Mutational analyses reveal a novel function of the nucleotide-binding domain of γ-tubulin in the regulation of basal body biogenesis

Shang, Yuhua; Tsao, Che-Chia; Gorovsky, Martin A.

doi:10.1083/jcb.200508184

Cited by 30 publications

(47 citation statements)

References 41 publications

(93 reference statements)

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“…etu1-Thr150Val results in a significant increase in basal body density implying that the NBD is part of mechanism to suppress overduplication of basal bodies. Previous mutational analysis of ctubulin isolated a mutant of the corresponding residue in the NBD that showed a very similar phenotype (Shang et al, 2005). Two mutations within the NBD of c-tubulin resulted in over-duplication and misorientation of newly formed basal bodies, but no defect in basal body stability as we observed for e-tubulin.…”

Section: Novel Role Of Nbd Of E-tubulin At the Basal Bodymentioning

confidence: 50%

“…3B), which does not possess many charged residues, and the large e-tubulin specific insertion, which was removed. Conserved residues within the NBD, shown previously to be involved with nucleotide binding (Löwe et al, 2001), were mutated to alanine, valine, or glycine, similar to mutations made in c-tubulin (Shang et al, 2005). In all, 23 different mutations were made in the domains of Etu1p (Fig.…”

Section: E-tubulin Is An Essential Protein In Tetrahymena Thermophilamentioning

confidence: 99%

“…Etu1p-T150V cells were arrested in G1 at 30˚C overnight then released into growth conditions at 30˚C and 38˚C to allow new basal body assembly. We performed immunofluorescence with antibodies against Cen1, which labels all basal bodies, and K-like antigen, which will label only mature basal bodies (Shang et al, 2005;Williams et al, 1990). These two antibodies allow us to distinguish between newly assembled basal bodies (2 K-like antigen) and older basal bodies (+ K-like antigen; Fig.…”

Section: Thr150 Of E-tubulin Is Essential For Basal Body Stabilitymentioning

confidence: 99%

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ε-tubulin is essential in Tetrahymena thermophila for the assembly and stability of basal bodies

Ross¹,

Clarissa²,

Giddings³

et al. 2013

Journal of Cell Science

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SummaryBasal bodies and centrioles are conserved microtubule-based organelles the improper assembly of which leads to a number of diseases, including ciliopathies and cancer. Tubulin family members are conserved components of these structures that are integral to their proper formation and function. We have identified the e-tubulin gene in Tetrahymena thermophila and detected the protein, through fluorescence of a tagged allele, to basal bodies. Immunoelectron microscopy has shown that e-tubulin localizes primarily to the core microtubule scaffold. A complete genomic knockout of e-tubulin has revealed that it is an essential gene required for the assembly and maintenance of the triplet microtubule blades of basal bodies. We have conducted site-directed mutagenesis of the e-tubulin gene and shown that residues within the nucleotide-binding domain, longitudinal interacting domains, and C-terminal tail are required for proper function. A single amino acid change of Thr150, a conserved residue in the nucleotide-binding domain, to Val is a conditional mutation that results in defects in the spatial and temporal assembly of basal bodies as well as their stability. We have genetically separated functions for the domains of e-tubulin and identified a novel role for the nucleotide-binding domain in the regulation of basal body assembly and stability.

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Section: Novel Role Of Nbd Of E-tubulin At the Basal Bodymentioning

confidence: 50%

Section: E-tubulin Is An Essential Protein In Tetrahymena Thermophilamentioning

confidence: 99%

Section: Thr150 Of E-tubulin Is Essential For Basal Body Stabilitymentioning

confidence: 99%

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ε-tubulin is essential in Tetrahymena thermophila for the assembly and stability of basal bodies

Ross¹,

Clarissa²,

Giddings³

et al. 2013

Journal of Cell Science

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“…Antibodies used were Tetrahymena Cen1 at 1:2000 (Stemm-Wolf et al, 2005), the kinetodesmal fiber antibody, FI-5D8 at 1:50, and the K-like antibody, FV-10D12 at 1:50 (Shang et al, 2005). Secondary antibodies were anti-rabbit Alexa Fluor 488, anti-mouse Alexa488 (Invitrogen, Grand Island, NY) anti-rabbit Texas Red, and anti-mouse Texas Red (Jackson ImmunoResearch, West Grove, PA) all used at 1:1000.…”

