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Biomedical subjects

A Matus

Publications and source records attributed to A Matus.

At least 19 recordsLinked to original sources
PubMed1992

Reorganisation of the microtubular cytoskeleton by embryonic microtubule-associated protein 2 (MAP2c).

Microtubule-associated protein 2c (MAP2c) is one of a set of embryonic MAP forms that are expressed during neuronal differentiation in the developing nervous system. We have investigated its mode of action by expressing recombinant protein in non-neuronal cell lines using cell cDNA transfection techniques. At every level of expression, all the MAP2c was bound to cellular microtubules. At low MAP2c levels, the microtubules retained their normal arrangement, radiating from the centrosomal microtubule-organising centre (MTOC) but at higher levels an increasing proportion of microtubules occurred independently of the MTOC. In most cells, radially oriented microtubules still attached to the MTOC co-existed with detached microtubules, suggesting that the primary effect of MAP2 is to increase the probability that tubulin polymerisation will occur independently of the MTOC. The MTOC-independent microtubules formed bundles whose distribution depended on their length in relation to the diameter of the transfected cell. Short bundles were attached to the cell cortex at one end and followed a straight course through the cytoplasm, whereas longer bundles followed a curved path around the periphery of the cell. By comparing these patterns to those produced by two chemical agents that stabilise microtubules, taxol and dimethyl sulphoxide, we conclude that effects of MAP2c arise from two sources. It stabilises microtubules without providing assembly initiation sites and as a result produces relatively few, long microtubule bundles. These bend only when they encounter the restraining influence of the cortical cytoskeleton of the cell, indicating that MAP2c also imparts stiffness to them. By conferring these properties of stability and stiffness to neuronal microtubules MAP2c contributes to supporting the structure of developing neurites.

Animals↗
PubMed1991

Phosphorylation determines the binding of microtubule-associated protein 2 (MAP2) to microtubules in living cells.

The influence of phosphorylation on the binding of microtubule-associated protein 2 (MAP2) to cellular microtubules was studied by microinjecting MAP2 in various phosphorylation states into rat-1 fibroblasts, which lack endogenous MAP2. Conventionally prepared brain MAP2, containing 10 mol of endogenous phosphate per mol (MAP2-P10), was completely bound to cellular microtubules within 2-3 min after injection. MAP2 prepared in the presence of phosphatase inhibitors, containing 25 mol/mol of phosphate (MAP2-P25), also bound completely. However, MAP2 whose phosphate content had been reduced to 2 mol phosphate per mol by treatment with alkaline phosphatase in vitro (MAP2-P2) did not initially bind to microtubules, suggesting that phosphorylation of certain sites in MAP2 is essential for binding to microtubules. MAP2-P10 was further phosphorylated in vitro via an endogenously bound protein kinase activity, adding 12 more phosphates, giving a total of 22 mol/mol. This preparation (MAP2-P10+12) also did not bind to microtubules. Assay of the binding of these preparations to taxol-stabilized tubulin polymers in vitro confirmed that their binding to tubulin depended on the state of phosphorylation, but the results obtained in microinjection experiments differed in some cases from in vitro binding. The results suggest that the site of phosphate incorporation rather than the amount is the critical factor in determining microtubule binding activity of MAP2. Furthermore, the interaction of MAP2 with cellular microtubules may be influenced by additional factors that are not evident in vitro.

Animals↗
PubMed1991

Microtubule-associated proteins and neuronal morphogenesis.

The microtubule-associated proteins (MAPs) are a set of structural proteins that bind to microtubules in vitro. Several of them occur at high levels in neurons where their expression is under strong developmental regulation, suggesting that they are involved in neuronal morphogenesis. Recently cDNAs for several of them have been cloned and sequenced revealing their primary structures and opening the way for genetic manipulation experiments aimed at determining their functions. Such experiments have shown that there are at least two classes of MAPs that are distinguished by the amino acid sequence motifs by which they bind to tubulin. One of these classes contains three known genes, two of which have been expressed in non-neuronal cells where the proteins cause bundling of microtubules and their rearrangement within the cytoplasm. The mechanism of this rearrangement is at present controversial. Another significant feature of these proteins is that several of them are differentially distributed within the neuronal cytoplasm; for example, some forms of MAP2 are selectively located in dendrites while in many situations MAP tau is limited to axons. In the case of MAP2, the mRNA that encodes the protein is also located in dendrites. This suggests that synthesis of MAP2 may be regulated locally in the dendritic cytoplasm. The molecular mechanism underlying the sorting of these proteins within neurons is unknown at present.

