scholarly journals B1-type cyclins control microtubule organization during cell division in Arabidopsis

2021 ◽  
Author(s):  
Mariana Romeiro Motta ◽  
Xin'Ai Zhao ◽  
Martine Pastuglia ◽  
Katia Belcram ◽  
Farshad Roodbarkelari ◽  
...  
Keyword(s):  
Double Mutant ◽  
Cell Cycle Control ◽  
Flowering Plants ◽  
Microtubule Organization ◽  
Complex Protein ◽  
Dividing Cells ◽  
Gamma Tubulin ◽  
Microtubule Networks

Flowering plants contain a large number of cyclin families, each containing multiple members, most of which have not been characterized to date. Here, we analyzed the role of the B1 subclass of mitotic cyclins in cell cycle control during Arabidopsis development. While we reveal CYCB1;5 to be a pseudogene, the remaining four members were found to be expressed in dividing cells. Mutant analyses showed a complex pattern of overlapping, development-specific requirements of B1-type cyclins with CYCB1;2 playing a central role. The double mutant cycb1;1 cycb1;2 is severely compromised in growth, yet viable beyond the seedling stage, hence representing a unique opportunity to study the function of B1-type cyclin activity at the organismic level. Immunolocalization of microtubules in cycb1;1 cycb1;2 and treating mutants with the microtubule drug oryzalin revealed a key role of B1-type cyclins in orchestrating mitotic microtubule networks. Subsequently, we identified the GAMMA-TUBULIN COMPLEX PROTEIN 3-INTERACING PROTEIN 1 (GIP1/MOZART) as an in vitro substrate of B1-type cyclin complexes and further genetic analyses support an important role in the regulation of GIP1 by CYCB1s.

scholarly journals Cytokinesis and Midzone Microtubule Organization inCaenorhabditis elegans Require the Kinesin-like Protein ZEN-4

1998 ◽  
Vol 9 (8) ◽  
pp. 2037-2049 ◽  
Author(s):  
William B. Raich ◽  
Adrienne N. Moran ◽  
Joel H. Rothman ◽  
Jeff Hardin
Keyword(s):  
Cell Division ◽  
Null Allele ◽  
Time Lapse ◽  
Equatorial Region ◽  
Microtubule Organization ◽  
Dividing Cells ◽  
Spindle Midzone ◽  
Cross Links ◽  
Complete Cell

Members of the MKLP1 subfamily of kinesin motor proteins localize to the equatorial region of the spindle midzone and are capable of bundling antiparallel microtubules in vitro. Despite these intriguing characteristics, it is unclear what role these kinesins play in dividing cells, particularly within the context of a developing embryo. Here, we report the identification of a null allele ofzen-4, an MKLP1 homologue in the nematodeCaenorhabditis elegans, and demonstrate that ZEN-4 is essential for cytokinesis. Embryos deprived of ZEN-4 form multinucleate single-celled embryos as they continue to cycle through mitosis but fail to complete cell division. Initiation of the cytokinetic furrow occurs at the normal time and place, but furrow propagation halts prematurely. Time-lapse recordings and microtubule staining reveal that the cytokinesis defect is preceded by the dissociation of the midzone microtubules. We show that ZEN-4 protein localizes to the spindle midzone during anaphase and persists at the midbody region throughout cytokinesis. We propose that ZEN-4 directly cross-links the midzone microtubules and suggest that these microtubules are required for the completion of cytokinesis.

scholarly journals gamma-tubulin is a minus end-specific microtubule binding protein.

