Automatic and Quantitative Measurement of Protein-Protein Colocalization in Live Cells

Abstract

We introduce a novel statistical approach that quantifies, for the first time, the amount of colocalization of two fluorescent-labeled proteins in an image automatically, removing the bias of visual interpretation. This is done by estimating simultaneously the maximum threshold of intensity for each color below which pixels do not show any statistical correlation. The sensitivity of the method was illustrated on simulated data by statistically confirming the existence of true colocalization in images with as little as 3% colocalization. This method was then tested on a large three-dimensional set of fixed cells cotransfected with CFP/YFP pairs of proteins that either co-compartmentalized, interacted, or were just randomly localized in the nucleolus. In this test, the algorithm successfully distinguished random color overlap from colocalization due to either co-compartmentalization or interaction, and results were verified by fluorescence resonance energy transfer. The accuracy and consistency of our algorithm was further illustrated by measuring, for the first time in live cells, the dissociation rate (k(d)) of the HIV-1 Rev/CRM1 export complex induced by the cytotoxin leptomycin B. Rev/CRM1 colocalization in nucleoli dropped exponentially after addition of leptomycin B at a rate of 1.25 x 10(-3) s(-1). More generally, this algorithm can be used to answer a variety of biological questions involving protein-protein interactions or co-compartmentalization and can be generalized to colocalization of more than two colors.

Tetraspanin Tspan9 regulates platelet collagen receptor GPVI lateral diffusion and activation

“…Background subtraction was performed in MATLAB using a rolling balling approach in which the radius of the ball was selected to be greater than image features (1 µm). A Costes’ approach was used to automate threshold selection for signal isolation and to determine P values for each z-stack [38], where the null hypothesis was randomly distributed signal within the region of interest. This region of interest was defined by manual segmentation of the platelets and collagen fibres using the interference reflectance channel.…”

Isthmin targets cell-surface GRP78 and triggers apoptosis via induction of mitochondrial dysfunction

“…Value of 1 represents perfect correlation, 0 means no correlation. 42,43 It has been reported that R p around 0.91 indicates that two images are almost identical, 44 and R p around 0.75 shows a relatively high degree of co-localization. 45 Tube formation assay.…”

Involvement of a Rac1-Dependent Macropinocytosis Pathway in Plasmid DNA Delivery by Electrotransfection

“…As shown by the images in Figure 3A, the majority of the electrotransfected pDNAs (blue color) co-localized with dextran (red color). We then quantified the correlation of the two channels by Manders' method, 57,58 which evaluates the fraction of all pixels in a z stack of confocal images from one channel that co-localizes with those from another channel by calculating the Manders' co-localization coefficient (MCC), which varies between 0 (no co-localization) and 1 (perfect co-localization). The Manders' M1 coefficient was 0.277 ± 0.062, indicating that approximately 28% of red pixels co-localized with blue pixels (see also Figure 3B).…”