Section: Fluorescence Imagingmentioning

confidence: 99%

“…Therefore, the orientation of new basal body assembly was assessed in sfr13D cells. The 10D12 antibody to the K-like protein was used as a marker for old basal bodies, and centrin was used to identify all basal bodies, including newly-assembled basal bodies (Pearson et al, 2009a;Shang et al, 2005;Williams et al, 1990). Cells were grown at 30˚C, synchronized at a time prior to cell division when increased basal body assembly occurs (by starvation for approximately one generation time), and released into media at either 30˚C or 38˚C.…”

mentioning

confidence: 99%

Sfr13 is a member of a large family of asymmetrically 1 localized Sfi1-repeat proteins and is important for basal body separation and stability inTetrahymena thermophila

Stemm-Wolf

Meehl

Winey

2013

Journal of Cell Science

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SummaryDirected fluid flow, which is achieved by the coordinated beating of motile cilia, is required for processes as diverse as cellular swimming, developmental patterning and mucus clearance. Cilia are nucleated, anchored and aligned at the plasma membrane by basal bodies, which are cylindrical microtubule-based structures with ninefold radial symmetry. In the unicellular ciliate Tetrahymena thermophila, two centrin family members associated with the basal body are important for both basal body organization and stabilization. We have identified a family of 13 proteins in Tetrahymena that contain centrin-binding repeats related to those identified in the Saccharomyces cerevisiae Sfi1 protein. We have named these proteins Sfr1-Sfr13 (for Sfi1-repeat). Nine of the Sfr proteins localize in unique polarized patterns surrounding the basal body, suggesting non-identical roles in basal body organization and association with basal body accessory structures. Furthermore, the Sfr proteins are found in distinct basal body populations in Tetrahymena cells, indicating that they are responsive to particular developmental programs. A complete genetic deletion of one of the family members, Sfr13, causes unstable basal bodies and defects in daughter basal body separation from the mother, phenotypes also observed with centrin disruption. It is likely that the other Sfr family members are involved in distinct centrin functions, providing specificity to the tasks that centrins perform at basal bodies.

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Multiple phosphorylation sites on γ‐tubulin are essential and contribute to the biogenesis of basal bodies in Tetrahymena

Joachimiak

Jerka‐Dziadosz

Krzemień‐Ojak

et al. 2018

Journal Cellular Physiology

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The mechanisms that regulate γ-tubulin, including its post-translational modifications, are poorly understood. γ-Tubulin is important for the duplication of centrioles and structurally similar basal bodies (BBs), organelles which contain a ring of nine triplet microtubules. The ciliate Tetrahymena thermophila carries hundreds of cilia in a single cell and provides an excellent model to specifically address the role of γ-tubulin in the BBs assembly and maintenance. The genome of Tetrahymena contains a single γ-tubulin gene. We show here that there are multiple isoforms of γ-tubulin that are likely generated by post-translational modifications. We identified evolutionarily conserved serine and threonine residues as potential phosphosites of γ-tubulin, including S80, S129, S131, T283, and S360. Several mutations that either prevent (S80A, S131A, T283A, S360A) or mimic (T283D) phosphorylation were conditionally lethal and at a higher temperature phenocopied a loss of γ-tubulin. Cells that overproduced S360D γ-tubulin displayed phenotypes consistent with defects in the microtubule-dependent functions, including an asymmetric division of the macronucleus and abnormalities in the pattern of BB rows, including gaps, fragmentation, and misalignment. In contrast, overexpression of S129D γ-tubulin affected the orientation, docking, and structure of the BBs, including a loss of either the B- or C-subfibers or the entire triplets. We conclude that conserved potentially phosphorylated amino acids of γ-tubulin are important for either the assembly or stability of BBs.

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Mutational analyses reveal a novel function of the nucleotide-binding domain of γ-tubulin in the regulation of basal body biogenesis

Cited by 30 publications

References 41 publications

ε-tubulin is essential in Tetrahymena thermophila for the assembly and stability of basal bodies

ε-tubulin is essential in Tetrahymena thermophila for the assembly and stability of basal bodies

Sfr13 is a member of a large family of asymmetrically 1 localized Sfi1-repeat proteins and is important for basal body separation and stability inTetrahymena thermophila

Multiple phosphorylation sites on γ‐tubulin are essential and contribute to the biogenesis of basal bodies in Tetrahymena

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