Association of fodrin with brain microtubules

1985 ◽  
Vol 63 (5) ◽  
pp. 372-381 ◽  
Author(s):  
Barbara L. Fach ◽  
Susan F. Graham ◽  
Robert A. B. Keates
Keyword(s):  
Bovine Brain ◽  
Polypeptide Composition ◽  
Brain Membrane ◽  
Subunit Exchange ◽  
Microtubule Associated Proteins ◽  
In Vitro Assembly ◽  
Microtubule Protein ◽  
Associated Proteins ◽  
Brain Microtubules

We have compared the polypeptide composition of microtubules isolated from bovine brain by the conventional in vitro reassembly method with those obtained by direct isolation of brain microtubules into a stabilizing buffer. The stabilizing buffer included 6.7 M glycerol to limit the rate of subunit exchange between assembled and unassembled states. The microtubule-associated proteins normally found by in vitro reassembly are also found in the stabilized preparation, but in smaller proportions. Fodrin, a brain membrane-associated protein believed to be homologous to spectrin, was found to be the most abundant component after tubulin in the stabilized microtubules. The ratio of tubulin to fodrin, 16:1 by mass, was almost constant at each stage of the preparation. Some actin was initially present in the stabilized microtubules, but was gradually lost during purification. When stabilized microtubules were diluted into cold aqueous buffer, they depolymerized and the recovered microtubule protein could then be purified by in vitro reassembly. The composition after this treatment resembled that of microtubules prepared initially by reassembly in vitro. The missing fodrin was found to be removed in the preliminary centrifugation and was unavailable for incorporation into growing microtubules during the in vitro assembly step. This suggests that the standard in vitro reassembly procedure for purification of microtubules may distort the composition of microtubule-associated proteins.

scholarly journals Identity and Origin of the ATPase activity associated with neuronal microtubules. I. The ATPase activity is associated with membrane vesicles.

1983 ◽  
Vol 96 (5) ◽  
pp. 1298-1305 ◽  
Author(s):  
D B Murphy ◽  
R R Hiebsch ◽  
K T Wallis
Keyword(s):  
Molecular Weight ◽  
Atpase Activity ◽  
High Molecular Weight ◽  
Brain Tissue ◽  
Membrane Vesicles ◽  
Microtubule Associated Proteins ◽  
In Vitro Assembly ◽  
Microtubule Protein ◽  
Associated Proteins

Microtubule protein purified from brain tissue by cycles of in vitro assembly-disassembly contains ATPase activity that has been postulated to be associated with microtubule-associated proteins (MAPs) and therefore significant for studies of microtubule-dependent motility. In this paper we demonstrate that greater than 90% of the ATPase activity is particulate in nature and may be derived from contaminating membrane vesicles. We also show that the MAPs (MAP-1, MAP-2, and tau factors) and other high molecular weight polypeptides do not contain significant amounts of ATPase activity. These findings do not support the concept of "brain dynein" or of MAPs with ATPase activity.

Microtubule associated proteins in microtubule preparations made with and without glycerol

1984 ◽  
Vol 62 (9) ◽  
pp. 803-813 ◽  
Author(s):  
Robert A. B. Keates
Keyword(s):  
Binding Assay ◽  
Critical Concentration ◽  
Brain Homogenate ◽  
Microtubule Assembly ◽  
Microtubule Associated Proteins ◽  
In Vitro Assembly ◽  
Microtubule Protein ◽  
Associated Proteins ◽  
The Difference

Preparation of microtubule protein in the presence or absence of glycerol results in differences in polymerization properties and content of microtubule associated proteins. The variation in properties appears to result from the reduced proportion of microtubule associated proteins in preparations made with glycerol. I have used the colchicine binding assay to monitor recovery of active tubulin and have found that a single factor can account for the difference. During the in vitro assembly of microtubules from the crude brain homogenate, glycerol promotes polymerization of the bulk of the tubulin, while less than half is incorporated into microtubules in the absence of glycerol. Assembly of partly purified microtubule protein is not enhanced by glycerol however. Microtubule associated proteins present in the crude homogenate are almost completely incorporated into the microtubules regardless of the presence of glycerol, and their high content in glycerol-free preparations appears to be the trivial result of low tubulin recovery. The high affinity of microtubule associated proteins for the assembled microtubules has other consequences for in vitro studies of microtubule assembly, and critical concentration plots to determine the polymerization equilibrium constant can be distorted unless the preparation used has a high content of microtubule associated proteins.

scholarly journals Interactions of tubulin and microtubule-associated proteins. Conformation and stability of the oligomeric species from glycerol-cycled microtubule protein of bovine brain

1982 ◽  
Vol 203 (3) ◽  
pp. 643-652 ◽  
Author(s):  
Stephen R. Martin ◽  
David C. Clark ◽  
Peter M. Bayley
Keyword(s):  
Ionic Strength ◽  
Bovine Brain ◽  
Microtubule Assembly ◽  
Microtubule Associated Proteins ◽  
Comparable Effect ◽  
Microtubule Protein ◽  
Associated Proteins ◽  
Ph Range ◽  
Ring Species

