Dictyostelium gamma-tubulin: molecular characterization and ultrastructural localization

The centrosome of Dictyostelium discoideum is a nucleus-associated body consisting of an electron-dense, three-layered core surrounded by an amorphous matrix, the corona. To elucidate the molecular and supramolecular architecture of this unique microtubule-organizing center, we have isolated and sequenced the gene encoding gamma-tubulin and have studied its localization in the Dictyostelium centrosome using immunofluorescence and postembedding immunoelectron microscopy. D. discoideum possesses a single copy of a gamma-tubulin gene that is related to, but more divergent from, other gamma-tubulins. The low-abundance gene product is localized to the centrosome in an intriguing pattern: it is highly concentrated in the corona in regularly spaced clusters whose distribution correlates with the patterning of dense nodules that are a prominent feature of the corona. These observations lend support to the notion that the corona is the functional homologue of the pericentriolar matrix of ‘higher’ eukaryotic centrosomes, and that nodules are the functional equivalent of gamma-tubulin ring complexes that serve as nucleation sites for microtubules in animal centrosomes.

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Structure of Centrosomes and Chromosomes Through IVEM Tomography

Microscopy and Microanalysis ◽

10.1017/s143192760000800x ◽

1997 ◽

Vol 3 (S2) ◽

pp. 223-224

Author(s):

D.A. Agard ◽

Vincent Guenbaut ◽

Michelle Moritz ◽

M.B. Braunfeld ◽

GuoFung Zhang ◽

...

Keyword(s):

Direct Proof ◽

Active Role ◽

Animal Cells ◽

Microtubule Organizing Center ◽

Nucleation Sites ◽

Organizing Center ◽

Ring Structures ◽

Ring Complexes ◽

Basic Level ◽

Drosophila Embryos

Centrosome structure: The centrosome is the major microtubule-organizing center of animal cells - it initiates spindle formation at the onset of mitosis and plays an active role in chromosome segregation. At the most basic level, microtubule (MT) formation is regulated at least in part, through the functional availability of nucleation sites on the centrosome. Functional centrosomes can be isolated from Drosophila embryos. When visualized with IVEM Tomography (IVEM-T) ring complexes are visualized throughout the peri-centriolar material (PCM) (1). γ-tubulin has been implicated in MT nucleation. Through immuno-IVEM-T we have shown that the ring structures within the PCM, contain y-tubulin and further that the only place γ-tubulin is found is at the minus (nucleating) end of MTs (2). This strongly implicates these γ-tubulin containing ring structures in MT polymerization, until our studies there was no direct proof that γ-tubulin was located at the site of MT nucleation. Furthermore Zheng et al have been able to isolate similar ring structures that are soluble from both Xenopus and Drosophila embryos (3).

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Three-Dimensional reconstruction of isolated centrosomes by automated electron tomography

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100169286 ◽

1994 ◽

Vol 52 ◽

pp. 310-311

Author(s):

M.B. Braunfeld ◽

M. Moritz ◽

B.M. Alberts ◽

J.W. Sedat ◽

D.A. Agard

Keyword(s):

Electron Tomography ◽

Three Dimensional ◽

Vital Role ◽

Complex Nature ◽

Animal Cells ◽

Microtubule Organizing Center ◽

Nucleation Sites ◽

Dimensional Reconstruction ◽

Organizing Center ◽

Resolution Model

In animal cells, the centrosome functions as the primary microtubule organizing center (MTOC). As such the centrosome plays a vital role in determining a cell's shape, migration, and perhaps most importantly, its division. Despite the obvious importance of this organelle little is known about centrosomal regulation, duplication, or how it nucleates microtubules. Furthermore, no high resolution model for centrosomal structure exists.We have used automated electron tomography, and reconstruction techniques in an attempt to better understand the complex nature of the centrosome. Additionally we hope to identify nucleation sites for microtubule growth.Centrosomes were isolated from early Drosophila embryos. Briefly, after large organelles and debris from homogenized embryos were pelleted, the resulting supernatant was separated on a sucrose velocity gradient. Fractions were collected and assayed for centrosome-mediated microtubule -nucleating activity by incubating with fluorescently-labeled tubulin subunits. The resulting microtubule asters were then spun onto coverslips and viewed by fluorescence microscopy.

