A high molecular mass phosphoprotein defined by a novel monoclonal antibody is closely associated with the intermicrotubule cross bridges in the Trypanosoma brucei cytoskeleton

1992 ◽  
Vol 103 (3) ◽  
pp. 665-675 ◽  
Author(s):  
A. Woods ◽  
A.J. Baines ◽  
K. Gull
Keyword(s):  
Molecular Mass ◽  
Trypanosoma Brucei ◽  
Minor Component ◽  
Relative Molecular Mass ◽  
Microtubule Associated Proteins ◽  
Heat Stable ◽  
Cytoplasmic Face ◽  
Associated Proteins ◽  
Cross Bridges ◽  
Main Component

The main component of the cell body cytoskeleton of Trypanosoma brucei is the highly organised array of stable, subpellicular microtubules on the cytoplasmic face of the plasma membrane. Although several microtubule associated proteins (MAPs) have been shown to be associated with this array, the mechanisms by which individual microtubules interact with one another and with the membrane are still largely undetermined. In this study we have used the T. brucei cytoskeleton as a complex immunogen for the production of monoclonal antibodies to define novel cytoskeletal antigens. Screening by immunofluorescence enabled the selection of an antibody, WCB-1, which detects an antigen associated specifically with the subpellicular microtubules and not with the flagellum microtubules. The antigen (WCB210) was shown to have a relative molecular mass of 210,000 by western blotting. Immunogold studies showed the epitope to be located on the membrane-facing side of the subpellicular cage; it appears to be closely associated with the cross-bridges lying between the microtubules. Unlike many MAPs this protein was shown not to be heat stable and is predicted to be a roughly globular monomer. Even though WCB210 is a very minor component of the cytoskeleton it is heavily phosphorylated. It is possible that this protein is involved in regulation of the subpellicular microtubule crossbridges by interaction with other 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.

Modulation of microtubule dynamic instability in vivo by brain microtubule associated proteins

1995 ◽  
Vol 108 (4) ◽  
pp. 1679-1689 ◽  
Author(s):  
R. Dhamodharan ◽  
P. Wadsworth
Keyword(s):  
Dynamic Behavior ◽  
Dynamic Instability ◽  
Average Rate ◽  
Stable Maps ◽  
Microtubule Dynamic ◽  
Unit Distance ◽  
Microtubule Associated Proteins ◽  
Heat Stable ◽  
Associated Proteins

Heat-stable brain microtubule associated proteins (MAPs) and purified microtubule associated protein 2 (MAP-2) were microinjected into cultured BSC-1 cells which had been previously injected with rhodamine-labeled tubulin. The dynamic instability behavior of individual microtubules was then examined using low-light-level fluorescence microscopy and quantitative microtubule tracking methods. Both MAP preparations suppressed microtubule dynamics in vivo, by reducing the average rate and extent of both growing and shortening events. The average duration of growing events was not affected. When measured as events/unit time, heat-stable MAPs and MAP-2 did not significantly alter the frequency of rescue; the frequency of catastrophe was decreased approximately two-fold by heat-stable MAPs and MAP-2. When transition frequencies were calculated as events/unit distance, both MAP preparations increased the frequency of rescue, without altering the frequency of catastrophe. The percentage of total time spent in the phases of growth, shrink and pause was determined. Both MAP-2 and heat-stable MAPs decreased the percentage of time spent shortening, increased the percentage of time spent paused, and had no effect on percentage of time spent growing. Heat-stable MAPs increased the average pause duration, decreased the average number of events per minute per microtubule and increased the probability that a paused microtubule would switch to growing rather than shortening. The results demonstrate that addition of MAPs to living cells reduces the dynamic behavior of individual microtubules primarily by suppressing the magnitude of dynamic events and increasing the time spent in pause, where no change in the microtubule length can be detected. The results further suggest that the expression of MAPs directly contributes to cell type-specific microtubule dynamic behavior.

scholarly journals Two Related Subpellicular Cytoskeleton-associated Proteins inTrypanosoma brucei Stabilize Microtubules

2002 ◽  
Vol 13 (3) ◽  
pp. 1058-1070 ◽  
Author(s):  
Cécile Vedrenne ◽  
Christiane Giroud ◽  
Derrick R. Robinson ◽  
Sébastien Besteiro ◽  
Christophe Bosc ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Molecular Weight ◽  
Trypanosoma Brucei ◽  
Cold Resistance ◽  
Chinese Hamster ◽  
Low Molecular Weight ◽  
Microtubule Bundle ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Asymmetric Cytokinesis

