Department of Obstetrics and Gynecology, Feinberg School of Medicine , Northwestern University , 250 E. Superior St., Suite 3-2303 , Chicago , Illinois 60611 , United States.
J Am Chem Soc. 2019 Oct 23;141(42):16696-16705. doi: 10.1021/jacs.9b06442. Epub 2019 Oct 15.
Previous work has shown that fluctuations in zinc content and subcellular localization play key roles in regulating cell cycle progression; however, a deep mechanistic understanding requires the determination of when, where, and how labile zinc pools are concentrated into or released from stores. Labile zinc ions can be difficult to detect with probes that require hydrolysis of toxic protecting groups or application at high concentrations that negatively impact cell function. We previously reported a BODIPY-based zinc probe, ZincBY-1, that can be used at working concentrations that are 20-200-fold lower than concentrations employed with other probes. To better understand how zinc pools can be visualized at such low probe concentrations, we modulated the photophysical properties via changes at the 5-position of the BODIPY core. One of these, ZincBY-4, exhibits an order of magnitude higher affinity for zinc, an 8-fold increase in brightness in response to zinc, and a 100 nm Stokes shift within cells. The larger Stokes shift of ZincBY-4 presents a unique opportunity for simultaneous imaging with GFP or fluorescein sensors upon single excitation. Finally, by creating a proxy for the cellular environment in spectrometer experiments, we show that the ZincBY series are highly effective at 50 nM because they can pass membranes and accumulate in regions of high zinc concentration within a cell. These features of the ZincBY probe class have widespread applications in imaging and for understanding the regulatory roles of zinc fluxes in live cells.
先前的工作表明，锌含量和亚细胞定位的波动在调控细胞周期进程中起着关键作用；然而，要深入了解其机制，就需要确定不稳定锌池何时、何地以及如何集中到储存库中或从储存库中释放。需要水解有毒保护基团或在对细胞功能产生负面影响的高浓度下使用的探针，才能检测到不稳定的锌离子。我们之前报道了一种基于 BODIPY 的锌探针，ZincBY-1，它可以在工作浓度下使用，其浓度比其他探针低 20-200 倍。为了更好地了解在如此低的探针浓度下如何可视化锌池，我们通过改变 BODIPY 核心的 5 位来调节其光物理性质。其中一种，ZincBY-4，对锌的亲和力高一个数量级，对锌的响应亮度增加 8 倍，在细胞内的斯托克斯位移增加 100nm。ZincBY-4 较大的斯托克斯位移为 GFP 或荧光素传感器在单次激发下同时成像提供了独特的机会。最后，通过在光谱仪实验中创建细胞环境的替代物，我们表明，ZincBY 系列在 50nM 时非常有效，因为它们可以穿过细胞膜，并在细胞内高锌浓度区域积累。ZincBY 探针类的这些特性在活细胞中的成像和理解锌通量的调节作用方面具有广泛的应用。