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Immunoprobe Localization by Correlative Microscopy

Microscopy and Microanalysis ◽

10.1017/s1431927600000337 ◽

2000 ◽

Vol 6 (3) ◽

pp. 195-201 ◽

Cited By ~ 2

Author(s):

Patricia G. Calarco

Keyword(s):

Electron Microscopy ◽

Light Microscopy ◽

Ultrastructural Level ◽

Correlative Microscopy ◽

Microtubule Organizing Center ◽

Large Size ◽

Organizing Center ◽

Gamma Tubulin ◽

High Level ◽

And Function

AbstractMammalian oocytes present challenges for optimal study by electron microscopy (EM) due to their high level of hydration, their large size, and their relatively undifferentiated cytoplasm. This is particularly true for immunoprobe localization which has led to a dependence on light microscopic (LM) techniques, such as immunofluorescence. This study presents correlative LM and EM data to describe an example of the failure of light microscopy to correctly predict the ultrastructure of one particular organelle. Immunoprobe localization of centrosome and microtubule organizing center (MTOC) antigens in the mammalian egg was made by immunofluorescence and post-embedding immuno-EM, with best EM results achieved in Lowicryl-embedded material. Centrosome and MTOC antigens were detected by 5051 and an antibody to gamma tubulin (γtubulin). Gamma tubulin is a highly conserved element of MTOCs in many species and, thus, is highly diagnostic for them; it is also considered essential for microtubule (MT) nucleation. Results indicate that prior to nuclear breakdown, 5051 antigens and γ-tubulin are found exclusively in a type of “organelle,” the multivesicular aggregate (MVA), that bears no resemblance to MTOCs at the ultrastructural level. Until recently, the MVA was considered an organelle without a known function, while standard MTOCs were presumed to be the entities that carry the proteins recognized by centrosome antibodies. LM localization of centrosomal antigens carried the presumption that standard MTOCs were the entities labeled. Whether or not other molecules are shown to co-localize to these MVA, the presence of γ-tubulin supports the contention that MVA, or their contents, serve as centrosomal precursors with a unique ultrastructure. Thus, dependence on LM techniques alone can lead to erroneous conclusions on organelle identity and function.

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Immunoprobe Localization by Correlative Microscopy

Microscopy and Microanalysis ◽

10.1007/s100059910021 ◽

2000 ◽

Vol 6 (3) ◽

pp. 195-201

Author(s):

Patricia G. Calarco

Keyword(s):

Electron Microscopy ◽

Light Microscopy ◽

Ultrastructural Level ◽

Correlative Microscopy ◽

Microtubule Organizing Center ◽

Large Size ◽

Organizing Center ◽

Gamma Tubulin ◽

High Level ◽

And Function

Abstract Mammalian oocytes present challenges for optimal study by electron microscopy (EM) due to their high level of hydration, their large size, and their relatively undifferentiated cytoplasm. This is particularly true for immunoprobe localization which has led to a dependence on light microscopic (LM) techniques, such as immunofluorescence. This study presents correlative LM and EM data to describe an example of the failure of light microscopy to correctly predict the ultrastructure of one particular organelle. Immunoprobe localization of centrosome and microtubule organizing center (MTOC) antigens in the mammalian egg was made by immunofluorescence and post-embedding immuno-EM, with best EM results achieved in Lowicryl-embedded material. Centrosome and MTOC antigens were detected by 5051 and an antibody to gamma tubulin (γtubulin). Gamma tubulin is a highly conserved element of MTOCs in many species and, thus, is highly diagnostic for them; it is also considered essential for microtubule (MT) nucleation. Results indicate that prior to nuclear breakdown, 5051 antigens and γ-tubulin are found exclusively in a type of “organelle,” the multivesicular aggregate (MVA), that bears no resemblance to MTOCs at the ultrastructural level. Until recently, the MVA was considered an organelle without a known function, while standard MTOCs were presumed to be the entities that carry the proteins recognized by centrosome antibodies. LM localization of centrosomal antigens carried the presumption that standard MTOCs were the entities labeled. Whether or not other molecules are shown to co-localize to these MVA, the presence of γ-tubulin supports the contention that MVA, or their contents, serve as centrosomal precursors with a unique ultrastructure. Thus, dependence on LM techniques alone can lead to erroneous conclusions on organelle identity and function.

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Drosophila gamma-tubulin is part of a complex containing two previously identified centrosomal MAPs.

The Journal of Cell Biology ◽

10.1083/jcb.121.4.823 ◽

1993 ◽

Vol 121 (4) ◽

pp. 823-835 ◽

Cited By ~ 65

Author(s):

J W Raff ◽

D R Kellogg ◽

B M Alberts

Keyword(s):