Animals↗
PubMed1990

Microtubule-associated protein 2 (MAP2) in Purkinje cell dendrites: evidence that factors other than binding to microtubules are involved in determining its cytoplasmic distribution.

We have studied the distribution of microtubule-associated protein 2 (MAP2) in the Purkinje cell dendrites of rats whose cerebella were exposed to X-irradiation during the second postnatal week. The Purkinje cells of such animals have abnormally elongated apical primary processes that branch in the other molecular layer rather than close to the cell body as in normal tissue. The results show that in these distorted dendrites the MAP2 distribution is "shifted" distally relative to the normal pattern, in which MAP2 is distributed evenly throughout the dendritic tree. Tubulin and other microtubule-associated proteins, such as MAP1, are not affected and remain evenly distributed throughout the dendritic tree despite the anatomical distortion. We conclude that the distribution of MAP2 in Purkinje cells is not determined solely by its binding to tubulin. Other factors must be involved and these appear to be related to dendritic morphology and possibly to branching.

Animals↗
PubMed1990

The expression of phosphorylated and non-phosphorylated forms of MAP5 in the amphibian CNS.

MAP5 is a microtubule-associated protein that in rat and quail is more abundant in the developing than in the adult brain. Previous studies in our laboratory have shown that MAP5 can be resolved into two forms by SDS-PAGE, MAP5a and MAP5b (Mr 300,000-320,000 Da) with MAP5a representing a highly phosphorylated form of this protein. In the present study, the relationship between MAP5 expression and neuronal growth and plasticity was investigated by assessing the amount and distribution of MAP5a and MAP5b in both the developing Xenopus brain and in different regions of the adult brain where neurons of varying growth potential and plasticity are present. In the larval and metamorphic Xenopus brain, like the neonatal rat brain, MAP5 is present in the highly phosphorylated form, MAP5a, and in concentrated in neuronal processes. In the adult Xenopus brain, MAP5a remains high in the optic tectum but, like the situation in the adult rat brain, is undetectable in the telencephalon. Immunohistochemistry showed that MAP5 was concentrated in the outer layer of the tectum, where ingrowing and plastic retinal ganglion cell axons are found. The correlation between MAP5 expression and phosphorylation and growth potential suggests that this molecule plays an important role in the regulation and organization of the neuronal cytoskeleton during neurite outgrowth and plasticity.

Animals↗
PubMed1990

Molecular cloning of microtubule-associated protein 1 (MAP1A) and microtubule-associated protein 5 (MAP1B): identification of distinct genes and their differential expression in developing brain.

cDNA clones encoding microtubule-associated proteins 1 (MAP1/MAP1A) and 5 (MAP5/MAP1B) were isolated and have been used to study their structural relationship as well as their regulated expression in developing rat brain. cDNA clones specific for MAP1 hybridized to a single 10-kb rat brain mRNA, and analysis of genomic DNA by Southern blotting indicated the existence of a single MAP1 gene. A second set of cDNAs specific for MAP5 hybridized to a single 11-kb mRNA in rat brain and also detected a single gene. By analysis of hybrid mouse-hamster cell lines, the MAP1 gene was located to mouse chromosome 2, designated Mtap-1, and the MAP5 gene to chromosome 13, designated Mtap-5. MAP1 and MAP5 mRNAs were expressed with different temporal patterns during rat brain development that mirrored the appearance of their protein products, suggesting that expression of these proteins is under transcriptional control. These results taken together demonstrate that although MAP1 and MAP5 have some properties that are similar, they are structurally distinct proteins whose transcription is differently regulated from separate genes.

Animals↗
PubMed1990

Microtubule-associated protein 3 (MAP3) expression in non-neuronal tissues.

