scholarly journals Cep152 interacts with Plk4 and is required for centriole duplication

2010 ◽  
Vol 191 (4) ◽  
pp. 721-729 ◽  
Author(s):  
Emily M. Hatch ◽  
Anita Kulukian ◽  
Andrew J. Holland ◽  
Don W. Cleveland ◽  
Tim Stearns
Keyword(s):  
Cell Cycle ◽  
Drosophila Melanogaster ◽  
The Other ◽  
Early Step ◽  
Centriole Duplication ◽  
Kinase Family ◽  
Centrosome Protein

Centrioles are microtubule-based structures that organize the centrosome and nucleate cilia. Centrioles duplicate once per cell cycle, and duplication requires Plk4, a member of the Polo-like kinase family; however, the mechanism linking Plk4 activity and centriole formation is unknown. In this study, we show in human and frog cells that Plk4 interacts with the centrosome protein Cep152, the orthologue of Drosophila melanogaster Asterless. The interaction requires the N-terminal 217 residues of Cep152 and the crypto Polo-box of Plk4. Cep152 and Plk4 colocalize at the centriole throughout the cell cycle. Overexpression of Cep152 (1–217) mislocalizes Plk4, but both Cep152 and Plk4 are able to localize to the centriole independently of the other. Depletion of Cep152 prevents both normal centriole duplication and Plk4-induced centriole amplification and results in a failure to localize Sas6 to the centriole, an early step in duplication. Cep152 can be phosphorylated by Plk4 in vitro, suggesting that Cep152 acts with Plk4 to initiate centriole formation.

Localization of Hematopoietic Stem Cells in Coculture with Mesenchymal Stromal Cells Impacts on Phenotype and Cell Cycle Status

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4774-4774
Author(s):  
Duohui Jing ◽  
Nael Alakel ◽  
Fernando Fierro ◽  
Katrin Mueller ◽  
Martin Bornhaeuser ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Division ◽  
Stromal Cells ◽  
The Other ◽  
Cell Contact ◽  
Adherent Cells ◽  
Hematopoietic Stem ◽  
Cell Fractions

Abstract Hematopoietic stem cells (HSC) are defined by their capacity of self-renewal and differentiation. In recent years it became clear that cell to cell contact mediated communication between mesenchymal stromal cells (MSC) and HSC is important for homeostasis of hematopoiesis. MSC play a crucial role in the so called bone marrow niche giving rise to the majority of marrow stromal cell lineages. In vitro we investigated the impact of MSC on CD34 purified HSC expansion and differentiation demonstrating a promoting impact of MSC on adherent HSC in comparison to non adherent HSC in terms of phenotype, migration capacity and clonogenicity. Performing phase contrast microscopy and confocal microscopy we are able to distinguish HSC which are located on the surface of a MSC monolayer (phase-bright cells) and HSC which are covered by MSC monolayer (phase-dim cells). Both HSC fractions and the non-adherent cells were isolated separately by performing serial washing steps. All three fractions were analyzed at fixed time points during the first week of co-culture in term of cell cycle progression, proliferation, maturation and cell division accompanied differentiation. First we performed propidium iodide (PI) staining for cell cycle analysis revealing that the phase-bright cells contained the highest percentage of G2 cells in comparison to the non adherent cells and the phase-dim cells; 13.9 ±1.0% vs 1.3 ±1.2% vs 2.7 ±2.0%, p<0.001. The data indicate the facilitating impact of MSC on HSC in performing mitosis which is however depending on the location of interaction. When HSC are released into supernatant (non adherent cells) or covered by MSC, G2 phase was significantly down-regulated. Next we studied the proliferation capacity of the separate cell fractions. Consistent with the data of cell cycle, cell number of phase-bright faction increased much faster than the other two fractions during the first 4 days suggesting that the MSC surface in vitro is the predominant location of HSC proliferation. Next we investigated the phenotype of HSC. According to FACS analysis results (CD34+CD38-) phase-dim cells revealed a more immature phenotype in comparison to the non adherent cells and the phase-bright cells. During the first four days 80% of phase-dim cells remained CD34+CD38-, while cells of the phase-bright- and the non adherent fraction exhibited a significant more mature phenotype. Performing cell division tracking using CFSE we were able to show that over time number of divisions of phase-dim cells were significantly diminished in comparison to the other two cell fractions in co-cultures. In addition, phase-dim cells started to lose CD34 at the 7th generation, while non-adherent and phase-bright cells already lost CD34 at the 4th generation. These data suggest that “stemness” of HSC was rather preserved in the cell fraction which was covered by MSC monolayer than in the cell fraction on the surface of MSC. In conclusion we demonstrate HSC in distinct locations in vitro showing different behaviors in terms of phenotype and proliferation. It becomes evident that not only the cell to cell contact matters but also the localization of contact. Further experiments are needed to investigate NOD/SCID repopulation potential of the different cell fractions.

