Title | Two-Color Spatial Cumulant Analysis Detects Heteromeric Interactions between Membrane Proteins. |
Publication Type | Journal Article |
Year of Publication | 2019 |
Authors | Foust, DJ, Godin, AG, Ustione, A, Wiseman, PW, Piston, DW |
Journal | Biophys J |
Volume | 117 |
Issue | 9 |
Pagination | 1764-1777 |
Date Published | 2019 Nov 05 |
ISSN | 1542-0086 |
Abstract | Fluorescence fluctuation spectroscopy can be used to measure the aggregation of fluorescently labeled molecules and is typically performed using time series data. Spatial intensity distribution analysis and fluorescence moment image analysis are established tools for measuring molecular brightnesses from single-color images collected with laser scanning microscopes. We have extended these tools for analysis of two-color images to resolve heteromeric interactions between molecules labeled with spectrally distinct chromophores. We call these new methods two-color spatial intensity distribution analysis and two-color spatial cumulant analysis (2c-SpCA). To implement these techniques on a hyperspectral imaging system, we developed a spectral shift filtering technique to remove artifacts due to intrinsic cross talk between detector bins. We determined that 2c-SpCA provides better resolution from samples containing multiple fluorescent species; hence, this technique was carried forward to study images of living cells. We used fluorescent heterodimers labeled with enhanced green fluorescent protein and mApple to quantify the effects of resonance energy transfer and incomplete maturation of mApple on brightness measurements. We show that 2c-SpCA can detect the interaction between two components of trimeric G-protein complexes. Thus, 2c-SpCA presents a robust and computationally expedient means of measuring heteromeric interactions in cellular environments. |
DOI | 10.1016/j.bpj.2019.09.028 |
Alternate Journal | Biophys. J. |
PubMed ID | 31606123 |
PubMed Central ID | PMC6838761 |
Grant List | T32 EB014855 / EB / NIBIB NIH HHS / United States R01 DK115972 / DK / NIDDK NIH HHS / United States T32 DK108742 / DK / NIDDK NIH HHS / United States P60 DK020579 / DK / NIDDK NIH HHS / United States P30 DK020579 / DK / NIDDK NIH HHS / United States R56 DK098659 / DK / NIDDK NIH HHS / United States R01 DK098659 / DK / NIDDK NIH HHS / United States |