ROLES OF THE MYELIN BASIC PROTEINS IN MYELINOGENESIS
ROLES OF THE MYELIN BASIC PROTEINS IN MYELINOGENESIS
批准号:
3418016
负责人:
DAVID R COLMAN
金额:
$17.62万
依托单位国家:
美国
项目类别:
财政年份:
1992
资助国家:
美国
项目状态:
已结题
起止时间:
1992-09-01 至 1993-03-31
关键词:
binding proteins complementary DNA developmental neurobiology gene expression genetic manipulation genetic mapping immunocytochemistry immunoelectron microscopy laboratory mouse myelin myelin basic proteins oligodendroglia protein sequence protein structure function radiotracer site directed mutagenesis tissue /cell culture transfection /expression vector
中文摘要
我们的长期目标是了解
成熟髓鞘的形成,在认识到这可能
建议修复过程中可以鼓励髓鞘后的方法,
杀伤性 实现这一目标的一个重要步骤是
精确定义髓鞘碱性蛋白所起的功能作用
(MBP)在髓鞘形成计划的过程中,该计划由
少突胶质细胞 对于下面概述的许多研究,我们将依赖于
我们已经开发和完善了cDNA转染系统,
在过去的两年中,采用特异性MBP抗血清和共聚焦显微镜,
免疫显微镜以绘制表达的
proteins. 我们的具体目标是:
I.鉴定14K和18.5K MBP内的功能结构域,
介导这些同种型与质膜的结合。
使用定点诱变,我们将(1)删除内部片段或
(2)截短14K和18.5K MBP cDNA的N或C末端,和
在非神经胶质细胞中表达突变的构建体,
少突胶质细胞 每个突变的细胞内分布
多肽将被免疫细胞化学定位,以确定最小
多肽长度和序列所需的膜协会。 在
平行地,(3)突变的多肽将被表达并在大肠杆菌中测试。
生物化学和超微结构实验的能力,结合,
来自颤抖的脑质膜的聚集体定向囊泡。
II.开始探索含外显子II的21.5K
和17KMBP从它们在细胞中的合成位点移位
细胞质穿过核孔复合体进入核基质。
在非神经胶质细胞和shiverer小鼠少突胶质细胞中,
在21.5K或17K MBP cDNA中,表达的蛋白质存在于21.5K或17K MBP cDNA中。
细胞质和核质。 值得注意的是,在正常发育的小鼠中,
在脑中,容易检测到少突胶质细胞,其核含有高浓度的
MBPs的浓度。 在许多细胞类型中,
一级氨基酸序列中含有的元素
作为核定位信号(NLS),
通过核孔复合体进入核室,
易位的蛋白质可以发挥调节功能。 我们有
在含有外显子II的MBP亚型中发现了一个区域,
与已知的NLS有很强的同源性。 我们将测试它的容量,
这些MBP中的其他序列作为瞬时NLS起作用,
转染子 严格定义NLS的三个标准,
其他系统将被应用:(1)NLS介导的MBP进入
核很可能是一个需要能量的过程,因此应该
对(a)冷却或(B)ATP耗竭的破坏敏感;(2)
MBP NLS序列的缺失或突变应引起细胞质内
突变蛋白的积累;和(3)当推定的MBP NLS
序列被工程化到不相关的非核蛋白质中,例如
牛血清白蛋白,嵌合蛋白应被易位到
原子核
英文摘要
Our long range goal is to understand the molecular mechanisms that underlie
the formation of the mature myelin sheath, in the realization that this may
suggest ways by which the repair process can be encouraged after myelin
destruction. An essential step towards achievement of this goal is to
precisely define the functional roles played by the myelin basic proteins
(MBPs) over the course of the myelinogenesis program that is implemented by
the oligodendrocyte. For many of the studies outlined below, we will rely
on the cDNA transfection systems we have developed and refined over the
past two years, that employ specific MBP antisera and confocal
immunomicroscopy to map intracellular distributions of the expressed
proteins. Our specific aims are:
I.To identify the functional domains within the 14K and 18.5K MBPs that
mediate association of these isoforms with the plasma membrane.
Using site-directed mutagenesis, we will (1) delete internal segments or
(2) truncate the N or C terminus of the 14K and 18.5K MBP cDNAs, and
express the mutated constructs in non-glial cells, and in shiverer
oligodendrocytes. the intracellular distribution of each mutated
polypeptide will be mapped immunocytochemically to determine minimum
polypeptide lengths and sequences required for membrane association. In
parallel, (3) the mutated polypeptides will be expressed and tested in
biochemical and ultrastructural experiments for the capacity to bind to and
aggregate oriented vesicles derived from shiverer brain plasma membranes.
II.To begin to explore the mechanisms by which the exon II-containing 21.5K
and 17K MBPs are translocated from their site of synthesis in the cell
cytoplasm across the nuclear pore complex into the nuclear matrix.
In non-glial cells, and in shiverer mouse oligodendrocytes transfected with
the 21.5K or 17K MBP cDNAs, the expressed proteins are found within the
cytoplasm and nucleoplasm. Significantly, in the normal developing mouse
brain, oligodendrocytes are readily detected whose nuclei contain high
concentrations of MBPs. In many cell types, proteins that are translocated
into the nucleus contain elements within their primary amino acid sequences
that act as nuclear localization signals (NLSs) which enable selective
entry through the nuclear pore complex into the nuclear compartment where
the translocated proteins can exert regulatory functions. We have
identified a region in the exon II-containing MBP isoforms that bears
strong homology to known NLSs. We will test the capacity of this, and
other sequences in these MBPs to function as authentic NLSs in transient
transfectants. Three criteria that are used to rigorously define NLSs in
other systems will be applied: (1) NLS-mediated entry of MBP into the
nucleus is likely to be an energy-requiring process, and therefore should
be sensitive to disruption by (a) chilling, or (b) ATP depletion; (2)
deletion or mutation of MBP NLS sequence(s) should cause cytoplasmic
accumulation of the mutated protein; and (3) when the putative MBP NLS
sequence is engineered into an unrelated non-nuclear protein, such as
bovine serum albumin, the chimeric protein should be translocated into the
nucleus.
