scholarly journals Drosophila kinesin-8 stabilises the kinetochore-microtubule interaction

2018 ◽  
Author(s):  
Tomoya Edzuka ◽  
Gohta Goshima
Keyword(s):  
Yeast Cell ◽  
Ectopic Expression ◽  
Cell Types ◽  
Binding Activity ◽  
S2 Cells ◽  
Tubulin Binding ◽  
Chromosome Alignment ◽  
Normal Frequency ◽  
Directed Motility

AbstractKinesin-8 is required for proper chromosome alignment in a variety of animal and yeast cell types. However, how this conserved motor protein controls chromosome alignment remains unclear, as multiple biochemical activities, including inconsistent ones between studies, have been identified for this motor family. Here, we show that Drosophila kinesin-8 Klp67A possesses both microtubule (MT) plus-end-stabilising and ‐destabilising activities in addition to commonly observed MT plus-end-directed motility and tubulin-binding activity in vitro, and is required for stable kinetochore-MT attachment during prometaphase in S2 cells In the absence of kinesin-8Klp67A, abnormally-long MTs interact in an “end-on” fashion with kinetochores at normal frequency. However, the interaction was not stable and, once-attached, MTs were frequently detached. This phenotype was rescued by ectopic expression of MT plus-end-stabilising factor CLASP, but not by artificial shortening of MTs. These results suggest that MT-stabilising activity of kinesin-8Klp67A is critical for stable kinetochore-MT attachment. Finally, human kinesin-8KIF18A was also shown important to ensure proper MT attachment.

scholarly journals Drosophila kinesin-8 stabilizes the kinetochore–microtubule interaction

2018 ◽  
Vol 218 (2) ◽  
pp. 474-488 ◽  
Author(s):  
Tomoya Edzuka ◽  
Gohta Goshima
Keyword(s):  
Yeast Cell ◽  
Ectopic Expression ◽  
Cell Types ◽  
Binding Activity ◽  
S2 Cells ◽  
Tubulin Binding ◽  
Chromosome Alignment ◽  
Normal Frequency ◽  
Directed Motility

Kinesin-8 is required for proper chromosome alignment in a variety of animal and yeast cell types. However, it is unclear how this motor protein family controls chromosome alignment, as multiple biochemical activities, including inconsistent ones between studies, have been identified. Here, we find that Drosophila kinesin-8 (Klp67A) possesses both microtubule (MT) plus end–stabilizing and –destabilizing activity, in addition to kinesin-8's commonly observed MT plus end–directed motility and tubulin-binding activity in vitro. We further show that Klp67A is required for stable kinetochore–MT attachment during prometaphase in S2 cells. In the absence of Klp67A, abnormally long MTs interact in an “end-on” fashion with kinetochores at normal frequency. However, the interaction is unstable, and MTs frequently become detached. This phenotype is rescued by ectopic expression of the MT plus end–stabilizing factor CLASP, but not by artificial shortening of MTs. We show that human kinesin-8 (KIF18A) is also important to ensure proper MT attachment. Overall, these results suggest that the MT-stabilizing activity of kinesin-8 is critical for stable kinetochore–MT attachment.

scholarly journals Ghrelin and Des-Acyl Ghrelin Promote Differentiation and Fusion of C2C12 Skeletal Muscle Cells

2007 ◽  
Vol 18 (3) ◽  
pp. 986-994 ◽  
Author(s):  
Nicoletta Filigheddu ◽  
Viola F. Gnocchi ◽  
Marco Coscia ◽  
Miriam Cappelli ◽  
Paolo E. Porporato ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Ectopic Expression ◽  
Cell Types ◽  
C2c12 Cells ◽  
Growth Hormone Secretagogue Receptor ◽  
Endocrine Activity ◽  
Growth Hormone Secretagogue ◽  
Inhibition Of Proliferation ◽  
Acyl Ghrelin

