A Closer Look at Cell Invasion with Biosensors

Abstract

The Rho family of small GTPases control cell motility and migration, and are understood to participate in the regulation of cancer metastasis. Specifically, Rac1 GTPase is overexpressed in several tumors. Invadopodia are F-actin-rich protrusions with proteolytic activity that are exclusive to invasive tumor cells, and are thought to be crucial for cellular invasion and metastatic phenotypes. Based on previous literature, I hypothesized that the Rho GTPase Rac1 functions in the invadopodia of tumor cells. To investigate, I developed a new, genetically-encoded intramolecular Rac1 FRET (Forster Resonance Energy Transfer) biosensor. This new sensor is a substantial improvement over previous-generation Rac1 biosensors because of its single-chain arrangement, ensuring an equimolar distribution of FRET donor and acceptor, and producing a more accurate readout. Additionally, this design maintains the correct C-terminal lipid modification of full-length Rac1, enabling proper interaction with upstream regulators.;I used this new biosensor to observe the real-time activation dynamics of Rac1 at a subcellular level in live cancer cells. The biosensor readout shows Rac1 activity is excluded from the core of invadopodia, up to the point when invadopodia disappear, suggesting that a lack of Rac1 activity is necessary for their maintenance, and Rac1 activation is involved in disassembly. Focally uncaging Rac1 at pre-formed invadopodia using a photoactivation approach confirmed this previously-unknown Rac1 function. I integrated these results with existing literature to build an invadopodia disassembly model, where a signaling cascade starts with the regulator TrioGEF activating Rac1, triggering kinase activity of p21-activated kinase (PAK1), which phosphorylates cortactin, causing a structural destabilization and invadopodia dissolution. This mechanism may be critical for the proper turnover of the invasive structures during invasion of tumor cells in vivo, where a balance of invasive proteolytic activity and locomotory protrusions must be carefully coordinated to achieve a maximally invasive phenotype.;As an additional project, I designed and developed a new biosensor for the tyrosine kinase Src, which is well known for being necessary in invadopodia formation but its activation dynamics have not been shown directly. The biosensor can be used in the future as a reporter for Src activity in live tumor cells.