Single Cell Analysis Reveals Multiple Requirements for Zinc in the Mammalian Cell Cycle

Abstract

8Despite recognition of the fundamental role of zinc (Zn 2+ ) for growth and proliferation, 9 mechanisms of how Zn 2+ deficiency arrests these processes remain enigmatic. We induced 10 subtle intracellular Zn 2+ perturbations and tracked asynchronously cycling cells throughout 11 division using fluorescent reporters, high throughput microscopy, and quantitative analysis. We 12 found that Zn 2+ deficiency induces quiescence and Zn 2+ resupply stimulates cell-cycle reentry. 13 By monitoring single cells after Zn 2+ deprivation, we found that depending on where cells were 14 in the cell cycle, they either went quiescent or entered the cell cycle but stalled in S phase. 15 Stalled cells were defective in DNA synthesis and had increased DNA damage levels, suggesting 16 a role for Zn 2+ in maintaining genome integrity. Finally, we found that Zn 2+ deficiency-induced 17 quiescence does not require the cell-cycle inhibitor p21. Overall, our study provides new 18 insights into when Zn 2+ is required during the mammalian cell cycle and the consequences Zn 2+ 19 deficiency. 20 21 of Zn 2+ deficiency are diverse and can be organism specific, one defining feature is universal: 1 Zn 2+ deficient cells fail to divide and proliferate normally, leading to organismal growth 2 impairment (5). Despite recognition of the fundamental role of Zn 2+ for proliferation, the 3 mechanisms of how Zn 2+ deficiency leads to cell-cycle arrest at the cellular and molecular level 4 remain poorly defined. 5Eukaryotic cell proliferation is governed by the cell-division cycle, a series of highly 6 choreographed steps that involve gap (G1), DNA replication (S-phase), gap (G2), and mitosis 7 (M) phases. Regulated transitions between proliferative and quiescent (i.e. reversible non-8 proliferative) states are essential for maintaining genome integrity and tissue homeostasis, 9 ensuring proper development, and preventing tumorigenesis. Given the essentiality of Zn 2+ for 10 growth and proliferation, a fundamental question is whether Zn 2+ serves as a nutrient, like amino 11 acids, whether it affects the rate of cell cycle progression, or whether it is required at a specific 12 phase of the cell cycle. Pioneering work by Chesters et al sought to define precisely when Zn 2+ 13is required in the mammalian cell cycle. By chelating Zn 2+ at different timepoints after release 14 from serum starvation-induced quiescence, they found that Zn 2+ was important for thymidine 15 incorporation and thus DNA synthesis, leading to the conclusion that Zn 2+ was required for the 16 G1 to S transition (6). Subsequent studies confirmed that treatment of mammalian cells with 17 high concentrations of metal chelators (DTPA and EDTA) seemed to compromise DNA 18 synthesis (7-10). However, later studies by Chesters et al suggested that after cells passed the 19 restriction point in mid-G1 there was no further Zn 2+ requirement for DNA synthesis in S phase, 20 but rather Zn 2+ was needed to transition from G2/M back into G1 (11). The restriction point is 21 classically defined as...

Single-cell image analysis to explore cell-to-cell heterogeneity in isogenic populations

“…At the individual cell level, cell-cycle stage can determine the outcome of a perturbation. For example, the cell-cycle stage of cells when they were deprived of zinc determined whether they either entered quiescence or began another cell cycle and then stalled in S phase (Lo et al, 2020).…”