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会议论文
GLIAL MEMBRANES AT THE NODE OF RANVIER PREVENT NEURITE OUTGROWTH
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批准号:7420807
-
项目类别:
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资助金额:$0.29万
-
财政年份:2006
-
负责人:DAVID R COLMAN
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依托单位:
ACTIN-BINDING PROTEINS IN A POSTSYNAPTIC PREPARATION: LASP-1 IS A COMPONENT OF
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批准号:7420656
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项目类别:
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资助金额:$0.29万
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财政年份:2006
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负责人:DAVID R COLMAN
-
依托单位:
ACTIN-BINDING PROTEINS IN A POSTSYNAPTIC PREPARATION: LASP-1 IS A COMPONENT OF
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批准号:7182312
-
项目类别:
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资助金额:$0.4万
-
财政年份:2005
-
负责人:DAVID R COLMAN
-
依托单位:
Cytoplasmic transport of mRNAs in the myelin sheath
-
批准号:6890388
-
项目类别:
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资助金额:$4.09万
-
财政年份:2003
-
负责人:DAVID R COLMAN
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依托单位:
Cytoplasmic transport of mRNAs in the myelin sheath
-
批准号:6755188
-
项目类别:
-
资助金额:$4.06万
-
财政年份:2003
-
负责人:DAVID R COLMAN
-
依托单位:
Cytoplasmic transport of mRNAs in the myelin sheath
-
批准号:6642458
-
项目类别:
-
资助金额:$4.03万
-
财政年份:2003
-
负责人:DAVID R COLMAN
-
依托单位:
Protein Components of the Synaptic Adhesive Scaffold
-
批准号:6339702
-
项目类别:
-
资助金额:$42.38万
-
财政年份:2001
-
负责人:DAVID R COLMAN
-
依托单位:
Protein Components of the Synaptic Adhesive Scaffold
-
批准号:6639781
-
项目类别:
-
资助金额:$42.38万
-
财政年份:2001
-
负责人:DAVID R COLMAN
-
依托单位:
Protein Components of the Synaptic Adhesive Scaffold
-
批准号:6540462
-
项目类别:
-
资助金额:$42.38万
-
财政年份:2001
-
负责人:DAVID R COLMAN
-
依托单位:
Protein Components of the Synaptic Adhesive Scaffold
-
批准号:6896374
-
项目类别:
-
资助金额:$42.38万
-
财政年份:2001
-
负责人:DAVID R COLMAN
-
依托单位:
Protein Components of the Synaptic Adhesive Scaffold
-
批准号:6761858
-
项目类别:
-
资助金额:$42.38万
-
财政年份:2001
-
负责人:DAVID R COLMAN
-
依托单位:
FUNCTIONAL ORGANIZATION OF THE MYELIN AXOGLIAL JUNCTION
-
批准号:6764142
-
项目类别:
-
资助金额:$21.6万
-
财政年份:2000
-
负责人:DAVID R COLMAN
-
依托单位:
FUNCTIONAL ORGANIZATION OF THE MYELIN AXOGLIAL JUNCTION
-
批准号:6197616
-
项目类别:
-
资助金额:$33.9万
-
财政年份:2000
-
负责人:DAVID R COLMAN
-
依托单位:
FUNCTIONAL ORGANIZATION OF THE MYELIN AXOGLIAL JUNCTION
-
批准号:6895741
-
项目类别:
-
资助金额:$21.6万
-
财政年份:2000
-
负责人:DAVID R COLMAN
-
依托单位:
FUNCTIONAL ORGANIZATION OF THE MYELIN AXOGLIAL JUNCTION
-
批准号:6540345
-
项目类别:
-
资助金额:$33.9万
-
财政年份:2000
-
负责人:DAVID R COLMAN
-
依托单位:
FUNCTIONAL ORGANIZATION OF THE MYELIN AXOGLIAL JUNCTION
-
批准号:6394535
-
项目类别:
-
资助金额:$33.9万
-
财政年份:2000
-
负责人:DAVID R COLMAN
-
依托单位:
CADHERINS EXPRESSED DURING NERVE MYELINATION
-
批准号:6112522
-
项目类别:
-
资助金额:$17.54万
-
财政年份:1999
-
负责人:DAVID R COLMAN
-
依托单位:
MYELIN PROTEIN ZERO--MECHANISMS OF MEMBRANE ADHESION
-
批准号:6394203
-
项目类别:
-
资助金额:$24.28万
-
财政年份:1999
-
负责人:DAVID R COLMAN
-
依托单位:
CORE--LABORATORY ANIMAL
-
批准号:6112525
-
项目类别:
-
资助金额:$17.54万
-
财政年份:1999
-
负责人:DAVID R COLMAN
-
依托单位:
MYELIN PROTEIN ZERO--MECHANISMS OF MEMBRANE ADHESION
-
批准号:6188301
-
项目类别:
-
资助金额:$23.57万
-
财政年份:1999
-
负责人:DAVID R COLMAN
-
依托单位:
海外基金