Recent advances in quantitative colocalization analysis: Focus on neuroscience

Zinchuk, Vadim S.; Grossenbacher-Zinchuk, Olga

doi:10.1016/j.proghi.2009.03.001

Cited by 157 publications

(139 citation statements)

References 93 publications

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“…To quantify the extent of colocalization of the CNGA3 C319R mutant channel with the plasma membrane, we calculated the Manders' overlap coefficient. 34 In comparison with the wild-type protein, the homomeric CNGA3 C319R mutant channel showed a significantly reduced Manders' overlap coefficient, indicating that the integration of CNGA3 C319R channels into the plasma membrane was reduced due to impaired protein folding and/ or trafficking (Figure 5c). Taken together with calcium imaging results, our functional analyses revealed that in vitro the p.Cys319Arg alteration in the S5 region severely affects the stability and function of the encoded mutant CNGA3 channel.…”

Section: Variant Of Cnga3 Causes Crd Rs Shaikh Et Almentioning

confidence: 99%

Homozygous missense variant in the human CNGA3 channel causes cone-rod dystrophy

et al. 2014

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We assessed a large consanguineous Pakistani family (PKAB157) segregating early onset low vision problems. Funduscopic and electroretinographic evaluation of affected individuals revealed juvenile cone-rod dystrophy (CRD) with maculopathy. Other clinical symptoms included loss of color discrimination, photophobia and nystagmus. Whole-exome sequencing, segregation and haplotype analyses demonstrated that a transition variant (c.955T4C; p.(Cys319Arg)) in CNGA3 co-segregated with the CRD phenotype in family PKAB157. The ability of CNGA3 channel to influx calcium in response to agonist, when expressed either alone or together with the wild-type CNGB3 subunit in HEK293 cells, was completely abolished due to p.Cys319Arg variant. Western blotting and immunolocalization studies suggest that a decreased channel density in the HEK293 cell membrane due to impaired folding and/or trafficking of the CNGA3 protein is the main pathogenic effect of the p.Cys319Arg variant. Mutant alleles of the human cone photoreceptor cyclic nucleotide-gated channel (CNGA3) are frequently associated with achromatopsia. In rare cases, variants in CNGA3 are also associated with cone dystrophy, Leber's congenital amaurosis and oligo cone trichromacy. The identification of predicted p.(Cys319Arg) missense variant in CNGA3 expands the repertoire of the known genetic causes of CRD and phenotypic spectrum of CNGA3 alleles.

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Section: Variant Of Cnga3 Causes Crd Rs Shaikh Et Almentioning

confidence: 99%

Homozygous missense variant in the human CNGA3 channel causes cone-rod dystrophy

et al. 2014

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“…Next, the proposed PPI 14 and a wellknown correlation coefficient 3,4 are used to estimate colocalization.…”

Section: Experimental Designmentioning

confidence: 99%

“…Visually, the overlap of the original colors of fluorophores will result in the appearance of a new, third, overlaid color, indicating colocalization. Observation of colocalization in fluorescence microscopy images was a significant advance for cell and molecular biological research 3,4 . Importantly, with the introduction of the ability to estimate the degree of this overlap quantitatively, it became possible to characterize it objectively as well [5][6][7] .…”

Section: Introductionmentioning

confidence: 99%

“…Unfortunately, it also remains flawed because of the doubts raised by many researchers about the eventual accuracy of the quantification of colocalization because of the magnitude of the change the procedure introduces to the images. Another problem is that uncertainty about background conditions in analyzed images forces researchers to use a number of different coefficients (such as the correlation coefficient (Rr), the overlap coefficient (R), the overlap coefficients k 1 − k 2 and the colocalization coefficients m 1 − m 2 ), which frequently produce contradictory results 4 . Previously, we reported a protocol in which quantification of colocalization was performed using an advanced thresholding procedure for background reduction with further coefficient calculations 7 .…”

Section: Introductionmentioning

confidence: 99%

“…Previously, we reported a protocol in which quantification of colocalization was performed using an advanced thresholding procedure for background reduction with further coefficient calculations 7 . Although the protocol proved to be very useful 4 , it relies on five different coefficients (two independent and two pairs for each image region of interest (ROI), plus one pair for each scattergram ROI), which can make it difficult to decide which one of them is best suited to the conditions of a particular experiment. In addition, it is essentially a 2D approach.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Quantifying spatial correlations of fluorescent markers using enhanced background reduction with protein proximity index and correlation coefficient estimations

Zinchuk

Grossenbacher-Zinchuk

et al. 2011

Nat Protoc

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Interactions of proteins are examined by detecting their overlap using fluorescent markers. The observed overlap is then quantified to serve as a measure of spatial correlation. A major drawback of this approach is that it can produce false values because of the properties of the image background. To remedy this, we provide a protocol to reduce the contribution of image background and then apply a protein proximity index (PPI) and correlation coefficient to estimate colocalization. Background heterogeneity is reduced by the median filtering procedure, comprising two steps, to reduce random noise and background, respectively. Alternatively, background can be reduced by advanced thresholding. PPI provides separate values for each channel to characterize the contribution of each protein, whereas correlation coefficient determines the overall colocalization. The protocol is demonstrated using computer-simulated and real biological images. It minimizes human bias and can be universally applied to various cell types in which there is a need to understand protein-protein interactions. Background reductions require 3-5 min per image. Quantifications take <1 min. The entire procedure takes approximately 15-30 min.Published in " " which should be cited to refer to this work.

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Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images

Zinchuk

Grossenbacher-Zinchuk

2011

CP Cell Biology

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Colocalization is an important finding in many cell biological studies. This unit describes a protocol for quantitative evaluation of images with colocalization based on the calculation of a number of specialized coefficients. First, images of double‐stained sections are subjected to background correction. Then, various coefficients are calculated. Meanings of the coefficients and a guide to interpretation of their results indicating either presence or absence of colocalization are given. Success in colocalization studies depends on the quality of analyzed images, proper preparation of the images for coefficients calculations, and correct interpretation of obtained results. This protocol helps to ensure reliability of colocalization coefficients calculations. Curr. Protoc. Cell Biol. 52:4.16.1‐4.16.16. © 2011 by John Wiley & Sons, Inc.

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Recent advances in quantitative colocalization analysis: Focus on neuroscience

Cited by 157 publications

References 93 publications

Homozygous missense variant in the human CNGA3 channel causes cone-rod dystrophy

Homozygous missense variant in the human CNGA3 channel causes cone-rod dystrophy

Quantifying spatial correlations of fluorescent markers using enhanced background reduction with protein proximity index and correlation coefficient estimations

Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images

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