Human cells experience a Zn2+ pulse in early G1.

Zinc is an essential micronutrient required for all domains of life. Cells maintain zinc homeostasis using a network of transporters, buffers, and transcription factors. Zinc is required for mammalian cell proliferation, and zinc homeostasis is remodeled during the cell cycle, but whether labile zinc changes in naturally cycling cells has not been established. We use genetically encoded fluorescent reporters, long-term time-lapse imaging, and computational tools to track labile zinc over the cell cycle in response to changes in growth media zinc and knockdown of the zinc-regulatory transcription factor MTF-1. Cells experience a pulse of labile zinc in early G1, whose magnitude varies with zinc in growth media. Knockdown of MTF-1 increases labile zinc and the zinc pulse. Our results suggest that cells need a minimum zinc pulse to proliferate and that if labile zinc levels are too high, cells pause proliferation until labile cellular zinc is lowered.

Single cell analysis reveals multiple requirements for zinc in the mammalian cell cycle.

Zinc is widely recognized as essential for growth and proliferation, yet the mechanisms of how zinc deficiency arrests these processes remain enigmatic. Here we induce subtle zinc perturbations and track asynchronously cycling cells throughout division using fluorescent reporters, high throughput microscopy, and quantitative analysis. Zinc deficiency induces quiescence and resupply stimulates synchronized cell-cycle reentry. Monitoring cells before and after zinc deprivation we found the position of cells within the cell cycle determined whether they either went quiescent or entered another cell cycle but stalled in S-phase. Stalled cells exhibited prolonged S-phase, were defective in DNA synthesis and had increased DNA damage levels, suggesting a role for zinc in maintaining genome integrity. Finally, we demonstrate zinc deficiency-induced quiescence occurs independently of DNA-damage response pathways, and is distinct from mitogen removal and spontaneous quiescence. This suggests a novel pathway to quiescence and reveals essential micronutrients play a role in cell cycle regulation.

Directed evolution of excited state lifetime and brightness in FusionRed using a microfluidic sorter.

Green fluorescent proteins (GFP) and their blue, cyan and red counterparts offer unprecedented advantages as biological markers owing to their genetic encodability and straightforward expression in different organisms. Although significant advancements have been made towards engineering the key photo-physical properties of red fluorescent proteins (RFPs), they continue to perform sub-optimally relative to GFP variants. Advanced engineering strategies are needed for further evolution of RFPs in the pursuit of improving their photo-physics. In this report, a microfluidic sorter that discriminates members of a cell-based library based on their excited state lifetime and fluorescence intensity is used for the directed evolution of the photo-physical properties of FusionRed. In-flow measurements of the fluorescence lifetime are performed in a frequency-domain approach with sub-millisecond sampling times. Promising clones are sorted by optical force trapping with an infrared laser. Using this microfluidic sorter, mutants are generated with longer lifetimes than their precursor, FusionRed. This improvement in the excited state lifetime of the mutants leads to an increase in their fluorescence quantum yield up to 1.8-fold. In the course of evolution, we also identified one key mutation (L177M), which generated a mutant (FusionRed-M) that displayed ∼2-fold higher brightness than its precursor upon expression in mammalian (HeLa) cells. Photo-physical and mutational analyses of clones isolated at the different stages of mutagenesis reveal the photo-physical evolution towards higher in vivo brightness.

Techniques for measuring cellular zinc.

The development and improvement of fluorescent Zn2+ sensors and Zn2+ imaging techniques have increased our insight into this biologically important ion. Application of these tools has identified an intracellular labile Zn2+ pool and cultivated further interest in defining the distribution and dynamics of labile Zn2+. The study of Zn2+ in live cells in real time using sensors is a powerful way to answer complex biological questions. In this review, we highlight newly engineered Zn2+ sensors, methods to test whether the sensors are accessing labile Zn2+, and recent studies that point to the challenges of using such sensors. Elemental mapping techniques can complement and strengthen data collected with sensors. Both mass spectrometry-based and X-ray fluorescence-based techniques yield highly specific, sensitive, and spatially resolved snapshots of metal distribution in cells. The study of Zn2+ has already led to new insight into all phases of life from fertilization of the egg to life-threatening cancers. In order to continue building new knowledge about Zn2+ biology it remains important to critically assess the available toolset for this endeavor.