Human cells experience a Zn2+ pulse in early G1

Abstract

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.

Keywords: CP: Cell biology; MTF-1; cell cycle; genetically encoded sensor; single-cell FRET; zinc; zinc dynamics; zinc homeostasis.

Figure 1.. Zn2⁺ levels influence cell proliferation over time

(A and B) Diagram of the ZapCV2 zinc-binding and zinc-dead sensors. Zinc-binding residues and alanine mutants are labeled. Illustration of sensor binding to zinc. Zinc binding induces a conformational change, which leads to increased FRET. (C) Proliferation of cells expressing the cytosolic zinc sensor ZapCV2 (solid line) and zinc-dead sensor (dashed line) in different medium conditions over 35 h. Each line represents the average of four wells normalized to initial plating density. Shaded areas represent standard deviation. (D and E) Single-cell FRET ratios of MCF10a cells expressing either NES-ZapCV2 or zinc-dead sensor. Red dots indicate a mitosis event. Each plotted trace must contain at least one mitosis event. 100 random traces across four wells are plotted in each condition. Zinc conditions: ZD3 (MM + 3 μM TPA), ZD2 (MM + 2 μM TPA), MM (minimal media), ZR15 (MM + 15 μM ZnCl₂), ZR30 (MM + 30 μM ZnCl₂), ZR50 (MM + 50 μM ZnCl₂).

Figure 2.. Tracking of labile zinc over the cell cycle reveals zinc dynamics around mitosis that is modulated by nutritional zinc

(A and B) Single-cell traces of cells expressing either NES-ZapCV2 or the NES-zinc-dead sensor after alignment to mitosis are represented in gray (n = 100 randomly selected cells per plot). Average FRET ratios of these traces are overlaid in color. (C) Averaged FRET ratio of NES-ZapCV2 and H2B-mCherry intensity in a 10 h window around mitosis. Resting FRET is calculated by averaging ten frames (4 h–2 h) approaching mitosis. Peak FRET is the maximum value within ten frames (2 h) of mitosis. (D) Plot as shown in (C) with cells expressing the NES-zinc-dead sensor. (E and F) Values of resting FRET (R) and peak FRET (P) in both zinc-binding and zinc-dead sensors. Mean values are shown as black bars. Each dot represents and individual cell track (n > 100 for each condition, one of three biological replicates is shown). Quantification of FRET change (ΔFRET) between resting and peak is shown below each group.

Figure 3.. MTF-1 knockdown results in less cell proliferation under high nutritional Zn2+ condition

(A) Western blots for MTF-1 and MT in MCF10a wild-type (WT), scrambled control (Scr Ctrl), and MTF-1 knockdown (KD) cells show lower MTF-1 expression in KD cells compared with WT and Scr Ctrl cells. MT levels are higher in Scr Ctrl cells than MTF-1 KD cells upon treatment with 40 μM Zn²⁺ for 48 h compared with the no zinc condition. Duplicates were run per condition. Blots for WT and Scr Ctrl cells were run on a single gel, whereas blot for MTF-1 KD cells was run on a separate gel. (B) CellTiter-Glo proliferation assay was performed on Scr Ctrl and MTF-1 KD cells grown for 48 h in different medium conditions. Cells were plated at a similar density in four biological replicates (n = 4) and technical triplicates. Intensity data were normalized to intensity of Scr Ctrl cells in MM condition. Each dot represents the mean of four technical replicates, and plot shows mean ± SEM of all biological replicates. Statistical analyses was performed using two-way ANOVA with Tukey-Kramer test (*p < 0.05, ****p < 0.0001). (C) Fluorescence images of Scr Ctrl and MTF-1 KD cells grown in MM, ZR30, and ZR50 media for 24 and 48 h shows lower proliferation in KD cells with ZR30 and ZR50 compared with Scr Ctrl cells. Scale bar: 100 μm.

