MICROTUBULE REASSEMBLY FROM NUCLEATING FRAGMENTS DURING THE REGROWTH OF AMPUTATED NEURITES
MICROTUBULE REASSEMBLY FROM NUCLEATING FRAGMENTS DURING THE REGROWTH OF AMPUTATED NEURITES
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DOI：
10.1083/jcb.103.3.917
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发表时间：
1986-09-01
影响因子：
7.8
通讯作者：
HEIDEMANN, SR
中科院分区：
文献类型：
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作者：
BAAS, PW;HEIDEMANN, SR
我们已经提出，抵抗解聚的稳定微管（MT）片段可以作为轴突中MT动力学局部控制的成核元素（Heidemann，S。R.，M. A. Hamborg，S. J.托马斯，B。宋，S. Lindley和D. Chu，1984，J. Cell Biol.，99：1289-1295）。在这里，我们报告的证据，支持这一建议在研究中的作用，MT的再生的神经突起从远端节段的切断小鸡感觉神经突起。切断的神经突塌陷成轴浆的“珠”，其快速再生（Shaw，G.，和D. Bray，1977，Exp. Cell Res.，104：55-62）。我们检查了未阻止的再生长和MT分解后的再生长，2小时）或诺考达唑（0.1 μ g/ml，15-20分钟）。在所有这些情况下，再生长以3.5-4.5 μ m/min发生，除了达到37 ℃的时间之外没有延迟时间。C或冲洗掉诺考达唑。未处理的珠的电子显微镜照片显示许多不同长度的MT，而冷和nocodazole处理的珠显示显着较短的MT。神经突从仅含有非常短的MT的珠的稳健再生长反对完整的MT从珠展开到生长的神经突中。在未处理的完整神经突中引起大量MT解聚的条件下裂解的冷处理珠的电子显微照片显示出与未裂解的冷处理珠中的那些类似的持久MT片段。我们将此解释为冷处理珠中的MT片段与大多数解聚的MT质量不同的证据。用更高剂量的诺考达唑（1.0 μ g/ml，15-20分钟）处理的塌陷的神经突完全没有MT，并且在两个病例中仅在15-20分钟延迟后重新生长，但在另外11个病例中从未重新生长。我们发现，在用1.0 μ g/ml诺考达唑处理的珠粒中，即使在除去药物后30分钟，MT也没有恢复。这些珠不能重新组装MT不太可能是由于诺考达唑的不完全去除，因为高得多的剂量（20 μ g/ml诺考达唑）可以从完整的神经突上快速冲洗掉。然而，用1.0 μ g/ml诺考达唑处理的珠可以通过用紫杉醇处理而被刺激以重新组装MT并再生神经突。我们的结论是，立即，强大的再生轴突从轴质的崩溃珠需要MT成核网站，以支持MT重组。我们的数据表明，珠内的微管蛋白可以正常延长现有的MT，但相对于脑微管蛋白在体外具有有限的自我成核能力。
We have proposed that stable microtubule (MT) fragments that resist depolymerization may serve as nucleating elements for the local control of MT dynamics in the axon (Heidemann, S. R., M. A. Hamborg, S. J. Thomas, B. Song, S. Lindley, and D. Chu, 1984, J. Cell Biol., 99:1289-1295). Here we report evidence that supports this proposal in studies on the role of MTs in the regrowth of neurites from the distal segments of amputated chick sensory neurites. Amputated neurites collapse to "beads" of axoplasm that rapidly regrow (Shaw, G., and D. Bray, 1977, Exp. Cell Res., 104:55-62). We examined both unarrested regrowth and regrowth after MT disassembly by either cold (-5.degree. C for 2 h) or nocodazole (0.1 .mu.g/ml for 15-20 min). In all these cases regrowth occurred at 3.5-4.5 .mu.m/min with no delay times other than the times to reach 37.degree. C or rinse out the nocodazole. Electron micrographs of untreated beads show many MTs of varying lengths, while those of cold- and nocodazole-treated beads show markedly shorter MTs. The robust regrowth of neurites from beads containing only very short MTs argues against unfurling of intact MTs from the bead into the growing neurite. Electron micrographs of cold-treated beads lysed under conditions that cause substantial MT depolymerization in untreated intact neurites show persistent MT fragments similar to those in unlysed cold-treated beads. We interpret this as evidence that the MT fragments in cold-treated beads are somehow distinct from the majority of the MT mass that had depolymerized. Collapsed neurites treated with a higher dose of nocodazole (1.0 .mu.g/ml for 15-20 min) were completely devoid of MTs and regrew only after a 15-20 min delay in two cases but never regrew in 11 other cases. We found that MTs did not return in beads treated with 1.0 .mu.g/ml nocodazole even 30 min after removal of the drug. It was unlikely that the inability of these beads to reassemble MTs was due to incomplete removal of nocodazole in that a much higher dose (20 .mu.g/ml nocodazole) could be quickly rinsed from intact neurites. Beads treated with 1.0 .mu.g/ml nocodazole could, however, be stimulated to reassemble MTs and regrow neurites by treatment with taxol. We conclude that the immediate, robust regrowth of neurites from collapsed beads of axoplasm requires MT nucleation sites to support MT reassembly. Our data suggest that tubulin within the bead can elongate existing MTs normally but has a limited capacity for self-nucleation relative to brain tubulin in vitro.