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A Matus

Publications and source records attributed to A Matus.

At least 73 records · Page 4Linked to original sources
PubMed1988

Selective localization of messenger RNA for cytoskeletal protein MAP2 in dendrites.

For nerve cells to develop their highly polarized form, appropriate structural molecules must be targeted to either axons or dendrites. This could be achieved by the synthesis of structural proteins in the cell body and their sorting to either axons or dendrites by specific transport mechanisms. For dendrites, an alternative possibility is that proteins could be synthesized locally in the dendritic cytoplasm. This is an attractive idea because it would allow regulation of the production of structural molecules in response to local demand during dendritic development. The feasibility of dendritic protein synthesis is suggested both by the existence of dendritic polyribosomes and by the recent demonstration that newly synthesized RNA is transported into the dendrites of neurons differentiating in culture. However, to date there has been no demonstration of the selective synthesis of an identified dendrite-specific protein in the dendritic cytoplasm. Here, we use in situ hybridization with specific complementary DNA probes to show that messenger RNA for the dendrite-specific microtubule-associated protein MAP2 (refs 3-5) is present in dendrites in the developing brain. By contrast the mRNA for tubulin, a protein present in both axons and dendrites is located exclusively in neuronal cell bodies.

Animals↗
PubMed1988

Single-shot cloning of multiple cDNAs coding for a set of related microtubule-associated proteins.

We describe a method for isolating multiple cDNA clones coding for a set of related proteins from bacteriophage lambda expression libraries in a single screening with polyclonal antiserum. The antiserum is raised against a tissue sub-fraction containing the proteins of interest; in the example presented this was brain microtubules. Each antibody-positive clone from the lambda expression library is plaque-purified and then grown in contact with nitrocellulose membrane that becomes coated with protein synthesized from the cloned cDNA. Each filter, containing the protein produced by a single lambda cDNA clone, is then used to affinity-select clone-specific antibodies from the original polyclonal antiserum. The monospecific antibody for each cDNA clone can be used on Western blots to identify the protein that the cDNA encodes and also to stain tissue sections. Using this method we have, in a single screening, obtained: (1) multiple cDNA clones representing different regions of a single large protein, (2) cDNA clones representing several functionally related proteins (microtubule-associated proteins), and (3) cDNA clones related to a novel protein species for which neither biochemical nor immunological data have previously been available.

Animals↗
PubMed1988

Denervation induces long-lasting changes in the distribution of microtubule proteins in hippocampal neurons.

The cellular distributions of tubulin and microtubule-associated protein 2 (MAP2) were examined in the dentate gyrus of the rat hippocampus after unilateral lesion of the entorhinal cortex, which destroys the major afferent pathway to dentate granule cells. Changes were observed in distribution of both tubulin and MAP2 in granule cell dendrites on the denervated side. After 24 h there was a noticeable increase in both anti-tubulin and anti-MAP2 staining in the outer two-thirds of the dentate molecular layer, corresponding to the area of denervation. This increased staining reached a maximum 1 week after the lesion. There was no change on the unlesioned side. During a subsequent second phase the region of increased anti-tubulin and anti-MAP2 staining became restricted, by 35 days after lesioning, to a narrow band mid-way through the molecular layer. This pattern remained the same until 6 months after the lesion, the longest time point examined. The results indicate that there is considerable plasticity in the microtubular cytoskeleton of dendrites in the adult brain and that rearrangements induced in it by axotomy can persist long after the immediate effects of denervation and subsequent re-innervation have subsided.

Animals↗
PubMed1988

Phylogenetic conservation of brain microtubule-associated proteins MAP2 and tau.

The major rat brain microtubule-associated proteins, MAP2 and tau, exhibit various properties that implicate them in the mechanisms underlying the growth of axons and dendrites during neuronal development. To determine if these properties represent fundamental morphogenetic mechanisms, we have examined the phylogenetic conservation of these proteins in Xenopus laevis, quail and rat with respect to their molecular form, cytological distribution and developmental expression. In all three species, the high-molecular weight form of MAP2 migrates as a pair of polypeptides (MAP2a and MAP2b); this doublet as well as the low-molecular weight form of MAP2 (MAP2c) and the tau proteins are markedly similar in size in the different classes of vertebrates. Immunohistochemical staining of the Xenopus and quail cerebellum showed that MAP2 is highly concentrated in dendrites whereas the tau proteins are predominantly confined to axons, exactly as they are in rat. The developmental regulation of these proteins in Xenopus and rat is also conserved. Between the larva and the adult (i.e. during metamorphosis) MAP2c undergoes a marked decrease while MAP2a undergoes a large increase. Thus, in both classes of vertebrates the timing of changes in MAP2 expression coincides with the maturation of neuronal morphology. Taken together, these conserved properties of MAP2 and tau in three phylogenetically divergent classes of vertebrates suggest that these proteins serve fundamental functions during neuronal morphogenesis.