Ghrelin is an acylated peptidyl gastric hormone acting on the pituitary and hypothalamus to stimulate appetite, adiposity, and growth hormone release, through activation of growth hormone secretagogue receptor (GHSR)-1a receptor. Moreover, ghrelin features several activities such as inhibition of apoptosis, regulation of differentiation, and stimulation or inhibition of proliferation of several cell types. Ghrelin acylation is absolutely required for both GHSR-1a binding and its central endocrine activities. However, the unacylated ghrelin form, des-acyl ghrelin, which does not bind GHSR-1a and is devoid of any endocrine activity, is far more abundant than ghrelin in plasma, and it shares with ghrelin some of its cellular activities. Inhere we show that both ghrelin and des-acyl ghrelin stimulate proliferating C2C12 skeletal myoblasts to differentiate and to fuse into multinucleated myotubes in vitro through activation of p38. Consistently, both ghrelin and des-acyl ghrelin inhibit C2C12 proliferation in growth medium. Moreover, the ectopic expression of ghrelin in C2C12 enhances differentiation and fusion of these myoblasts in differentiation medium. Finally, we show that C2C12 cells do not express GHSR-1a, but they do contain a common high-affinity binding site recognized by both acylated and des-acylated ghrelin, suggesting that the described activities on C2C12 are likely mediated by this novel, yet unidentified receptor for both ghrelin forms.

scholarly journals Mechanism of action of rigosertib does not involve tubulin binding

2019 ◽  
Author(s):  
Stacey J. Baker ◽  
Stephen C. Cosenza ◽  
Saikrishna Athuluri-Divakar ◽  
M.V. Ramana Reddy ◽  
Rodrigo Vasquez-Del Carpio ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Human Tumor ◽  
Binding Activity ◽  
Phase Iii ◽  
Clinical Grade ◽  
Wild Type ◽  
Binding Domains ◽  
Tubulin Binding

SUMMARYRigosertib is a novel benzyl styryl sulfone that inhibits the growth of a wide variety of human tumor cells in vitro and in vivo and is currently in Phase III clinical trials. We recently provided structural and biochemical evidence to show that rigosertib acts as a RAS-mimetic by binding to Ras Binding Domains (RBDs) of the RAF and PI3K family proteins and disrupts their binding to RAS. In a recent study, Jost et al (2017) attributed the mechanism of action of rigosertib to microtubule-binding. In these studies, rigosertib was obtained from a commercial vendor. We have been unable to replicate the reported results with clinical grade rigosertib, and hence compared the purity of clinical grade and commercially sourced rigosertib. We find that the commercially sourced rigosertib contains approximately 5% ON01500, a potent inhibitor of tubulin polymerization. Clinical grade rigosertib, which is free of this impurity, does not exhibit tubulin binding activity. In vivo, cell lines that express mutant β-tubulin (TUBBL240F) were also reported to be resistant to the effects of rigosertib. However, our studies showed that both wild-type and TUBBL240F-expressing cells failed to proliferate in the presence of rigosertib at concentrations that are lethal to wild-type cells. Morphologically, we find that rigosertib, at lethal concentrations, induced a senescence-like phenotype in the small percentage of both wild-type and TUBBL240F-expressing cells that survive in the presence of rigosertib. Our results suggest that TUBBL240F expressing cells are more prone to undergo senescence in the presence of rigosertib as well as BI2536, an unrelated ATP-competitive pan-PLK inhibitor. The appearance of these senescent cells could be incorrectly scored as resistant cells in flow cytometric assays using short term cultures.

scholarly journals Hydrozoan sperm-specific H2B histone variants stabilize chromatin and block transcription without enhancing chromatin condensation

2021 ◽  
Author(s):  
Anna Torok ◽  
Martin JG Browne ◽  
Jordina C Vilar ◽  
Indu Patwal ◽  
Timothy Q DuBuc ◽  
...  
Keyword(s):  
Sea Urchins ◽  
Chromatin Condensation ◽  
Ectopic Expression ◽  
Cell Types ◽  
Histone Variants ◽  
Sperm Chromatin ◽  
Chromatin Compaction ◽  
Cycle Arrest

