scholarly journals Structural basis of Stu2 recruitment to yeast kinetochores

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jacob A Zahm ◽  
Michael G Stewart ◽  
Joseph S Carrier ◽  
Stephen C Harrison ◽  
Matthew P Miller
Keyword(s):  
Cell Cycle Progression ◽  
Terminal Segment ◽  
Structural Basis ◽  
Wild Type ◽  
Cycle Progression ◽  
Delay Cell ◽  
Sister Chromatids

Chromosome segregation during cell division requires engagement of kinetochores of sister chromatids with microtubules emanating from opposite poles. As the corresponding microtubules shorten, these ‘bioriented’ sister kinetochores experience tension-dependent stabilization of microtubule attachments. The yeast XMAP215 family member and microtubule polymerase, Stu2, associates with kinetochores and contributes to tension-dependent stabilization in vitro. We show here that a C-terminal segment of Stu2 binds the four-way junction of the Ndc80 complex (Ndc80c) and that residues conserved both in yeast Stu2 orthologs and in their metazoan counterparts make specific contacts with Ndc80 and Spc24. Mutations that perturb this interaction prevent association of Stu2 with kinetochores, impair cell viability, produce biorientation defects, and delay cell cycle progression. Ectopic tethering of the mutant Stu2 species to the Ndc80c junction restores wild-type function in vivo. These findings show that the role of Stu2 in tension-sensing depends on its association with kinetochores by binding with Ndc80c.

scholarly journals Structural Basis of Stu2 Recruitment to Yeast Kinetochores

2020 ◽  
Author(s):  
Jacob A. Zahm ◽  
Michael G. Stewart ◽  
Joseph S. Carrier ◽  
Stephen C. Harrison ◽  
Matthew P. Miller
Keyword(s):  
Cell Cycle Progression ◽  
Spindle Assembly Checkpoint ◽  
Terminal Segment ◽  
Structural Basis ◽  
Amino Acid Residues ◽  
Checkpoint Signaling ◽  
Delay Cell ◽  
Sister Chromatids

ABSTRACTAccurate chromosome segregation during cell division requires engagement of the kinetochores of sister chromatids with microtubules emanating from opposite poles of the mitotic spindle. In yeast, these “bioriented” metaphase sister chromatids experience tension as the corresponding microtubules (one per sister chromatid) shorten. Spindle-assembly checkpoint signaling appears to cease from a kinetochore under tension, which also stabilizes kinetochore-microtubule attachment in single-kinetochore experiments in vitro. The microtubule polymerase, Stu2, the yeast member of the XMAP215/ch-TOG protein family, associates with kinetochores in cells and contributes to tension-dependent stabilization, both in vitro and in vivo. We show here that a C-terminal segment of Stu2 binds the four-way junction of the Ndc80 complex (Ndc80c) and that amino-acid residues conserved both in yeast Stu2 orthologs and in their metazoan counterparts make specific contacts with Ndc80 and Spc24. Mutations that perturb this interaction prevent association of Stu2 with kinetochores, impair cell viability, produce biorientation defects, and delay cell-cycle progression. Ectopic tethering of the mutant Stu2 species to the Ndc80c junction restores wild-type function. These findings show that the role of Stu2 in tension sensing depends on its association with kinetochores by binding with Ndc80c.

PI3K/Akt/FOXO3a Pathway Contributes to Thrombopoietin-Induced Proliferation of Primary Megakaryocytes In Vitro and In Vivo Via Modulation of p27Kip1.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 202-202
Author(s):  
Takafumi Nakao ◽  
Amy E Geddis ◽  
Norma E. Fox ◽  
Kenneth Kaushansky
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Cycle Progression ◽  
Wild Type ◽  
Cycle Progression ◽  
P27kip1 Expression ◽  
Cell Counts ◽  
Deficient Mice