Animals↗
PubMed1988

Different forms of microtubule-associated protein 2 are encoded by separate mRNA transcripts.

Brain microtubule-associated protein 2 (MAP2) consists of a pair of high molecular mass (280 kD) polypeptides, MAP2a and MAP2b, and a recently identified 70-kD protein, MAP2c, which is antigenically related to these high molecular mass MAP2's. Using cDNA clones we have analyzed the expression of these three proteins at the nucleic acid level. cDNA probes selective for the high molecular mass MAP2's a and b identified only a 9-kb mRNA, whereas a probe for sequence common to all three MAP2 isoforms, a, b, and c, recognized the 9-kb transcript and additionally a 6-kb mRNA. Southern blot analysis with cDNA probes indicated that there is only one MAP2 gene from which these two distinct mRNAs are derived. The 70-kD MAP2c protein is much more abundant in neurons of developing brain than those of adult tissues. Similarly the expression of the 6-kb MAP2c-related mRNA, is much greater in neonatal than adult rat brain, indicating that the developmental expression of MAP2 is determined by transcriptional regulation from a single MAP2 gene.

Animals↗
PubMed1988

PC12 cells express juvenile microtubule-associated proteins during nerve growth factor-induced neurite outgrowth.

Microtubule-associated proteins (MAPs) are believed to play an important role in regulating the growth of neuronal processes. The nerve growth factor-induced differentiation of PC12 pheochromocytoma cells is a widely used tissue culture model for studying this mechanism. We have found that contrary to previous suggestions, the major MAPs of adult brain, MAP1 and MAP2, are minor components of PC12 cells. Instead two novel MAPs characteristic of developing brain, MAP3 and MAP5, are present and increase more than 10-fold after nerve growth factor treatment; the timing of these increases coinciding with the bundling of microtubules and neurite outgrowth. Immunocytochemical staining showed that MAP3 and MAP5 are initially distributed throughout the cytoplasm. Subsequently MAP5 becomes associated with microtubules in both neurites and growth cones but MAP3 distribution remained diffuse. Thus MAP3 and MAP5, which are characteristic of developing neurons in the juvenile brain, are also induced in PC12 cells during neurite outgrowth in culture. In contrast MAP1, which is characteristic of mature neurons, does not increase during PC12 cell differentiation. These results provide evidence that one set of MAPs is expressed during neurite outgrowth and a different set during the maintenance of neuronal form. It also appears that the PC12 system is an appropriate model for studying the active neurite growth phase of neuronal differentiation but not for neuronal maturation.

Adrenal Gland Neoplasms↗
PubMed1988

A 70-kilodalton microtubule-associated protein (MAP2c), related to MAP2.

Microtubule-associated protein 2 (MAP2) from adult brain consists of a pair of high molecular mass (280 kilodaltons) polypeptides, MAP2a and MAP2b. Juvenile brain microtubules also contain a 70-kilodalton protein that cross-reacts with monoclonal antibodies against these high molecular weight MAP2s. We have analyzed the relationship between this 70-kilodalton protein and MAP2 by peptide mapping. Our results show that the 70-kilodalton species bears strong homology to the MAP2 molecules and that it is distinct from the tau MAPs. We propose the name MAP2c for this low molecular weight MAP2 species. MAP2c is developmentally regulated in brain, being more abundant in neonatal tissue than in the adult. In several cell lines, MAP2c is the sole MAP2 species expressed. We examined homogenates from both juvenile brain and MAP2c-containing cell lines for evidence of a protease activity that might be responsible for generating MAP2c from either MAP2a or MAP2b. No such activity was found, suggesting that MAP2c is an independently synthesized MAP2 species some 200 kilodaltons smaller than the previously recognized forms.

Adrenal Gland Neoplasms↗
PubMed1987

Age-related increase in a cathepsin D like protease that degrades brain microtubule-associated proteins.

