Role of Messenger RNA Location in Cell Orientation and Movement

9316508富尔顿细胞中的中间细丝与机械应变和抵抗力的能力有关。vimentin的mRNA是定位的，在发育过程中改变位置，在伤口愈合过程中在成纤维细胞中保留在中心体周围。这一提议背后的驱动假说是，正确的mRNA位置是静脉蛋白细丝组装和定位所必需的。该提案解决了三个问题：1)vimentin mRNA的3' UTR是否足以将mRNA引导到中心体区域？2)波形蛋白mRNA是否需要在中心体区域产生允许细胞定向和运动的波形蛋白丝？3)机械应力下的细胞是否对正确定位的vimentin mRNA和蛋白有更高的要求？构建合适的质粒并将其转染成纤维细胞，并通过原位杂交显示mRNA的位置。转染细胞的单层将被损伤，细胞在定向和迁移过程中的行为将被测量，无论有或没有机械应力。一些细胞骨架蛋白的mRNA是定位的，这表明这些蛋白的合成可能发生在适合功能或组装的位点。只有当细胞骨架mRNA定位错误时，细胞骨架蛋白的定位功能才会被发现。成纤维细胞在细胞定向和运动过程中的机械要求行为为mRNA定位的功能意义提供了强有力的测试。几种细胞骨架蛋白的信息集中在细胞的某些部分，这表明这些蛋白质可能需要在正确的地方制造才能发挥作用或组装。只有当细胞骨架mrna定位错误时，细胞骨架蛋白的信息定位功能才会被发现。单元格的指向和移动提供了一个很好的机会来了解消息位置的功能意义。* * *

9316508 Fulton Intermediate filaments in cells are associated with mechanical strain and the ability to resist forces. The mRNA for vimentin is localized, changes position during development and in fibroblasts remains around the centrosome during wound healing. The driving hypothesis behind this proposal is that correct mRNA location is required for the assembly and location of the vimentin filaments. The proposal addresses three questions: 1) Is the 3' UTR of vimentin mRNA sufficient to direct mRNA to the centrosomal region? 2) Does vimentin mRNA need to be in the centrosomal region to produce vimentin filaments that permit cellular orientation and movement? 3) Do cells under mechanical stress have greater requirements for properly located vimentin mRNA and protein?. Appropriate plasmids will be constructed and transfected into fibroblasts and the location of mRNA will be shown by in situ hybridization. Monolayers of transfected cells will be wounded and cell behavior during orientation and migration will be measured, either with or without mechanical stress. The mRNA's of several cytoskeletal proteins are localized, suggesting that synthesis of these proteins may occur at sites appropriate for function or assembly. The function of location for cytoskeletal proteins will only be known when cytoskeletal mRNA's are mislocalized. The mechanically demanding behavior of fibroblasts during cell orientation and movement offers a strong test of the functional significance for mRNA location. %%% The messages for several cytoskeletal proteins are concentrated in parts of the cell, suggesting that these proteins may need to be made at the right place for them to function or assemble. The function for message location for cytoskeletal proteins will only be known when cytoskeletal mRNAs are mislocalized. Cells pointing and moving offer a good opportunity to see the functional significance of message location. ***