We have determined the biochemical and immunocytochemical localization of the heterogeneous microtubule-associated protein tau using a monoclonal antibody that binds to all of the tau polypeptides in both bovine and rat brain. Using immunoblot assays and competitive enzyme-linked immunosorbent assays, we have shown tau to be more abundant in bovine white matter extracts and microtubules than in extracts and microtubules from an enriched gray matter region of the brain. On a per mole basis, twice-cycled microtubules from white matter contained three times more tau than did twice-cycled microtubules from gray matter. Immunohistochemical studies that compared the localization of tau with that of MAP2 and tubulin demonstrated that tau was restricted to axons, extending the results of the biochemical studies. Tau localization was not observed in glia, which indicated that, at least in brain, tau is neuron specific. These observations indicate that tau may help define a subpopulation of microtubules that is restricted to axons. Furthermore, the monoclonal antibody described in this report should prove very useful to investigators studying axonal sprouting and growth because it is an exclusive axonal marker.
Five beta-tubulin isotypes are expressed differentially during chicken brain development. One of these isotypes is encoded by the gene c beta 4 and has been assigned to an isotypic family designated as Class III (beta III). In the nervous system of higher vertebrates, beta III is synthesized exclusively by neurons. A beta III-specific monoclonal antibody was used to determine when during chick embryogenesis c beta 4 is expressed, the cellular localization of beta III, and the number of charge variants (isoforms) into which beta III can be resolved by isoelectric focusing. On Western blots, beta III is first detectable at stages 12-13. Thereafter, the relative abundance of beta III in brain increases steadily, apparently in conjunction with the rate of neural differentiation. The isotype was not detectable in non-neural tissue extracts from older embryos (days 10-14) and hatchlings. Western blots of protein separated by two-dimensional gel electrophoresis (2D-PAGE) reveal that the number of beta III isoforms increases from one to three during neural development. This evidence indicates that beta III is a substrate for developmentally regulated, multiple-site posttranslational modification. Immunocytochemical studies reveal that while c beta 4 expression is restricted predominantly to the nervous system, it is transiently expressed in some embryonic structures. More importantly, in the nervous system, immunoreactive cells were located primarily in the non-proliferative marginal zone of the neural epithelia. Regions containing primarily mitotic neuroblasts were virtually unstained. This localization pattern indicates that c beta 4 expression occurs either during or immediately following terminal mitosis, and suggests that beta III may have a unique role during early neuronal differentiation and neurite outgrowth.
Class III fi-tubulin, isolated from adult bovine brain, is resolved into at least seven charge variants on isoelectric focusing gels. To identify the posttranslational modifications responsible for this heterogeneity, a mixture of brain tubulins was treated with cyanogen bromide and the C-terminal fragments from the class III I3-tubulin isoforms were then isolated by binding them to the monoclonal antibody TuJ1. Combined use of tandem mass spectrometry and both subtractive and automated Edman degradation chemistry on the isolated peptides indicates that many of the isoforms differ by phosphorylation at Ser-444 plus attachment of one to six glutamic acid molecules to the side chain of the rst glutamate residue, Glu-438, in the C-terminal sequence Tyr-Glu-AspAsp-Glu-Glu-Glu-Ser-Glu-Ala-Gln-Gly-Pro-Lys.Microtubules, assembled from two similar 50-kDa proteins designated a-and f3-tubulin, are involved in a number of important biological processes including segregation of chromosomes during cell division, cell motility, organelle transport, and maintenance of cell shape (1). Brain tubulin exhibits a high degree of heterogeneity. Up to 21 charge variants have been observed by isoelectric focusing (IEF) techniques (2-4).Both a-and 83-tubulins are encoded by small multigene families (5). Since the number of charge variants exceeds the number of tubulin genes by approximately a factor of 2, the remaining isoforms are assumed to result from posttranslational modifications. a-Tubulin is reported to undergo acetylation (6-8), glutamylation (9), and the addition and removal of a C-terminal tyrosine residue (10, 11). Phosphorylation of a single seine residue in one of the five P-tubulin isotypes expressed in brain has also been described (12-16).Class III p8-tubulin, a vertebrate isotype found only in neurons and cells in the mammalian testis (17), undergoes a developmentally regulated increase in heterogeneity in the former but not the latter tissue (18,19). The testis and earliest embryonic rat brain isoforms cofocus on an IEF gel (19). At least six additional isoforms of neuron-specific class III ,B-tubulin are detected in the adult rat or bovine brain.Previous work localized the site of heterogeneity to the C-terminal 20 amino acids (19), a highly acidic region of the protein thought to be involved in the interaction of microtubules with both calcium ions (20) and microtubule-associated proteins (21-23).To further characterize both the nature and location of structural modifications responsible for the observed heterogeneity, class III /3-tubulin from bovine brain was subjected to amino acid sequence analysis by tandem mass spectrometry. We report that the neuron-specific class III /3-tubulin isoforms result in part from phosphorylation at Ser-444 and attachment of at least three and perhaps as many as six glutamic acid molecules to the side chain of the first glutamate residue, Glu-438, in the C-terminal sequence Tyr-GluAsp-Asp-Glu-Glu-Glu-Ser-Glu-Ala-Gln-Gly-Pro-Lys (YED-DEEESEAQGPK). MATERIALS AND METHODSIsolati...
