Differences in the abilities of human tau isoforms to promote microtubule assembly.

Three isoforms of human tau protein were compared for their abilities to induce microtubule assembly. The three isoforms, tau 3 (tau containing three microtubule-binding domains), tau 4 (tau containing four microtubule-binding domains) and tau 4L (tau containing four microtubule binding domains plus a 58-amino-acid insert near the N-terminus) were expressed in E. coli and purified using ammonium sulfate precipitation, ion exchange, and size exclusion chromatography. All three isoforms induced microtubule assembly at micromolar concentrations and showed similar critical concentrations for assembly of 0.4-0.45 microM. However, tau 4 induced microtubule formation at a rate five- to tenfold faster than either tau 3 or tau 4L. The rate of microtubule elongation seen with tau 4 was twofold greater than with tau 3 or tau 4L, suggesting that the faster rate of microtubule assembly seen with tau 4 was due, at least in part, to faster elongation. Tau 4 induced a greater number of microtubules to form at steady state than did tau 3 or tau 4L. The microtubules generated with each tau isoform had similar steady-state length distributions and were equally susceptible to cold-induced disassembly. These results indicate that the additional microtubule-binding domain in tau 4 enhances microtubule assembly, while the 58-amino-acid insert negates the stimulatory effect of the fourth microtubule-binding domain.     

Microtubule-associated protein tau is required for axonal neurite elaboration by neuroblastoma cells.

NB2a/d1 neuroblastoma cells constitutively express multiple isoforms of the microtubule-associated protein tau and incorporate this protein into the axonal neurites elaborated during serum deprivation. To examine whether or not tau played an essential role in axonal outgrowth, cells cultured in serum-free medium were treated at 24 h intervals with antisense- and sense-oriented cDNA oligonucleotides (25 or 36 mers that span or are upstream of tau initiation codon) and were simultaneously serum deprived. Oligonucleotide uptake was confirmed by determination of intracellular levels of radiolabeled oligonucleotides. Treatment for 48 h with tau antisense oligonucleotides reversibly inhibited the expression of tau and the number of neurite-bearing cells compared with treatment with sense oligonucleotides. By contrast, tubulin expression was not affected. When cells were treated with antisense oligonucleotide simultaneously with serum deprivation, the initial outgrowth of neurites was unaffected, but continued neurite elongation was prevented. By contrast, neurite outgrowth at 4 h was inhibited when cells were pretreated with tau antisense 24 h before serum deprivation. Furthermore, intracellular delivery of anti-tau antiserum prevented neurite outgrowth and, in cells that had previously been deprived of serum for 24 h, induced retraction of existing neurites. These findings indicate that both the initiation and the continued outgrowth of neurites are dependent on tau and that pre-existing cytoplasmic pools of tau can mediate initial neuritogenesis.     

Microtubule dynamics in axons and dendrites.

We have investigated the stability, alpha-tubulin composition, and polarity orientation of microtubules (MTs) in the axons and dendrites of cultured sympathetic neurons. MT stability was evaluated in terms of sensitivity to nocodazole, a potent anti-MT drug. Nocodazole sensitivity was assayed by quantifying the loss of MT polymer as a function of time in 2 micrograms/ml of the drug. MTs in the axon and the dendrite exhibit striking similarities in their drug sensitivity. In both types of neurites, the kinetics of MT loss are biphasic, and are consistent with the existence of two types of MT polymer that depolymerize with half-times of MT polymer that depolymerize with half-times of approximately 3.5 min and approximately 130 min. We define the more rapidly depolymerizing polymer as drug-labile and the more slowly depolymerizing polymer as drug-stable. The proportion of MT polymer that is drug-stable is greater in axons (58%) than in dendrites (25%). On the basis of current understanding of the mechanism of action of nocodazole, we suggest that the drug-labile and drug-stable polymer observed in both axons and dendrites correspond to two distinct types of polymer that differ in their relative rates of turnover in vivo. In a previous study, we established that in the axon, these drug-stable and drug-labile types of MT polymer exist in the form of distinct domains on individual MTs, with the labile domain situated at the plus end of the stable domain (Baas and Black, J Cell Biol 111:495-509, 1990). Because of the great difference in drug sensitivity between the drug-labile and drug-stable MT polymer, we were able to dissect them apart by appropriate treatments with nocodazole. This permitted us to evaluate the drug-labile and drug-stable polymer in terms of polarity orientation and relative content of alpha-tubulin variants generated by posttranslational detyrosination or acetylation. In both the axon and the dendrite, the modified as well as unmodified alpha-tubulins are present in both drug-labile and drug-stable polymer, but at different levels. Specifically, the modified forms of alpha-tubulin are enriched in the drug-stable MT polymer compared to the drug-labile MT polymer. In studies on MT polarity orientation, we demonstrate that in axons, MTs are uniformly plus-end-distal, whereas in dendrites, MTs are non uniform in their polarity orientation, with roughly equal levels of the MTs having each orientation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Maturation and aging of the axonal cytoskeleton: biochemical analysis of transported tubulin.

