Cross-linking of microtubules by microtubule-associated proteins (MAPs) from the brine shrimp, Artemia

1989 ◽  
Vol 93 (1) ◽  
pp. 29-39
Author(s):  
E.J. Campbell ◽  
S.A. MacKinlay ◽  
T.H. MacRae
Keyword(s):  
Molecular Weight ◽  
Brine Shrimp ◽  
Cross Linking ◽  
Cross Link ◽  
Independent Manner ◽  
Western Blots ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Tubulin Protein

Microtubules induced with taxol to assemble in cell-free extracts of the brine shrimp, Artemia, are cross-linked by microtubule-associated proteins (MAPs). When the MAPs, extracted from taxol-stabilized microtubules with 1 M-NaCl are co-assembled with purified Artemia or mammalian neural tubulin, reconstitution of cross-linking between microtubules occurs. The most prominent non-tubulin protein associated with reconstituted cross-linked microtubules has a molecular weight of 49,000 but we cannot yet exclude the possibility that other proteins may be responsible for the cross-linking. Cross-linkers are separated by varying distances while cross-linked microtubules, prepared under different conditions, are 6.9-7.7 nm apart. Cross-linking of microtubules by MAPs occurs whether MAPs are added to assembling tubulin or to microtubules, and it is not disrupted by ATP. The MAPs are heat-sensitive and do not stabilize microtubules to cold. Immunological characterization of Artemia MAPs on Western blots indicates that Artemia lack MAP 1, MAP 2 and tau. Our results clearly demonstrate that Artemia contain novel MAPs with the ability to cross-link microtubules from phylogenetically disparate organisms in an ATP-independent manner.

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.

A novel 49-kilodalton protein from Artemia cross-links microtubules in vitro

1992 ◽  
Vol 70 (10-11) ◽  
pp. 1055-1063 ◽  
Author(s):  
Jianshe Zhang ◽  
Thomas H. MacRae
Keyword(s):  
Protein A ◽  
Cross Linking ◽  
Amino Acid Residues ◽  
Microtubule Organization ◽  
Western Blots ◽  
Microtubule Associated Proteins ◽  
Amino Terminal ◽  
Apparent Homogeneity ◽  
Associated Proteins

A 49 kilodalton (kDa) protein, previously proposed to cross-link microtubules, was purified to apparent homogeneity from cell-free extracts of the brine shrimp Artemia. When incubated with tubulin under assembly conditions, the purified 49-kDa protein cross-linked the resulting microtubules. Preformed microtubules were also cross-linked when incubated with the 49-kDa protein. Upon centrifugation through sucrose cushions the 49-kDa protein cosedimented with microtubules, suggesting a stable association between the cross-linking protein and tubulin. Such microtubules were interconnected by particles which were circular, bilobed, or elongated in shape. Disruption of microtubule cross-linking and dissociation of the 49-kDa protein from microtubules occurred in the presence of ATP and 5′-adenylylimidodiphosphate (AMP–PNP), a nonhydrolyzable analogue of ATP. The 49-kDa protein was moderately resistant to heat, it did not stimulate tubulin assembly, and it did not react with antibodies to neural microtubule-associated proteins (MAPs) and kinesin. These observations indicate that the 49-kDa protein is different from many known MAPs, a conclusion strengthened by the inability of antibodies raised to the 49-kDa protein to recognize these proteins. The amino terminal 15 amino acid residues of the 49-kDa protein were determined by Edman digestion and an antibody raised to this peptide reacted with the 49-kDa protein on Western blots. Microtubule cross-linking was unaffected by the synthetic amino-terminal peptide, even when it was present at a fivefold molar excess over the 49-kDa protein. A search of three protein databanks revealed that the amino terminus of the 49-kDa protein is unique among published sequences. The findings verify our earlier proposal that Artemia contains a 49-kDa microtubule cross-linking protein and demonstrate that it has a novel set of characteristics. The 49-kDa protein has the potential to play an important role in microtubule organization and function.Key words: microtubule cross-linking, microtubule-associated proteins, Artemia.

scholarly journals Influence of phosphorylation on isoform composition and function of a microtubule-associated protein from developing Artemia

1995 ◽  
Vol 307 (2) ◽  
pp. 419-424 ◽  
Author(s):  
J Zhang ◽  
T H Macrae
Keyword(s):  
Brine Shrimp ◽  
Electrophoretic Analysis ◽  
Immunofluorescent Staining ◽  
Mobility Shift ◽  
Western Blots ◽  
Microtubule Associated Proteins ◽  
Apparent Homogeneity ◽  
Associated Proteins ◽  
And Function

