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.

Increased microtubule stability and alpha tubulin acetylation in cells transfected with microtubule-associated proteins MAP1B, MAP2 or tau

1992 ◽  
Vol 103 (4) ◽  
pp. 953-964 ◽  
Author(s):  
R. Takemura ◽  
S. Okabe ◽  
T. Umeyama ◽  
Y. Kanai ◽  
N.J. Cowan ◽  
...  
Keyword(s):  
Post Translational Modification ◽  
3T3 Cells ◽  
Microtubule Organization ◽  
Alpha Tubulin ◽  
Transfected Cells ◽  
Microtubule Associated Proteins ◽  
Cos Cells ◽  
Tubulin Acetylation ◽  
Microtubule Stability ◽  
Associated Proteins

We previously transfected MAP2, tau and MAP1B cDNA into fibroblasts and have studied the effect of expression of these microtubule-associated proteins on microtubule organization. In this study, we examined some additional characteristics of microtubule bundles and arrays formed in fibroblasts transfected with these microtubule-associated proteins. It was found that microtubule bundles formed in MAP2c- or tau-transfected cells were stabilized against microtubule depolymerizing reagents and were enriched in acetylated alpha tubulin. When mouse MAP1B cDNA was expressed following transfection into COS cells, MAP1B was localized along microtubule arrays, but no extensive reorganization of microtubules such as bundle formation was observed, in agreement with our previous finding using HeLa and 3T3 cells. However, stabilization of microtubules was indicated: (a) microtubules in MAP1B-transfected cells were stabilized against a microtubule depolymerizing reagent, although stabilization was less efficient than that seen in MAP2c- or tau-transfected cells, and (b) microtubules in MAP1B-transfected cells were enriched in acetylated alpha tubulin. These results suggest that neuronal microtubule-associated proteins introduced into fibroblasts by cDNA transfection stabilize microtubules and affect the state of post-translational modification of tubulin.

scholarly journals Intracellular Proadrenomedullin-Derived Peptides Decorate the Microtubules and Contribute to Cytoskeleton Function

Endocrinology ◽  
2008 ◽  
Vol 149 (6) ◽  
pp. 2888-2898 ◽  
Author(s):  
Dan L. Sackett ◽  
Laurent Ozbun ◽  
Enrique Zudaire ◽  
Lisa Wessner ◽  
John M. Chirgwin ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Morphological Changes ◽  
Microtubule Associated Proteins ◽  
Microtubule Stabilization ◽  
Gene Coding ◽  
Intracellular Compartments ◽  
Associated Proteins ◽  
G2 Phase ◽  
Interfering Rna

Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are secretory hormones, but it is not unusual to find them in intracellular compartments. Using yeast-2 hybrid technology, we found interactions between AM and several microtubule-associated proteins (MAPs), and between PAMP and tubulin. Expression of fluorescent-tagged AM and PAMP as well as immunofluorescence for the native peptides showed a complete decoration of the microtubules and colocalization with other MAPs. PAMP, but not AM, bound to tubulin in vitro and destabilized tubulin polymerization. Down-regulation of the gene coding for both AM and PAMP through small interfering RNA technology resulted in morphological changes, microtubule stabilization, increase in posttranslational modifications of tubulin such as acetylation and detyrosination, reduction in cell motility, and partial arrest at the G2 phase of the cell cycle, when compared with cells transfected with the same vector carrying a scrambled sequence. These results show that PAMP is a novel MAP, whereas AM may be exerting more subtle effects in regulating cytoskeleton function.

scholarly journals Identification and characterization of microtubule proteins from myxamoebae of Physarum polycephalum

1980 ◽  
Vol 189 (2) ◽  
pp. 305-312 ◽  
Author(s):  
A Roobol ◽  
C I Pogson ◽  
K Gull
Keyword(s):  
Physarum Polycephalum ◽  
Microtubule Assembly ◽  
Alpha Tubulin ◽  
Microtubule Associated Proteins ◽  
Cell Extracts ◽  
Microtubule Protein ◽  
Associated Proteins ◽  
Microtubule Formation

