Post-Translational Tubulin Modifications in Human Astrocyte Cultures (original) (raw)
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Post-Translational Tubulin Modifications in Differentiated Human Neural Stem Cells
ABSTRACTThe tubulin protein fulfills a variety of cellular functions that range from chromosomal separation to locomotion. Functional diversity is achieved through the expression of specific tubulin isotypes in different cell types or developmental time periods. Post-translational modifications (PTMs) of tubulin also are vital for specific intracellular tasks, such as binding and recruiting motor proteins. In neurons, the isotypic expression profile for tubulin is well characterized, and the importance of PTMs for proper neuronal function has gained recent attention due to their implication in neurodegenerative disorders. In contrast, the role of tubulin specializations in the specification of neural cell fate has received minimal attention and studies of tubulin PTMs and isotypes in neuroglia such as astrocytes are relatively few. To bridge this knowledge gap, we undertook an analysis of PTMs in neurons and astrocytes derived from the federally approved H9 hESC-derived human neural...
Expression of the Class III β-tubulin isotype in developing neurons in culture
Journal of Neuroscience Research, 1992
The expression of the class I11 P-tubulin isotype was studied in cultured brain neurons by means of a monoclonal antibody (TuJ1). The results obtained indicate that during early axonal outgrowth most of the class I11 p-tubulin is not incorporated into microtubules, a phenomenon which is also observed under conditions which alter the rate and extent of the neurite outgrowth response. On the other hand, a dramatic increase in its incorporation into microtubules is observed after the neurons have differentiated their neurites as axons and dendrites. In addition, the appearance of colchicine-resistant microtubules containing this isotype, a phenomenon which occurs late in neurite development, is highly coincident with the appearance of stable microtubules containing high molecular weight microtubule-associated proteins (MAPs). This pattern is different from that of the accumulation and incorporation of other P-tubulin isotypes into microtubules. Taken collectively, our results indicate that differences exist in the in vivo utilization of tubulin isotypes in developing brain neurons and suggest that the class I11 P-tubulin isotype is not a primary factor involved in the regulation of microtubule assembly during early neurite outgrowth, but that it may be important for maintaining further neurite elongation and/or determining some unique binding property of MAPs to specific microtubule subsets.
J Neuropathol Exp Neurol, 2008
Class III A-tubulin isotype (AIII-tubulin) is widely regarded as a neuronal marker in developmental neurobiology and stem cell research. To test the specificity of this marker protein, we determined its expression and distribution in primary cultures of glial fibrillary acidic protein (GFAP)-expressing astrocytes isolated from the cerebral hemispheres of 2 human fetuses at 18 to 20 weeks of gestation. Cells were maintained as monolayer cultures for 1 to 21 days without differentiation induction. By immunofluorescence microscopy, coexpression of AIII-tubulin and GFAP was detected in cells at all time points but in spatially distinct patterns. The numbers of GFAP + cells gradually decreased from Days 1 to 21 in vitro, whereas AIII-tubulin immunoreactivity was present in 100% of cells at all time points. A-III-tubulin mRNA and protein expression were demonstrated in cultured cells by reverse-transcriptase-polymerase chain reaction and immunoblotting, respectively. Glial fibrillary acidic protein þ /A-III-tubulin-positive cells coexpressed nestin and vimentin but lacked neurofilament proteins, CD133, and glutamateaspartate transporter. Weak cytoplasmic staining was detected with antibodies against microtubule-associated protein 2 isoforms. Confocal microscopy, performed on autopsy brain samples of human fetuses at 16 to 20 gestational weeks, revealed widespread colocalization of GFAP and AIII-tubulin in cells of the ventricular/ subventricular zones and the cortical plate. Our results indicate that in the midgestational human brain, AIII-tubulin is not neuron specific because it is constitutively expressed in GFAP + /nestin + presumptive fetal astrocytes.
?IV tubulin is selectively expressed by oligodendrocytes in the central nervous system
Glia, 2005
Oligodendrocyte differentiation and myelination involve dramatic changes in cell signaling pathways, gene expression patterns, cell shape, and cytoskeletal organization. In a pilot study investigating CNS angiogenesis, oligodendrocytes were intensely labeled by antisera directed against the C-terminal of Tie-2, a 140-kDa transmembrane receptor for angiopoietin. Immunoprecipitation of rat brain proteins with Tie-2 C-terminal antisera, however, produced a single spot of *55-kDa pI *5 by two-dimensional (2D) electrophoresis, which was identified as b-tubulin by mass spectrometry. Isotype-specific antibodies for b IV tubulin selectively labeled oligodendrocytes. First detected in premyelinating oligodendrocytes, b IV tubulin was abundant in myelinating oligodendrocyte perinuclear cytoplasm and processes extending to and along developing myelin internodes. b IV tubulin-positive MTs were diffusely distributed in oligodendrocyte perinuclear cytoplasm and not organized around the centrosome. b IV tubulin may play a role in establishing the oligodendrocyte MT network, which is essential for the transport of myelin proteins, lipids, and RNA during myelination. V V C 2005 Wiley-Liss, Inc.