scholarly journals Characterization of a Drosophila Centrosome Protein CP309 That Shares Homology with Kendrin and CG-NAP

2004 ◽  
Vol 15 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Shin-ichi Kawaguchi ◽  
Yixian Zheng
Keyword(s):  
Cell Cycle ◽  
Drosophila Melanogaster ◽  
Coiled Coil ◽  
Animal Cells ◽  
Microtubule Nucleation ◽  
Small Complex ◽  
Biochemical Studies ◽  
Ring Complex ◽  
Centrosome Protein

The centrosome in animal cells provides a major microtubule-nucleating site that regulates the microtubule cytoskeleton temporally and spatially throughout the cell cycle. We report the identification in Drosophila melanogaster of a large coiled-coil centrosome protein that can bind to calmodulin. Biochemical studies reveal that this novel Drosophila centrosome protein, centrosome protein of 309 kDa (CP309), cofractionates with the γ-tubulin ring complex and the centrosome-complementing activity. We show that CP309 is required for microtubule nucleation mediated by centrosomes and that it interacts with the γ-tubulin small complex. These findings suggest that the microtubule-nucleating activity of the centrosome requires the function of CP309.

scholarly journals Invadolysin

2004 ◽  
Vol 167 (4) ◽  
pp. 673-686 ◽  
Author(s):  
Brian McHugh ◽  
Sue A. Krause ◽  
Bin Yu ◽  
Anne-Marie Deans ◽  
Sarah Heasman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drosophila Melanogaster ◽  
Germ Cells ◽  
Leading Edge ◽  
Mitotic Progression ◽  
Wild Type ◽  
Directed Migration ◽  
New Family ◽  
Cytoplasmic Structures

The cell cycle is widely known to be regulated by networks of phosphorylation and ubiquitin-directed proteolysis. Here, we describe IX-14/invadolysin, a novel metalloprotease present only in metazoa, whose activity appears to be essential for mitotic progression. Mitotic neuroblasts of Drosophila melanogaster IX-14 mutant larvae exhibit increased levels of nuclear envelope proteins, monopolar and asymmetric spindles, and chromosomes that appear hypercondensed in length with a surrounding halo of loosely condensed chromatin. Zymography reveals that a protease activity, present in wild-type larval brains, is missing from homozygous tissue, and we show that IX-14/invadolysin cleaves lamin in vitro. The IX-14/invadolysin protein is predominantly found in cytoplasmic structures resembling invadopodia in fly and human cells, but is dramatically relocalized to the leading edge of migrating cells. Strikingly, we find that the directed migration of germ cells is affected in Drosophila IX-14 mutant embryos. Thus, invadolysin identifies a new family of conserved metalloproteases whose activity appears to be essential for the coordination of mitotic progression, but which also plays an unexpected role in cell migration.

scholarly journals Lactose synthetase activity in mouse mammary glands is controlled by thyroid hormones.