1. The conformation of bovine microtubule protein prepared by cycles of assembly and disassembly in the presence of glycerol has been studied by near-u.v. circular dichroism (c.d.) over a range of protein concentrations. The effects on the conformational properties of ionic strength and of a pH range from 6 to 7.5 have been correlated with the known oligomeric composition of microtubule protein preparations, as determined by the sedimentation behaviour of this preparation [Bayley, Charlwood, Clark & Martin (1982) Eur. J. Biochem.121, 579–585]. 2. The formation of 30S oligomeric ring species, either by decreasing ionic strength at pH6.5 or by changing pH in the presence of 0.1m-NaCl, correlates with a significant change in tubulin c.d. Formation of 18S oligomer by changing pH at ionic strength 0.2 produced no comparable effect. The c.d. of tubulin dimer itself is not affected by ionic strength and pH over the same range. 3. The results are interpreted as a small conformational adjustment between tubulin and specific microtubule-associated proteins on forming 30S oligomeric species, due to interaction with the high-molecular-weight-group proteins. The possible significance of this is discussed with respect to microtubule assembly in vitro. 4. By using this conformational parameter, together with equilibrium and kinetic light-scattering studies, the sensitivity of glycerol-cycled microtubule protein to dilution is shown to be strongly pH-dependent, the oligomers being much more stable at pH6.4 than at pH6.9. 5. Oligomeric complexes of tubulin with microtubule-associated proteins show marked stability under conditions similar to those for efficient microtubule assembly in vitro. Oligomeric material therefore must be incorporated directly during assembly in vitro from microtubule protein.

Tetrins: polypeptides that form bundled filaments in Tetrahymena

1990 ◽  
Vol 96 (2) ◽  
pp. 293-302
Author(s):  
J.E. Honts ◽  
N.E. Williams
Keyword(s):  
Intermediate Filaments ◽  
Tetrahymena Pyriformis ◽  
Ciliated Protozoan ◽  
Microtubule Associated Proteins ◽  
Fine Filament ◽  
Helical Content ◽  
In Vitro Assembly ◽  
Associated Proteins ◽  
Filament Bundles

The cortex of the ciliated protozoan Tetrahymena contains a number of fibrous elements, including a network of filaments that pervades the feeding organelle of this organism. The cluster of polypeptides (79–89K; K = 10(3) Mr) in Tetrahymena pyriformis GL-C that constitute these filaments has been purified by in vitro assembly after solubilization in 1.0 M KI. Four distinct sets of these polypeptides, designated ‘tetrins’, have been shown to be distinguishable from each other by immunochemical and biochemical criteria. The smallest filaments reassembled in vitro were 3–4 nm in diameter and these fine filaments were seen to be bundled together into thicker strands of varying diameters, similar to those within the cell. The thicker filament bundles were clearly distinguishable from intermediate filaments, but fine filaments in these bundles were superficially similar to the 2–5 nm filaments described as microtubule-associated proteins in other organisms. The ultrastructure of the tetrin filaments localized within the feeding organelle reveals a substantial presence of these filaments apart from microtubules. In addition, circular dichroism measurements indicate a relatively low alpha-helical content for these filaments and suggest that the tetrins may be substantially different from other fine filament proteins such as the tektins and giardins.

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.

scholarly journals Interactions between neurofilaments and microtubule-associated proteins: a possible mechanism for intraorganellar bridging.

1982 ◽  
Vol 95 (3) ◽  
pp. 982-986 ◽  
Author(s):  
J F Leterrier ◽  
R K Liem ◽  
M L Shelanski
Keyword(s):  
Spinal Cord ◽  
Molecular Weight ◽  
High Molecular Weight ◽  
Microtubule Assembly ◽  
Microtubule Associated Proteins ◽  
Saturable Binding ◽  
In Vitro Assembly ◽  
Associated Proteins ◽  
Brain And Spinal Cord

Mammalian neurofilaments prepared from brain and spinal cord by either of two methods partially inhibit the in vitro assembly of microtubules. This inhibition is shown to be due to the association of a complex of high molecular weight microtubule-associated proteins (MAP1 and MAP2) and tubulin with the neurofilament. Further analysis of the association reveals a saturable binding of purified brain MAPs to purified neurofilaments with a Kd of 10(-7) M. Purified astroglial filaments neither inhibit microtubule assembly nor show significant binding of MAPs. It is proposed that the MAPs might function as one element in a network of intraorganellar links in the cytoplasm.

scholarly journals Microtubule-associated proteins (MAPs) and the organization of actin filaments in vitro.

