Two-Color Spatial Cumulant Analysis Detects Heteromeric Interactions between Membrane Proteins.

TitleTwo-Color Spatial Cumulant Analysis Detects Heteromeric Interactions between Membrane Proteins.
Publication TypeJournal Article
Year of Publication2019
AuteursFoust, DJ, Godin, AG, Ustione, A, Wiseman, PW, Piston, DW
JournalBiophys J
Volume117
Issue9
Pagination1764-1777
Date Published2019 Nov 05
ISSN1542-0086
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.

DOI10.1016/j.bpj.2019.09.028
Alternate JournalBiophys. J.
PubMed ID31606123
PubMed Central IDPMC6838761
Grant ListT32 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