Investigating obscurin's modulation through the RhoA pathway.

Abstract

Obscurin is a large cytoskeletal protein found in epithelial and muscle cells. It is the second most mutated protein in breast and colorectal cancers and is significantly downregulated in pancreatic cancer. Obscurin activates ROCK via RhoA, which in turn activates myosin, stimulates F-actin polymerization, and modulates cellular motility and migration. Likewise, obscurin knockdown leads to an epithelial to mesenchymal transition (EMT), a hallmark of cancer progression. Obscurin is not known to be modulated in any way by any part of this downstream RhoA pathway, yet it seems likely that a cytoskeletal protein would be affected by cell motility and motion. Here we begin to describe the relationship between global obscurin structure and individual components of this downstream pathway, including cell architecture and cell motility. We show that our model system, MDCK cells, express either no or very little obscurin. Upon the introduction of mini-obscurin constructs containing a precalibrated FRET-based force sensor, we see that obscurin localizes around the cell membrane in a dose-dependent manner. In normal cells, all obscurin constructs under >5 pN of force. When either the actin or myosin cytoskeleton is disrupted, the cells show a characteristic round morphology and obscurin is under significantly less force. However, when ROCK is inhibited, obscurin tension remains unchanged yet cells show morphological differences. This suggests that obscurin tension is not directly modulated by the pathway which it controls. Instead some other external force, possibly physical or specifically directed at the actin/myosin cell architecture, modulates obscurin tension and thus influences global obscurin architecture. Experiments are underway to disambiguate this stimulus.