1981 ◽  
Vol 90 (2) ◽  
pp. 467-473 ◽  
Author(s):  
R F Sattilaro ◽  
W L Dentler ◽  
E L LeCluyse
Keyword(s):  
Actin Filaments ◽  
Muscle Actin ◽  
Actin Bundles ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Filament Bundles ◽  
Reversible Formation ◽  
Brain Microtubules ◽  
28 Nm

When purified muscle actin was mixed with microtubule-associated proteins (MAPs) prepared from brain microtubules assembled in vitro, actin filaments were organized into discrete bundles, 26 nm in diameter. MAP-2 was the principal protein necessary for the formation of the bundles. Analysis of MAP-actin bundle formation by sedimentation and electrophoresis revealed the bundles to be composed of approximately 20% MAP-2 and 80% actin by weight. Transverse striations were observed to occur at 28-nm intervals along negatively stained MAP-actin bundles, and short projections, approximately 12 nm long and spaced at 28-nm intervals, were resolved by high-resolution metal shadowing. The formation of MAP-actin bundles was inhibited by millimolar concentrations of ATP, AMP-PCP (beta, gamma-methylene-adenosine triphosphate), and pyrophosphate but not by AMP, ADP, or GTP. The addition of ATP to a solution containing MAP-actin bundles resulted in the dissociation of the bundles into individual actin filaments; discrete particles, presumably MAP-2, were periodically attached along the splayed filaments. These results demonstrate that MAPs can bind to actin filaments and can induce the reversible formation of actin filament bundles in vitro.

scholarly journals The periodic association of MAP2 with brain microtubules in vitro.

1979 ◽  
Vol 80 (2) ◽  
pp. 266-276 ◽  
Author(s):  
H Kim ◽  
L I Binder ◽  
J L Rosenbaum
Keyword(s):  
Critical Concentration ◽  
Microtubule Assembly ◽  
Molar Ratio ◽  
Thin Sections ◽  
Microtubule Associated Proteins ◽  
Heat Stable ◽  
Associated Proteins ◽  
Brain Microtubules ◽  
Brain Tubulin

Several high molecular weight polypeptides have been shown to quantitatively copurify with brain tubulin during cycles of in vitro assembly-disassembly. These microtubule-associated proteins (MAPs) have been shown to influence the rate and extent of microtubule assembly in vitro. We report here that a heat-stable fraction highly enriched for one of the MAPs, MAP2 (mol wt approximately 300,000 daltons), devoid of MAP1 (mol wt approximately 350,000 daltons), has been purified from calf neurotubules. This MAP2 fraction stoichiometrically promotes microtubule assembly, lowering the critical concentration for tubulin assembly to 0.05 mg/ml. Microtubules saturated with MAP2 contain MAP2 and tubulin in a molar ratio of approximately 1 mole of MAP2 to 9 moles of tubulin dimer. Electron microscopy of thin sections of the MAP2-saturated microtubules fixed in the presence of tannic acid demonstrates a striking axial periodicity of 32 +/- 8 nm.

scholarly journals Microtubule-associated proteins-dependent colchicine stability of acetylated cold-labile brain microtubules from the Atlantic cod, Gadus morhua.

1991 ◽  
Vol 113 (2) ◽  
pp. 331-338 ◽  
Author(s):  
M Billger ◽  
E Strömberg ◽  
M Wallin
Keyword(s):  
Posttranslational Modifications ◽  
Gadus Morhua ◽  
Atlantic Cod ◽  
Bovine Brain ◽  
General Effect ◽  
Acetylated Tubulin ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Stability ◽  
Brain Microtubules

Assembly of brain microtubule proteins isolated from the Atlantic cod, Gadus morhua, was found to be much less sensitive to colchicine than assembly of bovine brain microtubules, which was completely inhibited by low colchicine concentrations (10 microM). The degree of disassembly by colchicine was also less for cod microtubules. The lack of colchicine effect was not caused by a lower affinity of colchicine to cod tubulin, as colchicine bound to cod tubulin with a dissociation constant, Kd, and a binding ratio close to that of bovine tubulin. Cod brain tubulin was highly acetylated and mainly detyrosinated, as opposed to bovine tubulin. When cod tubulin, purified by means of phosphocellulose chromatography, was assembled by addition of DMSO in the absence of microtubule-associated proteins (MAPs), the microtubules became sensitive to low concentrations of colchicine. They were, however, slightly more stable to disassembly, indicating that posttranslational modifications induce a somewhat increased stability to colchicine. The stability was mainly MAPs dependent, as it increased markedly in the presence of MAPs. The stability was not caused by an extremely large amount of cod MAPs, since there were slightly less MAPs in cod than in bovine microtubules. When "hybrid" microtubules were assembled from cod tubulin and bovine MAPs, these microtubules became less sensitive to colchicine. This was not a general effect of MAPs, since bovine MAPs did not induce a colchicine stability of microtubules assembled from bovine tubulin. We can therefore conclude that MAPs can induce colchicine stability of colchicine labile acetylated tubulin.

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