FUNCTIONAL ROLES OF THE NF-H NEUROFILAMENT PROTEIN

神经细胞的细胞结构主要由两部分组成： 结构成分：微管和神经丝（NF）。 而 微管在神经突延伸中的功能以及作为 颗粒进入轴突的运输是公认的，没有同伴 NF的功能，神经元的中间丝，被证明。 然而，我们发现，亚基的合成水平 NF蛋白与哺乳动物神经的直径直接相关 纤维 这使我们提出NF基因表达是一个 轴突口径的主要决定因素，它本身指定 轴突的传导速度。 我们现在建议结合使用 体外和体内分子遗传学来测试这种假定的功能 的NF。 我们的方法将集中在NF-H多肽，最大的 三个NF亚基。 而其他亚基（NF-L和NF-M）是 更丰富，NF-H的长羧基末端尾部结构域具有长 据推测，交叉连接相邻的NF，并可能确定 NF的横向间距。 从克隆的小鼠NF-H基因出发， 将使用DNA转染来确定NF-H的组装特性 当单独表达或与NF-L和NF-M组合表达时。 因为在 轴突NF-H被严重磷酸化，其羧基末端结构域 携带>50个重复的假定磷酸化位点，我们还将 用这种缺失的突变型NF-H基因进行类似的实验， 其他领域。 最后，研究了这种突变型NF-H的体内性质。 将通过产生转基因小鼠来评估多肽， 含有专门设计增加或减少NF-H 表情

The cytoarchitecture of nerve cells is comprised principally of two structural components: microtubules and neurofilaments (NF). While the function of microtubules in neurite extension and as substrates for transport of particles into the axon is well accepted, no companion function for NF, the intermediate filaments of neurons, is proven. However, we have found that the level of synthesis of the subunit proteins of NF directly correlates with the diameters of mammalian nerve fibers. This has lead us to propose that NF gene expression is a principal determinator of axonal caliber, which itself specifies conduction velocity of the axon. We now propose to use a combination of in vitro and in vivo molecular genetics to test this putative function of NF. Our approach will focus on the NF-H polypeptide, the largest of the three NF subunits. While the other subunits (NF-L and NF-M) are more abundant, the long carboxy-terminal tail domain OF NF-H has long been speculated to cross link adjacent NFs and perhaps to determine the lateral spacing of NFs. Starting from the cloned mouse NF-H gene, we shall use DNA transfection to determine the assembly properties of NF-H when expressed alone or in combination with NF-L and NF-M. Since in axons NF-H is heavily phosphorylated and its carboxy-terminal domain carries >50 repeats of a putative phosphorylation site, we will also perform similar experiments with mutant NF-H genes deleted in this and other domains. Finally, the in vivo properties of such mutant NF-H polypeptides will be assessed by producing transgenic mice whose genomes contain genes specifically engineered to increase or decrease NF-H expression.