Protocol for measuring cell cycle Zn2+ dynamics using a FRET-based biosensor.

The exchangeable Zn2+ pool in cells is not static but responds to perturbations as well as fluctuates naturally through the cell cycle. Here, we present a protocol to carry out long-term live-cell imaging of cells expressing a cytosolic Zn2+ sensor. We then describe how to track cells using the published pipeline EllipTrack and how to analyze the single-cell traces to determine changes in labile Zn2+ in response to perturbation. For complete details on the use and execution of this protocol, please refer to Rakshit and Holtzen et al.1.

Protocol for measuring labile cytosolic Zn2+ using an in situ calibration of a genetically encoded FRET sensor.

Zinc (Zn2+) plays roles in structure, catalysis, and signaling. The majority of cellular Zn2+ is bound by proteins, but a fraction of total Zn2+ exists in a labile form. Here, we present a protocol for measuring labile cytosolic Zn2+ using an in situ calibration of a genetically encoded Förster resonance energy transfer (FRET) sensor. We describe steps for producing buffered Zn2+ solutions for performing an imaging-based calibration and analyzing the imaging data generated to determine labile Zn2+ concentration in single cells. For complete details on the use and execution of this protocol, please refer to Rakshit and Holtzen et al.1.

Transient Zn2+ deficiency induces replication stress and compromises daughter cell proliferation.

Cells must replicate their genome quickly and accurately, and they require metabolites and cofactors to do so. Ionic zinc (Zn2+) is an essential micronutrient that is required for hundreds of cellular processes, including DNA synthesis and adequate proliferation. Deficiency in this micronutrient impairs DNA synthesis and inhibits proliferation, but the mechanism is unknown. Using fluorescent reporters to track single cells via long-term live-cell imaging, we find that Zn2+ is required at the G1/S transition and during S phase for timely completion of S phase. A short pulse of Zn2+ deficiency impairs DNA synthesis and increases markers of replication stress. These markers of replication stress are reversed upon resupply of Zn2+. Finally, we find that if Zn2+ is chelated during the mother cell's S phase, daughter cells enter a transient quiescent state, maintained by sustained expression of p21, which disappears upon reentry into the cell cycle. In summary, short pulses of mild Zn2+ deficiency in S phase specifically induce replication stress, which causes downstream proliferation impairments in daughter cells.

Cellular zinc status alters chromatin accessibility and binding of transcription factor p53 to genomic sites.

Zinc (Zn2+) is an essential metal required by approximately 2500 proteins. Nearly half of these proteins act on DNA, including > 850 human transcription factors, polymerases, DNA damage response factors, and proteins involved in chromatin architecture. How these proteins acquire their essential Zn2+ cofactor and whether they are sensitive to changes in the labile Zn2+ pool in cells remain open questions. Here, we examine how changes in the labile Zn2+ pool affect chromatin accessibility and transcription factor binding to DNA. We observed both increases and decreases in accessibility in different chromatin regions via ATAC-seq upon treating MCF10A cells with elevated Zn2+ or the Zn2+-specific chelator tris(2-pyridylmethyl)amine (TPA). Transcription factor enrichment analysis was used to correlate changes in chromatin accessibility with transcription factor motifs, revealing 477 transcription factor motifs that were differentially enriched upon Zn2+ perturbation. 186 of these transcription factor motifs were enriched in Zn2+ and depleted in TPA, and the majority correspond to Zn2+ finger transcription factors. We selected TP53 as a candidate to examine how changes in motif enrichment correlate with changes in transcription factor occupancy by ChIP-qPCR. Using publicly available ChIP-seq and nascent transcription datasets, we narrowed the 50,000+ ATAC-seq peaks to 2164 TP53 targets and subsequently selected 6 high-probability TP53 binding sites for testing. ChIP-qPCR revealed that for 5 of the 6 targets, TP53 binding correlates with the local accessibility determined by ATAC-seq. These results demonstrate that changes in labile zinc directly alter chromatin accessibility and transcription factor binding to DNA.

Transient Zn2+ deficiency induces replication stress and compromises daughter cell proliferation.