1995 ◽  
Vol 131 (1) ◽  
pp. 207-214 ◽  
Author(s):  
Q Li ◽  
H C Joshi
Keyword(s):  
Chromosome Segregation ◽  
Structural Integrity ◽  
Cell Periphery ◽  
Vitro Assay ◽  
Interphase Cells ◽  
Gamma Tubulin ◽  
Slow Growing ◽  
Directional Transport

The role of microtubules in mediating chromosome segregation during mitosis is well-recognized. In addition, interphase cells depend upon a radial and uniform orientation of microtubules, which are intrinsically asymmetric polymers, for the directional transport of many cytoplasmic components and for the maintenance of the structural integrity of certain organelles. The slow growing minus ends of microtubules are linked to the centrosome ensuring extension of the fast growing plus ends toward the cell periphery. However, the molecular mechanism of this linkage is not clear. One hypothesis is that gamma-tubulin, located at the centrosome, binds to the minus ends of microtubules. To test this model, we synthesized radiolabeled gamma-tubulin in vitro. We demonstrate here biochemically a specific, saturable, and tight (Kd = 10(-10) M) interaction of gamma-tubulin and microtubule ends with a stoichiometry of 12.6 +/- 4.9 molecules of gamma-tubulin per microtubule. In addition, we designed an in vitro assay to visualize gamma-tubulin at the minus ends of axonemal microtubules. These data show that gamma-tubulin represents the first protein to bind microtubule minus ends and might be responsible for mediating the link between microtubules and the centrosome.

scholarly journals The structure of the Myo4p globular tail and its function in ASH1 mRNA localization

2010 ◽  
Vol 189 (3) ◽  
pp. 497-510 ◽  
Author(s):  
Alexander Heuck ◽  
Ingrid Fetka ◽  
Daniel N. Brewer ◽  
Daniela Hüls ◽  
Mary Munson ◽  
...  
Keyword(s):  
Mrna Localization ◽  
Surface Patch ◽  
X Ray ◽  
Membrane Tethering ◽  
Dividing Cells ◽  
Dependent Transport ◽  
Type V ◽  
Ash1 Mrna

Type V myosin (MyoV)–dependent transport of cargo is an essential process in eukaryotes. Studies on yeast and vertebrate MyoV showed that their globular tails mediate binding to the cargo complexes. In Saccharomyces cerevisiae, the MyoV motor Myo4p interacts with She3p to localize asymmetric synthesis of HO 1 (ASH1) mRNA into the bud of dividing cells. A recent study showed that localization of GFP-MS2–tethered ASH1 particles does not require the Myo4p globular tail, challenging the supposed role of this domain. We assessed ASH1 mRNA and Myo4p distribution more directly and found that their localization is impaired in cells expressing globular tail–lacking Myo4p. In vitro studies further show that the globular tail together with a more N-terminal linker region is required for efficient She3p binding. We also determined the x-ray structure of the Myo4p globular tail and identify a conserved surface patch important for She3p binding. The structure shows pronounced similarities to membrane-tethering complexes and indicates that Myo4p may not undergo auto-inhibition of its motor domain.

scholarly journals Role of the HSP70 Homologue from Chloroplasts in the Assembly of the Photosynthetic Apparatus

1993 ◽  
Author(s):  
Rachel Nechushtai ◽  
Parag Chitnis
Keyword(s):  
Protein Complexes ◽  
Photosynthetic Apparatus ◽  
Crop Productivity ◽  
Cdna Clones ◽  
Light Harvesting Complex ◽  
Complex Protein ◽  
Chlorophyll Protein Complexes ◽  
Chlorophyll Protein

The major goal of the proposed research was to study the role of a 70-kDa heat shock cognate protein from chloroplasts (ct-HSP70) in the assembly of chlorophyll-protein complexes. The latters are mostly important in allowing photosynthesis to occur. Photosynthesis is at the heart of crop productivity and the knowledge of the biogenesis of the photosynthetic apparatus is essential to manipulate the efficiency of photosynthesis. The characterization of the function of the ct-HSP70 was planned to be studied in vitro by assaying its capability to physically interact with the thylakoid proteins and to assist their assembly into thylakoid membranes. We planned to identify regions in the light-harvesting complex protein (LHCP) that interact with the ct-HSP70 and characterize the interaction between them. We also intended to isolate cDNA clones encoding ct-HSP70, sequence them, express one of them in E. coli and use the purified protein for functional assays. The research in this BARD proposal aimed at providing insights and aid in understanding the mechanism by which plants may respond to the heat stress. Since plants often experience increased temperatures.

