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 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.

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.

Unusual properties of a cold-labile fraction of Atlantic cod (Gadus morhua) brain microtubules

1989 ◽  
Vol 67 (11-12) ◽  
pp. 791-800 ◽  
Author(s):  
E. Strömberg ◽  
L. Serrano ◽  
J. Avila ◽  
M. Wallin
Keyword(s):  
Molecular Mass ◽  
Gadus Morhua ◽  
Atlantic Cod ◽  
Bovine Brain ◽  
Microtubule Assembly ◽  
Microtubule Associated Proteins ◽  
Heat Stable ◽  
Labile Fraction ◽  
Associated Proteins ◽  
Brain Microtubules

A cold-labile fraction of microtubules with unusual properties was isolated from the brain of the Atlantic cod (Gadus morhua). The yield was low, approximately six times lower than that for bovine brain microtubules. This was mainly caused by the presence of a large amount of cold-stable microtubules, which were not broken down during the disassembly step in the temperature-dependent assembly–disassembly isolation procedure and were therefore lost. The isolated cold-labile cod microtubules contained usually only a low amount of microtubule-associated proteins (MAPs). Three high molecular mass proteins were found, of which one was recognized as MAP2. Cod MAP2 differed from mammalian brain MAP2; it was not heat stable and had a slightly higher molecular mass. In contrast to mammalian MAPs, MAP1 was not found in the cold-labile fraction of microtubules. A new heat-labile MAP of higher molecular mass (400 kilodaltons) was however present, as well as a heat-stable protein of slightly lower molecular mass than MAP2. These MAPs showed similar tubulin-binding characteristics as bovine brain MAPs, since they coassembled with taxol-assembled bovine brain microtubules consisting of pure bovine tubulin. In spite of the fact that Ca2+ bound equally to cod and porcine tubulins, it did not inhibit cod microtubule assembly even at high concentrations (> 1 mM). In contrast, rings, spirals, and macrotubules were formed. The results show that there are major differences between this fraction of cod microtubules and microtubules from mammalian brain.Key words: microtubules, microtubule-associated proteins, calcium, cod.

scholarly journals Ultrastructural localization of the high molecular weight proteins associated with in vitro-assembled brain microtubules

1975 ◽  
Vol 65 (1) ◽  
pp. 237-241 ◽  
Author(s):  
WL Dentler ◽  
S Granett ◽  
JL Rosenbaum
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Ultrastructural Localization ◽  
Thin Sections ◽  
Microtubule Associated Proteins ◽  
Brain Extracts ◽  
Associated Proteins ◽  
High Molecular Weight Proteins ◽  
Brain Microtubules

Microtubules isolated from brain extracts by in vitro assembly (1, 19, 23) are composed principally of two tubulins and two high molecular weight proteins (microtubule-associated proteins [MAPS] 1 and 2) (2,5,7,20). Recently, it was demonstrated that in vitro-assembled brain microtubules (neurotubules) are coated with filaments (5, 7) which are similar to the filaments attached to neurotubules in situ (4, 15, 21, 24, 25), and it was suggested that the filaments are composed of the higher molecular weight MAPs (5, 7, 12). In this study, microtubules were assembled in the presence and absence of the MAPs, and thin sections of the microtubules were examined by electron microscopy. The results show that the filaments only occur on microtubules assembled in the presence of the MAPs and it is therefore concluded that the filaments are composed of the high molecular weight MAP's.

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 Proteins from morphologically differentiated neuroblastoma cells promote tubulin polymerization.

1978 ◽  
Vol 76 (2) ◽  
pp. 547-555 ◽  
Author(s):  
N W Seeds ◽  
R B Maccioni
Keyword(s):  
High Speed ◽  
Gradient Centrifugation ◽  
Neuroblastoma Cells ◽  
Morphological Differentiation ◽  
Tubulin Polymerization ◽  
Associated Proteins ◽  
Neurite Formation ◽  
Brain Microtubules ◽  
Brain Tubulin