Abstract Thrombopoietin (TPO), the primary regulator of megakaryocyte (MK) and platelet formation, modulates the activity of multiple signal transduction molecules, including those in the Jak/STAT, p42/p44 MAPK, and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. In the previous study, we reported that PI3K and Akt are necessary for TPO-induced cell cycle progression of primary MK progenitors. The absence of PI3K activity results in a block of transition from G1 to S phase in these cells (Geddis AE et al. JBC2001276:34473–34479). However, the molecular events secondary to the activation of PI3K/Akt responsible for MK proliferation remain unclear. In this study we show that FOXO3a and its downstream target p27Kip1 play an important role in TPO-induced proliferation of MK progenitors. TPO induces phosphorylation of Akt and FOXO3a in both UT-7/TPO, a megakaryocytic cell line, and primary murine MKs in a PI3K dependent fashion. Cell cycle progression of UT-7/TPO cells is blocked in G1 phase by inhibition of PI3K. We found that TPO down-modulates p27Kip1 expression at both the mRNA and protein levels in UT-7/TPO cells and primary MKs in a PI3K dependent fashion. UT-7/TPO stably expressing constitutively active Akt or a dominant-negative form of FOXO3a failed to induce p27Kip1 expression after TPO withdrawal. Induced expression of an active form of FOXO3a resulted in increased p27Kip1 expression in this cell line. In an attempt to assess whether FOXO3a has an effect of MK proliferation in vivo, we compared the number of MKs in Foxo3a-deficient mice and in wild type controls. Although peripheral blood cell counts of erythrocytes, neutrophils, monocytes and platelets were normal in the Foxo3a-deficient mice, total nucleated marrow cell count of Foxo3a-deficient mice were 60% increased compared with wild type controls. In addition, the increase of MKs was more profound than that of total nucleated marrow cells; CD41+ MKs from Foxo3a-deficient mice increased 2.1-fold, and mature MKs with 8N and greater ploidy increased 2.5-fold, compared with wild type controls. Taken together with the previous observation that p27Kip1-deficient mice also display increased numbers of MK progenitors, our findings strongly suggest that the effect of TPO on MK proliferation is mediated by PI3K/Akt-induced FOXO3a inactivation and subsequent p27Kip1 down-regulation in vitro and in vivo.

scholarly journals Epigenetic factors in atherogenesis: microRNA

2016 ◽  
Vol 62 (2) ◽  
pp. 134-140
Author(s):  
A.V. Smirnova ◽  
V.N. Sukhorukov ◽  
V.P. Karagodin ◽  
A.N. Orekhov
Keyword(s):  
Gene Expression ◽  
Antisense Oligonucleotides ◽  
Noncoding Rna ◽  
Cell Cycle Progression ◽  
Rna Sequences ◽  
Cycle Progression ◽  
Atherosclerosis Progression

MicroRNAs (miRNAs) are small (~22 nucleotides in length) noncoding RNA sequences regulating gene expression at posttranscriptional level. MicroRNAs bind complementarily to certain mRNA and cause gene silencing. The involvement of miRNAs in the regulation of lipid metabolism, inflammatory response, cell cycle progression and proliferation, oxidative stress, platelet activation, endothelial and vascular smooth muscle cells (VSMC) function, angiogenesis and plaque formation and rapture indicates important roles in the initiation and progression of atherosclerosis. The key role of microRNAs in pathophysiology of cardiovascular diseases (CVDs), including atherosclerosis, was demonstrated in recent studies. Creating antisense oligonucleotides is a novel technique for selective changes in gene expression both in vitro and in vivo. In this review, we draw attention to the role of miRNAs in atherosclerosis progression, using miRNA as the potential biomarkers and targets in the CVDs, as well as possible application of antisense oligonucleotides

scholarly journals E3 Ubiquitin Ligase TRIM29 Promotes Pancreatic Cancer Growth and Progression via Stabilizing Yes-associated Protein 1

2021 ◽  
Author(s):  
Xueqiang Deng ◽  
Xiaowei Fu ◽  
Hong Teng ◽  
Lu Fang ◽  
Bo Liang ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cell Cycle Progression ◽  
Nude Mouse Model ◽  
Cycle Progression ◽  
Signal Cascade ◽  
The Relationship ◽  
Precise Role

Abstract Background: Pancreatic cancer (PC) is one of the most fatal digestive system cancers. tripartite motif-29 (TRIM29) has been reported as oncogene in several human cancers. However, the precise role and underlying signal cascade of TRIM29 in PC progression remain unclear.Methods: Western blot, qRT-PCR and immunohistochemistry were used to analyze TRIM29 and Yes-associated protein 1 (YAP1) levels. CCK8 assays, EdU assays and flow cytometry were designed to explore the function and potential mechanism of TRIM29 and YAP1 in the proliferation of PC. Next, a nude mouse model of PC was established for validating the roles of TRIM29 and YAP1 in vivo. The relationship among TRIM29 and YAP1 was explored by co-immunoprecipitation and in vitro ubiquitination assay.Results: TRIM29 and YAP1 was significantly upregulated in PC patient samples, and TRIM29 expression was closely related to a malignant phenotype and poorer overall survival (OS) of PC patients. Functional assays revealed that TRIM29 knockdown suppresses cell growth, arrests cell cycle progression and promotes cell apoptosis of PC cells in vivo and in vitro. Furthermore, the rescue experiments demonstrated that TRIM29-induced proliferation is dependent on YAP1 in PC cells. Mechanistically, TRIM29 regulates YAP1 expression by directly binding to YAP1, and reduced its ubiquitination and degradation.Conclusion: Taken together, these results identify a novel mechanism used by PC growth, and provide insight regarding the role of TRIM29 in PC.

