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. Furthermore, 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 delta 1 labeled with green fluorescent protein on 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.
Bibliographical noteFunding Information:
This research was supported by the National Institutes of Health (NIH; R01 GM064589 and GM098550 ) and the National Science Foundation ( PHY-0957728 ). Melissa Gardner kindly loaned the objective collar heater used in this work. E.M.S. acknowledges support from the National Institute of Allergy and Infection Diseases , of NIH grant 5T32A1083196 (Minnesota Training Program in Virology), and a University of Minnesota Graduate School Doctoral Dissertation Fellowship .
© 2015 Elsevier Inc.
- Peripheral membrane protein
- Phospholipase C
- Pleckstrin homology domain
- Point spread function