Cells must replicate their genome quickly and accurately, and they require metabolites and cofactors to do so. Ionic zinc (Zn2+) is an essential micronutrient that is required for hundreds of cellular processes, including DNA synthesis and adequate proliferation. Deficiency in this micronutrient impairs DNA synthesis and inhibits proliferation, but the mechanism is unknown. Using fluorescent reporters to track single cells via long-term live-cell imaging, we find that Zn2+ is required at the G1/S transition and during S-phase for timely completion of S-phase. A short pulse of Zn2+ deficiency impairs DNA synthesis and increases markers of replication stress. These markers of replication stress are reversed upon resupply of Zn2+. Finally, we find that if Zn2+ is removed during the mother cell's S-phase, daughter cells enter a transient quiescent state, maintained by sustained expression of p21, which disappears upon reentry into the cell cycle. In summary, short pulses of mild Zn2+ deficiency in S-phase specifically induce replication stress, which causes downstream proliferation impairments in daughter cells.

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.

Targeting Riboswitches with Beta-Axial-Substituted Cobalamins.

RNA-targeting small-molecule therapeutics is an emerging field hindered by an incomplete understanding of the basic principles governing RNA-ligand interactions. One way to advance our knowledge in this area is to study model systems where these interactions are better understood, such as riboswitches. Riboswitches bind a wide array of small molecules with high affinity and selectivity, providing a wealth of information on how RNA recognizes ligands through diverse structures. The cobalamin-sensing riboswitch is a particularly useful model system, as similar sequences show highly specialized binding preferences for different biological forms of cobalamin. This riboswitch is also widely dispersed across bacteria and therefore holds strong potential as an antibiotic target. Many synthetic cobalamin forms have been developed for various purposes including therapeutics, but their interaction with cobalamin riboswitches is yet to be explored. In this study, we characterize the interactions of 11 cobalamin derivatives with three representative cobalamin riboswitches using in vitro binding experiments (both chemical footprinting and a fluorescence-based assay) and a cell-based reporter assay. The derivatives show productive interactions with two of the three riboswitches, demonstrating simultaneous plasticity and selectivity within these RNAs. The observed plasticity is partially achieved through a novel structural rearrangement within the ligand binding pocket, providing insight into how similar RNA structures can be targeted. As the derivatives also show in vivo functionality, they serve as several potential lead compounds for further drug development.

Genetically encoded fluorescent sensors for metals in biology.

Metal ions intersect a wide range of biological processes. Some metal ions are essential and hence absolutely required for the growth and health of an organism, others are toxic and there is great interest in understanding mechanisms of toxicity. Genetically encoded fluorescent sensors are powerful tools that enable the visualization, quantification, and tracking of dynamics of metal ions in biological systems. Here, we review recent advances in the development of genetically encoded fluorescent sensors for metal ions. We broadly focus on 5 classes of sensors: single fluorescent protein, FRET-based, chemigenetic, DNAzymes, and RNA-based. We highlight recent developments in the past few years and where these developments stand concerning the rest of the field.

Mycobacterium abscessus infection results in decrease of oxidative metabolism of lung airways cells and relaxation of the epithelial mucosal tight junctions.

Mycobacterium abscessus complex is a group of environmental pathogens that recently have been isolated more from patients with underlying lung diseases, such and COPD, bronchiectasis, and cystic fibrosis. The mechanisms involved in the pathogenesis of these diseases have only recently been investigated. Infection is associated with biofilm formation on the airway mucosa, invasion of the mucosal epithelial cells and a time-dependent impairment of the integrity of the monolayer. Using electron microscopy, it was shown that Mycobacterium abscessus induced lesions of the cell surface structures. Tight junction proteins claudin-1 and occludin-1 have increased transcription in cells exposed to Mycobacterium abscessus, in contrast to cells exposed to Mycobacterium avium. Infection of A549 alveolar epithelial cells by Mycobacterium abscessus reduced the oxidative metabolism of the cell, without inducing necrosis. A transposon library screen identified mutants that do not alter the metabolism of the A549 cells.Once the bacterium crosses the epithelial barrier, it may encounter sub-epithelial macrophages. Select mutants were used for infection assays to determine their effects on membrane integrity. Translocated select mutants were attenuated in macrophages compared to wild type Mycobacterium abscessus. In summary, the dynamics of Mycobacterium abscessus infection appears to be different from other non-tuberculous mycobacteria (NTMs). Future studies will attempt to address the mechanism involved in airway membrane lesions.

Single molecule microscopy to profile the effect of zinc status on transcription factor dynamics.

