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Fluorescence fluctuation spectroscopy (FFS) encompasses a bevy of techniques that involve analyzing fluorescence intensity fluctuations occurring due to fluorescently labeled molecules diffusing in and out of a microscope's focal region. Statistical analysis of these fluctuations may reveal the oligomerization (i.e., association) state of said molecules. We have recently developed a new FFS‐based method, termed Two‐Dimensional Fluorescence Intensity Fluctuation (2D FIF) spectrometry, which provides quantitative information on the size and stability of protein oligomers as a function of receptor concentration. This article describes protocols for employing FIF spectrometry to quantify the oligomerization of a membrane protein of interest, with specific instructions regarding cell preparation, image acquisition, and analysis of images given in detail. Application of the FIF Spectrometry Suite, a software package designed for applying FIF analysis on fluorescence images, is emphasized in the protocol. Also discussed in detail is the identification, removal, and/or analysis of inhomogeneous regions of the membrane that appear as bright spots. The 2D FIF approach is particularly suited to assess the effects of agonists and antagonists on the oligomeric size of membrane receptors of interest. © 2022 Wiley Periodicals LLC.

Basic Protocol 1: Preparation of live cells expressing protein constructs

Basic Protocol 2: Image acquisition and noise correction

Basic Protocol 3: Drawing and segmenting regions of interest

Basic Protocol 4: Calculating the molecular brightness and concentration of individual image segments

Basic Protocol 5: Combining data subsets using a manual procedure (Optional)

Alternate Protocol 1: Combining data subsets using the advanced FIF spectrometry suite (Optional; alternative to Basic Protocol 5)

Basic Protocol 6: Performing meta‐analysis of brightness spectrograms

Alternate Protocol 2: Performing meta‐analysis of brightness spectrograms (alternative to Basic Protocol 6)

Basic Protocol 7: Spot extraction and analysis using a manual procedure or by writing a program (Optional)

Alternate Protocol 3: Automated spot extraction and analysis (Optional; alternative to Protocol 7)

Support Protocol: Monomeric brightness determination

 

DLC1 locks talin R8 in a mechanically stable state, potentially preventing the inside-out activation of integrins. 

The quaternary organization of rhodopsin-like G protein-coupled receptors in native tissues is unknown. To address this we generated mice in which the M 1 muscarinic acetylcholine receptor was replaced with a C-terminally monomeric enhanced green fluorescent protein (mEGFP)–linked variant. Fluorescence imaging of brain slices demonstrated appropriate regional distribution, and using both anti-M 1 and anti–green fluorescent protein antisera the expressed transgene was detected in both cortex and hippocampus only as the full-length polypeptide. M 1 -mEGFP was expressed at levels equal to the M 1 receptor in wild-type mice and was expressed throughout cell bodies and projections in cultured neurons from these animals. Signaling and behavioral studies demonstrated M 1 -mEGFP was fully active. Application of fluorescence intensity fluctuation spectrometry to regions of interest within M 1 -mEGFP–expressing neurons quantified local levels of expression and showed the receptor was present as a mixture of monomers, dimers, and higher-order oligomeric complexes. Treatment with both an agonist and an antagonist ligand promoted monomerization of the M 1 -mEGFP receptor. The quaternary organization of a class A G protein-coupled receptor in situ was directly quantified in neurons in this study, which answers the much-debated question of the extent and potential ligand-induced regulation of basal quaternary organization of such a receptor in native tissue when present at endogenous expression levels.