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.

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.

scholarly journals Griseofulvin-induced aggregation of microtubule protein

1977 ◽  
Vol 167 (1) ◽  
pp. 39-43 ◽  
Author(s):  
A Roobol ◽  
K Gull ◽  
C I Pogson
Keyword(s):  
Microtubule Assembly ◽  
Microtubule Associated Proteins ◽  
Microtubule Protein ◽  
Associated Proteins ◽  
Induced Aggregation

Griseofulvin (7-chloro-2′,4,6-trimethoxy-6′-methylspiro[benzofuran-2(3H),1′-[2]cyclohexene]-3,4′-dione) induces aggregation of microtubule protein at 0 degrees C. This aggregate contains approx. 90% of the microtubule-associated proteins originally present in the microtubule protein. The supernatant obtained after removal of the griseofulvin-induced aggregate does not form microtubules on warming at 37 degrees C. Addition of the griseofulvin-aggregated protein to this supernatant and warming to 37 degrees C gives rise to a limited amount of microtubule assembly. The possible involvement of griseofulvin-induced aggregation of microtubule protein at 0 degrees C in the inhibition by griseofulvin of microtubule assembly in vitro is discussed.

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.

Taxol-induced assembly and characterization of microtubule proteins from developing brine shrimp (Artemia)

1986 ◽  
Vol 64 (3) ◽  
pp. 238-249 ◽  
Author(s):  
Parvaneh Rafiee ◽  
Sara Ann MacKinlay ◽  
Thomas H. MacRae
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Differentiation ◽  
Brine Shrimp ◽  
Electrophoretic Analysis ◽  
Microtubule Associated Proteins ◽  
Observable Change ◽  
Associated Proteins

Incubation of Artemia cell-free extracts with taxol, followed by centrifugation through sucrose cushions, yielded pellets composed of short, morphologically normal microtubules which exhibited a tendency to fray at their ends. Immunological staining of protein blots with polyclonal or monoclonal antibodies revealed that the major pellet protein is tubulin and that bovine neural tubulin and Artemia tubulin are antigenically distinct. By several criteria, but prinicipally by their taxol-induced coassembly with tubulin, many of the nontubulin pellet proteins are microtubule-associated proteins (MAP). In spite of extensive morphogenesis, hatching, and the eventual resumption of mitosis during development, no new MAP appear, with reduction in the number of MAP after hatching the only observable change in these proteins. We have yet to demonstrate a function for Artemia MAP but have shown that the rate and extent of assembly of Artemia tubulin, which polymerizes readily in vitro in the absence of MAP, are stimulated by bovine MAP. Electrophoretic analysis revealed that the taxol-assembled microtubules were composed of several isotubulins, these being identical to the isoforms in biochemically purified Artemia tubulin. In addition, a new Artemia α-tubulin was observed, and it was shown that the isotubulin population does not change during the period of development examined. Maintenance of identical isotubulin populations in developing organisms for extended periods, which suggests that all tubulins are functional, in concert with the lack of change in tubulin during cell differentiation, runs counter to the proposal that chemically distinct isotubulins are required for assembly of functionally specific microtubules.

Traces of brain microtubule-associated proteins affect dynamic properties of microtubules

1990 ◽  
Vol 68 (10) ◽  
pp. 1202-1209 ◽  
Author(s):  
Robert A. B. Keates
Keyword(s):  
Self Assembly ◽  
Dynamic Properties ◽  
Microtubule Assembly ◽  
Microtubule Associated Proteins ◽  
End Point ◽  
Associated Proteins ◽  
Polymerization Mixture ◽  
Low Levels

A method is described for measuring the quantities of stable and dynamic microtubules in a population in vitro. The method exploits the tendency of dynamic microtubules to depolymerize rapidly after being sheared. Stable microtubules, such as those protected by microtubule-associated proteins (MAPs), are broken to a smaller size by shearing, but do not depolymerize into subunits. The usual difficulty with this procedure is that the tubulin released from the dynamic microtubules rapidly repolymerizes before the end point of depolymerization can be measured. This has been overcome by including a small quantity of tubulin–colchicine complex in the mixture to block the repolymerization. For a total of 24 μM tubulin in a polymerization mixture, 10 μM of the sample polymerized originally under the conditions used. When 1.05 μM tubulin–colchicine complex was added at the time of shearing, the dynamic microtubules depolymerized, but the tubulin was released was unable to repolymerize and a small fraction of stable microtubules that resisted shear-induced depolymerization could then be detected. When traces of MAPs (0.23–2.8% by mass) were included in the tubulin mixture, the fraction of stable microtubules increased from 5% in the absence of added MAPs to 41% in the presence of 2.8% MAPs. All the MAPs in the mixture were found in the stable fraction and this stable fraction forms early during microtubule assembly. Calculations on the extent of enrichment of MAPs in the stable fraction indicated that as little as 4% MAPs in a microtubule protected it from shear-induced disassembly. The results suggest that low levels of MAPs may distribute nonrandomly in the microtubule population.Key words: dynamics, microtubules, tubulin, microtubule-associated proteins, self-assembly.

scholarly journals Mechanical properties of brain tubulin and microtubules.

