A practical guide to evaluating colocalization in biological microscopy

Dunn, Kenneth W.; Kamocka, Malgorzata M.; McDonald, John H.

doi:10.1152/ajpcell.00462.2010

Cited by 1,749 publications

(1,589 citation statements)

References 46 publications

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“…MOC has the following disadvantages: it does not reflect a substantial change and interpretation in data processing is difficult. However, it is possible to compare reciprocal association ratios for two fluorescent markers, although it is not as effective as PCC 22. All MOC values in Figure 3 were found to be between 0.5 and 1.0, which can be considered a positive range.…”

Section: Resultsmentioning

confidence: 98%

Enhanced Photodynamic Cancer Treatment by Mitochondria‐Targeting and Brominated Near‐Infrared Fluorophores

et al. 2017

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A noninvasive and selective therapy, photodynamic therapy (PDT) is widely researched in clinical fields; however, the lower efficiency of PDT can induce unexpected side effects. Mitochondria are extensively researched as target sites to maximize PDT effects because they play crucial roles in metabolism and can be used as cancer markers due to their high transmembrane potential. Here, a mitochondria targeting photodynamic therapeutic agent (MitDt) is developed. This photosensitizer is synthesized from heptamethine cyanine dyes, which are conjugated or modified as follows. The heptamethine meso‐position is conjugated with a triphenylphosphonium derivative for mitochondrial targeting, the N‐alkyl side chain is modified for regulation of charge balance and solubility, and the indolenine groups are brominated to enhance reactive oxygen species generation (ROS) after laser irradiation. The synthesized MitDt increases the cancer uptake efficiency due to the lipo‐cationic properties of the triphenylphosphonium, and the PDT effects of MitDt are amplified after laser irradiation because mitochondria are susceptible to ROS, the response to which triggers an apoptotic anticancer effect. Consequently, these hypotheses are demonstrated by in vitro and in vivo studies, and the results indicate strong potential for use of MitDts as efficient single‐molecule‐based PDT agents for cancer treatment.

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Section: Resultsmentioning

confidence: 98%

Enhanced Photodynamic Cancer Treatment by Mitochondria‐Targeting and Brominated Near‐Infrared Fluorophores

et al. 2017

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“…The images were converted to an 8-bit grayscale, and the background was subtracted from the region of interest using the region of interest plug-ins. The "Colocalization Threshold" plug-in algorithm determines the threshold automatically and reduces background for each channel to eliminate the bias (17). This algorithm generates two coefficients (Pearson and Manders) per dual-channel image to compute the degree of colocalization (see supplemental Fig.…”

Section: Methodsmentioning

confidence: 99%

αA-Crystallin and αB-Crystallin Reside in Separate Subcellular Compartments in the Developing Ocular Lens

Gangalum

Horwitz

Kohan

et al. 2012

Journal of Biological Chemistry

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Background:The small heat shock proteins, ␣A-crystallin and ␣B-crystallin are considered to be two subunits of one single monolithic lens protein, ␣-crystallin. Results: ␣A-Crystallin and ␣B-crystallin fractionate independent of each other and in two separate membrane compartments. Conclusion: ␣A-Crystallin and ␣B-crystallin are two independent proteins in the lens. Significance: These data provide functional insight into why ␣A-crystallin and ␣B-crystallin null mice have disparate phenotypes.

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“…Colocalization was quantified in a pixel-by-pixel basis for all confocal sections of the confocal stack. The Menders' coefficient was used (31). Microscopy image quantifications were performed with Zen software (Zeiss; see Supplementary Materials and Methods for additional details).…”

Section: Trafficking Assaysmentioning

confidence: 99%

Bispecific Antibodies and Antibody–Drug Conjugates (ADCs) Bridging HER2 and Prolactin Receptor Improve Efficacy of HER2 ADCs

Andreev

Thambi

Bay

et al. 2017

Molecular Cancer Therapeutics

126

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The properties of cell surface proteins targeted by antibodydrug conjugates (ADCs) have not been fully exploited; of particular importance are the rate of internalization and the route of intracellular trafficking. In this study, we compared the trafficking of HER2, which is the target of the clinically approved ADC ado-trastuzumab emtansine (T-DM1), with that of prolactin receptor (PRLR), another potential target in breast cancer. In contrast to HER2, we found that PRLR is rapidly and constitutively internalized, and traffics efficiently to lysosomes, where it is degraded. The PRLR cytoplasmic domain is necessary to promote rapid internalization and degradation, and when transferred to HER2, enhances HER2 degradation. In accordance with these findings, low levels of cell surface PRLR ($30,000 surface receptors per cell) are sufficient to mediate effective killing by PRLR ADC, whereas cell killing by HER2 ADC requires higher levels of cell surface HER2 ($10 6 surface receptors per cell). Noncovalently crosslinking HER2 to PRLR at the cell surface, using a bispecific antibody that binds to both receptors, dramatically enhances the degradation of HER2 as well as the cell killing activity of a noncompeting HER2 ADC. Furthermore, in breast cancer cells that coexpress HER2 and PRLR, a HER2xPRLR bispecific ADC kills more effectively than HER2 ADC. These results emphasize that intracellular trafficking of ADC targets is a key property for their activity and, further, that coupling an ADC target to a rapidly internalizing protein may be a useful approach to enhance internalization and cell killing activity of ADCs.

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A practical guide to evaluating colocalization in biological microscopy

Cited by 1,749 publications

References 46 publications

Enhanced Photodynamic Cancer Treatment by Mitochondria‐Targeting and Brominated Near‐Infrared Fluorophores

Enhanced Photodynamic Cancer Treatment by Mitochondria‐Targeting and Brominated Near‐Infrared Fluorophores

αA-Crystallin and αB-Crystallin Reside in Separate Subcellular Compartments in the Developing Ocular Lens

Bispecific Antibodies and Antibody–Drug Conjugates (ADCs) Bridging HER2 and Prolactin Receptor Improve Efficacy of HER2 ADCs

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