In microtubules isolated from brains of very old rats, two of the major microtubule-associated proteins, MAP1 and MAP2, are found only in degraded form. MAP1 is present as a piece whose molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is circa 50,000 smaller than the native protein, and MAP2 is extensively fragmented. The native forms of both proteins are present in tissue homogenates but are rapidly degraded during microtubule isolation. The proteolytic activity responsible for this degradation is cathepsin D like, being more active at acid pH than neutral and being completely blocked by pepstatin at 10(-7) M. Fractionation of aged brain supernatant by gel permeation chromatography showed that the MAP1 and MAP2 degrading activity elutes with a single peak of cathepsin D like activity. MAP1 and MAP2 are known to promote microtubule assembly, and their degradation by a protease whose levels increase with age could be related to defective microtubule assembly which is known to occur in age-related degenerative conditions such as Alzheimer's disease.

Aging↗
PubMed1987

Influence of monoclonal antibodies on microtubule assembly.

The influence on microtubule assembly in vitro of monoclonal antibodies against microtubule-associated proteins (MAPs) was studied. Light scattering was used for measuring net polymer formation and electron microscopy for determining the influence of antibodies on microtubule morphology. Control experiments showed that nonimmune mouse IgG had no effect on either the assembly or appearance of microtubules. The same was true for monoclonal antibodies against MAP1. At low levels, antibodies against MAP2 caused the aggregation of microtubules into bundles, an effect that did not occur with antibodies against any other MAP type studied. At increasing concentrations, anti-MAP2 progressively inhibited tubulin polymerization, producing irregular, shortened filaments. Anti-MAP5 produced a striking fragmentation of microtubules into very short pieces that were otherwise morphologically identical to control microtubules. The different effects of these antibodies show the potential of monoclonal antibodies for investigating MAP function and form an important adjunct to cellular microinjection experiments.

Animals↗
PubMed1986

The novel microtubule-associated protein MAP3 contributes to the in vitro assembly of brain microtubules.

MAP3 is a novel microtubule-associated protein found in brain and a variety of other tissues (Huber, G., Alaimo-Beuret, D., and Matus, A. (1985) J. Cell Biol. 100, 496-507). In this study, monoclonal antibodies were used to assess its influence on the polymerization of brain tubulin. When added to unpolymerized brain microtubules, anti-MAP3 IgG produced a dose-related inhibition of subsequent assembly. Under the same circumstances, nonimmune mouse IgG did not influence either the rate or the extent of tubulin polymerization. We also used immobilized antibodies to deplete brain MAPs selectively in either MAP3 or MAP1. MAP3-depleted MAPs showed a reproducible decrease in activity compared to control preparations that had been exposed to immobilized nonimmune IgG. MAP1-depleted MAPs did not differ significantly in performance from the nonimmune treated controls. We conclude that MAP3 contributes to the net assembly of brain microtubules observed in vitro. This may be particularly relevant in neonatal animals where brain MAP3 is more abundant than in the adult.

Animals↗
PubMed1986

MAP5: a novel brain microtubule-associated protein under strong developmental regulation.

A novel microtubule-associated protein, MAP5, is described, whose chemical properties and cytological distribution distinguish it from other known microtubule-associated proteins (MAPs). Its status as a MAP is indicated by the observations that (i) it co-assembles efficiently with microtubules in vitro, (ii) it is localized on microtubules in brain sections by immunogold staining with monoclonal antibody against MAP5 and (iii) immunoaffinity purified MAP5 stimulates tubulin polymerization. Immunoperoxidase staining of brain sections showed that MAP5 is present in neurons throughout the brain and that in them it is evenly distributed throughout axons, dendrites and cell bodies. In this respect it differs from previously described MAPs (1, 2, 3 and tau) which are differentially compartmentalized in brain neurons. MAP5 is not present in axon terminals, dendritic spines or other synaptic elements. It is present at substantially higher levels in neonatal brain than adult and it is more abundant than either MAP1 or MAP2a up to postnatal day 10. The fall in amount of MAP5, from juvenile to adult levels, is completed between postnatal days 10 and 20. This suggests that MAP5 is particularly important in modulating microtubule function during the formation of neuronal processes.

Animals↗
PubMed1986

Microtubule-associated protein 2 and tubulin are differently distributed in the dendrites of developing neurons.