Microtubule-associated protein 3 (MAP3, Mr 180,000), which in previous studies has been shown to be associated with glial processes and neurofilament-rich axons in rat brain, was examined in various non-neuronal rat tissues. Immunoblots of adult rat tissues (brain, liver, heart, spleen, adrenal medulla and kidney) showed that MAP3 is present in all organs tested. In addition we demonstrated that MAP3 is a heat-stable protein. Using immunohistochemistry, we established the localisation of MAP3 in various cell types. MAP3-containing cells appeared to have in common an asymmetric morphology with long processes that need structural support. In kidney MAP3 is limited to epithelial podocytes and in liver to Kupffer cells. In the adrenal gland, the cells of the cortex are devoid of MAP3 compared to the cells of the medulla. High concentrations of MAP3 are also found in cardiac muscle along the Z-disc and in the smooth muscle cells of the digestive tract. In spleen MAP3 is found in cells of the white pulp surrounding central blood vessels. A co-distribution of MAP3 with microtubules and intermediate filaments but not with microfilaments was found in each cell type examined. The widespread distribution pattern of MAP3 together with its molecular size and heat-stability indicate that MAP3 might be a member of the recently postulated family of homologous 200,000 Mr mammalian tissue MAPs. Potential functions for MAP3 in specific cell types are discussed.

Actins↗
PubMed1990

Microtubule-associated proteins and the determination of neuronal form.

1. The assembly of microtubules is essential for the maintenance of both the extension and the radial symmetry of axons and dendrites. Microtubule-associated proteins (MAPs) are implicated in this function because they promote tubulin polymerization and because they appear to be involved in cross-linking microtubules in the neuritic cytoplasm. 2. In a variety of species high molecular weight MAP2 is found only in dendrites and MAP tau is found only is axons, indicating that certain MAPs are associated with specific aspects of neuronal morphology. 3. All neuronal MAPs that have been studied are under strong developmental regulation with either their form or abundance changing between developing and adult brain. In both rat and Xenopus the change from "early" to "late" MAP forms occurs concurrently with the cessation of axon and dendrite growth and the maturation of neuronal morphology. 4. In situations where neuronal growth persists in the adult, such as retinal photoreceptor cells and the olfactory system, "early" MAPs continue to be expressed in the adult brain. 5. These results implicate MAPs in neuronal morphogenesis and suggest that "early" MAPs are involved in axon and dendrite growth whereas the "late" MAPs are involved in the stabilization of their mature form.

Animals↗
PubMed1989

Embryonic MAP2 lacks the cross-linking sidearm sequences and dendritic targeting signal of adult MAP2.

The most prominent microtubule-associated protein of the neuronal cytoskeleton is MAP2. In the brain it exists as a pair of high-molecular weight proteins, MAP2a and MAP2b, and a smaller form, MAP2c, which is particularly abundant in the developing brain. High-molecular weight MAP2 is expressed in dendrites, where its messenger RNA is also located, but is not found in axons; it has been shown to be present in fine filaments that crosslink dendritic microtubules. This correlates with the primary structure of high-molecular weight MAP2, which consists of a short carboxy-terminal tubulin-binding domain and a long amino-terminal arm, which forms a filamentous sidearm on reconstituted microtubules. Here we report that the high- and low-molecular weight forms of MAP2 are generated by alternative splicing and share the entire C-terminal tubulin-binding domain as well as a short N-terminal sequence. In contrast to high molecular weight MAP2, embryonic brain MAP2c lacks 1,342 amino acids from the filamentous sidearm domain. Furthermore, the mRNA for low molecular weight MAP2c is not present in dendrites, indicating that the dendritic targeting signal is specific for the high-molecular weight form.

Amino Acid Sequence↗
PubMed1989

Microtubule-associated proteins MAP5 and MAP1x: closely related components of the neuronal cytoskeleton with different cytoplasmic distributions in the developing brain.