Microtubule Organizing Center ◽

Substantial Fraction ◽

Beta Tubulin ◽

Microtubule Associated Proteins ◽

Organizing Center ◽

Associated Proteins ◽

Gamma Tubulin ◽

A Minor ◽

Drosophila Embryos

gamma-tubulin is a minor tubulin that is localized to the microtubule organizing center of many fungi and higher eucaryotic cells (Oakley, B. R., C. E. Oakley, Y. Yoon, and M. C. Jung. 1990. Cell. 61: 1289-1301; Stearns, T., L. Evans, and M. Kirschner. 1991. Cell. 65:825-836; Zheng, Y., M. K. Jung, and B. R. Oakley. 1991. Cell. 65:817-823). Here we show that gamma-tubulin is a component of a previously isolated complex of Drosophila proteins that contains at least two centrosomal microtubule-associated proteins called DMAP190 and DMAP60. Like DMAP190 and DMAP60, the gamma-tubulin in extracts of early Drosophila embryos binds to microtubules, although this binding may be indirect. Unlike DMAP190 and DMAP60, however, only 10-50% of the gamma-tubulin in the extract is able to bind to microtubules. We show that gamma-tubulin binds to a microtubule column as part of a complex, and a substantial fraction of this gamma-tubulin is tightly associated with DMAP60. As neither alpha- nor beta-tubulin bind to microtubule columns, the gamma-tubulin cannot be binding to such columns in the form of an alpha:gamma or beta:gamma heterodimer. These observations suggest that gamma-tubulin, DMAP60, and DMAP190 are components of a centrosomal complex that can interact with microtubules.

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Analysis of Tub4p, a yeast gamma-tubulin-like protein: implications for microtubule-organizing center function.

The Journal of Cell Biology ◽

10.1083/jcb.134.2.443 ◽

1996 ◽

Vol 134 (2) ◽

pp. 443-454 ◽

Cited By ~ 114

Author(s):

L G Marschall ◽

R L Jeng ◽

J Mulholland ◽

T Stearns

Keyword(s):

Fluorescent Protein ◽

Spindle Pole Body ◽

Spindle Pole ◽

Dependent Manner ◽

Protein Fusion ◽

Microtubule Organizing Center ◽

Pole Body ◽

Monopolar Spindle ◽

Organizing Center ◽

Gamma Tubulin

gamma-Tubulin is a conserved component of microtubule-organizing centers and is thought to be involved in microtubule nucleation. A recently discovered Saccharomyces cerevisiae gene (TUB4) encodes a tubulin that is related to, but divergent from, gamma-tubulins. TUB4 is essential for cell viability, and epitope-tagged Tub4 protein (Tub4p) is localized to the spindle pole body (Sobel, S.G., and M. Snyder. 1995.J. Cell Biol. 131:1775-1788). We have characterized the expression of TUB4, the association of Tub4p with the spindle pole body, and its role in microtubule organization. Tub4p is a minor protein in the cell, and expression of TUB4 is regulated in a cell cycle-dependent manner. Wild-type Tub4p is localized to the spindle pole body, and a Tub4p-green fluorescent protein fusion is able to associate with a preexisting spindle pole body, suggesting that there is dynamic exchange between cytoplasmic and spindle pole body forms of Tub4p. Perturbation of Tub4p function, either by conditional mutation or by depletion of the protein, results in spindle as well as spindle pole body defects, but does not eliminate the ability of microtubules to regrow from, or remain attached to, the spindle pole body. The spindle pole bodies in tub4 mutant cells duplicate but do not separate, resulting in a monopolar spindle. EM revealed that one spindle pole body of the duplicated pair appears to be defective for the nucleation of microtubules. These results offer insight into the role of gamma-tubulin in microtubule-organizing center function.

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CRISPR/Cas9 genome editing system confirms centriolin’s role in cytokinesis

BMC Research Notes ◽

10.1186/s13104-021-05898-w ◽

2022 ◽

Vol 15 (1) ◽

Author(s):

Eric Seronick ◽

Jae Son ◽

Cameron Michael ◽

Hannah Fogg ◽

Zeynep Gromley ◽

...

Keyword(s):

Cell Division ◽

Genome Editing ◽

Cell Cycle Progression ◽

Primary Cilia ◽

Effective Means ◽

Mrna Level ◽

Microtubule Organizing Center ◽

Gene Encoding ◽

Protein Levels ◽

Organizing Center

Abstract Objective In addition to its function as the microtubule organizing center of the cell, the centrosome has functions in many other cellular processes including primary cilia formation, DNA damage checkpoints, and cell cycle progression. But the role of individual components of the centrosome in these processes remains unclear. Previous studies used siRNA (small interfering RNA) to “knock down” protein levels of the centrosome component centriolin, resulting in failed cytokinesis. Since this approach was transient, only targeting centriolin at the mRNA level, we sought to confirm these findings by permanently disrupting the gene encoding centriolin using the CRISPR/Cas9 system of genome editing. Results This study provides evidence that the CRISPR/Cas9 system is capable of effectively reducing centriolin protein levels in the cell. Furthermore, this disruption leads to a failure of cytokinesis that is reminiscent of the phenotype previously reported for the siRNA-mediated disruption of centriolin. Furthermore, no additional defects in cell division were observed, consistent with results seen with previous siRNA studies. We conclude that the CRISPR/Cas9 system is an effective means of permanently removing the cellular pools of centriolin and that the disruption of centriolin at both the mRNA level and genomic level lead to similar cell division defects.

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