GATA-1 Overexpression Does Not Restore Deficient Erythropoiesis in Myelodysplastic Syndromes.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3430-3430
Author(s):  
Alexandra Rideau ◽  
Stephane Durual ◽  
Maciej Wiznerowics ◽  
Sylvie Ruault ◽  
Vincent Piguet ◽  
...  
Keyword(s):  
Myelodysplastic Syndromes ◽  
Culture System ◽  
Cell Cycle Control ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Differentiation Markers ◽  
Cd34 Cells ◽  
Erythroleukemic Cell

Abstract Introduction: Transcription factor GATA-1 is essential for erythroid and megakaryocytic maturation. A role of GATA-1 in cell cycle control is suggested by the fact that GATA-1 mutations are associated with hematopoietic precursor proliferation and leukemogenesis and that defective GATA-1 expression is observed in in vitro cultures of erythroid myelodysplastic precursors. In order to study more in detail a potential role of GATA-1 dysregulation in myelodysplastic syndromes (MDS), we constructed lentiviral vectors with the aim to overexpress GATA-1 protein or to inhibit its production in erythroid progenitors. Methods and Results: Using RNA interference technology we tested how GATA-1 inhibition interfered with erythroid differentiation. We selected one GATA-1 specific siRNA, which abolished expression of GATA-1 protein in K562 and HEL erythroleukemic cell lines, as verified by Western blot. Interestingly, we observed in parallel to the disappearance of GATA-1 protein, decreased proliferation rates (170x for K562 and 30x for HEL after 17 days of culture) and increased apoptosis. Normal CD34+ cells cultured in our culture system and transduced with the siRNA vector were practically blocked in their erythroid differentiation: 14 % glyco+/CD36- mature erythroid cells versus 81 % in untransduced and 80 % in cultures transduced with control lentivector (obtained after 14 days of culture). Differentiation into myeloid cells was not affected. To overexpress GATA-1 we cloned the wild-type as well as a mutated, caspase-resistant, form of GATA-1 in a pWPIR-ires-GFP bicistronic lentivector. Functionality of both lentivectors was validated in HeLa cells. For the study of GATA-1 in primary human hematopoietic cells we used an in vitro culture system in which CD34+ progenitors differentiate into mature red blood cells in the presence of erythropoietin, IL-3, and SCF. Transduction of CD34+ cells with lentivectors led to increase of GATA-1 mRNA (400-fold) measured by Realtime RT-PCR and to detection of protein. No difference was observed in cell numbers, expression of erythroid differentiation markers and survival between cells transduced with control vector and with pWPIR-GATA-1-ires-GFP. CD34+ cells from 3 patients with low-risk MDS in this culture system proliferated less (15x ± 13 amplification after 14 days of culture versus 72x ± 35 for normal precursors) differentiated less, and became apoptotic earlier than normal cells. However, overexpression of GATA-1 did not restore proliferation rate, nor did it lead to increased erythroid differentiation, or increase in survival. Conclusion: GATA-1 overexpression was not able to overcome defective erythroid differentiation of myelodysplastic progenitors, nor did it increase differentiation of normal erythroid progenitors. On the other hand, GATA-1 inhibition in normal erythroid precursors led to blockage of erythroid differentiation. We therefore assume that either factors upstream of GATA-1 or additional, GATA-1 independent factors, are responsible for the myelodysplastic phenotype.

scholarly journals Dynein Intermediate Chain Mediated Dynein–Dynactin Interaction Is Required for Interphase Microtubule Organization and Centrosome Replication and Separation inDictyostelium

1999 ◽  
Vol 147 (6) ◽  
pp. 1261-1274 ◽  
Author(s):  
Shuo Ma ◽  
Leda Triviños-Lagos ◽  
Ralph Gräf ◽  
Rex L. Chisholm
Keyword(s):  
Heavy Chain ◽  
Physiological Role ◽  
Cytoplasmic Dynein ◽  
Wild Type ◽  
Microtubule Organization ◽  
Dynein Intermediate Chain ◽  
Organizing Center ◽  
Intermediate Chain