Cell extracts of myxamoebae of Physarum polycephalum have been prepared in such a way that they do not inhibit assembly of brain microtubule protein in vitro even at high extract-protein concentration. Co-polymers of these extracts and brain tubulin have been purified to constant stoichiometry and amoebal components identified by radiolabelling. Amoebal tubulin has been identified as having an alpha-subunit, mol.wt. 54 000, which co-migrates with brain alpha-tubulin and a beta-subunit, mol.wt. 50 000, which co-migrates with Tetrahymena ciliary beta-tubulin. Non-tubulin amoebal proteins that co-purify with tubulin during co-polymer formation have been shown to be essential for microtubule formation in the absence of glycerol and appear to be rather more effective than brain microtubule-associated proteins in stimulating assembly. The mitotic inhibitor griseofulvin (7-chloro-2′,4,6-trimethoxy-6′-methylspiro[benzofuran-2(3H),1′-cyclohex-2′-ene] −3,4′-dione), which binds to brain microtubule-associated proteins and inhibits brain microtubule assembly in vitro, affected co-polymer microtubule protein in a similar way, but to a slightly greater extent.

Tubulin folding is altered by mutations in a putative GTP binding motif

1996 ◽  
Vol 109 (6) ◽  
pp. 1471-1478 ◽  
Author(s):  
J.C. Zabala ◽  
A. Fontalba ◽  
J. Avila
Keyword(s):  
Intramolecular Interaction ◽  
Terminal Region ◽  
Binding Motif ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Proposed Model ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Hydrolysis Of

Tubulins contain a glycine-rich loop, that has been implicated in microtubule dynamics by means of an intramolecular interaction with the carboxy-terminal region. As a further extension of the analysis of the role of the carboxy-terminal region in tubulin folding we have mutated the glycine-rich loop of tubulin subunits. An alpha-tubulin point mutant with a T150-->G substitution (the corresponding residue present in beta-tubulin) was able to incorporate into dimers and microtubules. On the other hand, four beta-tubulin point mutants, including the G148-->T substitution, did not incorporate into dimers, did not release monomers, but were able to form C900 and C300 complexes (intermediates in the process of tubulin folding). Three other mutants within this region (which approximately encompasses residues 137–152) were incapable of forming dimers and C300 complexes but gave rise to the formation of C900 complexes. These results suggest that tubulin goes through two sequential folding states during the folding process, first in association with TCP1-complexes (C900) prior to the transfer to C300 complexes. It is this second step that implies binding/hydrolysis of GTP, reinforcing our previous proposed model for tubulin folding and assembly.

scholarly journals Tubulin-tyrosine ligase has a binding site on beta-tubulin: a two-domain structure of the enzyme

1987 ◽  
Vol 104 (4) ◽  
pp. 1059-1067 ◽  
Author(s):  
J Wehland ◽  
K Weber
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Enzymatic Activity ◽  
Binding Site ◽  
Gradient Centrifugation ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Alpha Beta ◽  
Tubulin Tyrosine Ligase ◽  
Carboxy Terminal

Tubulin-tyrosine ligase and alpha beta-tubulin form a tight complex which is conveniently monitored by glycerol gradient centrifugation. Using two distinct ligase monoclonal antibodies, several subunit-specific tubulin monoclonal antibodies, and chemical cross-linking, a ligase-binding site was identified on beta-tubulin. This site is retained when the carboxy-terminal domains of both tubulin subunits are removed by subtilisin treatment. The ligase-tubulin complex is also formed when ligase is added to alpha beta-tubulin carrying the monoclonal antibody YL 1/2 which binds only to the carboxyl end of tyrosinated alpha-tubulin. The beta-tubulin-binding site described here explains the extreme substrate specificity of ligase, which does not act on other cellular proteins or carboxy-terminal peptides derived from detyrosinated alpha-tubulin. Differential accessibility of this site in tubulin and in microtubules seems to explain why ligase acts preferentially on unpolymerized tubulin. Ligase exposed to V8-protease is converted to a nicked derivative. This is devoid of enzymatic activity but still forms the complex with tubulin. Gel electrophoresis documents both 30- and a 14-kD domains, each which is immunologically and biochemically distinct and seems to cover the entire molecule. The two domains interact tightly under physiological conditions. The 30-kD domain carries the binding sites for beta-tubulin and ATP. The 14-kD domain can possibly form an additional part of the catalytic site as it harbors the epitope for the monoclonal antibody ID3 which inhibits enzymatic activity but not the formation of the ligase-tubulin complex.

