UBC Theses and Dissertations
Rho/ROCK dependent mRNA translocation to tumor cell pseudopodia Stuart, Heather Christine
The psuedopodial fraction of MSV-MDCK-INV cells was isolated and proteomic analysis by LS-MS/S revealed the enrichment of a variety of cytoskeletal and adhesion proteins, glycolytic enzymes, proteins required for translation, ubiquitin/proteasome associated proteins, RNA binding proteins and cell signaling proteins. The selective enrichment of these proteins indicated the potential existence of mRNA in the pseudopodial domain. Propidium iodide labeling demonstrated the presence of mRNA in the pseudopodia. The localization of mRNA in the pseudopodia could be inhibited by treatment with Rho-kinase inhibitor, Y27632. Further investigation of the involvement of Rho/OCK signaling in the pseudopodia demonstrated local activation of RhoA. This activation occurred preferentially to the activation of another RhoGTPase family member, Rac1 . However, Rac1 activation could be reversed with Y27632 treatment indicating communication between signaling components in the pseudopodia. Photobleaching of actin and mRNA in the pseudopodia in the presence of Y27632 showed a decreased recovery time for mRNA but not actin to the bleached regions. This supports a role for Rho-kinase in pseudopodial mRNA translocation. Microarray analysis of the pseudopodial fraction disclosed the presence of a number of upregulated mRNAs. In situ hybridization with anti-sense oligonucleotides to β-actin, RhoA, Shp-2, m-ras and Arp2/3 p41 subunit confirmed the presence of these mRNA in the pseudopodia. Active transport of mRNA via the cytoskeleton to intracellular domains has been documented in a number of cell types. It was shown here by treatment with nocodazole that mRNA localization to MSV-MDCK-INV cell pseudopodia occurs independently of microtubules. Therefore the MSV-MDCK-INV tumor cell model displaying distinct actin rich pseudopodial domains is characterized by distinct protein and mRNA complements. The regulation of these domains is mediated by local activation of a Rho/ROCK signaling pathway that contributes to mRNA localization in a microtubule independent manner.