Cytoplasmic dynein intermediate chain (IC) mediates dynein–dynactin interaction in vitro (Karki, S., and E.L. Holzbaur. 1995. J. Biol. Chem. 270:28806–28811; Vaughan, K.T., and R.B. Vallee. 1995. J. Cell Biol. 131:1507–1516). To investigate the physiological role of IC and dynein–dynactin interaction, we expressed IC truncations in wild-type Dictyostelium cells. ICΔC associated with dynactin but not with dynein heavy chain, whereas ICΔN truncations bound to dynein but bound dynactin poorly. Both mutations resulted in abnormal localization to the Golgi complex, confirming dynein function was disrupted. Striking disorganization of interphase microtubule (MT) networks was observed when mutant expression was induced. In a majority of cells, the MT networks collapsed into large bundles. We also observed cells with multiple cytoplasmic asters and MTs lacking an organizing center. These cells accumulated abnormal DNA content, suggesting a defect in mitosis. Striking defects in centrosome morphology were also observed in IC mutants, mostly larger than normal centrosomes. Ultrastructural analysis of centrosomes in IC mutants showed interphase accumulation of large centrosomes typical of prophase as well as unusually paired centrosomes, suggesting defects in centrosome replication and separation. These results suggest that dynactin-mediated cytoplasmic dynein function is required for the proper organization of interphase MT network as well as centrosome replication and separation in Dictyostelium.

scholarly journals Dissecting the roles of Haspin and VRK1 in Histone H3 threonine-3 phosphorylation during mitosis

2021 ◽  
Author(s):  
Tyrell N Cartwright ◽  
Rebecca J Harris ◽  
Stephanie K Meyer ◽  
Nikolaus A. Watson ◽  
Cheryl Tan ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Neuromuscular Disease ◽  
Kinase Activity ◽  
Histone H3 ◽  
Direct Role ◽  
Dividing Cells ◽  
Histone Kinase ◽  
Mitosis And Meiosis

Protein kinases that phosphorylate histones are ideally-placed to influence the behavior of chromosomes during cell division. Indeed, a number of conserved histone phosphorylation events occur prominently during mitosis and meiosis in most eukaryotes, including on histone H3 at threonine-3 (H3T3ph). At least two kinases, Haspin and VRK1 (NHK-1/ballchen in Drosophila), have been proposed to carry out this modification. Phosphorylation of H3 by Haspin has defined roles in mitosis, but the significance of VRK1 activity towards histones in dividing cells has been unclear. Here, using in vitro kinase assays, KiPIK screening, RNA interference, and CRISPR/Cas9 approaches, we were unable to substantiate a direct role for VRK1, or its homologue VRK2, in the phosphorylation of threonine-3 or serine-10 of Histone H3 in mitosis, although loss of VRK1 did slow cell proliferation. We conclude that the role of VRK1, and its more recently identified association with neuromuscular disease in humans, is unlikely to involve mitotic histone kinase activity. In contrast, Haspin is required to generate H3T3ph during mitosis.

The carboxy terminus of Tub4p is required for gamma-tubulin function in budding yeast

2000 ◽  
Vol 113 (21) ◽  
pp. 3871-3882 ◽  
Author(s):  
J. Vogel ◽  
M. Snyder
Keyword(s):  
Temperature Sensitive ◽  
Carboxy Terminus ◽  
Tail Region ◽  
Microtubule Organization ◽  
Electron Microscopic ◽  
Bud Neck ◽  
Cytoplasmic Microtubules ◽  
Gamma Tubulin ◽  
Carboxy Terminal