Changes in solubility and axonal transport of tubulin during maturation and aging have been investigated using sciatic motor fibers of rats at 4, 7, 14, 30, and 80 weeks of age. One to six weeks after injection of L-[35S]methionine into the spinal cord, labeled cytoskeletal proteins in consecutive segments of the sciatic nerve and the ventral roots were fractionated into soluble and insoluble forms by extraction in 1% Triton at low temperature. In 4-week-old rats, the two forms of tubulin were transported coordinately in a single wave with the average rate of 2 mm/day. At 7 weeks of age, two components in tubulin transport were observed to develop, possibly reflecting the maturation of the axonal cytoskeleton. The slower main component (1.5 mm/day) contained most of the insoluble form together with the neurofilament proteins and the faster component (3 mm/day) was enriched in the soluble form. Though significantly different in composition, the two components correspond to slow component a (SCa) and slow component b (SCb) originally defined in the optic system. A progressive decrease in transport rates of both SCa and SCb was observed with rats at 14, 30, and 80 weeks of age. In addition, there was a large decrease in the proportion of insoluble tubulin during the course of transport in animals older than 30 weeks. This loss of the insoluble form seems to be accounted for partly by the proteolytic degradation of the severely retarded SCa proteins. Changes in axonal transport of tubulin may thus reflect age-related changes in dynamics and turnover of the axonal cytoskeleton.     

低温冷冻对皮肤α-tubulin、β-tubulin和cytoskeratin表达的影响

目的:探讨低温冷冻对皮肤骨架蛋白中α-微管蛋白(α-tubu lin)、β-微管蛋白(β-tubu lin)和高分子量角蛋白(cytoskeratin)表达的影响,从而进一步揭示皮肤低温损伤的机制。方法:用-αtubu lin、β-tubu lin、cytoskeratin抗体对5种储存条件下的皮肤作免疫组化染色,采用计算机图像分析技术进行定量分析。并进一步采用透射电镜观察-αtubu lin、β-tubu lin、cytoskeratin的超微结构的变化。结果:α-tubu lin、β-tubu lin、cytoskeratin在4℃组、-20℃组、-80℃组均表达显著下降,且超微形态结构及分布改变明显。在-196℃组储存时三种成分含量下降较少,形态改变亦不显著。结论:皮肤的低温损伤与骨架蛋白-αtubu lin、β-tubu lin、cytoskeratin的破坏有关。     

Comparison of protein tyrosine phosphorylation and morphological changes induced by IL-2 and IL-3.

We constructed cell lines which can proliferate in response to IL-2 or IL-3 by introducing a wild-type and mutant forms of cDNAs encoding the human IL-2R p75 chain into an IL-3 dependent hematopoietic cell line which expresses the p55 chain of the IL-2R. We compared early events that were induced in these cells by IL-2 and IL-3. Analysis of protein tyrosine phosphorylation showed that two common protein bands, pp95 and pp90, were phosphorylated by stimulation of either IL-2 or IL-3, suggesting the possible sharing of part of a signal transduction pathway between IL-2R and IL-3R. Comparison of protein tyrosine phosphorylation profiles induced by IL-2 and IL-3 among a variety of cell lines revealed that the pp90 band is the common tyrosine phosphorylation substrate in the cell lines examined, although the general tyrosine phosphorylation pattern differed in each cell line. Mutant p75 molecules incapable of inducing tyrosine phosphorylation could bind and internalize IL-2, but could not support cell growth. We also found that swift changes of cytoskeletal protein organization are one of the early events caused by signal transduction through either IL-2R and IL-3R. Reorganization of cytoskeletal proteins seems to be associated with protein phosphorylation, as a significant portion of pp90 was found in a detergent-soluble fraction in IL-2 or IL-3 treated cells.     