Monoclonal antibodies were used to explore the relationship between two similarly sized microtubule-associated proteins (MAPs), MAP1x and MAP5. Although the proteins detected by anti-MAP1x and anti-MAP5 co-migrate in SDS-polyacrylamide gels, the patterns of antigenic proteolytic fragments (epitope maps) derived from them were completely different. The results suggest either that MAP1x is more stable than MAP5 or that the MAP1x epitope is situated close to one end of the molecule and gives rise to a very short proteolytic fragment. Immunoprecipitation from brain supernatants with either antibody brought down protein that cross-reacted with the other antibody, indicating that individual molecules bearing both epitopes exist in brain. Peptide maps of the proteins immunoprecipitated with the two antibodies showed that they are closely similar. Despite these similarities, the two antibodies gave different staining patterns on sections of developing rat brain, anti-MAP5 staining both axons and dendrites whereas anti-MAP1x stained only axons. We conclude that the MAP5 and MAP1x molecules are very similar, and possibly identical. The difference in staining patterns with the two antibodies could be because there are two proteins present in brain, one in immature axons bearing both the MAP5 and MAP1x epitopes and another with a wider distribution bearing only the MAP5 epitope. Alternatively, there may be a single protein bearing both epitopes, with the MAP1x epitope being masked in neuronal dendrites and mature axons by covalent modification or inter-molecular binding.

Aging↗
PubMed1989

In situ localization of microtubule-associated protein mRNA in the developing and adult rat brain.

We have used cDNA probes specific for three of the major brain microtubule-associated proteins (MAPs), MAP1, MAP2, and MAP5, to study the timing of appearance, relative abundance, and intracellular compartmentalization of MAP gene transcripts in developing rat brain. The MAP1 probe hybridizes throughout the brain, in both grey and white matter. MAP2 mRNA is detected only in grey matter and appears in cerebral neurons only after they have ceased dividing and have migrated to the cortical plate. The MAP5 cDNA hybridizes throughout the embryonic brain, but by P12, MAP5 mRNA distribution is restricted to relatively immature areas. MAP2 mRNA, found in dendrites in the developing brain, persists in some adult dendrites. MAP5 mRNA, like beta-tubulin mRNA, is found only in the cell bodies of developing neurons, indicating that the protein must be transported from the soma into processes. MAP1 mRNA is found only in the proximal regions of cortical pyramidal cell dendrites in both developing and adult brain. The diverse distributions of MAP gene transcripts emphasize the importance of these proteins in generating heterogeneity of microtubule function and indicate that MAP compartmentalization within neurons is regulated in part by differential mRNA transport.

Aging↗
PubMed1989

The adult rat olfactory system expresses microtubule-associated proteins found in the developing brain.

We have compared the expression and localization of neuronal microtubule-associated proteins (MAPs) MAP5, MAP2, and tau in the adult rat olfactory system and cerebral cortex. Each of these MAPS is known to exist as distinct "early" and "late" forms in the developing and adult brain, respectively. Because axonal growth and dendritic reinnervation continue in the adult olfactory system, it can serve as a test of whether expression of early MAP forms is necessary for neuronal growth and plasticity. We found that for all three MAPs, the early forms continue to be expressed in the adult olfactory system, whereas the cerebral cortex switches to the late forms during neuronal maturation (between 10 and 20 days of age in the rat). For MAP2, the 6 kb mRNA and low-molecular weight MAP2c, both of which are typically found in embryonic tissue, persist in the adult olfactory bulb. For MAP5, an early highly phosphorylated form, MAP5a, is present throughout the brain and disappears during maturation simultaneously with a several-fold drop in the overall level of MAP5. However, in the olfactory bulb, MAP5 levels do not fall, and MAP5a persists in the adult. The early form of tau is also prominent in the adult olfactory bulb and, by immunohistochemistry, is mainly confined to a subset of olfactory axons, the vomeronasal nerve. Thus, in the adult olfactory bulb, both MAP protein synthesis and phosphorylation conform to a pattern associated with the developing brain. Immunohistochemistry also showed that MAP5 is concentrated in the olfactory nerve axons and the mitral cell dendrites of the olfactory bulb, i.e., exactly those elements that are involved in the olfactory nerve innervation that takes place in the adult. These results suggest that the expression of the early MAP forms is closely associated with neurite outgrowth and plasticity.

Animals↗
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↗