Phosphorylation on carboxyl terminus domains of neurofilament proteins in retinal ganglion cell neurons in vivo: influences on regional neurofilament accumulation, interneurofilament spacing, and axon caliber.
Phosphorylation on carboxyl terminus domains of neurofilament proteins in retinal ganglion cell neurons in vivo: influences on regional neurofilament accumulation, interneurofilament spacing, and axon caliber.
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DOI：
10.1083/jcb.126.4.1031
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发表时间：
1994-08
期刊：
影响因子：
--
通讯作者：
Thayer CY
中科院分区：
文献类型：
--
作者：
Nixon RA;Paskevich PA;Sihag RK;Thayer CY
神经丝的高分子量亚基NF-H和NF-M具有明显长的羧基末端结构域，其在新形成的神经丝进入轴突后变得高度磷酸化。我们已经调查了这个过程中的功能，正常的，未受干扰的视网膜神经节细胞的成熟小鼠神经元。使用[35 S]甲硫氨酸或[32 P]正磷酸盐进行体内脉冲标记，并使用磷酸化依赖性神经丝表位的单克隆抗体进行免疫细胞化学，我们表明，运输的神经丝的NF-H和NF-M亚基开始在一个离散的、非常接近的区域内达到成熟的磷酸化状态，该区域沿着距离眼睛150微米的视神经轴突。在这个过渡区近端或远端的七个水平中的每一个的1，700 - 2，500个视神经轴突的超微结构形态测定表明，在150微米水平，轴突口径扩大了三倍，然后在远端保持不变。神经丝的数量在100微米和150微米之间几乎翻了一番，在1,200微米的水平上进一步增加了三倍。微管数量仅增加30- 35%。神经丝之间的最小间距也几乎增加了一倍，平均间距从30 nm增加到55 nm。这些结果表明，羧基末端磷酸化通过引发轴突内神经丝的局部积累以及通过增加神经丝之间的强制性横向间距来扩大轴突口径。髓鞘形成，也开始于150微米的水平，可能是一个重要的影响，这些事件，因为没有局部神经丝积累或口径扩张发生沿着无髓鞘视神经轴突。这些研究结果提供的证据表明，羧基末端磷酸化触发神经丝侧臂的径向延伸，是一个关键的调节影响神经丝运输和局部形成的一个固定的，但动态的轴突细胞骨架网络。
The high molecular weight subunits of neurofilaments, NF-H and NF-M, have distinctively long carboxyl-terminal domains that become highly phosphorylated after newly formed neurofilaments enter the axon. We have investigated the functions of this process in normal, unperturbed retinal ganglion cell neurons of mature mice. Using in vivo pulse labeling with [35S]methionine or [32P]orthophosphate and immunocytochemistry with monoclonal antibodies to phosphorylation- dependent neurofilament epitopes, we showed that NF-H and NF-M subunits of transported neurofilaments begin to attain a mature state of phosphorylation within a discrete, very proximal region along optic axons starting 150 microns from the eye. Ultrastructural morphometry of 1,700-2,500 optic axons at each of seven levels proximal or distal to this transition zone demonstrated a threefold expansion of axon caliber at the 150-microns level, which then remained constant distally. The numbers of neurofilaments nearly doubled between the 100- and 150- microns level and further increased a total of threefold by the 1,200- microns level. Microtubule numbers rose only 30-35%. The minimum spacing between neurofilaments also nearly doubled and the average spacing increased from 30 nm to 55 nm. These results show that carboxyl- terminal phosphorylation expands axon caliber by initiating the local accumulation of neurofilaments within axons as well as by increasing the obligatory lateral spacing between neurofilaments. Myelination, which also began at the 150-microns level, may be an important influence on these events because no local neurofilament accumulation or caliber expansion occurred along unmyelinated optic axons. These findings provide evidence that carboxyl-terminal phosphorylation triggers the radial extension of neurofilament sidearms and is a key regulatory influence on neurofilament transport and on the local formation of a stationary but dynamic axonal cytoskeletal network.