The role of gamma-tubulin in microtubule nucleation is well established, however, its function in other aspects of microtubule organization is unknown. The carboxy termini of alpha/beta-tubulins influence the assembly and stability of microtubules. We investigated the role of the carboxy terminus of yeast gamma-tubulin (Tub4p) in microtubule organization. This region consists of a conserved domain (DSYLD), and acidic tail. Cells expressing truncations lacking the DSYLD domain, tail or both regions are temperature sensitive for growth. Growth defects of tub4 mutants lacking either or both carboxy-terminal domains are suppressed by the microtubule destabilizing drug benomyl. tub4 carboxy-terminal mutants arrest as large budded cells with short bipolar spindles positioned at the bud neck. Electron microscopic analysis of wild-type and CTR mutant cells reveals that SPBs are tightly associated with the bud neck/cortex by cytoplasmic microtubules in mutants lacking the tail region (tub4-delta 444, tub4-delta 448). Mutants lacking the DSYLD residues (tub4-delta 444, tub4-delta DSYLD) form many cytoplasmic microtubules. We propose that the carboxy terminus of Tub4p is required for re-organization of the microtubules upon completion of nuclear migration, and facilitates spindle elongation into the bud.

scholarly journals Current Understanding of the Role of PPARγin Gastrointestinal Cancers

PPAR Research ◽  
2009 ◽  
Vol 2009 ◽  
pp. 1-8 ◽  
Author(s):  
Bing Zou ◽  
Liang Qiao ◽  
Benjamin C. Y. Wong
Keyword(s):  
Antitumor Effect ◽  
Cell Cycle Control ◽  
Control Cell ◽  
Multiple Roles ◽  
Gastrointestinal Cancers ◽  
Recent Developments ◽  
Positive Results ◽  
Prevention And Therapy

Numerous studies have indicated that PPARγplays multiple roles such as in inflammation, cell cycle control, cell proliferation, apoptosis, and carcinogenesis, thus PPARγcontributes to the homeostasis. Many in vitro studies have showed that ligand-induced activation of PPARγpossess antitumor effect in many cancers including CRC. However, the role of PPARγin gastrointestinal cancers, especially in colorectal cancer, is rather controversial. Nevertheless, some recent studies with the positive results on the possible application of PPARγligands, such as Bezafibrate or Rosiglitazone in gastrointestinal cancers, have suggested a potential usefulness of PPARγagonists in cancer prevention and therapy. In this review, the authors discuss the recent developments in the role of PPARγin gastrointestinal cancers.

scholarly journals Plant and mouse EB1 proteins have opposite intrinsic properties on the dynamic instability of microtubules.

2020 ◽  
Author(s):  
Arthur T Molines ◽  
Virginie Stoppin-Mellet ◽  
Isabelle Arnal ◽  
Frédéric M Coquelle
Keyword(s):  
Dynamic Instability ◽  
Cellular Organization ◽  
Cell Type ◽  
Intrinsic Properties ◽  
Microtubule Organization ◽  
Vitro Assay ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Microtubule Networks

Abstract Objective Most eukaryotic cells contain microtubule filaments, which play central roles in intra-cellular organization. However, microtubule networks have a wide variety of architectures from one cell type and organism to another. Nonetheless, the sequences of tubulins, of Microtubule Associated proteins (MAPs) and the structure of microtubules are usually well conserved throughout the evolution. MAPs being known to be responsible for regulating microtubule organization and dynamics, this raises the question of the conservation of their intrinsic properties. Indeed, knowing how the intrinsic properties of individual MAPs differ between organisms might enlighten our understanding of how distinct microtubule networks are built. End-Binding protein 1 (EB1), first described as a MAP in yeast, is conserved in plants and mammals. The intrinsic properties of the mammalian and the yeast EB1 proteins have been well described in the literature but, to our knowledge, the intrinsic properties of EB1 from plant and mammals have not been compared thus far.Results Here, using an in vitro assay, we discovered that plant and mammalian EB1 purified proteins have different intrinsic properties on microtubule dynamics. Indeed, the mammalian EB1 protein increases microtubules dynamic while the plant EB1 protein stabilizes them.

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