Interrogating Intracellular Zinc Chemistry with a Long Stokes Shift Zinc Probe ZincBY-4.

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.

Quantitative mapping of zinc fluxes in the mammalian egg reveals the origin of fertilization-induced zinc sparks.

Fertilization of a mammalian egg initiates a series of 'zinc sparks' that are necessary to induce the egg-to-embryo transition. Despite the importance of these zinc-efflux events little is known about their origin. To understand the molecular mechanism of the zinc spark we combined four physical approaches that resolve zinc distributions in single cells: a chemical probe for dynamic live-cell fluorescence imaging and a combination of scanning transmission electron microscopy with energy-dispersive spectroscopy, X-ray fluorescence microscopy and three-dimensional elemental tomography for high-resolution elemental mapping. We show that the zinc spark arises from a system of thousands of zinc-loaded vesicles, each of which contains, on average, 10(6) zinc atoms. These vesicles undergo dynamic movement during oocyte maturation and exocytosis at the time of fertilization. The discovery of these vesicles and the demonstration that zinc sparks originate from them provides a quantitative framework for understanding how zinc fluxes regulate cellular processes.