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Superiority of SpiroZin2 Versus FluoZin-3 for monitoring vesicular Zn²⁺ allows tracking of lysosomal Zn²⁺ pools.

ABSTRACT: Small-molecule fluorescent probes are powerful and ubiquitous tools for measuring the concentration and distribution of analytes in living cells. However, accurate characterization of these analytes requires rigorous evaluation of cell-to-cell heterogeneity in fluorescence intensities and intracellular distribution of probes. In this study, we perform a parallel and systematic comparison of two small-molecule fluorescent vesicular Zn²⁺ probes, FluoZin-3 AM and SpiroZin2, to evaluate each probe for measurement of vesicular Zn²⁺ pools. Our results reveal that SpiroZin2 is a specific lysosomal vesicular Zn²⁺ probe and affords uniform measurement of resting Zn²⁺ levels at the single cell level with proper calibration. In contrast, FluoZin-3 AM produces highly variable fluorescence intensities and non-specifically localizes in the cytosol and multiple vesicular compartments. We further applied SpiroZin2 to lactating mouse mammary epithelial cells and detected a transient increase of lysosomal free Zn²⁺ at 24-hour after lactation hormone treatment, which implies that lysosomes play a role in the regulation of Zn²⁺ homeostasis during lactation. This study demonstrates the need for critical characterization of small-molecule fluorescent probes to define the concentration and localization of analytes in different cell populations, and reveals SpiroZin2 to be capable of reporting diverse perturbations to lysosomal Zn²⁺.

SUBMITTER: Han Y

PROVIDER: S-EPMC6177427 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Superiority of SpiroZin2 Versus FluoZin-3 for monitoring vesicular Zn2+ allows tracking of lysosomal Zn2+ pools.

Han Yu Y Goldberg Jacob M JM Lippard Stephen J SJ Palmer Amy E AE

Scientific reports 20181009 1

Small-molecule fluorescent probes are powerful and ubiquitous tools for measuring the concentration and distribution of analytes in living cells. However, accurate characterization of these analytes requires rigorous evaluation of cell-to-cell heterogeneity in fluorescence intensities and intracellular distribution of probes. In this study, we perform a parallel and systematic comparison of two small-molecule fluorescent vesicular Zn2+ probes, FluoZin-3 AM and SpiroZin2, to evaluate e ...[more]

PMID: 30302024

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Project description:IntroductionAtherosclerosis is a major pathological condition that underlies many cardiovascular diseases (CVDs). Its etiology involves breach of tolerance to self, leading to clonal expansion of autoreactive apolipoprotein B (APOB)-reactive CD4+T cells that correlates with clinical CVD. The T-cell receptor (TCR) sequences that mediate activation of APOB-specific CD4+T cells are unknown.MethodsIn a previous study, we had profiled the hypervariable complementarity determining region 3 (CDR3) of CD4+T cells that respond to six immunodominant APOB epitopes in most donors. Here, we comprehensively analyze this dataset of 149,065 APOB-reactive and 199,211 non-reactive control CDR3s from six human leukocyte antigen-typed donors.ResultsWe identified 672 highly expanded (frequency threshold > 1.39E-03) clones that were significantly enriched in the APOB-reactive group as compared to the controls (log10 odds ratio ≥1, Fisher's test p < 0.01). Analysis of 114,755 naïve, 91,001 central memory (TCM) and 29,839 effector memory (TEM) CDR3 sequences from the same donors revealed that APOB+ clones can be traced to the complex repertoire of unenriched blood T cells. The fraction of APOB+ clones that overlapped with memory CDR3s ranged from 2.2% to 46% (average 16.4%). This was significantly higher than their overlap with the naïve pool, which ranged from 0.7% to 2% (average 1.36%). CDR3 motif analysis with the machine learning-based in-silico tool, GLIPHs (grouping of lymphocyte interactions by paratope hotspots), identified 532 APOB+ motifs. Analysis of naïve and memory CDR3 sequences with GLIPH revealed that ~40% (209 of 532) of these APOB+ motifs were enriched in the memory pool. Network analysis with Cytoscape revealed extensive sharing of the memory-affiliated APOB+ motifs across multiple donors. We identified six motifs that were present in TCM and TEM CDR3 sequences from >80% of the donors and were highly enriched in the APOB-reactive TCR repertoire.DiscussionThe identified APOB-reactive expanded CD4+T cell clones and conserved motifs can be used to annotate and track human atherosclerosis-related autoreactive CD4+T cells and measure their clonal expansion.

Dataset Information

Superiority of SpiroZin2 Versus FluoZin-3 for monitoring vesicular Zn²⁺ allows tracking of lysosomal Zn²⁺ pools.

Publications

Superiority of SpiroZin2 Versus FluoZin-3 for monitoring vesicular Zn<sup>2+</sup> allows tracking of lysosomal Zn<sup>2+</sup> pools.

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