EzColocalization: An ImageJ plugin for visualizing and measuring colocalization in cells and organisms

Stauffer, Weston; Sheng, Huanjie; Lim, Hyunseob

doi:10.1038/s41598-018-33592-8

Cited by 214 publications

(182 citation statements)

References 47 publications

(60 reference statements)

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“…Additional image processing was done using the Fiji (ImageJ) software package (56). PCC were calculated using the EzColocalization plugin for ImageJ (57) where, −1 = complete anticolocalization, 0 = noncolocalization, and 1 = complete colocalization.…”

Section: Methodsmentioning

confidence: 99%

Subdomain cryo-EM structure of nodaviral replication protein A crown complex provides mechanistic insights into RNA genome replication

Unchwaniwala

Zhan

Pennington

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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For positive-strand RNA [(+)RNA] viruses, the major target for antiviral therapies is genomic RNA replication, which occurs at poorly understood membrane-bound viral RNA replication complexes. Recent cryoelectron microscopy (cryo-EM) of nodavirus RNA replication complexes revealed that the viral double-stranded RNA replication template is coiled inside a 30- to 90-nm invagination of the outer mitochondrial membrane, whose necked aperture to the cytoplasm is gated by a 12-fold symmetric, 35-nm diameter “crown” complex that contains multifunctional viral RNA replication protein A. Here we report optimizing cryo-EM tomography and image processing to improve crown resolution from 33 to 8.5 Å. This resolves the crown into 12 distinct vertical segments, each with 3 major subdomains: A membrane-connected basal lobe and an apical lobe that together comprise the ∼19-nm-diameter central turret, and a leg emerging from the basal lobe that connects to the membrane at ∼35-nm diameter. Despite widely varying replication vesicle diameters, the resulting two rings of membrane interaction sites constrain the vesicle neck to a highly uniform shape. Labeling protein A with a His-tag that binds 5-nm Ni-nanogold allowed cryo-EM tomography mapping of the C terminus of protein A to the apical lobe, which correlates well with the predicted structure of the C-proximal polymerase domain of protein A. These and other results indicate that the crown contains 12 copies of protein A arranged basally to apically in an N-to-C orientation. Moreover, the apical polymerase localization has significant mechanistic implications for template RNA recruitment and (−) and (+)RNA synthesis.

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Section: Methodsmentioning

confidence: 99%

Subdomain cryo-EM structure of nodaviral replication protein A crown complex provides mechanistic insights into RNA genome replication

Unchwaniwala

Zhan

Pennington

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…and ImageJ (National Institutes of Health, Bethesda, MD) plugin (AIF and 4 0 ,6diamidino-2-phenylindole colocalization compared with Mander's coefficient of colocalization). 63 In addition, DNA was extracted from T84 cells using TRIzol (Invitrogen), 2-mg samples of DNA and 1 kb-plus DNA marker (Invitrogen) were loaded onto a 2% agarose gel with 0.5 mg/mL bromide. After electrophoresis, the gel was examined on a UV transilluminator and images were captured.…”

Section: Q30mentioning

confidence: 99%

Crohn’s Disease Pathobiont Adherent-Invasive E coli Disrupts Epithelial Mitochondrial Networks With Implications for Gut Permeability

Mancini

Rajeev

Jayme

et al. 2021

Cellular and Molecular Gastroenterology and Hepatology

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“…For deconvolution, 10-16 slices were captured with a 0.33 μm step size, exported and deconvolved using the Richard-Lucy algorithm 48,49 with the CUDA 50 deconvolution plugin in ImageJ (version 1.52g, Wayne Rasband, National Institute of Health, USA). Pearson's correlation coefficient was calculated using the EzColocalization ImageJ plugin 51 .…”

Section: Discussionmentioning

confidence: 99%

A molecular sensor to quantify the localization of proteins, DNA and nanoparticles in cells

et al. 2020

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Intracellular trafficking governs receptor signaling, pathogenesis, immune responses and fate of nanomedicines. These processes are typically tracked by observing colocalization of fluorescent markers using confocal microscopy. However, this method is low throughput, limited by the resolution of microscopy, and can miss fleeting interactions. To address this, we developed a localization sensor composed of a quenched SNAP-tag substrate (SNAPSwitch) that can be conjugated to biomolecules using click chemistry. SNAPSwitch enables quantitative detection of trafficking to locations of interest within live cells using flow cytometry. Using SNAPSwitch, we followed the trafficking of DNA complexes from endosomes into the cytosol and nucleus. We show that antibodies against the transferrin or hyaluronan receptor are initially sorted into different compartments following endocytosis. In addition, we can resolve which side of the cellular membrane material was located. These results demonstrate SNAPSwitch is a high-throughput and broadly applicable tool to quantitatively track localization of materials in cells.

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EzColocalization: An ImageJ plugin for visualizing and measuring colocalization in cells and organisms

Cited by 214 publications

References 47 publications

Subdomain cryo-EM structure of nodaviral replication protein A crown complex provides mechanistic insights into RNA genome replication

Subdomain cryo-EM structure of nodaviral replication protein A crown complex provides mechanistic insights into RNA genome replication

Crohn’s Disease Pathobiont Adherent-Invasive E coli Disrupts Epithelial Mitochondrial Networks With Implications for Gut Permeability

A molecular sensor to quantify the localization of proteins, DNA and nanoparticles in cells

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