Cytoplasmic Structuring by Non-motor Microtubule Associated Proteins (MAPs)

[603730] Olmsted提出的研究的长期目标是了解非马达的微管相关蛋白如何参与微管的功能和组织以及细胞质的结构。这些研究的重点是MAP 4，这是一种微管相关蛋白，在小鼠的早期发育和特定成年组织中表达。目前的假设是，这种无处不在的蛋白质的功能是与环境相关的；循环细胞中的map4结构和功能可能与分化组织中的map4结构和功能不同。基本方法将是剖析由选择性剪接或翻译后修饰自然产生的变异如何影响细胞内MAP 4的行为。在嵌合体中，map4构建体在对应于特征翻译后修饰的位点上进行修饰，或反映在特定组织中观察到的初级序列变异。用绿色荧光蛋白或其光谱变体标记的MAP 4构建物转染活细胞后，将进行分析，以推断MAP结构的变化如何影响间期和有丝分裂细胞的微管动力学。细胞质组织的其他方面，包括细胞骨架元件或主要膜细胞器的分布，也将进行分析，以评估MAP 4的特定结构域是否可能与非微管结构相互作用。这些研究应该提供关于这种普遍存在的蛋白质是否根据其表达位置具有多种作用的基本信息。微管是真核细胞中三个主要的细胞骨架系统之一，也是已知参与细胞内运动现象的两个这样的系统之一，作为atp驱动的分子马达（动力蛋白和动力蛋白）运动的特定“轨迹”。除了马达蛋白外，其他特异性蛋白（“微管相关蛋白”或MAPs）已知与微管结合，尽管它们的功能还远未确定。奥姆斯特德博士将对其中一种被称为MAP-4的map进行功能研究。由于选择性剪接（细胞“读取”基因的不同方式）和/或翻译后修饰，MAP-4蛋白的结构在不同的组织中是不同的；然而，这些变异的功能意义尚不清楚。这些功能研究将采用最先进的方法，如定点诱变和“绿色荧光蛋白”（一种标记物，可以让人们在显微镜下确切地看到MAP-4蛋白在细胞中的位置）。越来越多的证据表明，这些不同的细胞骨架系统不是独立的，而是在细胞内的功能方面相互依赖的。这些研究的结果有望帮助我们了解MAP-4的功能及其在协调微管功能与各种细胞结构（包括其他细胞骨架系统）中的可能作用。＊＊＊

9603730 Olmsted The long range goal of the proposed research is to understand how microtubule- associated proteins that are not motors participate in the function and organization of microtubules and the structuring of the cytoplasm. These studies focus on MAP 4, a microtubule-associated protein that is expressed during early development and in specific adult tissues in mice. The working hypothesis is that the functions of this ubiquitous protein are context-specific; MAP 4 structure and functions in cycling cells may differ from those in differentiated tissues. The basic approach will be to dissect how variations which naturally arise either by alternative splicing or by post-translational modification affect MAP 4 behavior within cells. Chimeras will be generated in which MAP 4 constructs are modified at sites corresponding to characterized post-translational modifications or which reflect primary sequence variations observed in specific tissues. Living cells transfected with MAP 4 constructs tagged with green fluorescent protein or its spectral variants will be analyzed to deduce how such changes in MAP structure affect microtubule dynamics in interphase and mitotic cells. Other aspects of cytoplasmic organization, including the distribution of cytoskeletal elements or major membranous organelles, will also be analyzed to assess whether specific domains of MAP 4 may interact with non-microtubular structures. These studies should provide fundamental information on whether this ubiquitous protein has multiple roles depending on its place of expression. Microtubules comprise one of the three major cytoskeletal systems in eukaryotic cells, and one of two such systems known to be involved in intracellular motility phenomena by serving as specific "tracks" along which ATP-powered molecular motors (dyneins and kinesins) move. In addition to the motor proteins, other specific proteins ("Microtubule- Associated Proteins, or MAPs) are known bind to microtubules, although their functions are far from established. Dr. Olmsted will carry out functional studies of one of these MAPs, termed MAP-4. The structure of the MAP-4 protein is known to vary among different tissues, as a result of alternative splicing (different ways in which the cell "reads" the gene) and/or posttranslational modifications; however, the functional significance of these variations is unknown. These functional studies will employ state-of-the-art approaches such as site-directed mutagenesis and "green fluorescent protein" (a marker that allows one to literally see, in a microscope, exactly where the MAP-4 protein is located within the cell). Evidence increasingly suggests that these various cytoskeletal systems are not independent, but rather are dependent upon one another in terms of their function within cells. The results of these studies are expected to help us understand MAP-4 function and its possible role in coordinating microtubule function with various cellular structures, including other cytoskeletal systems. ***