Figure 4.. MTF-1 KD cells have higher resting zinc than Scr Ctrl cells

FRET calibration experiments on MCF10a Scr Ctrl and MTF-1 KD cells stably expressing H2B-HaloTag and NES-ZapCV2 FRET sensor were carried out to quantify the labile zinc pool. (A and B) Scatterplots showing a direct comparison of cytosolic resting Zn²⁺ [${\text{Zn}}_{\text{rest}}$] in Scr Ctrl and MTF-1 KD cells grown for 24 h in MM and ZR50 media, respectively. Fractional saturation (FS) and dynamic range (DR) of the sensor were calculated, and [Zn²⁺] was quantified from each experiment as described in the STAR Methods section (Figures S1 and S4). Each dot on the scatterplot represents [${\text{Zn}}_{\text{rest}}$] of a single cell plotted on a log scale. Only values within FS: 0–1 and DR: 1.35–1.65 ranges were considered for calculating [${\text{Zn}}_{\text{rest}}$]. For statistical analysis, cells were pooled from two biological replicates for Scr Ctrl cells in MM (131 cells) and ZR50 (200 cells) and for MTF-1 KD cells in MM (193 cells) and ZR50 (82 cells). Statistical analyses were performed using non-parametric unpaired two-tailed Mann Whitney test (with 99% confidence level) (n = 3 biological replicates, ****p ≤ 0.0001). (C and D) Cumulative fractional distribution plots of cytosolic [${\text{Zn}}_{\text{rest}}$] for Scr Ctrl cells grown in MM and ZR50 and for MTF-1 KD cells grown in MM and ZR50 conditions, respectively. Values along the x axis are truncated for better display of distribution of data points.

Figure 5.. Depletion of MTF-1 changes labile zinc dynamics after perturbation of available media zinc

(A and B) Mean traces of FRET ratio before and after zinc perturbation in MTF-1 KD or Scr Ctrl cells. Cells were grown in MM, then changed to different zinc media (indicated by vertical dotted line). One representative biological replicate is shown, and the remaining biological replicates are shown in Figure S5. (C) Parameters used to identify changes in free zinc dynamics after perturbation. Maximum FRET, ending FRET, and ${\text{t}}_{\max }$ are calculated from 20 subsampled mean FRET traces. (D) Maximum FRET after media change between different zinc levels and MTF-1 status. (E) Time to FRET maximum after media change. (F) Time for FRET ratio trace to decay to half the maximum value after media change (${\text{t}}_{1/2}$). (G) Ending FRET ratio after media change. Ending FRET is calculated by taking the average of the final 30 time points of the mean FRET traces. (H) Difference in cytosolic zinc at end of timelapse. Difference calculated by subtracting the cytosolic zinc at the end of the timelapse from the cytosolic zinc level before the media change. Comparisons conducted with one-way ANOVA, with follow-up t tests, which are plotted. *p < 0.05, ****p < 0.0001. NS indicates p >0.05. Figure created with BioRender. Cells in this experiment were expressing NES-ZapCV2, H2B-HaloTag, and either a Scr shRNA or an MTF-1 KD shRNA. Cells were plated with similar density in two biological replicates (n = 2) across five wells in each replicate.

Figure 6.. MTF-1 KD compromises cell proliferation in high Zn2⁺

(A and B) Proliferation of MCF10a Scr Ctrl and MTF-1 KD cells expressing NES-ZapCV2 and H2B-HaloTag in different zinc conditions over 48 h. Each line represents the average of four wells normalized to initial plating density. Shaded areas represent standard deviation. (C and D) Histograms show the number of mitosis events of MCF10a Scr Ctrl and MTF-1 KD cells expressing NES-ZapCV2 over time. Scr Ctrl or MTF-1 KD cells were plated with similar cell density in two biological replicates (n = 2) across five wells in each replicate.

Figure 7.. MTF-1 KD increases the magnitude of the Zn2⁺ pulse and alters Zn2⁺ dynamics following mitosis

(A and B) Average FRET ratios of all traces aligned to mitosis showing late mitosis events (from the last 120–200 frames into the time series). Horizontal dashed line refers to the resting FRET ratio before mitosis, and vertical dashed line denotes alignment to mitosis (t = 0). (C and D) Corresponding conversion of average FRET ratio to [Zn²⁺] from all the late mitosis events considering a mean DR of 1.55 for ZapCV2 sensor expressed in Scr Ctrl and MTF-1 KD cells. The dashed line denotes the resting to [Zn²⁺] level (n = 2, two biological replicates across five wells in each replicate).