scholarly journals Novel Long Noncoding RNA miR205HG Functions as an Esophageal Tumor-Suppressive Hedgehog Inhibitor

Cancers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1707
Author(s):  
Jee Hoon Song ◽  
Alan H. Tieu ◽  
Yulan Cheng ◽  
Ke Ma ◽  
Venkata S. Akshintala ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Noncoding Rna ◽  
Cell Cycle Progression ◽  
Esophageal Tumor ◽  
Cycle Progression ◽  
In Vivo Experiments ◽  
Hh Signaling

Barrett’s esophagus (BE) is a precursor to esophageal adenocarcinoma (EAC). Recently, long noncoding RNAs (lncRNAs) have been identified as key regulators of biological pathways. However, involvement of lncRNAs in the development of BE and EAC has not been well-studied. The aims of the current study were: (1) to study involvement of the lncRNA, miR205HG, in the development of BE and EAC; (2) to clarify the role of miR205HG in in vitro and in vivo experiments; and (3) to investigate the mechanism of miR205HG involving the Hedgehog (Hh) signaling pathway. These experiments revealed that miR205HG was downregulated in EAC vs. normal esophageal epithelia (NE) as well as in EAC cell lines, and its forced overexpression inhibited EAC cell proliferation and cell cycle progression in vitro. Similarly, overexpression of miR205HG inhibited xenograft tumor growth in mice In vivo. Finally, we show that one mechanism of action of miR205HG involves the Hh signaling pathway: miR205HG and Hh expression levels were inversely correlated in both EAC (r = −0.73) and BE (r = −0.83) tissues, and in vitro studies revealed details of Hh signaling inhibition induced by miR205HG. In conclusion, these findings establish that lncRNA miR205HG functions as a tumor suppressor in the development of BE and EAC, at least in part through its effect on the Hh signaling pathway.

p38α Activates Purine Metabolism to Initiate Hematopoietic Stem Cell Cycling

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 776-776
Author(s):  
Daiki Karigane ◽  
Shinichiro Okamoto ◽  
Toshio Suda ◽  
Keiyo Takubo
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Purine Metabolism ◽  
Stress Conditions ◽  
Wild Type ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Deficient Mice

