A general method to quantify ligand-driven oligomerization from fluorescence-based images
Authors: Ionel Popa
Dammar Badu
Michael R Stoneman
Graeme Milligan
Richard J Ward
Annie Eis
Valerică Raicu
Gabriel Biener
John D Pediani
Journal: Nature Methods (2019)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Fluorescence intensity fluctuation spectrometry; protein oligomers; abundance; stability; monomers
Affiliations: Physics Department, University of Wisconsin-Milwaukee, Milwaukee, WI, USA., Centre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK., Physics Department, University of Wisconsin-Milwaukee, Milwaukee, WI, USA. ., Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI, USA. .

Abstract:  Here, we introduce fluorescence intensity fluctuation spectrometry for determining the identity, abundance and stability of protein oligomers. This approach was tested on monomers and oligomers of known sizes and was used to uncover the oligomeric states of the epidermal growth factor receptor and the secretin receptor in the presence and absence of their agonist ligands. This method is fast and is scalable for high-throughput screening of drugs targeting protein-protein interactions. 

Retracted: No

PubPeer comments:
 I am confused by how the authors can get different brightness peaks from systems (membranes) which are clearly mixtures of many molecules, especially since in their Methods section they state that they extract a single brightness value per ROI. If the system is in equilibrium, shouldn t they extract always the same brightness from all the ROIs? And if not, how can they apply an FCS-derived method? , 
 You are right to expect single peaks for uniform mixtures of oligomers of different sizes. Cell membranes, however, are inghomogeneous and present lateral organization on all relevant size scales [Heberle, F. A. Feigenson, G. W. Phase separation in lipid membranes. Cold Spring Harb Perspect Biol 3, a004630 (2011); Sadegh, S. et al. Plasma Membrane is Compartmentalized by a Self-Similar Cortical Actin Meshwork. Phys Rev X 7, 011031 (2017); Demurtas, D. et al. Direct visualization of dispersed lipid bicontinuous cubic phases by cryo-electron tomography. Nat Commun 6, 8915 (2015)]. As one zooms in on a membrane different levels of inhomogeneity will be seen, following a fractal or self-similar pattern [Sadegh, S. et al. Plasma Membrane is Compartmentalized by a Self-Similar Cortical Actin Meshwork. Phys Rev X 7, 011031 (2017)]. Thus, even if the concentration of a large region of interest may be characterized by a single average number, locally (i.e., in each segment of an ROI) the concentration deviates from the average values sometimes by significant amounts. That means there is a distribution of oligomer sizes between different image segments of the membrane. , 
 #1 Hi Oligo, I ran into this paper a couple of weeks ago. We work a lot with oligomerisation of receptor tyrosine kinases. I tried running some sims with a software we have in the lab, simFCS. Unless the software is bogus, if you mix monoms and dimers, no way you are gonna see different brightness peaks. The conclusion seems that the membrane is heaps inhomogeneous, which is cool in a way, but also kinda hard to believe. Also I do not get the argument from the author: one thing is the variance, another the average. if you get a lot of variance from one zoom to the other, then you should get a broad peak, not many peaks (many different averages? But maybe I am just too stupid a biologist to understand this stuff. Anyway, cool that the topic of membrane receptor oligomerisation is making a comeback to big journals like Nature Method. Best X. , 
 I personally find Supplementary Figure 2 misleading: in the spectrograms of panel c, the authors always show the extreme cases, either pure monomer, dimer or pure aggregate. The point, would have been to show that in mixed cases (coexistence of monomers and dimers) one has multiple peaks in the brightness histogram. This is the basic claim about 2D-FIF: when multiple oligomeric species are present together, then one can resolve multiple bands in the spectrograms. I am inclined to think, reading also others comments, that probably they never saw multiple clear peaks even in their in-vitro, purified protein controls. I wonder why none of the reviewers asked for this basic controls. Concerned Scientist ,