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.

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.

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 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 Analysis of the spatial organization of microtubule-associated proteins.

1986 ◽  
Vol 103 (2) ◽  
pp. 559-569 ◽  
Author(s):  
C G Jensen ◽  
B H Smaill
Keyword(s):  
Spatial Organization ◽  
Binary Sequence ◽  
Peak Frequency ◽  
Map Projections ◽  
Microtubule Associated Proteins ◽  
Confidence Levels ◽  
Associated Proteins ◽  
Brain Microtubules ◽  
Significant Match

We have developed microdensitometer-computer correlation techniques to analyze the arrangement of microtubule arms and bridges (i.e., microtubule-associated proteins [MAPs]). A microdensitometer was used to scan immediately adjacent to the wall of longitudinally sectioned microtubules in positive transparency electron micrographs. Signal enhancement procedures were applied to the digitized densitometer output to produce a binary sequence representing the apparent axial spacing of MAP projections. These enhanced records were analyzed in two ways. (a) Autocorrelograms were formed for each record and correlogram peaks from a group of scans were pooled to construct a peak frequency histogram. (b) Cross-correlation was used to optimize the match between each enhanced record and templates predicted by different models of MAP organization. Seven symmetrical superlattices were considered as well as single axial repeats. The analyses were repeated with randomly generated records to establish confidence levels. Using the above methods, we analyzed the intrarow bridges of the Saccinobaculus axostyle and the MAP2 projections associated with brain microtubules synthesized in vitro. We confirmed a strict 16-nm axial repeat for axostyle bridges. For 26 MAP2 records, the only significant match was to a 12-dimer superlattice model (P less than 0.002). However, we also found some axial distances between MAP2 projections which were compatible with the additional spacings predicted by a 6-dimer superlattice. Therefore, we propose that MAP2 projections are arranged in a "saturated 12-dimer, unsaturated 6-dimer" superlattice, which may be characteristic of a wide variety of MAPs.

Association of p34cdc2/cyclin B complex with microtubules in starfish oocytes

1993 ◽  
Vol 105 (4) ◽  
pp. 873-881 ◽  
Author(s):  
K. Ookata ◽  
S. Hisanaga ◽  
E. Okumura ◽  
T. Kishimoto
Keyword(s):  
Complex Form ◽  
Cyclin B ◽  
Microtubule Associated Proteins ◽  
Inactive Form ◽  
Microtubular Cytoskeleton ◽  
Associated Proteins ◽  
Meiotic Spindles ◽  
Microtubule Networks ◽  
Brain Microtubules

The microtubular cytoskeleton exhibits a dramatic reorganization, progressing from interphase radial arrays to a mitotic spindle at the G2/M transition. Although this reorganization has been suspected to be caused by maturation promoting factor (MPF: p34cdc2/cyclin B complex), little is known about how p34cdc2 kinase controls microtubule networks. We provide evidence of the direct association of the p34cdc2/cyclin B complex with microtubules in starfish oocytes. Anti-cyclin B staining of detergent-treated oocytes, isolated asters and meiotic spindles revealed fluorescence associated with microtubule fibers, chromosomes and centrosomes. Microtubules prepared from starfish oocytes were associated with cyclin B and p34cdc2 proteins. Microtubule-bound p34cdc2 and cyclin B were released from microtubules by a high-salt solution and possessed a complex form as shown by the adsorption to suc1-beads and by immunoprecipitation with the anti-cyclin B antibody. The p34cdc2/cyclin B complex associated to microtubules had high histone H1 kinase activity at meiotic metaphase. However, it was not necessary for the p34cdc2/cyclin B complex to be active for microtubule binding, as an inactive form in immature oocytes was also observed to bind to microtubules. The coprecipitation of suc1-column purified p34cdc2/cyclin B with purified porcine brain microtubules in the presence of starfish oocyte microtubule-associated proteins (MAPs) indicates that the association of p34cdc2/cyclin B with microtubules in vitro is mediated by MAPs.