We have followed the appearance of two microtubule proteins, tubulin and microtubule-associated protein 2, in rat hippocampal neurons differentiating in cell culture. Double-label immunofluorescence staining showed that from day 1 in vitro onward tubulin appeared as filaments but that microtubule-associated protein 2 remained distributed throughout the cytoplasm. This difference persisted throughout development and was also detectable in cells that had reached morphological maturity. When cells were treated with the microtubule-depolymerizing agent nocodazole, the depolymerized tubulin became spread throughout the cytoplasm so that its distribution was then identical to microtubule associated protein 2. At the same time, multiple side branches began to emerge along the dendrites. When cells which had been exposed to nocodazole were allowed to recover before staining, the tubulin was again present as filaments but the microtubule-associated protein 2 remained distributed throughout the dendritic cytoplasm. Under these conditions the previously extended proximal side branches were resorbed into the main process. These results suggest that cellular microtubule-associated protein 2 is not necessarily exclusively associated with microtubules. Neuronal dendrites in particular appear to contain this protein at levels in excess of the capacity of microtubular microtubule-associated protein 2 binding sites. In view of the known effectiveness of microtubule-associated protein 2 as a promoter of tubulin polymerization, its abundance in dendrites suggests that it acts to ensure total polymerization of dendritic microtubules. In this way it would contribute both to the support of the growing process and the suppression of adventitious sidebranching.

Animals↗
PubMed1986

Distribution of calpains I and II in rat brain.

Calpains I and II are calcium-dependent proteases that have been implicated in several aspects of brain function, including neurofilament turnover, Wallerian degeneration, and excitatory synaptic transmission. In this study, specific affinity-purified antibodies against each of the enzymes were used to determine their cellular distribution in rat brain. Differences between the two were found throughout the brain, with calpain I being located primarily in neurons, whereas calpain II was more prominent in glial cells. In myelinated axons, calpain II was present at low levels but calpain I was not detectable. In all brain areas, both enzymes were concentrated in cell bodies, with lesser amounts in neuronal and glial processes. Calpain I was only detectable proximally in dendrites and was not found in spiny branchlets of either pyramidal or Purkinje cells. These results suggest that calpain II is the likely form of the enzyme involved in calcium-activated proteolytic phenomena in axons. They do not support the existence of a role for calpain at excitatory axospinous synapses.

Animals↗
PubMed1985

Changes in the cytoplasmic distribution of microtubule-associated protein 2 during the differentiation of cultured cerebellar granule cells.

The distribution of microtubule-associated protein 2 in cultured cerebellar granule cell neurons was followed by immunohistochemical staining with specific antibodies. During differentiation in vitro, the neurites of these cells pass through a series of developmental stages. At first the emergent processes contain only trace levels of microtubule-associated protein 2 which is most concentrated in and near the cell body. When the neurites are between two and five cell diameters long they exhibit both microtubule-associated protein 2 and tubulin, apparently evenly distributed, throughout their length. Subsequently microtubule-associated protein 2 is limited to an initial, usually varicose portion of the neurite whereas its long distal extension contains abundant tubulin but is apparently devoid of microtubule-associated protein 2. Thus microtubule-associated protein 2 and tubulin are not necessarily co-distributed with a single neuronal process. In both morphological appearance and in the different distributions of microtubule-associated protein 2 and tubulin they contain, these processes show a mixture of axonal and dendritic properties. Since these same cells do not develop their characteristic dendritic arborizations, our results suggest that when removed from the developing brain, cerebellar granule neurons achieve part but not all of their normal morphological and cytoskeletal differentiation.

Animals↗
PubMed1985

Differential expression of distinct microtubule-associated proteins during brain development.

The levels of three different microtubule-associated proteins (MAP1, -2, and -3) in brain were found to undergo large changes during postnatal development. MAP1 was barely detectable at birth but thereafter steadily increased, reaching adult levels by postnatal day 20 (P20). Both MAP2 and MAP3 showed differential expression patterns of their component peptides. At birth, MAP2 was represented by the smaller of two Mr 280,000 peptides (MAP2b) and three antigenically related Mr 70,000 peptides. The larger of the Mr 280,000 peptides (MAP2a) first appeared between P10 and P20, and the Mr 70,000 components disappeared at the same time. Of the two MAP3 peptides, the larger (MAP3a) was present in the late embryo, several days before MAP3b appeared. Between P10 and P20, both MAP3 components underwent a striking decrease in abundance (a factor of 10), which correlated with their disappearance from all neuronal compartments except neurofilament-containing axons. These developmental changes in expression are different and characteristic for each of the three MAPs, yet in each case they are detectable in brain homogenates, indicating that they occur concurrently throughout the brain.

Animals↗