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

1993 ◽  
Vol 121 (4) ◽  
pp. 823-835 ◽  
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.

scholarly journals Cytokinesis in Prokaryotes and Eukaryotes: Common Principles and Different Solutions

2001 ◽  
Vol 65 (2) ◽  
pp. 319-333 ◽  
Author(s):  
Nanne Nanninga
Keyword(s):  
Dna Replication ◽  
Cellular Structures ◽  
Microtubule Organizing Center ◽  
Microtubule Associated Proteins ◽  
Origin Of Dna Replication ◽  
Organizing Center ◽  
Associated Proteins ◽  
Eukaryotic Origin ◽  
Common Principle ◽  
Macromolecular Composition

SUMMARY Cytokinesis requires duplication of cellular structures followed by bipolarization of the predivisional cell. As a common principle, this applies to prokaryotes as well as eukaryotes. With respect to eukaryotes, the discussion has focused mainly on Saccharomyces cerevisiae and on Schizosaccharomyces pombe. Escherichia coli and to a lesser extent Bacillus subtilis have been used as prokaryotic examples. To establish a bipolar cell, duplication of a eukaryotic origin of DNA replication as well as its genome is not sufficient. Duplication of the microtubule-organizing center is required as a prelude to mitosis, and it is here that the dynamic cytoskeleton with all its associated proteins comes to the fore. In prokaryotes, a cytoskeleton that pervades the cytoplasm appears to be absent. DNA replication and the concomitant DNA segregation seem to occur without help from extensive cytosolic supramacromolecular assemblies but with help from the elongating cellular envelope. Prokaryotic cytokinesis proceeds through a contracting ring, which has a roughly 100-fold-smaller circumference than its eukaryotic counterpart. Although the ring contains proteins that can be considered as predecessors of actin, tubulin, and microtubule-associated proteins, its macromolecular composition is essentially different.

A protein related to brain microtubule-associated protein MAP1B is a component of the mammalian centrosome

1994 ◽  
Vol 107 (2) ◽  
pp. 601-611 ◽  
Author(s):  
J.E. Dominguez ◽  
B. Buendia ◽  
C. Lopez-Otin ◽  
C. Antony ◽  
E. Karsenti ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cultured Cells ◽  
Polyclonal Antibodies ◽  
Microtubule Organizing Center ◽  
Microtubule Associated Proteins ◽  
Organizing Center ◽  
Associated Proteins ◽  
Pericentriolar Material ◽  
Peptide Maps

The centrosome is the main microtubule organizing center of mammalian cells. Structurally, it is composed of a pair of centrioles surrounded by a fibro-granular material (the pericentriolar material) from which microtubules are nucleated. However, the nature of centrosomal molecules involved in microtubules nucleation is still obscure. Since brain microtubule-associated proteins (MAPs) lower the critical tubulin concentration required for microtubule nucleation in tubulin solution in vitro, we have examined their possible association with centrosomes. By immunofluorescence, monoclonal and polyclonal antibodies raised against MAP1B stain the centrosome in cultured cells as well as purified centrosomes, whereas antibodies raised against MAP2 give a completely negative reaction. The MAP1B-related antigen is localized to the pericentriolar material as revealed by immunoelectron microscopy. In preparations of purified centrosomes analyzed on poly-acrylamide gels, a protein that migrates as brain MAP1B is present. After blotting on nitrocellulose, it is decorated by anti-MAP1B antibodies and the amino acid sequence of proteolytic fragments of this protein is similar to brain MAP1B. Moreover, brain MAP1B and its centrosomal counterpart share the same phosphorylation features and have similar peptide maps. These data strongly suggest that a protein homologue to MAP1B is present in centrosomes and it is a good candidate for being involved in the nucleating activity of the pericentriolar material.

subito Encodes a Kinesin-like Protein Required for Meiotic Spindle Pole Formation in Drosophila melanogaster

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1489-1501 ◽  
Author(s):  
Kelly L Giunta ◽  
Janet K Jang ◽  
Elizabeth A Manheim ◽  
Gayathri Subramanian ◽  
Kim S McKim
Keyword(s):  
Drosophila Melanogaster ◽  
Spindle Pole ◽  
Meiotic Spindle ◽  
Microtubule Organizing Center ◽  
Bipolar Spindle ◽  
Microtubule Associated Proteins ◽  
Organizing Center ◽  
Associated Proteins ◽  
Similar Phenotype ◽  
Meiosis I

Abstract The female meiotic spindle lacks a centrosome or microtubule-organizing center in many organisms. During cell division, these spindles are organized by the chromosomes and microtubule-associated proteins. Previous studies in Drosophila melanogaster implicated at least one kinesin motor protein, NCD, in tapering the microtubules into a bipolar spindle. We have identified a second Drosophila kinesin-like protein, SUB, that is required for meiotic spindle function. At meiosis I in males and females, sub mutations affect only the segregation of homologous chromosomes. In female meiosis, sub mutations have a similar phenotype to ncd; even though chromosomes are joined by chiasmata they fail to segregate at meiosis I. Cytological analyses have revealed that sub is required for bipolar spindle formation. In sub mutations, we observed spindles that were unipolar, multipolar, or frayed with no defined poles. On the basis of these phenotypes and the observation that sub mutations genetically interact with ncd, we propose that SUB is one member of a group of microtubule-associated proteins required for bipolar spindle assembly in the absence of the centrosomes. sub is also required for the early embryonic divisions but is otherwise dispensable for most mitotic divisions.

Immunoprobe Localization by Correlative Microscopy

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

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.

scholarly journals MAP 1C is a microtubule-activated ATPase which translocates microtubules in vitro and has dynein-like properties.

