scholarly journals MOZART1 and γ-tubulin complex receptors are both required to turn γ-TuSC into an active microtubule nucleation template

2016 ◽  
Vol 215 (6) ◽  
pp. 823-840 ◽  
Author(s):  
Tien-chen Lin ◽  
Annett Neuner ◽  
Dirk Flemming ◽  
Peng Liu ◽  
Takumi Chinen ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Human Cells ◽  
Molecular Function ◽  
Microtubule Nucleation ◽  
Small Complex ◽  
Ring Complex

MOZART1/Mzt1 is required for the localization of γ-tubulin complexes to microtubule (MT)–organizing centers from yeast to human cells. Nevertheless, the molecular function of MOZART1/Mzt1 is largely unknown. Taking advantage of the minimal MT nucleation system of Candida albicans, we reconstituted the interactions of Mzt1, γ-tubulin small complex (γ-TuSC), and γ-tubulin complex receptors (γ-TuCRs) Spc72 and Spc110 in vitro. With affinity measurements, domain deletion, and swapping, we show that Spc110 and Mzt1 bind to distinct regions of the γ-TuSC. In contrast, both Mzt1 and γ-TuSC interact with the conserved CM1 motif of Spc110/Spc72. Spc110/Spc72 and Mzt1 constitute “oligomerization chaperones,” cooperatively promoting and directing γ-TuSC oligomerization into MT nucleation-competent rings. Consistent with the functions of Mzt1, human MOZART1 directly interacts with the CM1-containing region of the γ-TuCR CEP215. MOZART1 depletion in human cells destabilizes the large γ-tubulin ring complex and abolishes CEP215CM1-induced ectopic MT nucleation. Together, we reveal conserved functions of MOZART1/Mzt1 through interactions with γ-tubulin complex subunits and γ-TuCRs.

scholarly journals Recruitment of the γ-Tubulin Ring Complex to Drosophila Salt-stripped Centrosome Scaffolds

1998 ◽  
Vol 142 (3) ◽  
pp. 775-786 ◽  
Author(s):  
Michelle Moritz ◽  
Yixian Zheng ◽  
Bruce M. Alberts ◽  
Karen Oegema
Keyword(s):  
Soluble Fraction ◽  
Protein Complexes ◽  
Drosophila Embryo ◽  
Microtubule Nucleation ◽  
Complementation Assay ◽  
Embryo Extract ◽  
Ring Complex ◽  
Centrosomal Proteins

Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that lack the centrosomal proteins CP190, CP60, centrosomin, and γ-tubulin. To clarify the role of these proteins in microtubule nucleation by centrosomes and to identify additional centrosome components required for nucleation, we have developed an in vitro complementation assay for centrosome function. Centrosome aster formation is reconstituted when these inactive, salt-stripped centrosome scaffolds are supplemented with a soluble fraction of a Drosophila embryo extract. The CP60 and CP190 can be removed from this extract without effect, whereas removing the γ-tubulin destroys the complementing activity. Consistent with these results, we find no evidence that these three proteins form a complex together. Instead, γ-tubulin is found in two distinct protein complexes of 240,000 and ∼3,000,000 D. The larger complex, which is analogous to the Xenopus γ-tubulin ring complex (γTuRC) (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578–583), is necessary but not sufficient for complementation. An additional factor found in the extract is required. These results provide the first evidence that the γTuRC is required for microtubule nucleation at the centrosome.

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.

Enhanced antifungal activity of Ni-doped ZnO nanostructures under dark conditions

RSC Advances ◽  
2016 ◽  
Vol 6 (110) ◽  
pp. 108468-108476 ◽  
Author(s):  
R. Saravana Kumar ◽  
S. H. S. Dananjaya ◽  
Mahanama De Zoysa ◽  
Minyang Yang
Keyword(s):  
Candida Albicans ◽  
Antifungal Activity ◽  
Zno Nanoparticles ◽  
Human Cells ◽  
Zno Nanostructures ◽  
Doped Zno ◽  
Dark Conditions

In this work we synthesized undoped and Ni-doped ZnO nanoparticles by reflux method, and investigated their cytotoxicity behavior using human cells (HEp2 and HEK293T) and antifungal activity against pathogenic Candida albicans fungi in vitro.