Many animals achieve sperm chromatin compaction and stabilisation during spermatogenesis by replacing canonical histones with sperm nuclear basic proteins (SNBPs) such as protamines. A number of animals including hydrozoan cnidarians and echinoid sea urchins lack protamines and have instead evolved a distinctive family of sperm-specific histone H2Bs (spH2Bs) with extended N-termini rich in SPKK-related motifs. Sperm packaging in echinoids such as sea urchins is regulated by spH2Bs and their sperm is negatively buoyant for fertilization on the sea floor. Hydroid cnidarians also package sperm with spH2Bs but undertake broadcast spawning and their sperm properties are poorly characterised. We show that sperm chromatin from the hydroid Hydractinia possesses higher stability than its somatic equivalent, with reduced accessibility of sperm chromatin to transposase Tn5 integration in vivo and to endonucleases in vitro. However, nuclear dimensions are only moderately reduced in mature Hydractinia sperm compared to other cell types. Ectopic expression of spH2B in the background of H2B knockdown resulted in downregulation of global transcription and cell cycle arrest in embryos without altering their nuclear density. Taken together, spH2B variants containing SPKK-related motifs act to stabilise chromatin and silence transcription in Hydractinia sperm without significant chromatin compaction. This is consistent with a contribution of spH2B to sperm buoyancy as a reproductive adaptation.

scholarly journals DPre: computational identification of differentiation bias and genes underlying cell type conversions

2019 ◽  
Author(s):  
Simon Steffens ◽  
Xiuling Fu ◽  
Fangfang He ◽  
Yuhao Li ◽  
Isaac A Babarinde ◽  
...  
Keyword(s):  
Ectopic Expression ◽  
Large Cell ◽  
Cell Types ◽  
Supplementary Information ◽  
Computational Techniques ◽  
Cell Type ◽  
Mixed Cell ◽  
Directional Bias ◽  
Differentiated Cells

Abstract Summary Cells are generally resistant to cell type conversions, but can be converted by the application of growth factors, chemical inhibitors and ectopic expression of genes. However, it remains difficult to accurately identify the destination cell type or differentiation bias when these techniques are used to alter cell type. Consequently, there is demand for computational techniques that can help researchers understand both the cell type and differentiation bias. While advanced tools identifying cell types exist for single cell data and the deconvolution of mixed cell populations, the problem of exploring partially differentiated cells of indeterminate transcriptional identity has not been addressed. To fill this gap, we developed driver-predictor, which relies on scoring per gene transcriptional similarity between RNA-Seq datasets to reveal directional bias of differentiation. By comparing against large cell type transcriptome libraries or a desired target expression profile, the tool enables the user to visualize both the changes in transcriptional identity as well as the genes accounting for the cell type changes. This software will be a powerful tool for researchers to explore in vitro experiments that involve cell type conversions. Availability and implementation Source code is open source under the MIT license and is freely available on https://github.com/LoaloaF/DPre. Supplementary information Supplementary data are available at Bioinformatics online.

Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms

Development ◽  
1998 ◽  
Vol 125 (11) ◽  
pp. 2149-2158 ◽  
Author(s):  
J.S. Britton ◽  
B.A. Edgar
Keyword(s):  
Cell Cycle ◽  
Fat Body ◽  
Environmental Control ◽  
Ectopic Expression ◽  
Cell Types ◽  
Quiescent State ◽  
Independent Manner ◽  
Cell Cycle Activation ◽  
E2f Transcription Factor

