scholarly journals Yeast Bim1p Promotes the G1-specific Dynamics of Microtubules

1999 ◽  
Vol 145 (5) ◽  
pp. 993-1007 ◽  
Author(s):  
Jennifer S. Tirnauer ◽  
Eileen O'Toole ◽  
Lisbeth Berrueta ◽  
Barbara E. Bierer ◽  
David Pellman
Keyword(s):  
Cell Cycle ◽  
Dynamic Instability ◽  
Microtubule Dynamics ◽  
Yeast Protein ◽  
Gene Encoding ◽  
Cytoplasmic Microtubule ◽  
A Cell ◽  
Cytoplasmic Microtubules

Microtubule dynamics vary during the cell cycle, and microtubules appear to be more dynamic in vivo than in vitro. Proteins that promote dynamic instability are therefore central to microtubule behavior in living cells. Here, we report that a yeast protein of the highly conserved EB1 family, Bim1p, promotes cytoplasmic microtubule dynamics specifically during G1. During G1, microtubules in cells lacking BIM1 showed reduced dynamicity due to a slower shrinkage rate, fewer rescues and catastrophes, and more time spent in an attenuated/paused state. Human EB1 was identified as an interacting partner for the adenomatous polyposis coli (APC) tumor suppressor protein. Like human EB1, Bim1p localizes to dots at the distal ends of cytoplasmic microtubules. This localization, together with data from electron microscopy and a synthetic interaction with the gene encoding the kinesin Kar3p, suggests that Bim1p acts at the microtubule plus end. Our in vivo data provide evidence of a cell cycle–specific microtubule-binding protein that promotes microtubule dynamicity.

scholarly journals β-Tubulin C354 Mutations that Severely Decrease Microtubule Dynamics Do Not Prevent Nuclear Migration in Yeast

2002 ◽  
Vol 13 (8) ◽  
pp. 2919-2932 ◽  
Author(s):  
Mohan L. Gupta ◽  
Claudia J. Bode ◽  
Douglas A. Thrower ◽  
Chad G. Pearson ◽  
Kathy A. Suprenant ◽  
...  
Keyword(s):  
Essential Gene ◽  
Dynamic Properties ◽  
Microtubule Dynamics ◽  
Cytoplasmic Microtubule ◽  
Bud Emergence ◽  
Spindle Elongation ◽  
Mutant Cells ◽  
Β Tubulin

Microtubule dynamics are influenced by interactions of microtubules with cellular factors and by changes in the primary sequence of the tubulin molecule. Mutations of yeast β-tubulin C354, which is located near the binding site of some antimitotic compounds, reduce microtubule dynamicity greater than 90% in vivo and in vitro. The resulting intrinsically stable microtubules allowed us to determine which, if any, cellular processes are dependent on dynamic microtubules. The average number of cytoplasmic microtubules decreased from 3 in wild-type to 1 in mutant cells. The single microtubule effectively located the bud site before bud emergence. Although spindles were positioned near the bud neck at the onset of anaphase, the mutant cells were deficient in preanaphase spindle alignment along the mother-bud axis. Spindle microtubule dynamics and spindle elongation rates were also severely depressed in the mutants. The pattern and extent of cytoplasmic microtubule dynamics modulation through the cell cycle may reveal the minimum dynamic properties required to support growth. The ability to alter intrinsic microtubule dynamics and determine the in vivo phenotype of cells expressing the mutant tubulin provides a critical advance in assessing the dynamic requirements of an essential gene function.

Abstract 76: Meox1 is a Cell-cycle Oscillator Required for Mitotic Transition and Proliferation in Cardiac Fibroblasts

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Shuichiro Higo ◽  
Yoshihiro Asano ◽  
Yuki Masumura ◽  
Yasushi Sakata ◽  
Masafumi Kitakaze ◽  
...  
Keyword(s):  
Heart Failure ◽  
Cell Cycle ◽  
Cardiac Fibroblasts ◽  
Tissue Fibrosis ◽  
Cell Cycle Synchronization ◽  
M Phase ◽  
A Cell ◽  
End Stage