Class III A-tubulin isotype (AIII-tubulin) is widely regarded as a neuronal marker in developmental neurobiology and stem cell research. To test the specificity of this marker protein, we determined its expression and distribution in primary cultures of glial fibrillary acidic protein (GFAP)-expressing astrocytes isolated from the cerebral hemispheres of 2 human fetuses at 18 to 20 weeks of gestation. Cells were maintained as monolayer cultures for 1 to 21 days without differentiation induction. By immunofluorescence microscopy, coexpression of AIII-tubulin and GFAP was detected in cells at all time points but in spatially distinct patterns. The numbers of GFAP + cells gradually decreased from Days 1 to 21 in vitro, whereas AIII-tubulin immunoreactivity was present in 100% of cells at all time points. A-III-tubulin mRNA and protein expression were demonstrated in cultured cells by reverse-transcriptase-polymerase chain reaction and immunoblotting, respectively. Glial fibrillary acidic protein þ /A-III-tubulin-positive cells coexpressed nestin and vimentin but lacked neurofilament proteins, CD133, and glutamateaspartate transporter. Weak cytoplasmic staining was detected with antibodies against microtubule-associated protein 2 isoforms. Confocal microscopy, performed on autopsy brain samples of human fetuses at 16 to 20 gestational weeks, revealed widespread colocalization of GFAP and AIII-tubulin in cells of the ventricular/ subventricular zones and the cortical plate. Our results indicate that in the midgestational human brain, AIII-tubulin is not neuron specific because it is constitutively expressed in GFAP + /nestin + presumptive fetal astrocytes.
Journal of Neuropathology and Experimental Neurology, 2008
Class III A-tubulin isotype (AIII-tubulin) is widely regarded as a neuronal marker in developmental neurobiology and stem cell research. To test the specificity of this marker protein, we determined its expression and distribution in primary cultures of glial fibrillary acidic protein (GFAP)-expressing astrocytes isolated from the cerebral hemispheres of 2 human fetuses at 18 to 20 weeks of gestation. Cells were maintained as monolayer cultures for 1 to 21 days without differentiation induction. By immunofluorescence microscopy, coexpression of AIII-tubulin and GFAP was detected in cells at all time points but in spatially distinct patterns. The numbers of GFAP + cells gradually decreased from Days 1 to 21 in vitro, whereas AIII-tubulin immunoreactivity was present in 100% of cells at all time points. A-III-tubulin mRNA and protein expression were demonstrated in cultured cells by reverse-transcriptase-polymerase chain reaction and immunoblotting, respectively. Glial fibrillary acidic protein þ /A-III-tubulin-positive cells coexpressed nestin and vimentin but lacked neurofilament proteins, CD133, and glutamateaspartate transporter. Weak cytoplasmic staining was detected with antibodies against microtubule-associated protein 2 isoforms. Confocal microscopy, performed on autopsy brain samples of human fetuses at 16 to 20 gestational weeks, revealed widespread colocalization of GFAP and AIII-tubulin in cells of the ventricular/ subventricular zones and the cortical plate. Our results indicate that in the midgestational human brain, AIII-tubulin is not neuron specific because it is constitutively expressed in GFAP + /nestin + presumptive fetal astrocytes.
Journal of Cellular Biochemistry, 1991
We describe the presence of a-tubulin and MAP2 acetyltransferase activities in mouse brain. The enzyrne(s) copurified with microtubules through two cycles of assembly-disassembly. Incubation of microtubule proteins with [3H]acetyl CoA resulted in a strong labeling of both a-tubulin and MAPZ. To determine the site of the modification, tubulin was purified and digested with GIu-C endoproteinase. A unique radioactive peptide was detected and purified by HPLC. Edman degradation sequencing showed that this peptide contained EN-acetyllysine at position 40 of the a-tubulin molecule. This result demonstrates that mouse brain a-tubulin was acetylated at the same site as in Chlamydomonas. lsoelectric focusing analysis showed that acetylated a-tubulin was resolved into five isoelectric variants, denoted a3 and a5 to a8. This heterogeneity is not due to acetylation of other sites but results from a single acetylation of Lys4' of an heterogeneous population of a-tubulin isoforms. These isoforms are produced by posttranslational addition of one to five glutamyl units. Thus, neuronal a-tubulin is extensively modified by a combination of modifications including acetylation, glutamylation, tyrosylation, and other yet unknown modifications.
Association of brain g-tubulins with ab-tubulin dimers
Biochemical …, 2002
g-Tubulin is necessary for nucleation and polar orientation of microtubules in vivo. The molecular mechanism of microtubule nucleation by g-tubulin and the regulation of this process are not fully understood. Here we show that there are two g-tubulin forms in brain that are present in complexes of various sizes. Large complexes tend to dissociate in the presence of high-salt concentration. Both g-tubulins copolymerized with tubulin dimers and multiple g-tubulin bands were identified in microtubule protein preparations under conditions of non-denaturing electrophoresis. Immunoprecipitation experiments with monoclonal antibodies against g-tubulin and a-tubulin revealed interactions of both g-tubulin forms with tubulin dimers irrespective of the size of complexes. We suggest that besides small and large g-tubulin complexes, other molecular g-tubulin form(s) exist in brain extracts. Twodimensional electrophoresis revealed multiple charge variants of g-tubulin both in brain extracts and in microtubule protein preparations. Post-translational modification(s) of gtubulins might therefore play an important role in the regulation of microtubule nucleation in neuronal cells.