scholarly journals Complex regulation and functional versatility of mammalian alpha- and beta-tubulin isotypes during the differentiation of testis and muscle cells.

1988 ◽  
Vol 106 (6) ◽  
pp. 2023-2033 ◽  
Author(s):  
S A Lewis ◽  
N J Cowan
Keyword(s):  
Cell Types ◽  
Beta Tubulin ◽  
Specific Expression ◽  
Alpha Tubulin ◽  
Tubulin Isotypes ◽  
Tubulin Isotype ◽  
A Cell ◽  
Complex Regulation ◽  
Carboxy Terminal ◽  
Different Cell Types

In the accompanying paper (Gu, W., S. A. Lewis, and N. J. Cowan. 1988. J. Cell Biol. 106: 2011-2022), we report the generation of three antisera, each of which uniquely recognizes a different mammalian alpha-tubulin isotype, plus a fourth antibody that distinguishes between microtubules containing the tyrosinated and nontyrosinated form of the only known mammalian alpha-tubulin gene product that lacks an encoded carboxy-terminal tyrosine residue. These sera, together with five sera we raised that distinguish among the known mammalian beta-tubulin isotypes, have been used to study patterns of tubulin isotype-specific expression in muscle and testis, two tissues in which characteristic developmental changes are accompanied by dramatic rearrangements in microtubule structures. As in the case of cells in culture, there is no evidence to suggest that there is subcellular sorting of different tubulin isotypes among different kinds of microtubule, even in a cell type (the developing spermatid) that simultaneously contains such functionally distinct structures as the manchette and the flagellum. On the other hand, the patterns of expression of the various tubulin isotypes show marked and distinctive differences in different cell types and, in at least one case, evidence is presented for regulation at the translational or posttranslational level. The significance of these observations is discussed in terms of the existence of the mammalian alpha- and beta-tubulin multigene families.

scholarly journals Exploring neurodegeneration at the atomic level

2018 ◽  
Vol 40 (5) ◽  
pp. 9-11
Author(s):  
Adam Tozer
Keyword(s):  
Amino Acids ◽  
Central Nervous System ◽  
Nervous System ◽  
Atomic Level ◽  
Tau Proteins ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Central Nervous System ◽  
The Brain ◽  
Cell Maintenance

Tau proteins are microtubule-associated proteins essential for the correct functioning of neurons. This small family of proteins, 352–441 amino acids in length, are abundant in the brain and exist to stabilize microtubules in neurons and glia (non-neuronal cells of the central nervous system) to ensure correct trafficking of cellular cargo and cell maintenance.

TAU PROTEINS AND OTHER MICROTUBULE-ASSOCIATED PROTEINS (MAP) ARE POTENTIAL INTRACELLULAR TARGETS FOR LIPOPOLYSACCHARIDES (LPS)

1998 ◽  
Vol 26 (Supplement) ◽  
pp. 77A
Author(s):  
Stefan RuBwurm ◽  
Konrad J. Bohm ◽  
Nagib Ghaleb ◽  
Eberhard Unger ◽  
Konrad Reinhart
Keyword(s):  
Tau Proteins ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Intracellular Targets

scholarly journals Generation of microtubule stability subclasses by microtubule-associated proteins: implications for the microtubule "dynamic instability" model.

