Electron-microscopic demonstration of α-tubulin immunoreactivity in astroglia

Summary Vibratome sections of the cerebral cortex, hippocampus and cerebellum were immunostained for -tubulin using the TU-Ol monoclonal antibody. In all three regions, electron microscopy of the immunostained preparations revealed — in addition to the previously described reaction of pyramidal apical dendritic microtubules — consistent staining of the ribosomal apparatus of astrocytes.     

Distribution of α-Tocopherol Stereoisomers in Rats

The distribution of α-tocopherol (α-Toc) stereoisomers in the tissues of rats fed on a diet containing all-rac-α-tocopheryl acetate was investigated by a newly revised HPLC. The concentrations of 2R-isomers of α-Toc in blood and tissues of the rats were significantly higher than those of 2S-isomers. In most tissues, the levels of 2S-isomers were in order SRS> (SSS +SSR)/2 > SRR.     

Distinct subcellular localization of β-tubulin epitopes in the adult mouse brain

 A panel of monoclonal antibodies specific of α-tubulin (TU-01, TU-09) and β-tubulin (TU-06, TU-13) subunits was used to study the location of N-terminal structural domains of tubulin in adult mouse brain. The specificity of antibodies was confirmed b immunoblotting experiments. Immunohistochemical staining of vibratome sections from cerebral cortex, cerebellum, hippocampus, and corpus callosum showed that antibodies TU-01, TU-09, and TU13 reacted with neuronal and glial cells and their processes, whereas the TU-06 antibody stained only the perikarya. Dendrites and axons were either unstained or their staining was very weak. As the TU-06 epitope is located on the N-terminal structural domain of β-tubulin, the observed staining pattern cannot be interpreted as evidence of a distinct subcellular localization of β-tubulin isotypes or known post-translational modifications. The limited distribution of the epitope could, rather, reflect differences between the conformations of tubulin molecules in microtubules of somata and neurites or, alternatively, a specific masking of the corresponding region on the N-terminal domain of β-tubulin by interacting protein(s) in dendrites and axons. Accepted: 11 November 1996     

Posttranslational acetylation of α-tubulin constrains protofilament number in native microtubules

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Highlights? α-TAT mutants have short microtubules and variable protofilament number ? α-tubulin K40 acetylation promotes interprotofilament salt bridges ? α-tubulin K40 acetylation is a key constraint on protofilament number in vivo     

A truncated form of α-tubulin detected in purified proteasome complexes

The 26S proteasome is a multisubunit protein complex responsible for selective protein degradation in the cell. A number of proteins with known and unknown functions were shown to be permanently or temporarily associated with 26S proteasomes. Identification of proteins that interact with proteasomes is an important step in the understanding of the proteasome functions in the cell and the mechanisms of their regulation. Using MALDI–ICR mass spectrometry, we have shown that some proteins of the cytoskeleton, such as actin, α-actinin 4, and α- and β-tubulins are associated with proteasomes obtained by affinity purification from the human myelogenous leukemia cell line K562. Western blot analysis showed that a truncated form of α-tubulin was associated with the purified proteasomes. The presence of the α-tubulin isoform in complex with affinity purified proteasomes was also observed in the human embryonic kidney cell line 293.     

Regional distribution of α-neo-endorphin in rat brain and pituitary

α-Neo-endorphin was isolated as the first form of “big” Leu-enkephalin and its complete amino acid sequence has recently been established. Using an antiserum raised against synthetic α-neo-endorphin, a highly sentitive and specific radioimmunoassay was developed. The antiserum practically possesses no cross-reactivity to Leu-enkephalin, dynorphin[1–13] and PH-8P, and very little to β-neo-endorphin. Distribution of α-neo-endorphin has been determined in rat brain and pituitary by the use of the highly specific antiserum. The highest concentration was observed at posterior lobe of pituitary. Furthermore, immunoreactive α-neo-endorphin was characterized by gel-filtration and high performance liquid chromatography, and shown to be identical with authentic α-neo-endorphin.     

Distribution of an enzyme which acetylates α-melanocyte stimulating hormone in rat brain and pituitary gland and effects of arcuate nucleus lesions

This study describes the distribution of an α-melanocyte stimulating hormone (α-MSH) acetyltransferase (MAT) in rat brain and pituitary gland. Highest activities of MAT were found in the neurointermediate lobe of the pituitary gland with the anterior lobe containing slightly less. Within the brain, lowest MAT activities were measured in the hypothalamus, the region which contained the highest concentrations of α-MSH. Relatively high enzyme activities of MAT were measured in the hippocampus, cortex and cerebellum—regions with very low α-MSH concentrations. The fact that MAT activity levels did not parallel α-MSH concentrations indicates that MAT was not solely localized to α-MSH synthesizing neurons or endocrine cells. Furthermore, arcuate nucleus lesions which depleted brain α-MSH failed to deplete MAT activity. Although MAT was not solely localized to α-MSH synthesizing cells, it may have functional significance for α-MSH acetylation due to compartmentalization with α-MSH in α-MSH synthesizing endocrine cells and neurons. Alternatively a second regionally specific MAT may exist.     

