Investigation of Rho Signaling Pathways in 3-D Collagen Matrices with Multidimensional Microscopy and Visualization Techniques

摘要

Spatial-temporal dynamics of proteins involved in cell-cell communication, cell-extracellular matrix interactions, and ultimately tissue organization are difficult to study using conventional biochemical approaches. Recent progress in the development of 3D culture models has provided a more physiologically relevant growth environment, in which breast cancer cells imbedded within floating collagen matrices undergo morphogenesis, in part, through contraction of the surrounding matrix. The importance of rho kinase (ROCK) generated contractility in this process has previously been demonstrated through antibody staining of cells imbedded in collagen matrices of differing rigidities and treatment with pharmacological ROCK inhibitors. We are stably transfecting T47D human breast cancer cells, cultured within floating collagen matrices, with fluorescent fusion proteins, and observing them through the course of morphogenesis (5-11 days). Spectral Lifetime Imaging Microscopy (SLIM) is used to separate second harmonic generated (SHG) signals from intrinsic and extrinsic fluorescence signals. Together these multidimensional signals map targeted regions of intercellular protein environment and their interaction with the extracellular matrix and can be applied to further studies of cell adhesion and motility.