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 Tubulin isoform composition tunes microtubule dynamics

2017 ◽  
Vol 28 (25) ◽  
pp. 3564-3572 ◽  
Author(s):  
Annapurna Vemu ◽  
Joseph Atherton ◽  
Jeffrey O. Spector ◽  
Carolyn A. Moores ◽  
Antonina Roll-Mecak
Keyword(s):  
Cell Types ◽  
Microtubule Dynamics ◽  
Microtubule Associated Proteins ◽  
Cryo Electron Microscopy ◽  
Associated Proteins ◽  
Composition Characteristic ◽  
In Trans ◽  
Immortalized Cell ◽  
Brain Microtubules

Microtubules polymerize and depolymerize stochastically, a behavior essential for cell division, motility, and differentiation. While many studies advanced our understanding of how microtubule-associated proteins tune microtubule dynamics in trans, we have yet to understand how tubulin genetic diversity regulates microtubule functions. The majority of in vitro dynamics studies are performed with tubulin purified from brain tissue. This preparation is not representative of tubulin found in many cell types. Here we report the 4.2-Å cryo-electron microscopy (cryo-EM) structure and in vitro dynamics parameters of α1B/βI+βIVb microtubules assembled from tubulin purified from a human embryonic kidney cell line with isoform composition characteristic of fibroblasts and many immortalized cell lines. We find that these microtubules grow faster and transition to depolymerization less frequently compared with brain microtubules. Cryo-EM reveals that the dynamic ends of α1B/βI+βIVb microtubules are less tapered and that these tubulin heterodimers display lower curvatures. Interestingly, analysis of EB1 distributions at dynamic ends suggests no differences in GTP cap sizes. Last, we show that the addition of recombinant α1A/βIII tubulin, a neuronal isotype overexpressed in many tumors, proportionally tunes the dynamics of α1B/βI+βIVb microtubules. Our study is an important step toward understanding how tubulin isoform composition tunes microtubule dynamics.

Analysis of the Mechanism of Microtubule Dynamic Instability Using a UV Microbeam to Sever Elongating Microtubules

1988 ◽  
Vol 46 ◽  
pp. 122-123
Author(s):  
R.A Walker ◽  
S. Inoue ◽  
E.D. Salmon
Keyword(s):  
Dynamic Instability ◽  
Microtubule Dynamic ◽  
Gtp Hydrolysis ◽  
Abrupt Transition ◽  
Microtubule Associated Proteins ◽  
Asymmetrical Structure ◽  
Associated Proteins

Microtubules polymerized in vitro from tubulin purified free of microtubule-associated proteins exhibit dynamic instability (1,2,3). Free microtubule ends exist in persistent phases of elongation or rapid shortening with infrequent, but, abrupt transitions between these phases. The abrupt transition from elongation to rapid shortening is termed catastrophe and the abrupt transition from rapid shortening to elongation is termed rescue. A microtubule is an asymmetrical structure. The plus end grows faster than the minus end. The frequency of catastrophe of the plus end is somewhat greater than the minus end, while the frequency of rescue of the plus end in much lower than for the minus end (4).The mechanism of catastrophe is controversial, but for both the plus and minus microtubule ends, catastrophe is thought to be dependent on GTP hydrolysis. Microtubule elongation occurs by the association of tubulin-GTP subunits to the growing end. Sometime after incorporation into an elongating microtubule end, the GTP is hydrolyzed to GDP, yielding a core of tubulin-GDP capped by tubulin-GTP (“GTP-cap”).

Molecular architecture and functions of the neuronal cytoskeleton

1990 ◽  
Vol 48 (3) ◽  
pp. 2-3
Author(s):  
Nobutaka Hirokawa
Keyword(s):  
Major Elements ◽  
Molecular Structures ◽  
Organelle Transport ◽  
Molecular Architecture ◽  
Neuronal Morphogenesis ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
And Function ◽  
Small Clusters

In this symposium I will present our studies about the molecular architecture and function of the cytomatrix of the nerve cells. The nerve cell is a highly polarized cell composed of highly branched dendrites, cell body, and a single long axon along the direction of the impulse propagation. Each part of the neuron takes characteristic shapes for which the cytoskeleton provides the framework. The neuronal cytoskeletons play important roles on neuronal morphogenesis, organelle transport and the synaptic transmission. In the axon neurofilaments (NF) form dense arrays, while microtubules (MT) are arranged as small clusters among the NFs. On the other hand, MTs are distributed uniformly, whereas NFs tend to run solitarily or form small fascicles in the dendrites Quick freeze deep etch electron microscopy revealed various kinds of strands among MTs, NFs and membranous organelles (MO). These structures form major elements of the cytomatrix in the neuron. To investigate molecular nature and function of these filaments first we studied molecular structures of microtubule associated proteins (MAP1A, MAP1B, MAP2, MAP2C and tau), and microtubules reconstituted from MAPs and tubulin in vitro. These MAPs were all fibrous molecules with different length and formed arm like projections from the microtubule surface.

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