The subpellicular microtubules of the trypanosome cytoskeleton are cross-linked to each other and the plasma membrane, creating a cage-like structure. We have isolated, from Trypanosoma brucei, two related low-molecular-weight cytoskeleton-associated proteins (15- and 17-kDa), called CAP15 and CAP17, which are differentially expressed during the life cycle. Immunolabeling shows a corset-like colocalization of both CAPs and tubulin. Western blot and electron microscope analyses show CAP15 and CAP17 labeling on detergent-extracted cytoskeletons. However, the localization of both proteins is restricted to the anterior, microtubule minus, and less dynamic half of the corset. CAP15 and CAP17 share properties of microtubule-associated proteins when expressed in heterologous cells (Chinese hamster ovary and HeLa), colocalization with their microtubules, induction of microtubule bundle formation, cold resistance, and insensitivity to nocodazole. When overexpressed inT. brucei, both CAP15 and CAP17 cover the whole subpellicular corset and induce morphological disorders, cell cycle-based abnormalities, and subsequent asymmetric cytokinesis.

Creatine kinase isoenzyme of high relative molecular mass in serum of a cancer patient.

1978 ◽  
Vol 24 (11) ◽  
pp. 2054-2057 ◽  
Author(s):  
H Yuu ◽  
Y Takagi ◽  
O Senju ◽  
J Hosoya ◽  
K Gomi ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Molecular Mass ◽  
Left Breast ◽  
Creatine Kinase Activity ◽  
Relative Molecular Mass ◽  
Anion Exchange Column ◽  
Creatine Kinase Isoenzyme ◽  
Heat Stable ◽  
Immunological Investigation ◽  
Therapeutic Radiation

Abstract We describe an atypical serum creatine kinase isoenzyme in the serum of a woman with cancer of the left breast. This isoenzyme migrated toward the cathode, closely following the MM isoenzyme on agarose gel electrophoresis. Its relative molecular mass was estimated to be about 325,000, fourfold that of normal creatine kinase. It is more heat-stable and is inhibited more by urea than the normal MM isoenzyme. Isoenzyme monomer B activity was observed to be 20 U/liter in the serum, as measured with use of an antibody against the M monomer. On anion-exchange column analysis, creatine kinase activity was observed only in the MM fraction, in spite of the fact that B activity was observed in the patient's serum. Results of the immunological investigation make it unlikely that the atypical isoenzyme is linked to immunoglobulin or beta-lipoprotein. It may have been present as the result of modification of normal creatine kinase by the therapeutic radiation the patient was receiving.

Immunoreactive forms of erythrocyte spectrin and ankyrin in brain

1982 ◽  
Vol 299 (1095) ◽  
pp. 301-312 ◽  
Keyword(s):  
Molecular Mass ◽  
Erythrocyte Membranes ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Molecular Masses ◽  
Low Ionic Strength ◽  
Polypeptide Chains ◽  
The Brain ◽  
Total Membrane ◽  
Rotary Shadowing

Polypeptides immunologically related to erythrocyte spectrin and ankyrin have been detected in brain. The cross-reacting proteins include soluble as well as membrane-associated forms. A class of soluble cross-reacting polypeptides have been identified as high molecular mass microtubule-associated proteins (MAPS). MAP1, a group of polypeptides of molecular mass ca . 370 kDa contains a component that cross-reacts with anti-ankyrin IgG. MAP2, a polypeptide of molecular mass 300 kDa cross-reacts with anti-spectrin IgG, with the shared antigenic sites localized to the α chain of spectrin. The functional basis for structural homology between MAP1 and ankyrin may involve association with tubulin, since erythrocyte ankyrin binds to microtubules polymerized from pure brain tubulin. Spectrin did not associate with microtubules, but does have in common with MAP2 the ability to bind to actin (Brenner & Korn 1979; Sattilaro et al . 1981) and the shape of a flexible rod as visualized by rotary shadowing (Shotton et al . 1979; Voter & Erickson 1981). Immunoreactive forms of spectrin and ankyrin are also present in membrane fractions. A homologue of spectrin which constitutes 3% of the total membrane protein has been purified from low ionic strength extracts of membranes. This protein contains two non-identical polypeptide chains of molecular masses of 260 and 265 kDa, binds to F-actin, and displaces binding of erythrocyte spectrin to erythrocyte membranes. The brain protein pas been visualized by rotary shadowing as an extended rod-like molecule 195 nm in length. These studies indicate that the organization of proteins in the membrane-cytoskeleton complex of erythrocytes has direct relevance to other types of cells, and suggest the existence of families of proteins related to spectrin and ankyrin.

The Repetitive Microtubule-Associated Proteins MARP-1 and MARP-2 of Trypanosoma brucei

1994 ◽  
Vol 112 (3) ◽  
pp. 241-251 ◽  
Author(s):  
Marianne Affolter ◽  
Andrew Hemphill ◽  
Isabel Roditi ◽  
Norbert Müller ◽  
Thomas Seebeck
Keyword(s):  
Trypanosoma Brucei ◽  
Microtubule Associated Proteins ◽  
Associated Proteins

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.

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