Semi-constitutive expression of an Arabidopsis thaliana α-tubulin gene

In Arabidopsis tissues, the pool of tubulin protein is provided by the expression of multiple -tubulin and -tubulin genes. Previous evidence suggested that the TUA2 -tubulin gene was expressed in all organs of mature plants. We now report a more detailed analysis of TUA2 expression during plant development. Chimeric genes containing TUA2 5-flanking DNA fused to the -glucuronidase (GUS) coding region were used to create transgenic Arabidopsis plants. Second-generation progeny of regenerated plants were analyzed by histochemical assay to localize GUS expression. GUS activity was seen throughout plant development and in nearly all tissues. The blue product of GUS activity accumulated to the highest levels in tissues with actively dividing and elongating cells. GUS activity was not detected in a few plant tissues, suggesting that, though widely expressed, the TUA2 promoter is not constitutively active.     

Tubulin evolution: Two major types of α-tubulin

Summary Tubulin subunits have been isolated from a variety of protists and marine invertebrates. The sources were: sperm tails of a tunicate (Ciona intestinalis), an abalone (Haliotis rufescens) and a sea anemone (Tealia crassicornis), the gill cilia of a clam (Mercenaria mercenaria), the cilia of a ciliate (Tetrahymena pyriformis) and the cytoplasm of a slime mold (Physarum polycephalum). All the -tubulins, as characterised by their electropherograms after limited proteolytic cleavage withStaphylococcus aureus protease, were fairly similar. In contrast, two markedly different peptide patterns were found for the -tubulins of (a) metazoan axonemes and (b) protistan axonemes, plant axonemes and slime mold cytoplasm.Metazoan axonemal -tubulin peptide patterns could be further divided into two similar but distinct subtypes which did not correlate with the taxonomic divisions of deuterostomia and protostomia, or to different tubulins within an axoneme, or to different tubulins of flagella and cilia. We have postulated that these small differences may be accounted for by a simple glutamicaspartic acid exchange at a particular position in the -tubulin sequence. Identical peptide patterns were observed for sea urchin and sea anemone sperm tail tubulins, proving that the metazoan type of axonemal tubulin arose before the divergence of bilateral and radial symmetric organisms.The close similarity of the slime mold cytoplasmic -tubulin peptide pattern to protistan and plant axonemal -tubulin patterns suggests that the same type of tubulin might be used to form both axonemal and cytoplasmic types of microtubules in protists and plants. The large structural constraints imposed upon this tubulin molecule probably allowed very little change in its primary structure, thus explaining the similarity of tubulins from organisms which diverged at such an early time in eukaryote history. Duplication and modification of the tubulin gene may then have led to the development of specific axonemal and cytoplasmic microtubules during the evolution of the metazoa.     

Relationship between the Golgi complex and microtubules enriched in detyrosinated or acetylated α-tubulin: studies on cells recovering from nocodazole and cells in the terminal phase of cytokinesis

Double immunofluorescence microscopy was used to study the relationship between the Golgi complex and microtubules enriched in posttranslationally modified tubulins in cultured mouse L929 fibroblasts. In interphase cells, the elements of the Golgi complex were grouped around the microtubule-organizing center. From here, tyrosinated microtubules extended to the periphery of the cells, whereas the distribution of detyrosinated and acetylated microtubules largely overlapped with that of the Golgi complex. Treatment of cells with 10 M nocodazole led to the disruption of all microtubules and dispersion of the Golgi elements. Following withdrawal of the drug, tyrosinated microtubules reformed first, followed by acetylated and then detyrosinated microtubules. In parallel, the Golgi elements moved back toward the juxtanuclear region and reestablished a close spatial relationship first with the acetylated and later also with the detyrosinated microtubules. Long-term recovery in the presence of 0.15 or 0.3 M nocodazole allowed partial reformation of tyrosinated and acetylated microtubules, whereas no or only a few detyrosinated microtubules were detected. At the same time, the Golgi elements were grouped closer together around or on one side of the nucleus in close relation to acetylated microtubules. In synchronized cells released from a mitotic block, a radiating array of tyrosinated microtubules was first formed, followed by acetylated and detyrosinated microtubules. The Golgi elements initially came together in a few groups and thereafter took an overall morphology similar to that in interphase cells. During this reunification, they showed a close spatial relationship to acetylated microtubules, whereas detyrosinated microtubules appeared only later. Microtubules enriched in acetylated and/or detyrosinated tubulin thus appear to take part in establishing and maintaining the organization of the Golgi elements within an interconnected supraorganellar system. Whether the acetylation and detyrosination of tubulin are directly involved in this process or merely represent two modifications within this subpopulation of microtubules remains unknown.On leave of absence from the Department of Histology and Embryology, Institute of Biostructure, Medical School, Warsaw, Poland     

