Electrostatic interactions drive molecular assembly and organization in the plasma membrane. Specific protein-lipid interactions, however, are difficult to resolve. Here we report on a unique approach to investigate these interactions with time-resolved fluorescence spectroscopy. The experiments were performed on a model membrane system consisting of a supported lipid bilayer with an asymmetric distribution of PIP2 in the upper leaflet of the bilayer. The bilayer also contained nickel-chelating lipids that bind to a histidine-tagged peptide of interest. Both the peptide and the lipid were labeled with orthogonal fluorescent probes, so that diffusion and binding could be measured with two-color, pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). Our PIE-FCCS data showed significant lipid-peptide cross-correlation between PIP2 lipids and membrane-bound cationic peptides. Cross-correlation is a direct indication of lipid-peptide binding and complexation. Together with mobility data, we quantified the degree of binding, which offers new insight into this class of lipid-peptide interactions. Overall, this is the first report of lipid-peptide cross-correlation by FCCS, and provides a new route to quantifying the interactions between proteins and lipid membranes, a key interface in cell signaling.
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Four-color fluorescence cross-correlation spectroscopy with one laser and one camera
The diffusion and reorganization of phospholipids and membrane-associated proteins are fundamental for cellular function. Fluorescence cross-correlation spectroscopy (FCCS) measures diffusion and molecular interactions at nanomolar concentration in biological systems. We have developed an economical method to simultaneously monitor diffusion and complexation with the use of super-continuum laser and spectral deconvolution from a single detector. Customizable excitation wavelengths were chosen from the wide-band source and spectral fitting of the emitted light revealed the interactions for up to four chromatically overlapping fluorophores simultaneously. This method was applied to perform four-color FCCS that we demonstrated with polystyrene nanoparticles, lipid vesicles, and membrane-bound molecules. Up to four individually customizable excitation channels were selected from the broad-spectrum fiber laser to excite the diffusers within a diffraction-limited spot. The fluorescence emission passed through a cleanup filter and a dispersive prism prior to being collected by a sCMOS or EMCCD camera with up to 1.8 kHz frame rates. The emission intensity versus time of each fluorophore was extracted through a linear least-square fitting of each camera frame and temporally correlated via custom software. Auto- and cross-correlation functions enabled the measurement of the diffusion rates and binding partners. We have measured the induced aggregation of nanobeads and lipid vesicles in solution upon increasing the buffer salinity. Because of the adaptability of investigating four fluorophores simultaneously with a cost-effective method, this technique will have wide application for examining macromolecular complex formation in model and living systems.
more » « less- Award ID(s):
- 1652316
- NSF-PAR ID:
- 10427467
- Publisher / Repository:
- Optical Society of America
- Date Published:
- Journal Name:
- Biomedical Optics Express
- Volume:
- 14
- Issue:
- 7
- ISSN:
- 2156-7085
- Page Range / eLocation ID:
- Article No. 3812
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Background Commonly used methods to measure whole gut transit time in rodents have yet to combine high sensitivity, objectivity, and automation. We have developed a novel method using oral gavage of non‐toxic fluorescent dye particles and their detection by fluorescence imaging to enable unbiased automated detection of gut transit time simultaneously in 8 cages.
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