The distribution and subcellular localization of tubulin and MAP2 in brain tissue were analyzed by immunocytochemistry with monoclonal hybridoma antibodies prepared against Chinese hamster brain tubulin and MAP2. We examined three anti-tubulin hybridoma antibodies (Tu3B, Tu9B, Tu12) specific for beta-tubulin, and two anti-MAP2 hybridoma antibodies (AP9,AP13). The specificity of each of the monoclonal antibodies was characterized by staining nitrocellulose electrophoretic blots of SDS-polyacrylamide gels of whole brain or hippocampal extracts. Each hybridoma antibody bound only its respective antigen in these preparations. Polyclonal antisera against tubulin were also examined. Sections reacted with antisera against tubulin or monoclonal antibodies against beta-tubulin revealed a wide variety of stained cellular compartments. The reaction product was found to decorate dendritic and axonal microtubles in neurons; glial cells were also stained. MAP2 immunoreactivity was found only in neurons. In the case of one of the monoclonal antibodies (AP9), staining was preferentially associated with dendritic processes. However, light but significant staining of axonal processes was seen with AP13. Within dendrites, MAP2 was found associated with dendritic microtubules and postsynaptic densities (psd), both in shaft and spine synapses. In addition, strong immunoreactivity for MAP2 was found within the cytoplasm of dendritic spines. There was little or no immunoreactivity for tubulin in the spine cytoplasm, although the psd was stained. The localization of MAP2 in dendritic spines and in the psd suggests that this protein may have a biological role independent of its association with microtubules. The observations on differential staining of the hybridoma antibodies against MAP2 suggest that there may be distinct subtypes or states of MAP2 within neurons.
The major limitation to our understanding of the clinical importance of enterotoxigenic Escherichia coli in diarrheal illness has been the lack of a simple rapid assay for the enterotoxin produced by certain E. coli. On the basis of the activation of adenylate cyclase by heat-labile enterotoxin of E. coli (LT) and by cholera toxin (CT) in intestinal and other tissues, cultured Chinese hamster ovary (CHO) cells with known morphological responses to dibutyryl cyclic adenosine 5'-monophosphate (AMP) were exposed to these enterotoxins. Crude culture filtrates of LT-producing E. coli and CT stimulated cyclic AMP accumulation and cell elongation in CHO cells. The similarity of time course, concentration dependence, and potentiation by phosphodiesterase inhibitors suggested cyclic AMP mediation of the morphological change. Heat inactivated CT and LT in this system. Choleragenoid inhibited CT; antiserum against CT inhibited both enterotoxin effects. In contrast to culture filtrates of 16 strains of E. coli known to produce LT, culture filtrates from 13 E. coli that do not produce LT did not alter CHO cell morphology. The morphological change is a simple, specific assay for these enterotoxins and detect 3 x 10-17 mol of CT or a 1: 250 dilution of crude culture filtrate of LT-producing E. coli 334.
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