1979 ◽  
Vol 82 (3) ◽  
pp. 675-681 ◽  
Author(s):  
B K Vonderhaar
Keyword(s):  
Cell Cycle ◽  
Thyroid Hormones ◽  
Dna Synthesis ◽  
Milk Protein ◽  
Mammary Glands ◽  
Mouse Mammary Gland ◽  
The Other ◽  
Synthetase Activity

Epithelial cells in explants from the mammary glands of euthyroid mature virgin mice are proliferatively dormant. They must undergo DNA synthesis and traverse the cell cycle in vitro before they are able to differentiate fully in response to insulin, hydrocortisone, and prolactin, and synthesize enzymatically active alpha-lactalbumin (measured as lactose synthetase activity). In contrast, glands from hyperthyroid mature virgin mice do not require DNA synthesis in vitro to differentiate. Explants from the euthyroid virgin tissue overcome their dependence on DNA synthesis when 10(-9) M 3,5,3'-triiodo-L-thyronine is added directly to the cultures in addition to the other three hormones. Explants from involuted mammary glands from euthyroid primiparous mice do not require DNA synthesis in vitro to make the milk protein even though they, like explants from mature euthyroid virgin tissue, are proliferatively dormant and do not contain detectable lactose synthetase activity in vivo. Glands from primiparous animals made mildly hypothyroid by ingestion of 0.1% thiouracil in drinking water during 7 wk of involution remain morphologically indistinguishable from glands of their euthyroid counterparts. However, explants from the glands of these hypothyroid animals revert to a state of dependence on DNA synthesis to differentiate functionally. These observations suggest that the dependence on DNA synthesis and cell cycle traversal for hormonal induction of lactose synthetase activity in the mouse mammary gland is controlled by thyroid hormones.

scholarly journals Two Polo-like kinase 4 binding domains in Asterless perform distinct roles in regulating kinase stability

2015 ◽  
Vol 208 (4) ◽  
pp. 401-414 ◽  
Author(s):  
Joseph E. Klebba ◽  
Brian J. Galletta ◽  
Jonathan Nye ◽  
Karen M. Plevock ◽  
Daniel W. Buster ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drosophila Melanogaster ◽  
Binding Domain ◽  
Terminal Region ◽  
Scaffolding Protein ◽  
Binding Partner ◽  
Centriole Duplication ◽  
Binding Domains ◽  
C Terminus ◽  
N Terminus

Plk4 (Polo-like kinase 4) and its binding partner Asterless (Asl) are essential, conserved centriole assembly factors that induce centriole amplification when overexpressed. Previous studies found that Asl acts as a scaffolding protein; its N terminus binds Plk4’s tandem Polo box cassette (PB1-PB2) and targets Plk4 to centrioles to initiate centriole duplication. However, how Asl overexpression drives centriole amplification is unknown. In this paper, we investigated the Asl–Plk4 interaction in Drosophila melanogaster cells. Surprisingly, the N-terminal region of Asl is not required for centriole duplication, but a previously unidentified Plk4-binding domain in the C terminus is required. Mechanistic analyses of the different Asl regions revealed that they act uniquely during the cell cycle: the Asl N terminus promotes Plk4 homodimerization and autophosphorylation during interphase, whereas the Asl C terminus stabilizes Plk4 during mitosis. Therefore, Asl affects Plk4 in multiple ways to regulate centriole duplication. Asl not only targets Plk4 to centrioles but also modulates Plk4 stability and activity, explaining the ability of overexpressed Asl to drive centriole amplification.

scholarly journals Motor domain phosphorylation and regulation of the Drosophila kinesin 13, KLP10A

2009 ◽  
Vol 186 (4) ◽  
pp. 481-490 ◽  
Author(s):  
Vito Mennella ◽  
Dong-Yan Tan ◽  
Daniel W. Buster ◽  
Ana B. Asenjo ◽  
Uttama Rath ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Cycle ◽  
Molecular Dynamics ◽  
Drosophila Melanogaster ◽  
Structural Features ◽  
Motor Domain ◽  
Α Helix ◽  
Dynamics Simulations