Background: Tissue fibrosis plays important roles in the pathogenesis of chronic diseases, including heart failure. The mechanism underlying interstitial fibroblast proliferation is a promising analytical target for therapeutic applications. Here we developed quantitative epigenome profiling to identify a critical regulator in interstitial cell populations that emerges during the progression of heart failure. Methods and Results: We subjected pressure-overloaded hearts of mice to trimethylated histone H3 lysine 4 (H3K4me3) ChIP-sequence and RNA-sequence. Expression analysis followed by quantitative H3K4me3 profiling identified 45 fibrosis-related genes with significant H3K4me3 enrichment in failing hearts, including Meox1 transcription factor. Meox1 emerged in the interstitial fibrotic region in failing heart, and intriguingly Meox1 was expressed in the limited population of cardiac fibroblasts both in vivo and in vitro. Meox1-positive fibroblasts were increased in response to a paracrine signal from cardiomyocytes, and knockdown of Meox1 completely inhibited the reactive proliferation of cardiac fibroblasts stimulated by conditioned medium from cardiomyocytes. Gene expression profiling combined with siRNAs clarified that Meox1 depletion resulted in down regulation in the mitosis-related genes including Aurora B kinase. Indeed, Meox1 depletion decreased the cells under mitosis, but conversely increased the proportion of DNA synthesizing cells, thereby inhibited mitotic transition. The cell-cycle synchronization analysis and promoter analysis using live-cell imaging clarified that Meox1 oscillated throughout the cell-cycle and specifically emerged in G2/M phase. Finally, we revealed that Meox1 heterogenously expressed in the interstitial fibrotic are of human ventricular heart tissues from patients with end-stage heart failure. Notably, Meox1 expression was significantly correlated with the fibrosis-related genes in diseased ventricular heart tissues (n=15), suggesting the pathological relevance in clinical settings. Conclusion: Our findings identify a novel cell-cycle regulator and propose that Meox1 is a potential target for therapies aimed at preventing tissue fibrosis.

scholarly journals Control of Microtubule Dynamics by Stu2p Is Essential for Spindle Orientation and Metaphase Chromosome Alignment in Yeast

2001 ◽  
Vol 12 (9) ◽  
pp. 2870-2880 ◽  
Author(s):  
Karena A. Kosco ◽  
Chad G. Pearson ◽  
Paul S. Maddox ◽  
Peijing Jeremy Wang ◽  
Ian R. Adams ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Microtubule Dynamics ◽  
Spindle Orientation ◽  
Cytoplasmic Microtubule ◽  
Chromosome Alignment ◽  
In Vivo Analysis ◽  
Cytoplasmic Microtubules ◽  
Green Fluorescent

Stu2p is a member of a conserved family of microtubule-binding proteins and an essential protein in yeast. Here, we report the first in vivo analysis of microtubule dynamics in cells lacking a member of this protein family. For these studies, we have used a conditional Stu2p depletion strain expressing α-tubulin fused to green fluorescent protein. Depletion of Stu2p leads to fewer and less dynamic cytoplasmic microtubules in both G1 and preanaphase cells. The reduction in cytoplasmic microtubule dynamics is due primarily to decreases in both the catastrophe and rescue frequencies and an increase in the fraction of time microtubules spend pausing. These changes have significant consequences for the cell because they impede the ability of cytoplasmic microtubules to orient the spindle. In addition, recovery of fluorescence after photobleaching indicates that kinetochore microtubules are no longer dynamic in the absence of Stu2p. This deficiency is correlated with a failure to properly align chromosomes at metaphase. Overall, we provide evidence that Stu2p promotes the dynamics of microtubule plus-ends in vivo and that these dynamics are critical for microtubule interactions with kinetochores and cortical sites in the cytoplasm.

scholarly journals THAP1 modulates oligodendrocyte maturation by regulating ECM degradation in lysosomes

2021 ◽  
Vol 118 (31) ◽  
pp. e2100862118
Author(s):  
Dhananjay Yellajoshyula ◽  
Samuel S. Pappas ◽  
Abigail E. Rogers ◽  
Biswa Choudhury ◽  
Xylena Reed ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Extracellular Matrix ◽  
Nervous System ◽  
Oligodendrocyte Progenitor Cells ◽  
Gene Encoding ◽  
A Cell ◽  
Ecm Degradation ◽  
Oligodendrocyte Development

Mechanisms controlling myelination during central nervous system (CNS) maturation play a pivotal role in the development and refinement of CNS circuits. The transcription factor THAP1 is essential for timing the inception of myelination during CNS maturation through a cell-autonomous role in the oligodendrocyte lineage. Here, we demonstrate that THAP1 modulates the extracellular matrix (ECM) composition by regulating glycosaminoglycan (GAG) catabolism within oligodendrocyte progenitor cells (OPCs). Thap1−/− OPCs accumulate and secrete excess GAGs, inhibiting their maturation through an autoinhibitory mechanism. THAP1 controls GAG metabolism by binding to and regulating the GusB gene encoding β-glucuronidase, a GAG-catabolic lysosomal enzyme. Applying GAG-degrading enzymes or overexpressing β-glucuronidase rescues Thap1−/− OL maturation deficits in vitro and in vivo. Our studies establish lysosomal GAG catabolism within OPCs as a critical mechanism regulating oligodendrocyte development.