1985 ◽  
Vol 101 (5) ◽  
pp. 1680-1689 ◽  
Author(s):  
D Job ◽  
M Pabion ◽  
R L Margolis
Keyword(s):  
Dynamic Instability ◽  
Protein Activity ◽  
Microtubule Dynamic ◽  
Microtubule Associated Proteins ◽  
Microtubule Stability ◽  
Low Concentrations ◽  
Protein Map ◽  
Associated Proteins ◽  
Specific Influence ◽  
Assay Conditions

We have developed a method to distinguish microtubule associated protein (MAP)-containing regions from MAP-free regions within a microtubule, or within microtubule sub-populations. In this method, we measure the MAP-dependent stabilization of microtubule regions to dilution-induced disassembly of the polymer. The appropriate microtubule regions are identified by assembly in the presence of [3H]GTP, and assayed by filter trapping and quantitation of microtubule regions that contain label. We find that MAPs bind very rapidly to polymer binding sites and that they do not exchange from these sites measurably once bound. Also, very low concentrations of MAPs yield measurable stabilization of local microtubule regions. Unlike the stable tubule only polypeptide (STOP) proteins, MAPs do not exhibit any sliding behavior under our assay conditions. These results predict the presence of different stability subclasses of microtubules when MAPs are present in less than saturating amounts. The data can readily account for the observed "dynamic instability" of microtubules through unequal MAP distributions. Further, we report that MAP dependent stabilization is quantitatively reversed by MAP phosphorylation, but that calmodulin, in large excess, has no specific influence on MAP protein activity when MAPs are on microtubules.

Assembly properties of altered beta-tubulin polypeptides containing disrupted autoregulatory domains

1989 ◽  
Vol 9 (8) ◽  
pp. 3418-3428
Author(s):  
W Gu ◽  
N J Cowan
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Tubulin Isotype ◽  
The Stability ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

beta-Tubulin synthesis in eucaryotic cells is subject to control by an autoregulatory posttranscriptional mechanism in which the first four amino acids of the beta-tubulin polypeptide act either directly or indirectly to control the stability of beta-tubulin mRNA. To investigate the contribution of this amino-terminal domain to microtubule assembly and dynamics, we introduced a series of deletions encompassing amino acids 2 to 5 of a single mammalian beta-tubulin isotype, M beta 1. Constructs carrying such deletions were inserted into an expression vector, and the ability of the altered polypeptide to coassemble into microtubules was tested by using an anti-M beta 1-specific antibody. We show that the M beta 1 beta-tubulin polypeptide was competent for coassembly into microtubules in transient transfection experiments and in stably transfected cell lines when it lacked either amino acid 2 or amino acids 2 and 3. The capacity of these mutant beta-tubulins to coassemble into polymerized microtubules was only slightly diminished relative to that of unaltered beta-tubulin, and their expression did not influence the viability or growth properties of cell lines carrying these deletions. However, more extensive amino-terminal deletions either severely compromised or abolished the capacity for coassembly. In analogous experiments in which alterations were introduced into the amino-terminal domain of a mammalian alpha-tubulin isotype, M alpha 4, deletion of amino acid 2 did not affect the ability of the altered polypeptide to coassemble, although removal of additional amino-terminal residues essentially abolished the capacity for competent coassembly. The stability of the altered assembly-competent alpha- and beta-tubulin polypeptides was measured in pulse-chase experiments and found to be indistinguishable from the stability of the corresponding unaltered polypeptides. An assembly-competent M alpha 4 polypeptide carrying a deletion encompassing the 12 carboxy-terminal amino acids also had a half-life indistinguishable from that of the wild-type alpha-tubulin molecule. These data suggest that the universally conserved amino terminus of beta-tubulin acts largely in a regulatory role and that the carboxy-terminal domain of alpha-tubulin is not essential for coassembly in mammalian cells in vivo.

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