Microtubule (MT)-destabilizing kinesin 13s perform fundamental roles throughout the cell cycle. In this study, we show that the Drosophila melanogaster kinesin 13, KLP10A, is phosphorylated in vivo at a conserved serine (S573) positioned within the α-helix 5 of the motor domain. In vitro, a phosphomimic KLP10A S573E mutant displays a reduced capacity to depolymerize MTs but normal affinity for the MT lattice. In cells, replacement of endogenous KLP10A with KLP10A S573E dampens MT plus end dynamics throughout the cell cycle, whereas a nonphosphorylatable S573A mutant apparently enhances activity during mitosis. Electron microscopy suggests that KLP10A S573 phosphorylation alters its association with the MT lattice, whereas molecular dynamics simulations reveal how KLP10A phosphorylation can alter the kinesin–MT interface without changing important structural features within the motor’s core. Finally, we identify casein kinase 1α as a possible candidate for KLP10A phosphorylation. We propose a model in which phosphorylation of the KLP10A motor domain provides a regulatory switch controlling the time and place of MT depolymerization.

scholarly journals Nafamostat mesilate, a nuclear factor kappa B inhibitor, enhances the antitumor action of radiotherapy on gallbladder cancer cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257019
Author(s):  
Naoki Takada ◽  
Hiroshi Sugano ◽  
Yoshihiro Shirai ◽  
Nobuhiro Saito ◽  
Ryoga Hamura ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Combination Treatment ◽  
Nuclear Factor Kappa B ◽  
The Other ◽  
Antitumor Action ◽  
Nafamostat Mesilate ◽  
Nuclear Factor ◽  
Radiation Induced

Nuclear factor kappa B (NF-κB) is a transcriptional factor that can be activated by radiotherapy and chemotherapy. The synthetic protease inhibitor nafamostat mesilate (NM) inhibits NF-κB activity and exerts antitumor actions in various types of cancer. In the present study, we hypothesized that NM might enhance the antitumor action of radiotherapy on gallbladder cancer (GBC) cells by inhibiting radiation-induced NF-κB activity. Thus, we investigated the correlation between radiotherapy and NF-κB activity in GBC cells. We assessed the in vitro effects of radiotherapy with or without NM on NF-κB activity, apoptosis of GBC cells (NOZ and OCUG-1), induction of apoptotic cascade, cell cycle progression, and viability of GBC cells using four treatment groups: 1) radiation (5 Gy) alone; 2) NM (80 μg/mL and 40 μg/mL, respectively) alone; 3) combination (radiation and NM); and 4) vehicle (control). The same experiments were performed in vivo using a xenograft GBC mouse model. In vitro, NM inhibited radiation-induced NF-κB activity. Combination treatment significantly attenuated cell viability and increased cell apoptosis and G2/M phase cell cycle arrest compared with those in the other groups for NOZ and OCUG-1 cells. Moreover, combination treatment upregulated the expression of apoptotic proteins compared with that after the other treatments. In vivo, NM improved the antitumor action of radiation and increased the population of Ki-67-positive cells. Overall, NM enhanced the antitumor action of radiotherapy on GBC cells by suppressing radiation-induced NF-κB activity. Thus, the combination of radiotherapy and NM may be useful for the treatment of locally advanced unresectable GBC.

scholarly journals Gross genetic dissection and interaction of the chromosomal region 95E;96F of Drosophila melanogaster.

Genetics ◽  
1989 ◽  
Vol 123 (2) ◽  
pp. 371-377
Author(s):  
C González ◽  
I Molina ◽  
J Casal ◽  
P Ripoll
Keyword(s):  
Cell Cycle ◽  
Drosophila Melanogaster ◽  
Abdominal Segment ◽  
Chromosomal Region ◽  
The Other ◽  
Genetic Dissection ◽  
Metaphase Arrest ◽  
Distal Breakpoint ◽  
Drastic Reduction