scholarly journals The microtubule polymerase Stu2 promotes oligomerization of the γ-TuSC for cytoplasmic microtubule nucleation

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Judith Gunzelmann ◽  
Diana Rüthnick ◽  
Tien-chen Lin ◽  
Wanlu Zhang ◽  
Annett Neuner ◽  
...  
Keyword(s):  
Budding Yeast ◽  
Spindle Pole Body ◽  
Family Members ◽  
Spindle Pole ◽  
Microtubule Nucleation ◽  
Cytoplasmic Microtubule ◽  
Pole Body ◽  
Cytoplasmic Microtubules

Stu2/XMAP215/ZYG-9/Dis1/Alp14/Msps/ch-TOG family members in association with with γ-tubulin complexes nucleate microtubules, but we know little about the interplay of these nucleation factors. Here, we show that the budding yeast Stu2 in complex with the γ-tubulin receptor Spc72 nucleates microtubules in vitro without the small γ-tubulin complex (γ-TuSC). Upon γ-TuSC addition, Stu2 facilitates Spc72–γ-TuSC interaction by binding to Spc72 and γ-TuSC. Stu2 together with Spc72–γ-TuSC increases microtubule nucleation in a process that is dependent on the TOG domains of Stu2. Importantly, these activities are also important for microtubule nucleation in vivo. Stu2 stabilizes Spc72–γ-TuSC at the minus end of cytoplasmic microtubules (cMTs) and an in vivo assay indicates that cMT nucleation requires the TOG domains of Stu2. Upon γ-tubulin depletion, we observed efficient cMT nucleation away from the spindle pole body (SPB), which was dependent on Stu2. Thus, γ-TuSC restricts cMT assembly to the SPB whereas Stu2 nucleates cMTs together with γ-TuSC and stabilizes γ-TuSC at the cMT minus end.

KDM1A and KDM3A promote tumor growth by upregulating cell cycle-associated genes in pancreatic cancer

2021 ◽  
pp. 153537022110234
Author(s):  
Xuyang Hou ◽  
Qiuguo Li ◽  
Leping Yang ◽  
Zhulin Yang ◽  
Jun He ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Pancreatic Cancer ◽  
Tumor Growth ◽  
Clinical Stage ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Pancreatic Cancer Cells ◽  
A Cell

Pancreatic cancer is a highly malignant cancer of the pancreas with a very poor prognosis. Methylation of histone lysine residues is essential for regulating cancer physiology and pathophysiology, mediated by a set of methyltransferases (KMTs) and demethylases (KDMs). This study surveyed the expression of methylation regulators functioning at lysine 9 of histone 3 (H3K9) in pancreatic lesions and explored the underlying mechanisms. We analyzed KDM1A and KDM3A expression in clinical samples by immunohistochemical staining and searching the TCGA PAAD program and GEO datasets. Next, we identified the variation in tumor growth in vitro and in vivo after knockdown of KDM1A or KDM3A and explored the downstream regulators of KDM1A and KDM3A via RNA-seq, and gain- and loss-of-function assays. Eleven H3K9 methylation regulators were highly expressed in pancreatic cancer, and only KDM1A and KDM3A expression positively correlated with the clinicopathological characteristics in pancreatic cancer. High expression of KDM1A or KDM3A positively correlated with pathological grade, lymphatic metastasis, invasion, and clinical stage. Kaplan–Meier analysis indicated that a higher level of KDM1A or KDM3A led to a shorter survival period. Knockdown of KDM1A or KDM3A led to markedly impaired tumor growth in vitro and in vivo. Mechanistically, CCNA2, a cell cycle-associated gene was partially responsible for KDM1A knockdown-mediated effect and CDK6, also a cell cycle-associated gene was partially responsible for KDM3A knockdown-mediated effect on pancreatic cancer cells. Our study demonstrates that KDM1A and KDM3A are highly expressed in pancreatic cancer and are intimately correlated with clinicopathological factors and prognosis. The mechanism of action of KDM1A or KDM3A was both linked to the regulation of cell cycle-associated genes, such as CCNA2 or CDK6, respectively, by an H3K9-dependent pathway.

