Raster Image Correlation Spectroscopy [RICS] Analysis of HeLa cells

The objective of the project is to use the RICS analysis technique in complement with confocal microscopy to determine the diffusion coefficient of the selectively labeled green fluorescent protein (GFP), GFP-EGFR and GFP-p53 in cervical cancer cells. This is a collaboration work with MD Anderson Cancer Center. The application of the study is to lay the foundation for further study in understanding the cell metabolism, subcellular morphologic and dynamic biochemical processes to aid in the diagnosis and to potentially screen cancers. Fluorescence microscopy techniques have been developed for the study of cellular processes and molecular signal pathway. However, the spatial resolution to distinguish and resolve the interactions of single molecular complexes or molecule in cells is limited by the wavelength. Hence, indirect image correlation methods complementary to the imaging techniques were developed to obtain the dynamic information within the cell. RICS is one such mathematical image processing method to determine the dynamics of the cell. HeLa cells are transfected with GFP to highlight the protein of interest. These samples were imaged with confocal microscope, Olympus FV1000 with a 60 x 1.2 NA water objective in the pseudo photon counting mode with an excitation of 488 nm argon ion laser. About 100 frames of scan area 256x256 pixels were collected from each sample at scan speed of 12.5 seconds per pixel. The stacks of images were processed with SimFCS software. The images were subjected to immobile subtraction algorithm to remove the immobile features. Consequently, each frame in the stack is subjected to 2D-correlation; then, the average 2D-spatial correlation is calculated. This 2D-spatial correlated data constitutes as RICS data which is then displayed and analyzed by fitting it to specific models. This generates a spatial temporal map of the molecular dynamics of fluorescently labeled probes within the cell. In summary, we apply RICS techniques based on correlation spectroscopy to the image data and quantify diffusion coefficient of protein of interest in cancerous cells with different treatments. This is expected to better understand cellular dynamics of cancerous cells and build better diagnostic biosensor devices for early screening

Mechanical Signaling Induced Cellular Remodeling Studied By Integrated Optical And Atomic Force Microscopy

Vascular wall composition and mechanics are important for cardiovascular physiology and pathology. The reciprocal interaction between cells and their microenvironment influence cellular adaptation to external mechanical cues through the remodeling of cytoskeletal structures and cell–matrix adhesions to ensure normal cell function. We proposed to investigate the relationship between the cytoskeletal tension development and cell adhesion to the matrix in the context of cellular contraction and migration. Our studies aimed to understand how cells sense, respond, and adapt to external mechanical forces in order to induce vascular remodeling in cardiovascular disease. Integration of atomic force microscopy with total internal reflection fluorescence and spinning-disk confocal microscopy enabled acquisition of complementary structural and functional measurements on live vascular smooth muscle cells expressing key mutant proteins with important roles in defining contractile and migratory cellular properties. Single ligand–receptor interaction measurements showed that RhoA and c-Src activation have different effects on cytoskeletal tension development, inducing two distinct force–stiffness functional regimes for α5β1-integrin binding to fibronectin. In addition, c-Src was associated with regulation of myosin light chain phosphorylation, suggesting a c-Src-dependent modulation of RhoA pathway through activation of downstream effectors. These data were in good agreement with fluorescence measurements that showed a modest effect of Src activation on stress fibers formation, in contrast with RhoA activation that had a significant effect. On the other hand, α-actin null cells exhibited increased FAK activation and cell stiffness. Our results suggest that the absence of α-actin may induce compensatory effects of up-regulation of other contractile proteins and activation of focal adhesion proteins in order to encourage cell migration and proliferation. In addition, our findings suggest that Nck regulates directional cell migration in part through modulation of cytoskeletal tension and cell-matrix adhesion strength, which has an important role in coordination of cytoskeletal mechanics through a mechanism that also involves the RhoA pathway. Thus, our findings suggest that the contractile state of the cell is determined by cytoskeletal tension, which is controlled by a regulatory network involving RhoA and activation state of actomyosin apparatus. In turn, the cytoskeletal tension state modulates integrin α5β1–fibronectin adhesion force. The results of this study suggest a central role for cytoskeletal tension in modulating cytoskeletal dynamics and cell adhesion to the matrix

Smooth muscle hyperplasia due to loss of smooth muscle α-actin is driven by activation of focal adhesion kinase, altered p53 localization and increased levels of platelet-derived growth factor receptor-β

Mutations in ACTA2, encoding the smooth muscle cell (SMC)-specific isoform of α-actin (α-SMA), cause thoracic aortic aneurysms and dissections and occlusive vascular diseases, including early onset coronary artery disease and stroke. We have shown that occlusive arterial lesions in patients with heterozygous ACTA2 missense mutations show increased numbers of medial or neointimal SMCs. The contribution of SMC hyperplasia to these vascular diseases and the pathways responsible for linking disruption of α-SMA filaments to hyperplasia are unknown. Here, we show that the loss of Acta2 in mice recapitulates the SMC hyperplasia observed in ACTA2 mutant SMCs and determine the cellular pathways responsible for SMC hyperplasia. Acta2−/− mice showed increased neointimal formation following vascular injury in vivo, and SMCs explanted from these mice demonstrated increased proliferation and migration. Loss of α-SMA induced hyperplasia through focal adhesion (FA) rearrangement, FA kinase activation, re-localization of p53 from the nucleus to the cytoplasm and increased expression and ligand-independent activation of platelet-derived growth factor receptor beta (Pdgfr-β). Disruption of α-SMA in wild-type SMCs also induced similar cellular changes. Imatinib mesylate inhibited Pdgfr-β activation and Acta2−/− SMC proliferation in vitro and neointimal formation with vascular injury in vivo. Loss of α-SMA leads to SMC hyperplasia in vivo and in vitro through a mechanism involving FAK, p53 and Pdgfr-β, supporting the hypothesis that SMC hyperplasia contributes to occlusive lesions in patients with ACTA2 missense mutation