Proteomic analysis of importin α-interacting proteins in adult mouse brain

Many transport factors, such as importins and exportins, have been identified, and the molecular mechanisms underlying nucleocytoplasmic transport have been characterized. The specific molecules that are carried by each transport factor and the temporal profiles that characterize the movements of various proteins into or out of the nucleus, however, have yet to be elucidated. Here, we used a proteomic approach to identify molecules that are transported into the nuclei of adult mouse brain cells via importin α5. We identified 48 proteins in total, among which we chose seven to characterize more extensively: acidic (leucine-rich) nuclear phosphoprotein 32 family member A (Anp32a), far upstream element binding protein 1 (FUBP1), thyroid hormone receptor β1 (TRβ1), transaldolase 1, CDC42 effector protein 4 (CDC42-ep4), Coronin 1B, and brain-specific creatine kinase (CK-B). Analyses using green fluorescent protein (GFP)-fused proteins showed that Anp32a, FUBP1, and TRβ1 were localized in the nucleus, whereas transaldolase 1, CDC42-ep4, CK-B, and Coronin 1B were distributed in both the cytoplasm and nucleus. Using a digitonin-permeabilized in vitro transport assay, we demonstrated that, with the exception of CK-B, these proteins were transported into the nucleus by importin α5 together with importin β and Ran. Further, we found that leptomycin B (LMB) treatment increased nuclear CK-B-GFP signals, suggesting that CK-B enters the nucleus and is then exported in a CRM1-dependent manner. Thus, we identified a comprehensive set of candidate proteins that are transported into the nucleus in a manner dependent on importin α5, which enhances our understanding of nucleocytoplasmic signaling in neural cells.     

Assessment of α-synuclein secretion in mouse and human brain parenchyma

Genetic, biochemical, and animal model studies strongly suggest a central role for α-synuclein in the pathogenesis of Parkinson's disease. α-synuclein lacks a signal peptide sequence and has thus been considered a cytosolic protein. Recent data has suggested that the protein may be released from cells via a non-classical secretory pathway and may therefore exert paracrine effects in the extracellular environment. However, proof that α-synuclein is actually secreted into the brain extracellular space in vivo has not been obtained. We developed a novel highly sensitive ELISA in conjugation with an in vivo microdialysis technique to measure α-synuclein in brain interstitial fluid. We show for the first time that α-synuclein is readily detected in the interstitial fluid of both α-synuclein transgenic mice and human patients with traumatic brain injury. Our data suggest that α-synuclein is physiologically secreted by neurons in vivo. This interstitial fluid pool of the protein may have a role in the propagation of synuclein pathology and progression of Parkinson's disease.     

A tandem of α-tubulin genes preferentially expressed in radicular tissues from Zea mays

The identification of a cDNA (MR19) corresponding to a maize -tubulin and homologous genomic clones (MG19/6 and MG19/14) is described. The cDNA has been isolated by differential screening of a cDNA maize root library. We have found two -tubulin genes in a tandem arrangement in the genomic clones, separated by approximately 1.5 kbp. One of the genes (gene I) contains an identical nucleotide sequence which corresponds to the cDNA clone. The two deduced proteins from DNA sequences are very similar (only two conservative replacements in 451 amino acids) and they share a high homology as compared with the published -tubulin sequences from other systems and in particular with the Arabidopsis thaliana and Chlamydomonas reinhardtii sequences reported. The structure of both genes is also very similar; it includes two introns, of 1.7 kbp and 0.8 kbp respectively, in each gene and only one intron placed at a homologous position in relation to Arabidopsis thaliana genes. By using specific 3 probes it appears that both genes are preferentially expressed in the radicular system of the plant. The -tubulin gene family of Zea mays seems to be represented by at least 3 or 4 members.     

Distribution of α1-(PI) phenotypes in chromosome abnormalities

Summary PI phenotypes (including subtypes) were determined for 168 individuals with chromosomal abnormalities ascertained in Adelaide. These included patients with mosaicism, trisomy 21, trisomy 13, trisomy 18, and various sex chromosome aberrations (45,X, 47,XXX, 47,XXY, 47,XYY, and 48,XXXY). Data did not support an existing proposition that mildly deficient PI phenotypes predispose to abnormal chromosome segregation during mitosis or meiosis. Phenotypic distributions of each group were statistically similar to control populations of cord bloods and bloods donors.     

Regional heterogeneity in the ratio of α-MSH:β-endorphin in rat brain

The molar ratio of α-MSH:β-endorphin varies markedly among discrete microdissected regions of rat brain ranging from 0.57 in the median eminence to 2.74 in the lateral septum. This finding demonstrates that α-MSH and β-endorphin (β-END) are not uniformly distributed in a 1:1 molar ratio in rat brain as one might predict based on the consideration that the two peptides are synthesized in equimolar amounts as part of a common precursor molecule, pro-opiomelanocortin. The data indicate instead that the concentrations of α-MSH and β-END, the two predominant peptides expressed by opiomelantropinergic neurons, are independently regulated in rat brain. The heterogeneity of α-MSH:β-END ratios suggests that the regulation of α-MSH and β-END is regionally specific and may impart functional selectivity to the multisecretory opiomelanotropinergic neuronal system.