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.

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.

scholarly journals Yeast proteins associated with microtubules in vitro and in vivo.

1992 ◽  
Vol 3 (1) ◽  
pp. 29-47 ◽  
Author(s):  
G Barnes ◽  
K A Louie ◽  
D Botstein
Keyword(s):  
Self Assembly ◽  
Synthetic Peptides ◽  
Immunofluorescence Microscopy ◽  
The Self ◽  
Microtubule Associated Proteins ◽  
Amino Terminal ◽  
Novel Proteins ◽  
Associated Proteins

Conditions were established for the self-assembly of milligram amounts of purified Saccharomyces cerevisiae tubulin. Microtubules assembled with pure yeast tubulin were not stabilized by taxol; hybrid microtubules containing substoichiometric amounts of bovine tubulin were stabilized. Yeast microtubule-associated proteins (MAPs) were identified on affinity matrices made from hybrid and all-bovine microtubules. About 25 yeast MAPs were isolated. The amino-terminal sequences of several of these were determined: three were known metabolic enzymes, two were GTP-binding proteins (including the product of the SAR1 gene), and three were novel proteins not found in sequence databases. Affinity-purified antisera were generated against synthetic peptides corresponding to two of the apparently novel proteins (38 and 50 kDa). Immunofluorescence microscopy showed that both these proteins colocalize with intra- and extranuclear microtubules in vivo.

scholarly journals Plk Phosphorylation Regulates the Microtubule-Stabilizing Protein TCTP

2002 ◽  
Vol 22 (17) ◽  
pp. 6209-6221 ◽  
Author(s):  
Frederic R. Yarm
Keyword(s):  
Protein A ◽  
Phosphorylation Site ◽  
Mitotic Cell ◽  
Structural Protein ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Mutant Phenotypes ◽  
And Function

ABSTRACT The mitotic polo-like kinases have been implicated in the formation and function of bipolar spindles on the basis of their respective localizations and mutant phenotypes. To date, this putative regulation has been limited to a kinesin-like motor protein, a centrosomal structural protein, and two microtubule-associated proteins (MAPs). In this study, another spindle-regulating protein, the mammalian non-MAP microtubule-binding and -stabilizing protein, the translationally controlled tumor protein (TCTP), was identified as a putative Plk-interacting clone by a two-hybrid screen. Plk phosphorylates TCTP on two serine residues in vitro and cofractionates with the majority of kinase activity toward TCTP in mitotic cell lysates. In addition, these sites were demonstrated to be phosphorylated in vivo. Overexpression of a Plk phosphorylation site-deficient mutant of TCTP induced a dramatic increase in the number of multinucleate cells, rounded cells with condensed ball-like nuclei, and cells undergoing cell death, similar to both the reported anti-Plk antibody microinjection and the low-concentration taxol treatment phenotypes. These results suggest that phosphorylation decreases the microtubule-stabilizing activity of TCTP and promotes the increase in microtubule dynamics that occurs after metaphase.

scholarly journals Plant and mouse EB1 proteins have opposite intrinsic properties on the dynamic instability of microtubules.

2020 ◽  
Author(s):  
Arthur T Molines ◽  
Virginie Stoppin-Mellet ◽  
Isabelle Arnal ◽  
Frédéric M Coquelle
Keyword(s):  
Dynamic Instability ◽  
Cellular Organization ◽  
Cell Type ◽  
Intrinsic Properties ◽  
Microtubule Organization ◽  
Vitro Assay ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Microtubule Networks