scholarly journals The cryo-EM structure of a γ-TuSC elucidates architecture and regulation of minimal microtubule nucleation systems

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Erik Zupa ◽  
Anjun Zheng ◽  
Annett Neuner ◽  
Martin Würtz ◽  
Peng Liu ◽  
...  
Keyword(s):  
De Novo ◽  
Relative Positioning ◽  
Microtubule Nucleation ◽  
Cryo Electron Microscopy ◽  
Small Complex ◽  
Ring Complex ◽  
Regulation Mechanisms ◽  
Microtubule Formation ◽  
Nucleation Activity ◽  
Higher Eukaryotes

Abstract The nucleation of microtubules from αβ-tubulin subunits is mediated by γ-tubulin complexes, which vary in composition across organisms. Aiming to understand how de novo microtubule formation is achieved and regulated by a minimal microtubule nucleation system, we here determined the cryo-electron microscopy structure of the heterotetrameric γ-tubulin small complex (γ-TuSC) from C. albicans at near-atomic resolution. Compared to the vertebrate γ-tubulin ring complex (γ-TuRC), we observed a vastly remodeled interface between the SPC/GCP-γ-tubulin spokes, which stabilizes the complex and defines the γ-tubulin arrangement. The relative positioning of γ-tubulin subunits indicates that a conformational rearrangement of the complex is required for microtubule nucleation activity, which follows opposing directionality as predicted for the vertebrate γ-TuRC. Collectively, our data suggest that the assembly and regulation mechanisms of γ-tubulin complexes fundamentally differ between the microtubule nucleation systems in lower and higher eukaryotes.

scholarly journals γ-Tubulin–γ-Tubulin Interactions as the Basis for the Formation of a Meshwork

2018 ◽  
Vol 19 (10) ◽  
pp. 3245 ◽  
Author(s):  
Catalina Rosselló ◽  
Lisa Lindström ◽  
Greta Eklund ◽  
Matthieu Corvaisier ◽  
Maria Kristensson
Keyword(s):  
Present Review ◽  
Cellular Functions ◽  
Microtubule Nucleation ◽  
Small Complex ◽  
Ring Complex ◽  
Ring Shaped Structure

In cytoplasm, protein γ-tubulin joins with various γ-tubulin complex proteins (GCPs) to form a heterotetramer γ-tubulin small complex (γ-TuSC) that can grow into a ring-shaped structure called the γ-tubulin ring complex (γ-TuRC). Both γ-TuSC and γ-TuRC are required for microtubule nucleation. Recent knowledge on γ-tubulin with regard to its cellular functions beyond participation in its creation of microtubules suggests that this protein forms a cellular meshwork. The present review summarizes the recognized functions of γ-tubulin and aims to unite the current views on this protein.

scholarly journals An extended γ-tubulin ring functions as a stable platform in microtubule nucleation

2012 ◽  
Vol 197 (1) ◽  
pp. 59-74 ◽  
Author(s):  
Sarah Erlemann ◽  
Annett Neuner ◽  
Linda Gombos ◽  
Romain Gibeaux ◽  
Claude Antony ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Fluorescence Recovery After Photobleaching ◽  
Nucleation Site ◽  
Fluorescence Recovery ◽  
Microtubule Nucleation ◽  
Nucleation Sites ◽  
Small Complex ◽  
Tubulin Binding

γ-Tubulin complexes are essential for microtubule (MT) nucleation. The γ-tubulin small complex (γ-TuSC) consists of two molecules of γ-tubulin and one molecule each of Spc97 and Spc98. In vitro, γ-TuSCs oligomerize into spirals of 13 γ-tubulin molecules per turn. However, the properties and numbers of γ-TuSCs at MT nucleation sites in vivo are unclear. In this paper, we show by fluorescence recovery after photobleaching analysis that γ-tubulin was stably integrated into MT nucleation sites and was further stabilized by tubulin binding. Importantly, tubulin showed a stronger interaction with the nucleation site than with the MT plus end, which probably provides the basis for MT nucleation. Quantitative analysis of γ-TuSCs on single MT minus ends argued for nucleation sites consisting of approximately seven γ-TuSCs with approximately three additional γ-tubulin molecules. Nucleation and anchoring of MTs required the same number of γ-tubulin molecules. We suggest that a spiral of seven γ-TuSCs with a slight surplus of γ-tubulin nucleates MTs in vivo.