1988 ◽  
Vol 106 (4) ◽  
pp. 1205-1211 ◽  
Author(s):  
M Sato ◽  
W H Schwartz ◽  
S C Selden ◽  
T D Pollard
Keyword(s):  
Mechanical Properties ◽  
Viscoelastic Properties ◽  
Microtubule Assembly ◽  
Cross Link ◽  
Microtubule Associated Proteins ◽  
Heat Stable ◽  
Microtubule Protein ◽  
Associated Proteins ◽  
Steady Shearing ◽  
Brain Tubulin

We measured the elasticity and viscosity of brain tubulin solutions under various conditions with a cone and plate rheometer using both oscillatory and steady shearing modes. Microtubules composed of purified tubulin, purified tubulin with taxol and 3x cycled microtubule protein from pig, cow, and chicken behaved as mechanically indistinguishable viscoelastic materials. Microtubules composed of pure tubulin and heat stable microtubule-associated proteins were also similar but did not recover their mechanical properties after shearing like other samples, even after 60 min. All of the other microtubule samples were more rigid after flow orientation, suggesting that the mechanical properties of anisotropic arrays of microtubules may be substantially greater than those of randomly arranged microtubules. These experiments confirm that MAPs do not cross link microtubules. Surprisingly, under conditions where microtubule assembly is strongly inhibited (either 5 degrees or at 37 degrees C with colchicine or Ca++) tubulin was mechanically indistinguishable from microtubules at 10-20 microM concentration. By electron microscopy and ultracentrifugation these samples were devoid of microtubules or other obvious structures. However, these mechanical data are strong evidence that tubulin will spontaneously assemble into alternate structures (aggregates) in nonpolymerizing conditions. Because unpolymerized tubulin is found in significant quantities in the cytoplasm, it may contribute significantly to the viscoelastic properties of cytoplasm, especially at low deformation rates.

scholarly journals Alf1p, a CLIP-170 Domain-containing Protein, Is Functionally and Physically Associated with α-Tubulin

1999 ◽  
Vol 144 (1) ◽  
pp. 113-124 ◽  
Author(s):  
Becket Feierbach ◽  
Eva Nogales ◽  
Kenneth H. Downing ◽  
Tim Stearns
Keyword(s):  
Fluorescent Protein ◽  
Null Alleles ◽  
Microtubule Associated Proteins ◽  
Cytoplasmic Face ◽  
Associated Proteins ◽  
Green Fluorescent ◽  
Β Tubulin

Tubulin is a heterodimer of α- and β-tubulin polypeptides. Assembly of the tubulin heterodimer in vitro requires the CCT chaperonin complex, and a set of five proteins referred to as the tubulin cofactors (Tian, F., Y. Huang, H. Rommelaere, J. Vandekerckhove, C. Ampe, and N.J. Cowan. 1996. Cell. 86:287–296; Tian, G., S.A. Lewis, B. Feierbach, T. Stearns, H. Rommelaere, C. Ampe, and N.J. Cowan. 1997. J. Cell Biol. 138:821–832). We report the characterization of Alf1p, the yeast ortholog of mammalian cofactor B. Alf1p interacts with α-tubulin in both two-hybrid and immunoprecipitation assays. Alf1p and cofactor B contain a single CLIP-170 domain, which is found in several microtubule-associated proteins. Mutation of the CLIP-170 domain in Alf1p disrupts the interaction with α-tubulin. Mutations in α-tubulin that disrupt the interaction with Alf1p map to a domain on the cytoplasmic face of α-tubulin; this domain is distinct from the region of interaction between α-tubulin and β-tubulin. Alf1p-green fluorescent protein (GFP) is able to associate with microtubules in vivo, and this localization is abolished either by mutation of the CLIP-170 domain in Alf1p, or by mutation of the Alf1p-binding domain in α-tubulin. Analysis of double mutants constructed between null alleles of ALF1 and PAC2, which encodes the other yeast α-tubulin cofactor, suggests that Alf1p and Pac2p act in the same pathway leading to functional α-tubulin. The phenotype of overexpression of ALF1 suggests that Alf1p can act to sequester α-tubulin from interaction with β-tubulin, raising the possibility that it plays a regulatory role in the formation of the tubulin heterodimer.

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.

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.

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