Abstract Hematopoietic stem cells (HSCs) maintain quiescence by activating specific metabolic pathways, including glycolysis. However, how stress hematopoiesis, including bone marrow transplantation (BMT), induces metabolic changes in HSCs remains unclear. Here, we report a critical role for the p38MAPK family isoform p38α in initiating HSC proliferation during stress hematopoiesis in mouse. First, we identified p38α as the major p38MAPK isozyme highly expressed in HSCs and we also performed conditional knockout of p38α in mice. This mouse showed no overt difference relative to wild type mouse. However, treatment of p38α-deficient mice with 5-FU exhibited defective recovery of hematopoiesis, and the survival rate were lower in p38α-deficient mice than wild-type mice (42.9%, N=7, p38α-deficient mice, vs 100%, wild-type mice, N=6, p=0.03) and loss of p38α in HSCs showed a defective transplantation capacity in primary and secondary transplantation. To gain further insight into p38MAPK function during hematological stress, we evaluated the time course of p38MAPK activation in stressful contexts by intracellular flow cytometry. We found that p38MAPK was immediately phosphorylated in HSCs after hematological stress and returned to normal in a short period, suggesting that p38α functions rapidly after hematological stresses to activate downstream events. To identify events downstream of p38α after hematological stress, we initially evaluated mechanisms such as homing, apoptosis, and ROS generation immediately after BMT. However, defects seen in p38α-deficient HSCs after hematological stress could not be explained by these mechanisms. Therefore we next focused on cell cycle. In CFSE assay, p38α loss resulted in defective recovery from hematological stress and a delay in initiating cycling of HSCs. In addition, p38α-deficient HSCs showed lower BrdU incorporation in vivo (p=0.045) and EdU incorporation in vitro (p=0.003). Transcriptome analysis of transplanted wild-type or p38α-deficient HSCs suggested that p38α-deficient HSCs showed lower enrichment of genes related to HSC-related markers and proliferation. Taken together, loss of p38α resulted in defective HSC cell cycle progression in stressed settings such as transplantation. Given that altered metabolic activities can change cell cycle status, we asked whether p38α regulation of a particular metabolic pathway could initiate HSC cycling under stress conditions. To do so, we collected p38α-deficient or wild-type LSK cells either at steady state or after BMT and extracted metabolites for metabolome analysis using mass spectrometry. Among metabolites surveyed, we focused on changes in glycine and aspartic acid, which are required for purine biosynthesis. Levels of both increased in p38α-deficient as compared with wild-type LSK cells after BMT. Also, mice transplanted with p38α-deficient compared with wild-type LSK cells showed lower levels of allantoin, a product of purine catabolism. These findings suggest that p38α loss suppresses purine metabolism during stress hematopoiesis. Next, we evaluated mRNAs encoding key enzymes functioning in purine metabolism by qPCR. Expression of both inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), and guanosine monophosphate synthetase (GMPS) was significantly decreased in p38α-deficient HSCs relative to wild-type HSCs on day 1 after BMT. To assess how changes in purine metabolism could affect the HSC response to stress, we treated HSCs with cytokines in the presence or absence of mycophenolic acid (MPA), an IMPDH2 inhibitor. MPA treatment significantly suppressed colony formation capacity of HSCs in a dose-dependent manner. Also, EdU incorporation into HSCs was reduced by MPA dose-dependently. Finally, isolated HSCs were cultured with or without MPA for 3 days and then transplanted into recipients along with competitor cells. PB chimerism was dose-dependently decreased in recipients of MPA-treated cells. These findings suggest that purine metabolism directly maintains proliferation capacity of HSCs in stress conditions. In summary, expression of purine-synthesizing enzymes decreased in p38α-deficient HSCs after transplantation, an activity correlated with defective cell cycle progression in vitro and in vivo. Overall, this is the first report of p38α-regulated changes in purine metabolism associated with HSC stress and cell cycle initiation. Disclosures No relevant conflicts of interest to declare.

scholarly journals E3 ubiquitin ligase TRIM29 promotes pancreatic cancer growth and progression via stabilizing Yes-associated protein 1

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Xueqiang Deng ◽  
Xiaowei Fu ◽  
Hong Teng ◽  
Lu Fang ◽  
Bo Liang ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cell Cycle Progression ◽  
Nude Mouse Model ◽  
Cycle Progression ◽  
Signal Cascade ◽  
The Relationship ◽  
Precise Role

Abstract Background Pancreatic cancer (PC) is one of the most fatal digestive system cancers. tripartite motif-29 (TRIM29) has been reported as oncogene in several human cancers. However, the precise role and underlying signal cascade of TRIM29 in PC progression remain unclear. Methods Western blot, qRT-PCR and immunohistochemistry were used to analyze TRIM29 and Yes-associated protein 1 (YAP1) levels. CCK8 assays, EdU assays and flow cytometry were designed to explore the function and potential mechanism of TRIM29 and YAP1 in the proliferation of PC. Next, a nude mouse model of PC was established for validating the roles of TRIM29 and YAP1 in vivo. The relationship among TRIM29 and YAP1 was explored by co-immunoprecipitation and in vitro ubiquitination assay. Results TRIM29 and YAP1 was significantly upregulated in PC patient samples, and TRIM29 expression was closely related to a malignant phenotype and poorer overall survival (OS) of PC patients. Functional assays revealed that TRIM29 knockdown suppresses cell growth, arrests cell cycle progression and promotes cell apoptosis of PC cells in vivo and in vitro. Furthermore, the rescue experiments demonstrated that TRIM29-induced proliferation is dependent on YAP1 in PC cells. Mechanistically, TRIM29 regulates YAP1 expression by directly binding to YAP1, and reduced its ubiquitination and degradation. Conclusion Taken together, these results identify a novel mechanism used by PC growth, and provide insight regarding the role of TRIM29 in PC.

scholarly journals A Circular RNA from the MDM2 Locus Controls Cell Cycle Progression by Suppressing p53 Levels

2020 ◽  
Vol 40 (9) ◽  
Author(s):  
Ritu Chaudhary ◽  
Bruna R. Muys ◽  
Ioannis Grammatikakis ◽  
Supriyo De ◽  
Kotb Abdelmohsen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
P53 Protein ◽  
Circular Rnas ◽  
Small Interfering Rnas ◽  
Cycle Progression ◽  
Protein Levels