The regulation of transcription is a complex process that involves binding of transcription factors (TFs) to specific sequences, recruitment of cofactors and chromatin remodelers, assembly of the pre-initiation complex and recruitment of RNA polymerase II. Increasing evidence suggests that TFs are highly dynamic and interact only transiently with DNA. Single molecule microscopy techniques are powerful approaches for tracking individual TF molecules as they diffuse in the nucleus and interact with DNA. Here we employ multifocus microscopy and highly inclined laminated optical sheet microscopy to track TF dynamics in response to perturbations in labile zinc inside cells. We sought to define whether zinc-dependent TFs sense changes in the labile zinc pool by determining whether their dynamics and DNA binding can be modulated by zinc. We used fluorescently tagged versions of the glucocorticoid receptor (GR), with two C4 zinc finger domains, and CCCTC-binding factor (CTCF), with eleven C2H2 zinc finger domains. We found that GR was largely insensitive to perturbations of zinc, whereas CTCF was significantly affected by zinc depletion and its dwell time was affected by zinc elevation. These results indicate that at least some transcription factors are sensitive to zinc dynamics, revealing a potential new layer of transcriptional regulation.

Directed Evolution of a Bright Variant of mCherry: Suppression of Nonradiative Decay by Fluorescence Lifetime Selections.

The approximately linear scaling of fluorescence quantum yield (ϕ) with fluorescence lifetime (τ) in fluorescent proteins (FPs) has inspired engineering of brighter fluorophores based on screening for increased lifetimes. Several recently developed FPs such as mTurquoise2, mScarlet, and FusionRed-MQV which have become useful for live cell imaging are products of lifetime selection strategies. However, the underlying photophysical basis of the improved brightness has not been scrutinized. In this study, we focused on understanding the outcome of lifetime-based directed evolution of mCherry, which is a popular red-FP (RFP). We identified four positions (W143, I161, Q163, and I197) near the FP chromophore that can be mutated to create mCherry-XL (eXtended Lifetime: ϕ = 0.70; τ = 3.9 ns). The 3-fold higher quantum yield of mCherry-XL is on par with that of the brightest RFP to date, mScarlet. We examined selected variants within the evolution trajectory and found a near-linear scaling of lifetime with quantum yield and consistent blue-shifts of the absorption and emission spectra. We find that the improvement in brightness is primarily due to a decrease in the nonradiative decay of the excited state. In addition, our analysis revealed the decrease in nonradiative rate is not limited to the blue-shift of the energy gap and changes in the excited state reorganization energy. Our findings suggest that nonradiative mechanisms beyond the scope of energy-gap models such the Englman-Jortner model are suppressed in this lifetime evolution trajectory.

MLN4924 Inhibits Defective Ribosomal Product Antigen Presentation Independently of Direct NEDDylation of Protein Antigens.

Successful direct MHC class I Ag presentation is dependent on the protein degradation machinery of the cell to generate antigenic peptides that can be loaded onto MHC class I molecules for surveillance by CD8+ T cells of the immune system. Most often this process involves the ubiquitin (Ub)-proteasome system; however, other Ub-like proteins have also been implicated in protein degradation and direct Ag presentation. In this article, we examine the role of neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8) in direct Ag presentation in mouse cells. NEDD8 is the Ub-like protein with highest similarity to Ub, and fusion of NEDD8 to the N terminus of a target protein can lead to the degradation of target proteins. We find that appending NEDD8 to the N terminus of the model Ag OVA resulted in degradation by both the proteasome and the autophagy protein degradation pathways, but only proteasomal degradation, involving the proteasomal subunit NEDD8 ultimate buster 1, resulted in peptide presentation. When directly compared with Ub, NEDD8 fusion was less efficient at generating peptides. However, inactivation of the NEDD8-conugation machinery by treating cells with MLN4924 inhibited the presentation of peptides from the defective ribosomal product-derived form of a model Ag. These results demonstrate that NEDD8 activity in the cell is important for direct Ag presentation, but not by directly targeting proteins for degradation.

Virulent Mycobacterium avium subspecies hominissuis subverts macrophages during early stages of infection.

