Measurement of co‐localization of objects in dual‐colour confocal images

Manders, Erik M. M.; Verbeek, Fons J.; Aten, Jacob A.

doi:10.1111/j.1365-2818.1993.tb03313.x

Cited by 1,910 publications

(1,894 citation statements)

References 18 publications

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“…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.…”

Section: Methodsmentioning

confidence: 99%

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

Zhang

et al. 2014

Cell Death Differ

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Isthmin (ISM) is a secreted 60-kDa protein that potently induces endothelial cell (EC) apoptosis. It suppresses tumor growth and angiogenesis in mice when stably overexpressed in cancer cells. Although avb5 integrin serves as a low-affinity receptor for ISM, the mechanism by which ISM mediates antiangiogenesis and apoptosis in ECs remain to be fully resolved. In this work, we report the identification of cell-surface glucose-regulated protein 78 kDa (GRP78) as a high-affinity receptor for ISM (K d ¼ 8.6 nM). We demonstrated that ISM-GRP78 interaction triggers apoptosis not only in activated ECs but also in cancer cells expressing high level of cell-surface GRP78. Normal cells and benign tumor cells tend to express low level of cell-surface GRP78 and are resistant to ISM-induced apoptosis. Upon binding to GRP78, ISM is internalized into ECs through clathrin-dependent endocytosis that is essential for its proapoptotic activity. Once inside the cell, ISM co-targets with GRP78 to mitochondria where it interacts with ADP/ATP carriers on the inner membrane and blocks ATP transport from mitochondria to cytosol, thereby causing apoptosis. Hence, ISM is a novel proapoptotic ligand that targets cell-surface GRP78 to trigger apoptosis by inducing mitochondrial dysfunction. The restricted and high-level expression of cell-surface GRP78 on cancer cells and cancer ECs make them uniquely susceptible to ISM-targeted apoptosis. Indeed, systemic delivery of recombinant ISM potently suppressed subcutaneous 4T1 breast carcinoma and B16 melanoma growth in mice by eliciting apoptosis selectively in the cancer cells and cancer ECs. Together, this work reveals a novel ISM-GRP78 apoptosis pathway and demonstrates the potential of ISM as a cancer-specific and dual-targeting anticancer agent.

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Section: Methodsmentioning

confidence: 99%

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

Zhang

et al. 2014

Cell Death Differ

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“…This region of interest was defined by manual segmentation of the platelets and collagen fibres using the interference reflectance channel. The Manders’ coefficients (M1 and M2) were used as the co-localisation measure [39]. …”

Section: Methodsmentioning

confidence: 99%

Tetraspanin Tspan9 regulates platelet collagen receptor GPVI lateral diffusion and activation

et al. 2016

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The tetraspanins are a superfamily of four-transmembrane proteins, which regulate the trafficking, lateral diffusion and clustering of the transmembrane proteins with which they interact. We have previously shown that tetraspanin Tspan9 is expressed on platelets. Here we have characterised gene-trap mice lacking Tspan9. The mice were viable with normal platelet numbers and size. Tspan9-deficient platelets were specifically defective in aggregation and secretion induced by the platelet collagen receptor GPVI, despite normal surface GPVI expression levels. A GPVI activation defect was suggested by partially impaired GPVI-induced protein tyrosine phosphorylation. In mechanistic experiments, Tspan9 and GPVI co-immunoprecipitated and co-localised, but super-resolution imaging revealed no defects in collagen-induced GPVI clustering on Tspan9-deficient platelets. However, single particle tracking using total internal reflection fluorescence microscopy showed that GPVI lateral diffusion was reduced by approximately 50% in the absence of Tspan9. Therefore, Tspan9 plays a fine-tuning role in platelet activation by regulating GPVI membrane dynamics.

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“…55 Colocalization coefficients ranging from 0 (none of the signal above threshold in that channel exists as colocalized with the other channel) to 1 (all of the signal above threshold in that channel exists as colocalized with the other channel) for each of the selected objects were then obtained. A colocalization coefficient of 0.5 or higher was considered to be indicative of colocalization.…”

Section: ■ Methodsmentioning

confidence: 99%

APP+, a Fluorescent Analogue of the Neurotoxin MPP+, Is a Marker of Catecholamine Neurons in Brain Tissue, but Not a Fluorescent False Neurotransmitter

Karpowicz

Dunn

Sulzer

et al. 2013

ACS Chem. Neurosci.

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ABSTRACT:We have previously introduced fluorescent false neurotransmitters (FFNs) as optical reporters that enable visualization of individual dopaminergic presynaptic terminals and their activity in the brain. In this context, we examined the fluorescent pyridinium dye 4-(4-dimethylamino)phenyl-1-methylpyridinium (APP+), a fluorescent analogue of the dopaminergic neurotoxin MPP+, in acute mouse brain tissue. APP+ is a substrate for the dopamine transporter (DAT), norepinephrine transporter (NET), and serotonin transporter (SERT), and as such represented a candidate for the development of new FFN probes. Here we report that APP+ labels cell bodies of catecholaminergic neurons in the midbrain in a DAT-and NET-dependent manner, as well as fine dopaminergic axonal processes in the dorsal striatum. APP+ destaining from presynaptic terminals in the dorsal striatum was also examined under the conditions inducing depolarization and exocytotic neurotransmitter release. Application of KCl led to a small but significant degree of destaining (approximately 15% compared to control), which stands in contrast to a nearly complete destaining of the new generation FFN agent, FFN102. Electrical stimulation of brain slices at 10 Hz afforded no significant change in the APP+ signal. These results indicate that the majority of the APP+ signal in axonal processes originates from labeled organelles including mitochondria, whereas only a minor component of the APP+ signal represents the releasable synaptic vesicular pool. These results also show that APP+ may serve as a useful probe for identifying catecholaminergic innervations in the brain, although it is a poor candidate for the development of FFNs.

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Measurement of co‐localization of objects in dual‐colour confocal images

Cited by 1,910 publications

References 18 publications

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

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

Tetraspanin Tspan9 regulates platelet collagen receptor GPVI lateral diffusion and activation

APP+, a Fluorescent Analogue of the Neurotoxin MPP+, Is a Marker of Catecholamine Neurons in Brain Tissue, but Not a Fluorescent False Neurotransmitter

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