Abstract Making use of deficiencies, inversions and translocations, we have genetically dissected the region 95E to 96F of Drosophila melanogaster. We localized cytologically the loci abnormal spindle (asp: 3-85.2: 96A20-25;96B1-10) and M(3)96C2 (96C1;96C5). We have also found several new phenotypes associated with lesions in the 95E to 97B region: (1) Minute(3)96A (M(3)96A) is a haplo-insufficient phenotype of thin and short bristles presented by individuals deficient for the region 95E6-8;96A1-5. (2) abdominal-one reduced (aor) shows two different phenotypes associated with the distal breakpoint of In(3R)Ubx7L (89E;96A1-7). One is the increase of the Ubx phenotype, but its effect requires the presence of lesions in Ubx. The other phenotype is a drastic reduction or disappearance of the first abdominal segment. Both phenotypes might be due to lesions in the same gene. (3) metaphase arrest (mar) is associated with the breakpoint of the T(Y;3)B197 (96B1-10) and produces a phenotype typical of mitotic mutants with arrest of the cell cycle during prometaphase or metaphase. There is another region localized in 97B which interacts with asp: in a background homozygous for asp, three doses of this region enhance the asp phenotype.

scholarly journals An ordered pattern of Ana2 phosphorylation by Plk4 is required for centriole assembly

2017 ◽  
Author(s):  
Tiffany A. McLamarrah ◽  
Daniel W. Buster ◽  
Brian J. Galletta ◽  
Cody J. Boese ◽  
John M. Ryniawec ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Coiled Coil ◽  
Kinase Domain ◽  
Early Step ◽  
Centriole Duplication ◽  
Physiological Relevance ◽  
Phosphorylation Pattern ◽  
Centriole Assembly ◽  
Assembly Site

AbstractPolo-like kinase 4 (Plk4) initiates an early step in centriole assembly by phosphorylating Ana2/STIL, a structural component of the procentriole. Here, we show that Plk4 binding to the central coiled-coil (CC) of Ana2 is a conserved event, involving Polo-box 3 and a previously unidentified putative CC located adjacent to the kinase domain. Ana2 binding stimulates Plk4 kinase activity in vitro, and, in turn, is phosphorylated along its length. Previous studies showed that Plk4 phosphorylates the C-terminal STAN domain of Ana2/STIL, triggering binding and recruitment of the cartwheel protein Sas6 to the procentriole assembly site. However, the physiological relevance of N-terminal phosphorylation was unknown. We found that Plk4 first phosphorylates the extreme N–terminus of Ana2 which is critical for subsequent STAN domain modification. Phosphorylation of the central region then breaks the Plk4-Ana2 interaction. This phosphorylation pattern is important for centriole assembly and integrity because replacement of endogenous Ana2 with phospho-Ana2 mutants disrupts distinct steps in Ana2 function and inhibits centriole duplication.

Maytansine-Induced Mitotic Arrest in Human Lymphoblasts

1977 ◽  
Vol 35 ◽  
pp. 460-461
Author(s):  
Tai-Te Chao ◽  
John Sullivan ◽  
Awtar Krishan
Keyword(s):  
Electron Microscopy ◽  
Cell Cycle ◽  
Transmission Electron Microscopy ◽  
Tubulin Polymerization ◽  
Vinca Alkaloids ◽  
Antimitotic Activity ◽  
Transmission Electron ◽  
Flow Microfluorometry ◽  
Brain Tubulin

Maytansine, a novel ansa macrolide (1), has potent anti-tumor and antimitotic activity (2, 3). It blocks cell cycle traverse in mitosis with resultant accumulation of metaphase cells (4). Inhibition of brain tubulin polymerization in vitro by maytansine has also been reported (3). The C-mitotic effect of this drug is similar to that of the well known Vinca- alkaloids, vinblastine and vincristine. This study was carried out to examine the effects of maytansine on the cell cycle traverse and the fine struc- I ture of human lymphoblasts.Log-phase cultures of CCRF-CEM human lymphoblasts were exposed to maytansine concentrations from 10-6 M to 10-10 M for 18 hrs. Aliquots of cells were removed for cell cycle analysis by flow microfluorometry (FMF) (5) and also processed for transmission electron microscopy (TEM). FMF analysis of cells treated with 10-8 M maytansine showed a reduction in the number of G1 cells and a corresponding build-up of cells with G2/M DNA content.

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