ABSTRACT Circular RNAs (circRNAs) are a class of noncoding RNAs produced by a noncanonical form of alternative splicing called back-splicing. To investigate a potential role of circRNAs in the p53 pathway, we analyzed RNA sequencing (RNA-seq) data from colorectal cancer cell lines (HCT116, RKO, and SW48) that were untreated or treated with a DNA-damaging agent. Surprisingly, unlike the strong p53-dependent induction of hundreds of p53-induced mRNAs upon DNA damage, only a few circRNAs were upregulated from p53-induced genes. circ-MDM2, an annotated circRNA from the MDM2 locus, was one of the handful of circRNAs that originated from a p53-induced gene. Given the central role of MDM2 in suppressing p53 protein levels and p53 activity, we investigated the function of circ-MDM2. Knocking down circ-MDM2 with small interfering RNAs (siRNAs) that targeted circ-MDM2 did not alter MDM2 mRNA or MDM2 protein levels but resulted in increased basal p53 levels and growth defects in vitro and in vivo. Consistent with these results, transcriptome profiling showed increased expression of several direct p53 targets, reduced retinoblastoma protein (Rb) phosphorylation, and defects in G1-S progression upon silencing circ-MDM2. Our results on the initial characterization of circ-MDM2 identify a new player from the MDM2 locus that suppresses p53 levels and cell cycle progression.

scholarly journals Nuclear Chaperone ASF1 is Required for Gametogenesis in Arabidopsis thaliana

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Yunsook Min ◽  
Jennifer M. Frost ◽  
Yeonhee Choi
Keyword(s):  
Arabidopsis Thaliana ◽  
Cell Cycle Progression ◽  
Critical Role ◽  
Cycle Progression ◽  
Male Side ◽  
Development Of Gametophytes ◽  
Mutant Pollen

Abstract Sexual reproduction in flowering plants is distinct from that in animals since gametogenesis requires production of haploid spores, which divide and differentiate into specialised gametophyte structures. Anti-Silencing Function 1 (ASF1) is a histone H3/H4 chaperone involved in chromatin remodeling during cell division, which we have found plays a critical role in gametophyte development in Arabidopsis thaliana. Using mutant alleles for the two ASF1 homologs, asf1a and asf1b, we show that ASF1 is required for successful development of gametophytes and acquisition of fertilisation competency. On the female side, reproductive failure is caused by aberrant development of ovules, leading to gamete degeneration. On the male side, we show both in vitro and in vivo that asf1 mutant pollen tube growth is stunted, limiting fertilisation to ovules nearest the stigma. Consistent with ASF1 importance in gametogenesis, we show that ASF1A and ASF1B are expressed throughout female and male gametogenesis. We show that the gametogenesis defects can be corrected by ASF1A and ASF1B transgenes, and that ASF1A and ASF1B act redundantly. Thus, in contrast to the role of ASF1 in sporophytic cell cycle progression, our data indicate that during reproduction, ASF1 is required for the precise nuclei differentiation necessary for gametophyte maturation and fertilisation.

scholarly journals A Review on the Role of miR-1246 in the Pathoetiology of Different Cancers

2022 ◽  
Vol 8 ◽  
Author(s):  
Soudeh Ghafouri-Fard ◽  
Tayyebeh Khoshbakht ◽  
Bashdar Mahmud Hussen ◽  
Mohammad Taheri ◽  
Mohammad Samadian
Keyword(s):  
Cell Cycle Progression ◽  
High Throughput Sequencing ◽  
Embryonic Stem ◽  
Clinical Samples ◽  
Cycle Progression ◽  
Current Review ◽  
Liver Cancers

miR-1246 is a microRNA firstly recognized through application of a high throughput sequencing technique in human embryonic stem cells. Subsequent studies have shown the role of this microRNA in the carcinogenesis. miR-1246 has been found to exert oncogenic roles in colorectal, breast, renal, oral, laryngeal, pancreatic and ovarian cancers as well as melanoma and glioma. In lung, cervical and liver cancers, studies have reported contradictory results regarding the role of miR-1246. miR-1246 has been reported to regulate activity of RAF/MEK/ERK, GSK3β, Wnt/β-catenin, JAK/STAT, PI3K/AKT, THBS2/MMP and NOTCH2 pathways. In addition to affecting cell cycle progression and proliferation, miR-1246 can influence stemness and resistance of cancer cells to therapeutics. In the current review, we describe the summary of in vitro and in vivo studies about the influence of miR-1246 in carcinogenesis in addition to studies that measured expression levels of miR-1246 in clinical samples.

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