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Visualization of long-term Mg2+ dynamics in apoptotic cells using a novel targetable fluorescent probe.

ABSTRACT: Mg2+ plays important roles in many physiological processes. However, the underlying molecular mechanisms, especially in the apoptotic pathway, remain unclear due to the diffusion of Mg2+ probes, which hinders long-term imaging in specific organelles. We developed an immobilized Mg2+ probe, MGH, which is covalently conjugated with the HaloTag protein in various organelles. HaloTag-coupled MGH enabled long-term imaging of intracellular local Mg2+ dynamics for 24 h. To exploit this remarkable property, MGH was applied to the investigation of intracellular Mg2+ dynamics during apoptosis. Time-lapse imaging revealed an increase in the Mg2+ concentration after apoptotic cell shrinkage. Combined imaging analyses of intracellular Mg2+ and ATP concentrations strongly suggested that this Mg2+ concentration increase was caused by the dissociation of Mg2+ from ATP, along with a decrease in the intracellular ATP concentration. Thus, this protein-coupled Mg2+ probe could be a new chemical tool to elucidate intracellular Mg2+ dynamics with high spatiotemporal resolution.

SUBMITTER: Matsui Y

PROVIDER: S-EPMC5858021 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Visualization of long-term Mg2+ dynamics in apoptotic cells using a novel targetable fluorescent probe.

Matsui Yusuke Y Matsui Yusuke Y Funato Yosuke Y Imamura Hiromi H Miki Hiroaki H Mizukami Shin S Kikuchi Kazuya K

Chemical science 20171020 12

Mg2+ plays important roles in many physiological processes. However, the underlying molecular mechanisms, especially in the apoptotic pathway, remain unclear due to the diffusion of Mg2+ probes, which hinders long-term imaging in specific organelles. We developed an immobilized Mg2+ probe, MGH, which is covalently conjugated with the HaloTag protein in various organelles. HaloTag-coupled MGH enabled long-term imaging of intracellular local Mg2+ dynamic ...[more]

PMID: 29619172

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Project description:Keratinocyte carcinomas, including basal and squamous cell carcinomas, are the most common human cancers worldwide. While 75% of all keratinocyte carcinoma (4 million annual cases in the United States) are treated with conventional excision, this surgical modality has much lower cure rates than Mohs micrographic surgery, likely due to the bread-loaf histopathologic assessment that visualizes <1% of the tissue margins. A quenched protease-activated fluorescent probe 6qcNIR, which produces a signal only in the protease-rich tumor microenvironment, was topically applied to 90 specimens ex vivo immediately following excision. "Puzzle-fit" analysis was used to correlate the fluorescent images with histology. Probe-dependent fluorescent images correlated with cancer determined by conventional histology. Point-of-care fluorescent detection of skin cancer had a clinically relevant sensitivity of 0.73 and corresponding specificity of 0.88. Importantly, clinicians were effectively trained to read fluorescent images within 15 minutes with reliability and confidence, resulting in sensitivities of 62%-78% and specificities of 92%-97%. Fluorescent imaging using 6qcNIR allows 100% tumor margin assessment by generating en face images that correlate with histology and may be used to overcome the limitations of conventional bread-loaf histology. The utility of 6qcNIR was validated in a busy real-world clinical setting, and clinicians were trained to effectively read fluorescent margins with a short guided instruction, highlighting clinical adaptability. When used in conventional excision, this approach may result in higher cure rates at a lower cost by allowing same-day reexcision when needed, reducing patient anxiety and improving compliance by expediting postsurgical specimen assessment. SIGNIFICANCE: A fluorescent-probe-tumor-visualization platform was developed and validated in human keratinocyte carcinoma excision specimens that may provide simple, rapid, and global assessment of margins during skin cancer excision, allowing same-day reexcision when needed.

Dataset Information

Visualization of long-term Mg2+ dynamics in apoptotic cells using a novel targetable fluorescent probe.

Publications

Visualization of long-term Mg<sup>2+</sup> dynamics in apoptotic cells using a novel targetable fluorescent probe.

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