Supplement request for Cellular mechanotransduction - from the immune response to transcriptional regulation

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Cell-cell and cell-substrate interactions, mediated by adhesion and signaling receptors, are highly dynamic and subject to cytoskeletal movements that impart substantial mechanical force at the interface. How cells combine mechanical and biochemical signals to carry out specific functions is not well understood. Supported by this MIRA award, we are examining how mechanical cues are relayed to the nucleus to regulate gene expression in a functionally appropriate manner and how mechanical cues interact with tissue-specific cues. This requires an imaging system capable of multicolor single-molecule imaging with high signal to noise ratio deep within the cell nucleus. Highly Inclined Laminated Optical Sheet (HILO) Microscopy is an imaging technology that allows for visualization of single molecules deep within the cell interior with nanometer scale resolution. Single molecule imaging of transcription factor mobility and binding yields a dynamic view of gene regulation at the single cell level, thus complementing high throughput genomic studies. In preliminary studies, we have shown that transcription factors and histones exhibit multiple low mobility states, indicative of a complex interaction between the heterogeneous dynamics of chromatin and TF binding to these chromatin mobility modes. In order to study how cells integrate mechanical and chemical cues, we are using hydrogels of tunable stiffness to examine the regulation of transcription by mechanical cues. While conventional Total Internal Reflection Fluorescence (TIRF) capable microscopes can be adapted to HILO imaging of cells on glass coverslips, imaging cells on hydrogels (30-50 micron thickness) poses a challenge due to the thickness and the excess scattering of light within the intervening gel. Furthermore, the relatively low power lasers within our setup limits the signal to noise ratios necessary for tracking single molecules at fast imaging rates, making the analysis of TF diffusion and chromatin dynamics at sub-second timescales difficult. This supplement request is to upgrade the HILO microscopy capabilities of our current TIRF microscope by adding an advanced azimuthal TIRF module, higher power lasers and pixel-registered multicolor single molecule imaging modules. As single molecule imaging is directly related to all of our original Aims, this enhanced capability will inform all our proposed studies. Moreover, the high speed imaging capability will enable us to apply our newly developed algorithms to study transcription factor mobility and chromatin interactions. Our studies of mechanosensing and the regulation of transcription by mechanical properties of the cellular microenvironment and the underlying pathways will advance our understanding of breast and other cancers.