In newly hatched Drosophila larvae, quiescent cells reenter the cell cycle in response to dietary amino acids. To understand this process, we varied larval nutrition and monitored effects on cell cycle initiation and maintenance in the mitotic neuroblasts and imaginal disc cells, as well as the endoreplicating cells in other larval tissues. After cell cycle activation, mitotic and endoreplicating cells respond differently to the withdrawal of nutrition: mitotic cells continue to proliferate in a nutrition-independent manner, while most endoreplicating cells reenter a quiescent state. We also show that ectopic expression of Drosophila Cyclin E or the E2F transcription factor can drive quiescent endoreplicating cells, but not quiescent imaginal neuroblasts, into S-phase. Conversely, we demonstrate that quiescent imaginal neuroblasts, but not quiescent endoreplicating cells, can be induced to enter the cell cycle when co-cultured with larval fat body in vitro. These results demonstrate a fundamental difference in the control of cell cycle activation and maintenance in these two cell types, and imply the existence of a novel mitogen generated by the larval fat body in response to nutrition.

scholarly journals Crescerin uses a TOG domain array to regulate microtubules in the primary cilium

2015 ◽  
Vol 26 (23) ◽  
pp. 4248-4264 ◽  
Author(s):  
Alakananda Das ◽  
Daniel J. Dickinson ◽  
Cameron C. Wood ◽  
Bob Goldstein ◽  
Kevin C. Slep
Keyword(s):  
Crystal Structure ◽  
Binding Activity ◽  
Structure And Function ◽  
Sensory Disorders ◽  
Tubulin Binding ◽  
Sensory Cilia ◽  
And Function ◽  
Binding Determinants ◽  
Extracellular Environment

Eukaryotic cilia are cell-surface projections critical for sensing the extracellular environment. Defects in cilia structure and function result in a broad range of developmental and sensory disorders. However, mechanisms that regulate the microtubule (MT)-based scaffold forming the cilia core are poorly understood. TOG domain array–containing proteins ch-TOG and CLASP are key regulators of cytoplasmic MTs. Whether TOG array proteins also regulate ciliary MTs is unknown. Here we identify the conserved Crescerin protein family as a cilia-specific, TOG array-containing MT regulator. We present the crystal structure of mammalian Crescerin1 TOG2, revealing a canonical TOG fold with conserved tubulin-binding determinants. Crescerin1's TOG domains possess inherent MT-binding activity and promote MT polymerization in vitro. Using Cas9-triggered homologous recombination in Caenorhabditis elegans, we demonstrate that the worm Crescerin family member CHE-12 requires TOG domain–dependent tubulin-binding activity for sensory cilia development. Thus, Crescerin expands the TOG domain array–based MT regulatory paradigm beyond ch-TOG and CLASP, representing a distinct regulator of cilia structure.

Proadipogenic effect of leptin on rat preadipocytes in vitro: activation of MAPK and STAT3 signaling pathways

2002 ◽  
Vol 282 (4) ◽  
pp. C853-C863 ◽  
Author(s):  
F. Machinal-Quélin ◽  
M. N. Dieudonné ◽  
M. C. Leneveu ◽  
R. Pecquery ◽  
Y. Giudicelli
Keyword(s):  
Subcutaneous Fat ◽  
Cell Types ◽  
Binding Activity ◽  
Mitogen Activated Protein Kinase ◽  
Cell Counting ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Peroxisome Proliferator Activated Receptor ◽  
Stat3 Phosphorylation

Because leptin has recently been shown to induce proliferation and/or differentiation of different cell types through different pathways, the aim of the present study was to investigate, in vitro, the influence of leptin on adipogenesis in rat preadipocytes. A prerequisite to this study was to identify leptin receptors (Ob-Ra and Ob-Rb) in preadipocytes from femoral subcutaneous fat. We observed that expressions of Ob-Ra and Ob-Rb increase during adipogenesis. Furthermore, leptin induces an increase of p42/p44 mitogen-activated protein kinase phosphorylated isoforms in both confluent and differentiated preadipocytes and of STAT3 phosphorylation only in confluent preadipocytes. Moreover, exposure to leptin promoted activator protein-1 complex DNA binding activity in confluent preadipocytes. Finally, exposure of primary cultured preadipocytes from the subcutaneous area to leptin (10 nM) resulted in an increased proliferation ([3H]thymidine incorporation and cell counting) and differentiation (glycerol-3-phosphate dehydrogenase activity and mRNA levels of lipoprotein lipase, peroxisome proliferator-activated receptor-γ2, and c-fos). Altogether, these results indicate that, in vitro at least, leptin through its functional receptors exerts a proadipogenic action in subcutaneous preadipocytes.