scholarly journals CDK5RAP2 stimulates microtubule nucleation by the γ-tubulin ring complex

2010 ◽  
Vol 191 (6) ◽  
pp. 1089-1095 ◽  
Author(s):  
Yuk-Kwan Choi ◽  
Pengfei Liu ◽  
Siu Kwan Sze ◽  
Chao Dai ◽  
Robert Z. Qi
Keyword(s):  
Drosophila Melanogaster ◽  
Rna Interference ◽  
Schizosaccharomyces Pombe ◽  
Binding Domain ◽  
Deficient Mutant ◽  
Microtubule Nucleation ◽  
Ring Complex ◽  
Regulatory Functions

CDK5RAP2 is a human microcephaly protein that contains a γ-tubulin complex (γ-TuC)–binding domain conserved in Drosophila melanogaster centrosomin and Schizosaccharomyces pombe Mto1p and Pcp1p, which are γ-TuC–tethering proteins. In this study, we show that this domain within CDK5RAP2 associates with the γ-tubulin ring complex (γ-TuRC) to stimulate its microtubule-nucleating activity and is therefore referred to as the γ-TuRC–mediated nucleation activator (γ-TuNA). γ-TuNA but not its γ-TuC–binding-deficient mutant stimulates microtubule nucleation by purified γ-TuRC in vitro and induces extensive, γ-TuRC-dependent nucleation of microtubules in a microtubule regrowth assay. γ-TuRC bound to γ-TuNA contains NME7, FAM128A/B, and actin in addition to γ-tubulin and GCP2–6. RNA interference–mediated depletion of CDK5RAP2 impairs both centrosomal and acentrosomal microtubule nucleation, although γ-TuRC assembly is unaffected. Collectively, these results suggest that the γ-TuNA found in CDK5RAP2 has regulatory functions in γ-TuRC–mediated microtubule nucleation.

scholarly journals In vitro Effects of Lemongrass Extract on Candida albicans Biofilms, Human Cells Viability, and Denture Surface

2016 ◽  
Vol 6 ◽  
Author(s):  
Petrus L. B. Madeira ◽  
Letícia T. Carvalho ◽  
Marco A. B. Paschoal ◽  
Eduardo M. de Sousa ◽  
Eduardo B. Moffa ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Human Cells ◽  
In Vitro Effects ◽  
Candida Albicans Biofilms

scholarly journals Higher-order oligomerization of Spc110p drives γ-tubulin ring complex assembly

2016 ◽  
Vol 27 (14) ◽  
pp. 2245-2258 ◽  
Author(s):  
Andrew S. Lyon ◽  
Geneviève Morin ◽  
Michelle Moritz ◽  
King Clyde B. Yabut ◽  
Tamira Vojnar ◽  
...  
Keyword(s):  
Spindle Pole Body ◽  
Spindle Pole ◽  
Higher Order ◽  
Cellular Processes ◽  
Pole Body ◽  
Small Complex ◽  
Ring Complex ◽  
The Stability

The microtubule (MT) cytoskeleton plays important roles in many cellular processes. In vivo, MT nucleation is controlled by the γ-tubulin ring complex (γTuRC), a 2.1-MDa complex composed of γ-tubulin small complex (γTuSC) subunits. The mechanisms underlying the assembly of γTuRC are largely unknown. In yeast, the conserved protein Spc110p both stimulates the assembly of the γTuRC and anchors the γTuRC to the spindle pole body. Using a quantitative in vitro FRET assay, we show that γTuRC assembly is critically dependent on the oligomerization state of Spc110p, with higher-order oligomers dramatically enhancing the stability of assembled γTuRCs. Our in vitro findings were confirmed with a novel in vivo γTuSC recruitment assay. We conclude that precise spatial control over MT nucleation is achieved by coupling localization and higher-order oligomerization of the receptor for γTuRC.

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