A novel 49 kDa protein, which exhibits nucleotide-dependent cross-linking of microtubules in vitro and localizes to ordered microtubule arrays by immunofluorescent staining, has been purified to apparent homogeneity from the brine shrimp, Artemia. Electrophoretic analysis involving isoelectric focusing and two-dimensional gels, supplemented by staining of Western blots with affinity-purified antibody, revealed that the 49 kDa protein consists of five isoforms with pI values of 6.0-6.2. The amount of 49 kDa protein increased slightly, but its isoform composition did not change significantly, during development of Artemia gastrula to third-instar larvae. Treatment with alkaline phosphatase caused the 49 kDa protein to undergo a mobility shift on gel electrophoresis, and, by use of an antibody to phosphoserine, at least two isoforms of the protein were shown to be phosphorylated. The serine phosphate, presumably added by a post-translational mechanism, did not influence binding of the 49 kDa protein to microtubules. Under conditions in which microtubules were cross-linked, the 49 kDa protein failed to interact with actin filaments. Our results demonstrate that the 49 kDa protein, like other structural microtubule-associated proteins such as tau and MAP2, is composed of several isoforms, some of which are phosphorylated. This protein has the potential to regulate the spatial distribution of microtubules within cells but does not link microfilaments to one another or to microtubules.

Microtubule motors and other maps

1990 ◽  
Vol 48 (3) ◽  
pp. 1-1
Author(s):  
Richard B. Vallee
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Cytoplasmic Dynein ◽  
Organelle Transport ◽  
Structural Components ◽  
Microtubule Associated Proteins ◽  
Microtubule Motors ◽  
Associated Proteins ◽  
The Subject ◽  
Intracellular Motility

Microtubules are involved in a number of forms of intracellular motility, including mitosis and bidirectional organelle transport. Purified microtubules from brain and other sources contain tubulin and a diversity of microtubule associated proteins (MAPs). Some of the high molecular weight MAPs - MAP 1A, 1B, 2A, and 2B - are long, fibrous molecules that serve as structural components of the cytamatrix. Three MAPs have recently been identified that show microtubule activated ATPase activity and produce force in association with microtubules. These proteins - kinesin, cytoplasmic dynein, and dynamin - are referred to as cytoplasmic motors. The latter two will be the subject of this talk.Cytoplasmic dynein was first identified as one of the high molecular weight brain MAPs, MAP 1C. It was determined to be structurally equivalent to ciliary and flagellar dynein, and to produce force toward the minus ends of microtubules, opposite to kinesin.

scholarly journals Microtubules and microtubule-associated proteins from the nematode Caenorhabditis elegans: periodic cross-links connect microtubules in vitro.

1986 ◽  
Vol 103 (1) ◽  
pp. 23-31 ◽  
Author(s):  
E J Aamodt ◽  
J G Culotti
Keyword(s):  
Caenorhabditis Elegans ◽  
Apparent Molecular Weight ◽  
Cross Link ◽  
Nematode Caenorhabditis Elegans ◽  
Microtubule Associated Proteins ◽  
C Elegans ◽  
Associated Proteins ◽  
Cross Links ◽  
The Cross

The nematode Caenorhabditis elegans should be an excellent model system in which to study the role of microtubules in mitosis, embryogenesis, morphogenesis, and nerve function. It may be studied by the use of biochemical, genetic, molecular biological, and cell biological approaches. We have purified microtubules and microtubule-associated proteins (MAPs) from C. elegans by the use of the anti-tumor drug taxol (Vallee, R. B., 1982, J. Cell Biol., 92:435-44). Approximately 0.2 mg of microtubules and 0.03 mg of MAPs were isolated from each gram of C. elegans. The C. elegans microtubules were smaller in diameter than bovine microtubules assembled in vitro in the same buffer. They contained primarily 9-11 protofilaments, while the bovine microtubules contained 13 protofilaments. The principal MAP had an apparent molecular weight of 32,000 and the minor MAPs were 30,000, 45,000, 47,000, 50,000, 57,000, and 100,000-110,000 mol wt as determined by SDS-gel electrophoresis. The microtubules were observed, by electron microscopy of negatively stained preparations, to be connected by stretches of highly periodic cross-links. The cross-links connected the adjacent protofilaments of aligned microtubules, and occurred at a frequency of one cross-link every 7.7 +/- 0.9 nm, or one cross-link per tubulin dimer along the protofilament. The cross-links were removed when the MAPs were extracted from the microtubules with 0.4 M NaCl. The cross-links then re-formed when the microtubules and the MAPs were recombined in a low salt buffer. These results strongly suggest that the cross-links are composed of MAPs.

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.

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