1987 ◽  
Vol 105 (3) ◽  
pp. 1273-1282 ◽  
Author(s):  
B M Paschal ◽  
H S Shpetner ◽  
R B Vallee
Keyword(s):  
Atpase Activity ◽  
Heavy Chain ◽  
Sedimentation Coefficient ◽  
Minor Component ◽  
High Molecular Mass ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Axonemal Dynein ◽  
A Minor

We observe that one of the high molecular mass microtubule-associated proteins (MAPs) from brain exhibits nucleotide-dependent binding to microtubules. We identify the protein as MAP IC, which was previously described in this laboratory as a minor component of standard microtubule preparations (Bloom, G.S., T. Schoenfeld, and R.B. Vallee, 1984, J. Cell Biol., 98:320-330). We find that MAP 1C is enriched in microtubules prepared in the absence of nucleotide. Kinesin is also found in these preparations, but can be specifically extracted with GTP. A fraction highly enriched in MAP 1C can be prepared by subsequent extraction of the microtubules with ATP. Two activities cofractionate with MAP 1C upon further purification, a microtubule-activated ATPase activity and a microtubule-translocating activity. These activities indicate a role for the protein in cytoplasmic motility. MAP 1C coelectrophoreses with the beta heavy chain of Chlamydomonas flagellar dynein, and has a sedimentation coefficient of 20S. Exposure to ultraviolet light in the presence of vanadate and ATP results in the production of two large fragments of MAP 1C. These characteristics suggest that MAP 1C may be a cytoplasmic analogue of axonemal dynein.

scholarly journals Stabilization and bundling of subtilisin-treated microtubules induced by microtubule associated proteins

1995 ◽  
Vol 108 (1) ◽  
pp. 357-367 ◽  
Author(s):  
Y. Saoudi ◽  
I. Paintrand ◽  
L. Multigner ◽  
D. Job
Keyword(s):  
Tau Proteins ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Microtubule Associated Proteins ◽  
Microtubule Stabilization ◽  
Microtubule Stability ◽  
Associated Proteins ◽  
Carboxy Terminal ◽  
Normal Sensitivity ◽  
Marked Resistance

The acidic carboxy-terminal regions of alpha- and beta-tubulin subunits are currently thought to be centrally involved in microtubule stability and in microtubule association with a variety of proteins (MAPs) such as MAP2 and tau proteins. Here, pure tubulin microtubules were exposed to subtilisin to produce polymers composed of cleaved tubulin subunits lacking carboxy termini. Polymer exposure to subtilisin was achieved in buffer conditions compatible with further tests of microtubule stability. Microtubules composed of normal alpha-tubulin and cleaved beta-tubulin were indistinguishable from control microtubules with regard to resistance to dilution-induced disassembly, to cold temperature-induced disassembly and to Ca(2+)-induced disassembly. Microtubules composed of cleaved alpha- and beta-tubulins showed normal sensitivity to dilution-induced disassembly and to low temperature-induced disassembly, but marked resistance to Ca(2+)-induced disassembly. Polymers composed of normal alpha-tubulin and cleaved beta-tubulin or of cleaved alpha- and beta-tubulins were stabilized in the presence of added MAP2, myelin basic protein and histone H1. Cleavage of tubulin carboxy termini greatly potentiated microtubule stabilization by tau proteins. We show that this potentiation of polymer stabilization can be ascribed to tau-induced microtubule bundling. In our working conditions, such bundling upon association with tau proteins occurred only in the case of microtubules composed of cleaved alpha- and beta-tubulins and triggered apparent microtubule cross-stabilization among the bundled polymers. These results, as well as immunofluorescence analysis, which directly showed interactions between subtilisin-treated microtubules and MAPs, suggest that the carboxy termini of alpha- and beta-tubulins are not primarily involved in the binding of MAPs onto microtubules. However, interactions between tubulin carboxy termini and MAPs remain possible and might be involved in the regulation of MAP-induced microtubule bundling.

Tubulin polyglutamylase: isozymic variants and regulation during the cell cycle in HeLa cells

1999 ◽  
Vol 112 (23) ◽  
pp. 4281-4289 ◽  
Author(s):  
C. Regnard ◽  
E. Desbruyeres ◽  
P. Denoulet ◽  
B. Edde
Keyword(s):  
Cell Cycle ◽  
Hela Cells ◽  
Molecular Motors ◽  
Dependent Manner ◽  
Proliferating Cells ◽  
Beta Tubulin ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
A Cell ◽  
Cycle Dependent

Polyglutamylation is a posttranslational modification of tubulin that is very common in neurons and ciliated or flagellated cells. It was proposed to regulate the binding of microtubule associated proteins (MAPs) and molecular motors as a function of the length of the polyglutamyl side-chain. Though much less common, this modification of tubulin also occurs in proliferating cells like HeLa cells where it is associated with centrioles and with the mitotic spindle. Recently, we partially purified tubulin polyglutamylase from mouse brain and described its enzymatic properties. In this work, we focused on tubulin polyglutamylase activity from HeLa cells. Our results support the existence of a tubulin polyglutamylase family composed of several isozymic variants specific for alpha- or beta-tubulin subunits. In the latter case, the specificity probably also concerns the different beta-tubulin isotypes. Interestingly, we found that tubulin polyglutamylase activity is regulated in a cell cycle dependent manner and peaks in G(2)-phase while the level of glutamylated tubulin peaks in mitosis. Consistent results were obtained by treating the cells with hydroxyurea, nocodazole or taxotere. In particular, in mitotic cells, tubulin polyglutamylase activity was always low while glutamylation level was high. Finally, tubulin polyglutamylase activity and the level of glutamylated tubulin appeared to be inversely related. This paradox suggests a complex regulation of both tubulin polyglutamylase and the reverse deglutamylase activity.

Immunoprobe Localization by Correlative Microscopy

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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