scholarly journals Epigenetic Manipulation Facilitates the Generation of Skeletal Muscle Cells from Pluripotent Stem Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
Tomohiko Akiyama ◽  
Shunichi Wakabayashi ◽  
Atsumi Soma ◽  
Saeko Sato ◽  
Yuhki Nakatake ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Myogenic Differentiation ◽  
Ectopic Expression ◽  
Cell Types ◽  
Culture Conditions ◽  
The Body ◽  
Specific Cell ◽  
Modifying Factors

Human pluripotent stem cells (hPSCs) have the capacity to differentiate into essentially all cell types in the body. Such differentiation can be directed to specific cell types by appropriate cell culture conditions or overexpressing lineage-defining transcription factors (TFs). Especially, for the activation of myogenic program, early studies have shown the effectiveness of enforced expression of TFs associated with myogenic differentiation, such as PAX7 and MYOD1. However, the efficiency of direct differentiation was rather low, most likely due to chromatin features unique to hPSCs, which hinder the access of TFs to genes involved in muscle differentiation. Indeed, recent studies have demonstrated that ectopic expression of epigenetic-modifying factors such as a histone demethylase and an ATP-dependent remodeling factor significantly enhances myogenic differentiation from hPSCs. In this article, we review the recent progress for in vitro generation of skeletal muscles from hPSCs through forced epigenetic and transcriptional manipulation.

scholarly journals Actopaxin, a New Focal Adhesion Protein That Binds Paxillin Ld Motifs and Actin and Regulates Cell Adhesion

2000 ◽  
Vol 151 (7) ◽  
pp. 1435-1448 ◽  
Author(s):  
Sotiris N. Nikolopoulos ◽  
Christopher E. Turner
Keyword(s):  
Cell Adhesion ◽  
Actin Cytoskeleton ◽  
Focal Adhesion ◽  
Substantial Reduction ◽  
Ectopic Expression ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Cell Types ◽  
Focal Adhesion Protein

Paxillin is a focal adhesion adapter protein involved in the integration of growth factor– and adhesion-mediated signal transduction pathways. Paxillin LD motifs have been demonstrated to bind to several proteins associated with remodeling of the actin cytoskeleton including the focal adhesion kinase, vinculin, and a complex of proteins comprising p95PKL, PIX, and PAK (Turner, C.E., M.C. Brown, J.A. Perrotta, M.C. Riedy, S.N. Nikolopoulos, A.R. McDonald, S. Bagrodia, S. Thomas, and P.S. Leventhal. 1999. J. Cell Biol. 145:851–863). In this study, we report the cloning and initial characterization of a new paxillin LD motif–binding protein, actopaxin. Analysis of the deduced amino acid sequence of actopaxin reveals a 42-kD protein with two calponin homology domains and a paxillin-binding subdomain (PBS). Western blotting identifies actopaxin as a widely expressed protein. Actopaxin binds directly to both F-actin and paxillin LD1 and LD4 motifs. It exhibits robust focal adhesion localization in several cultured cell types but is not found along the length of the associated actin-rich stress fibers. Similar to paxillin, it is absent from actin-rich cell–cell adherens junctions. Also, actopaxin colocalizes with paxillin to rudimentary focal complexes at the leading edge of migrating cells. An actopaxin PBS mutant incapable of binding paxillin in vitro cannot target to focal adhesions when expressed in fibroblasts. In addition, ectopic expression of the PBS mutant and/or the COOH terminus of actopaxin in HeLa cells resulted in substantial reduction in adhesion to collagen. Together, these results suggest an important role for actopaxin in integrin-dependent remodeling of the actin cytoskeleton during cell motility and cell adhesion.

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