Crystal Containing Membrane Bound Intracellular Inclusion Bodies in the Epithelium of Experimentally Infected Sinus Mucosa

The sinus mucosa of 16 rabbits was experimentally infected with Bacteroides fragilis. This paper describes and discusses large inclusion bodies, which were found in abundance by light and electron microscopy inside ciliated cells of the sinus epithelium in 3 of the studied animals. The spindle-shaped inclusions were located in the apical portion of the cytoplasm. They were bound by a trilaminar membrane with several coils to the interior as well as to the exterior. The interior of an inclusion body consisted to a large extent of electron-lucent, floccular substance, but fibrogranular aggregates and rod-shaped crystals with a line periodicity center-to-center of about 15 nm were also conspicuous. These peculiar formations may be constituted by abnormally stored material from defective synthesis of cilia.     

Noggin signaling from Xenopus animal blastomere lineages promotes a neural fate in neighboring vegetal blastomere lineages.

In Xenopus, localized factors begin to regionalize embryonic fates prior to the inductive interactions that occur during gastrulation. We previously reported that an animal-to-vegetal signal that occurs prior to gastrulation promotes primary spinal neuron fate in vegetal equatorial (C-tier) blastomere lineages. Herein we demonstrate that maternal mRNA encoding noggin is enriched in animal tiers and at low concentrations in the C-tier, suggesting that the neural fates of C-tier blastomeres may be responsive to early signaling from their neighboring cells. In support of this hypothesis, experimental alteration of the levels of Noggin from animal equatorial (B-tier) or BMP4 from vegetal (D-tier) blastomeres significantly affects the numbers of primary spinal neurons derived from their neighboring C-tier blastomeres. These effects are duplicated in blastomere explants isolated at cleavage stages and cultured in the absence of gastrulation interactions. Co-culture with animal blastomeres enhanced the expression of zygotic neural markers in C-tier blastomere explants, whereas co-culture with vegetal blastomeres repressed them. The expression of these markers in C-tier explants was promoted when Noggin was transiently added to the culture during cleavage/morula stages, and repressed with the transient addition of BMP4. Reduction of Noggin translation in B-tier blastomeres by antisense morpholino oligonucleotides significantly reduced the efficacy of neural marker induction in C-tier explants. These experiments indicate that early anti-BMP signaling from the animal hemisphere recruits vegetal equatorial cells into the neural precursor pool prior to interactions that occur during gastrulation.     

Extraskeletal myxoid chondrosarcoma characterized by microtubular aggregates in the rough endoplasmic reticulum and tubulin immunoreactivity

A case is reported of a 66-year-old female with an extraskeletal myxoid chondrosarcoma which had originated in the lateral region of the right knee. The tumour tissue of the primary, recurrent, and metastatic deposits in the lungs was examined by electron microscopy and immunohistochemistry. Almost all the sarcoma cells in every tumour specimen harboured immunoreactivity to both alpha- and beta-subunits of S-100 protein. A large population of cells in the subcutaneous tumour at autopsy had numerous parallel arrays of microtubules within the rough endoplasmic reticulum in addition to the well-described ultrastructural features indicative of chondroblastic origin. These structures were present in round to polygonal, but not in fibroblastic, tumour cells. Tubulin immunoreactivity in the tumour cells showed the same tendency, being frequently positive in the large cells of the subcutaneous tumour but weakly positive in the fibroblastic and medium-sized cells of the recurrent and metastatic tumours. The parallel arrays of intracisternal microtubules therefore may be composed of tubulin protein, as in ordinary cytoplasmic microtubules.     

细胞骨架蛋白tubulin α、β在不同级别胶质瘤中的表达及其与预后的相关性

目的探讨不同病理学分级胶质瘤中细胞骨架蛋白tubulin α、β的表达,评估tubulin α、β表达水平与胶质瘤的恶性程度及其与预后的相关性.方法应用免疫组化方法检测57例不同病理级别人胶质瘤组织中tubulin α、β表达情况,并分析tubulin α、β表达水平与病理分级及患者术后生存时间的相关性.结果不同级别胶质瘤tubulin α、β表达水平不同,高级别（Ⅲ、Ⅳ级）与低级别（Ⅰ、Ⅱ级）胶质瘤tubulin α、β表达水平的差异有显著性（P〈0.05）,且tubulin α、β表达水平与患者术后生存时间呈正相关性.结论 tubulin α、β在胶质瘤恶性进展方面可能具有重要意义,对指导胶质瘤的分期、组织学分级和判断患者的预后有一定参考价值.