Clonal cells (N18) of the mouse neuroblastoma C-1300 can be induced to undergo a morphological differentiation characterized by the outgrowth of very long neurites (> 150 microns) that contain many microtubules. Because the marked increase in the number and length of microtubules is apparently not due to an increase in the concentration of tubulin subunits, the possible role of additional macromolecules in the regulation of tubulin polymerization during neurite formation by N18 cells was examined. Using an in vitro system where the polymerization of low concentrations (< 4 mg/ml) of purified brain tubulin requires microtubule-associated proteins (MAPs), high-speed supernates (250,000 g) from neuroblastoma and glioma cells were assayed for their ability to replace MAPs in the polymerization of brain tubulin. Only the supernates from "differentiated" N18 cells were polymerization competent. Electron microscope observations of these supernates failed to demonstrate the presence of nucleation structures (rings or disks). The active factor(s) sedimented at approximately 7S on sucrose gradient centrifugation and eluted from 4B Sepharose in the region of 170,000 mol wt proteins. Furthermore, the inactive supernates from other cells did not inhibit polymerization when tested in the presence of limiting MAPs. Thus, microtubule formation accompanying neurite outgrowth in neuroblastoma cells appears to be regulated by the presence of additional macromolecular factor(s) that may be functionally equivalent to the MAPs found with brain 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 Microtubule assembly in cytoplasmic extracts of Xenopus oocytes and eggs.

1987 ◽  
Vol 105 (5) ◽  
pp. 2191-2201 ◽  
Author(s):  
D L Gard ◽  
M W Kirschner
Keyword(s):  
Xenopus Oocytes ◽  
Critical Concentration ◽  
Specific Inhibitor ◽  
Bovine Brain ◽  
High Rate ◽  
Microtubule Assembly ◽  
Microtubule Networks ◽  
Brain Tubulin

We have investigated the differences in microtubule assembly in cytoplasm from Xenopus oocytes and eggs in vitro. Extracts of activated eggs could be prepared that assembled extensive microtubule networks in vitro using Tetrahymena axonemes or mammalian centrosomes as nucleation centers. Assembly occurred predominantly from the plus-end of the microtubule with a rate constant of 2 microns.min-1.microM-1 (57 s-1.microM-1). At the in vivo tubulin concentration, this corresponds to the extraordinarily high rate of 40-50 microns.min-1. Microtubule disassembly rates in these extracts were -4.5 microns.min-1 (128 s-1) at the plus-end and -6.9 microns.min-1 (196 s-1) at the minus-end. The critical concentration for plus-end microtubule assembly was 0.4 microM. These extracts also promoted the plus-end assembly of microtubules from bovine brain tubulin, suggesting the presence of an assembly promoting factor in the egg. In contrast to activated eggs, assembly was never observed in extracts prepared from oocytes, even at tubulin concentrations as high as 20 microM. Addition of oocyte extract to egg extracts or to purified brain tubulin inhibited microtubule assembly. These results suggest that there is a plus-end-specific inhibitor of microtubule assembly in the oocyte and a plus-end-specific promoter of assembly in the eggs. These factors may serve to regulate microtubule assembly during early development in Xenopus.

Microtubules in immature oocytes of Xenopus laevis

1985 ◽  
Vol 77 (1) ◽  
pp. 129-141
Author(s):  
S.R. Heidemann ◽  
M.A. Hamborg ◽  
J.E. Balasz ◽  
S. Lindley
Keyword(s):  
Light Microscopy ◽  
Microscopic Observation ◽  
Electron Microscopic Observation ◽  
Microtubule Assembly ◽  
Electron Microscopic ◽  
Immature Oocytes ◽  
Large Numbers ◽  
Brain Microtubules ◽  
Brain Tubulin

Previous work indicated that immature oocytes of Xenopus were incapable of assembling microtubules but that competence was achieved during maturation. We report here that small numbers of microtubules do exist in immature oocytes. Consistent with this finding, ultrastructural observations indicate that brain microtubules injected into immature oocytes persist in large numbers for at least 30 min. We report that the tubulin dimers of mature and immature oocytes are equally capable of assembling with brain tubulin in vitro. We confirmed previous results that injection of taxol into immature oocytes has no effect when assayed by light microscopy. However, ultrastructural observations suggest that some microtubule assembly is stimulated by taxol. We tested for the ability of immature oocytes to elongate microtubules from ‘seeds’ by injecting deciliated pellicles of Tetrahymena. No elongation was observed either by light or electron microscopic observation. We conclude that the immature oocyte is capable of very limited microtubule assembly and that a marked increase in assembly competence occurs during maturation. Our data suggest that the change in assembly competence during maturation is due to the release, activation or synthesis of a stimulatory co-factor.

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.

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