scholarly journals CycleFlow quantifies cell-cycle heterogeneity in vivo

2020 ◽  
Author(s):  
Adrien Jolly ◽  
Ann-Kathrin Fanti ◽  
Ines Gräßer ◽  
Nils B. Becker ◽  
Thomas Höfer
Keyword(s):  
Cell Cycle ◽  
Cycle Length ◽  
Proliferating Cells ◽  
Average Cell ◽  
Quiescent Cells ◽  
A Cell ◽  
Cell Cycle Length ◽  
Combined Measurements

AbstractWhile the average cell-cycle length in a cell population can be derived from pulse-chase experiments, proliferative heterogeneity has been difficult to quantify. Here we describe CycleFlow, a broadly applicable method that applies Bayesian inference to combined measurements of EdU incorporation and DNA content. CycleFlow accurately quantifies the fraction of proliferating versus quiescent cells and the durations of cell-cycle phases of the proliferating cells in vitro and in vivo.

scholarly journals Overexpression of 14-3-3σ Modulates Cholangiocarcinoma Cell Survival by PI3K/Akt Signaling

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Qiao Wu ◽  
Hua Fan ◽  
Ren Lang ◽  
Xianliang Li ◽  
Xingmao Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Physiological Function ◽  
Tumor Stage ◽  
Akt Signaling ◽  
Cellular Processes ◽  
Cholangiocarcinoma Cell ◽  
A Cell ◽  
Xenograft Models

The protein 14-3-3σ is involved in numerous cellular processes through its ability to bind phosphorylated serine/threonine residues. It is a key regulator of the cell cycle involving in G2 arrest by p53. Deregulation of 14-3-3σ expression has been associated with a large variety of human cancers. However, its physiological function and therapeutic significance have rarely been investigated in cholangiocarcinoma. Using immunohistochemistry (IHC), we evaluated 14-3-3σ expression in 65 human extrahepatic cholangiocarcinomas. As a result, we found that 14-3-3σ is expressed in the tissue of 56 patients (86.2%), and its expression is positively correlated with tumor size, lymph node metastasis, and tumor stage. We also explored the significance of 14-3-3σ and found that 14-3-3σ exerts cell type-dependent effects on cell proliferation through PI3K/Akt signaling in both in vitro and in vivo xenograft models. These results suggest that 14-3-3σ assumes a constitutive role in tumorigenesis rather than acting as a cell cycle regulator in cholangiocarcinoma, which makes 14-3-3σ a new potential target for therapeutic intervention.

scholarly journals Cell Cycle-Dependent Changes in Microtubule Dynamics in Living Cells Expressing Green Fluorescent Protein-α Tubulin

2001 ◽  
Vol 12 (4) ◽  
pp. 971-980 ◽  
Author(s):  
Nasser M. Rusan ◽  
Carey J. Fagerstrom ◽  
Anne-Marie C. Yvon ◽  
Patricia Wadsworth
Keyword(s):  
Cell Cycle ◽  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Dynamic Instability ◽  
Microtubule Dynamics ◽  
A Cell ◽  
Cycle Dependent ◽  
Green Fluorescent ◽  
Quantitative Immunoblotting

LLCPK-1 cells were transfected with a green fluorescent protein (GFP)-α tubulin construct and a cell line permanently expressing GFP-α tubulin was established (LLCPK-1α). The mitotic index and doubling time for LLCPK-1α were not significantly different from parental cells. Quantitative immunoblotting showed that 17% of the tubulin in LLCPK-1α cells was GFP-tubulin; the level of unlabeled tubulin was reduced to 82% of that in parental cells. The parameters of microtubule dynamic instability were compared for interphase LLCPK-1α and parental cells injected with rhodamine-labeled tubulin. Dynamic instability was very similar in the two cases, demonstrating that LLCPK-1α cells are a useful tool for analysis of microtubule dynamics throughout the cell cycle. Comparison of astral microtubule behavior in mitosis with microtubule behavior in interphase demonstrated that the frequency of catastrophe increased twofold and that the frequency of rescue decreased nearly fourfold in mitotic compared with interphase cells. The percentage of time that microtubules spent in an attenuated state, or pause, was also dramatically reduced, from 73.5% in interphase to 11.4% in mitosis. The rates of microtubule elongation and rapid shortening were not changed; overall dynamicity increased 3.6-fold in mitosis. Microtubule release from the centrosome and a subset of differentially stable astral microtubules were also observed. The results provide the first quantitative measurements of mitotic microtubule dynamics in mammalian cells.

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