Abstract Objective Most eukaryotic cells contain microtubule filaments, which play central roles in intra-cellular organization. However, microtubule networks have a wide variety of architectures from one cell type and organism to another. Nonetheless, the sequences of tubulins, of Microtubule Associated proteins (MAPs) and the structure of microtubules are usually well conserved throughout the evolution. MAPs being known to be responsible for regulating microtubule organization and dynamics, this raises the question of the conservation of their intrinsic properties. Indeed, knowing how the intrinsic properties of individual MAPs differ between organisms might enlighten our understanding of how distinct microtubule networks are built. End-Binding protein 1 (EB1), first described as a MAP in yeast, is conserved in plants and mammals. The intrinsic properties of the mammalian and the yeast EB1 proteins have been well described in the literature but, to our knowledge, the intrinsic properties of EB1 from plant and mammals have not been compared thus far.Results Here, using an in vitro assay, we discovered that plant and mammalian EB1 purified proteins have different intrinsic properties on microtubule dynamics. Indeed, the mammalian EB1 protein increases microtubules dynamic while the plant EB1 protein stabilizes them.

scholarly journals Plant and mouse EB1 proteins have opposite intrinsic properties on the dynamic instability of microtubules.

2020 ◽  
Author(s):  
Arthur T Molines ◽  
Virginie Stoppin-Mellet ◽  
Isabelle Arnal ◽  
Frédéric M Coquelle
Keyword(s):  
Dynamic Instability ◽  
Eukaryotic Cells ◽  
Cellular Organization ◽  
Cell Type ◽  
Intrinsic Properties ◽  
Microtubule Organization ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Microtubule Networks

Abstract Objective Most eukaryotic cells contain microtubule filaments, which play central roles in intra-cellular organization. However, microtubule networks have a wide variety of architectures from one cell type and organism to another. Nonetheless, the sequences of tubulins, of Microtubule Associated proteins (MAPs) and the structure of microtubules are usually well conserved throughout the evolution. MAPs being known to be responsible for regulating microtubule organization and dynamics, this raises the question of the conservation of their intrinsic properties. Indeed, knowing how the intrinsic properties of individual MAPs differ between organisms might enlighten our understanding of how distinct microtubule networks are built. End-Binding protein 1 (EB1), first described as a MAP in yeast, is conserved in plants and mammals. The intrinsic properties of the mammalian and the yeast EB1 proteins have been well described in the literature but, to our knowledge, the intrinsic properties of EB1 from plant and mammals have not been compared thus far.Results Here, using an in vitro assay, we discovered that plant and mammalian EB1 purified proteins have different intrinsic properties on microtubule dynamics. Indeed, the mammalian EB1 protein increases microtubules dynamic while the plant EB1 protein stabilizes them.

scholarly journals 270K microtubule-associated protein cross-reacting with anti-MAP2 IgG in the crayfish peripheral nerve axon.

1986 ◽  
Vol 103 (1) ◽  
pp. 33-39 ◽  
Author(s):  
N Hirokawa
Keyword(s):  
Cross Linking ◽  
Flexible Structure ◽  
High Molecular Weight Protein ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Weight Protein ◽  
The Cross ◽  
Cross Bridges ◽  
Brain Tubulin

MAPs (microtubule-associated proteins) were isolated from crayfish walking leg nerves. A major MAP was identified as a high molecular weight protein (270K). This protein co-migrated with mammalian MAP2, stimulated the polymerization of rat brain tubulin into microtubules, and was heat resistant. Rotary shadowing revealed that the 270K MAP is a long thin flexible structure. It formed cross-bridges of fine strands, linking microtubules with each other in vitro. These strands resemble the cross-bridges between microtubules observed in the crayfish axon permeabilized with saponin and quick-frozen, deep-etched. Antibodies against mammalian MAP2 cross-reacted with this crayfish MAP and stained the axoplasm of the walking leg nerves. Thus MAPs, especially the 270K MAP, appear to be a major component of the cross-linking strands between microtubules observed in the crayfish axon.

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.

scholarly journals Stu2p binds tubulin and undergoes an open-to-closed conformational change

2006 ◽  
Vol 172 (7) ◽  
pp. 1009-1022 ◽  
Author(s):  
Jawdat Al-Bassam ◽  
Mark van Breugel ◽  
Stephen C. Harrison ◽  
Anthony Hyman
Keyword(s):  
Conformational Change ◽  
Conformational Transition ◽  
Budding Yeast ◽  
Microtubule Dynamics ◽  
Open Structure ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Common

Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat–containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.

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