Z-scan fluorescence profile deconvolution of cytosolic and membrane-associated protein populations
Analytical Biochemistry, 2015
The Extracellular Matrix (ECM) is composed of collagen, non-collagenous glycoproteins and proteoglycans. These components are secreted from cells to create an ECM meshwork that surrounds cells and tissues. The ECM regulates many aspects of cellular function, including the cells' dynamic behavior, cytoskeletal organization and intercellular communication. Fibronectin and Laminin constitute the majority of the dynamic portion of the extracellular matrix of tissues and they are essential components of the culturing technique of many cell types.
This study introduces a technique that characterizes the spatial distribution of peripheral membrane proteins that associate reversibly with the plasma membrane. An axial scan through the cell generates a z-scan intensity profile of a fluorescently labeled peripheral membrane protein. This profile is analytically separated into membrane and cytoplasmic components by accounting for both the cell geometry and the point spread function. We experimentally validated the technique and characterized both the resolvability and stability of z-scan measurements. Further, using the cellular brightness of green fluorescent protein, we were able to convert the fluorescence intensities into concentrations at the membrane and in the cytoplasm. We applied the technique to study the translocation of the pleckstrin homology domain of phospholipase C-delta1 labeled with green fluorescent protein upon ionomycin treatment. Analysis of the z-scan fluorescence profiles revealed protein-specific cell height changes and allowed for comparison between the observed fluorescence changes and predictions based on the cellular surface area to volume ratio. The quantitative capability of z-scan fluorescence profile deconvolution offers opportunities for investigating peripheral membrane proteins in the living cell that were previously not accessible.
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