of histone H3; PI, propidium iodide; SFA, saturated fatty acids; PUFA, polyunsaturated fatty acids; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; UFA, unsaturated fatty acids

Lyz2-Cre-Mediated Genetic Deletion of Septin7 Reveals a Role of Septins in Macrophage Cytokinesis and Kras-Driven Tumorigenesis.

By crossing septin7-floxed mice with Lyz2-Cre mice carrying the Cre recombinase inserted in the Lysozyme-M (Lyz2) gene locus we aimed the specific deletion of septin7 in myeloid cells, such as monocytes, macrophages and granulocytes. Septin7 flox/flox :Lyz2-Cre mice show no alterations in the myeloid compartment. Septin7-deleted macrophages (BMDMs) were isolated and analyzed. The lack of Septin7 expression was confirmed and a constitutive double-nucleation was detected in Septin7-deficient BMDMs indicating a defect in macrophage cytokinesis. However, phagocytic function of macrophages as judged by uptake of labelled E. coli particles and LPS-stimulated macrophage activation as judged by induction of TNF mRNA expression and TNF secretion were not compromised. In addition to myeloid cells, Lyz2-Cre is also active in type II pneumocytes (AT2 cells). We monitored lung adenocarcinoma formation in these mice by crossing them with the conditional knock-in Kras-LSL-G12D allele. Interestingly, we found that control mice without septin7 depletion die after 3–5 weeks, while the Septin7-deficient animals survived 11 weeks or even longer. Control mice sacrificed in the age of 4 weeks display a bronchiolo-alveolar hyperplasia with multiple adenomas, whereas the Septin7-deficient animals of the same age are normal or show only a weak multifocal brochiolo-alveolar hyperplasia. Our findings indicate an essential role of Septin7 in macrophage cytokinesis but not in macrophage function. Furthermore, septin7 seems absolutely essential for oncogenic Kras-driven lung tumorigenesis making it a potential target for anti-tumor interventions.

Deletion of gmfA induces keratocyte-like migration in Dictyostelium.

Glia maturation factor (GMF) has been established as an inactivating factor of the actin‐related protein 2/3 (Arp2/3) complex, which regulates actin assembly. Regulation of actin assembly and reorganization is crucial for various cellular events, such as cell migration, cell division, and development. Here, to examine the roles of ADF‐H domain‐containing protein (also known as glia maturation factor; GmfA), the product of a single GMF homologous gene in Dictyostelium, gmfA‐null cells were generated. They had moderate defects in cell growth and cytokinesis. Interestingly, they showed a keratocyte‐like fan shape with a broader pseudopod, where Arp3 accumulated at higher levels than in wild‐type cells. They migrated with higher persistence, but their velocities were comparable to those of wild‐type cells. The polar pseudopods during cell division were also broader than those in wild‐type cells. However, GmfA did not localize at the pseudopods in migrating cells or the polar pseudopods in dividing cells. Adhesions of mutant cells to the substratum were much stronger than that of wild‐type cells. Although the mutant cells showed chemotaxis comparable to that of wild‐type cells, they formed disconnected streams during the aggregation stage; however, they finally formed normal fruiting bodies. These results suggest that GmfA plays a crucial role in cell migration.