Virulent non-tuberculous Mycobacteria (NTMs) successfully reside and multiply within the phagosomes of phagocytic cells such as monocytes and macrophages. Macrophages play a very important role in the innate clearance of intracellular pathogens including NTMs. Attenuated Mycobacterium avium subsp. hominissuis 100 enters macrophages but is incapable of escaping these cells via canonical mycobacteria escape mechanisms. Alternatively, virulent Mycobacterium avium subsp. hominissuis 104 and Mycobacterium abscessus subsp. abscessus are able to modify macrophages to suit their growth, survival and ultimately escape from macrophages, while non-virulent Mycobacterium smegmatis is readily killed by macrophages. In this study we focused on early infection of macrophages with NTMs to determine the phenotypic response of macrophages, M1 or M2 differentiation, and phosphorylation alterations that can affect cellular response to invading bacteria. Our findings indicate that infection of the macrophage with MAH 100 and M. smegmatis favours the development of M1 macrophage, a pro-inflammatory phenotype associated with the killing of intracellular pathogens, while infection of the macrophage with MAH 104 and M. abscessus favoured the development of M2 macrophage, an anti-inflammatory phenotype associated with the healing process. Interference with the host post-translational mechanisms, such as protein phosphorylation, is a key strategy used by many intracellular bacterial pathogens to modulate macrophage phenotype and subvert macrophage function. By comparing protein phosphorylation patterns of infected macrophages, we observed that uptake of both MAH 100 and M. smegmatis resulted in MARCKS-related protein phosphorylation, which has been associated with macrophage activation. In contrast, in macrophages infected with MAH 104 and M. abscessus, methionine adenosyltransferase IIß, an enzyme that catalyses the biosynthesis of S-adenosylmethionine, a methyl donor for DNA methylation. Inhibition of DNA methylation with 5-aza-2 deoxycytidine, significantly impaired the survival of MAH 104 in macrophages. Our findings suggest that the virulent MAH 104 and M. abscessus enhance its survival in the macrophage possibly through interference with the epigenome responses.

Stage-specific differential expression of zinc transporter SLC30A and SLC39A family proteins during prostate tumorigenesis.

Prostate cancer (PCa) initiation and progression uniquely modify the prostate milieu to aid unrestrained cell proliferation. One salient modification is the loss of the ability of prostate epithelial cells to accumulate high concentrations of zinc; however, molecular alterations associated with loss of zinc accumulating capability in malignant prostate cells remain poorly understood. Herein, we assessed the stage-specific expression of zinc transporters (ZNTs) belonging to the ZNT (SLC30A) and Zrt- and Irt-like protein (ZIP) (SLC39A) solute-carrier family in the prostate tissues of different genetically engineered mouse models (GEMM) of PCa (TMPRSS2-ERG.Ptenflox/flox , Hi-Myc+/- , and transgenic adenocarcinoma of mouse prostate), their age-matched wild-type controls, and 104 prostate core biopsies from human patients with different pathological lesions. Employing immunohistochemistry, differences in the levels of protein expression and spatial distribution of ZNT were evaluated as a function of the tumor stage. Results indicated that the expression of zinc importers (ZIP1, ZIP2, and ZIP3), which function to sequester zinc from circulation and prostatic fluid, was low to negligible in the membranes of the malignant prostate cells in both GEMM and human prostate tissues. Regarding zinc exporters (ZNT1, ZNT2, ZNT9, and ZNT10) that export excess zinc into the extracellular spaces or intracellular organelles, their expression was low in normal prostate glands of mice and humans; however, it was significantly upregulated in prostate adenocarcinoma lesions in GEMM and PCa patients. Together, our findings provide new insights into altered expression of ZNTs during the progression of PCa and indicate that changes in zinc homeostasis could possibly be an early-initiation event during prostate tumorigenesis and a likely prevention/intervention target.

Characterization of Global Gene Expression, Regulation of Metal Ions, and Infection Outcomes in Immune-Competent 129S6 Mouse Macrophages.

Nutritional immunity involves cellular and physiological responses to invading pathogens, such as limiting iron, increasing exposure to bactericidal copper, and altering zinc to restrict the growth of pathogens. Here, we examine infection of bone marrow-derived macrophages from 129S6/SvEvTac mice by Salmonella enterica serovar Typhimurium. The 129S6/SvEvTac mice possess a functional Slc11a1 (Nramp-1), a phagosomal transporter of divalent cations that plays an important role in modulating metal availability to the pathogen. We carried out global RNA sequencing upon treatment with live or heat-killed Salmonella at 2 h and 18 h postinfection and observed widespread changes in metal transport, metal-dependent genes, and metal homeostasis genes, suggesting significant remodeling of iron, copper, and zinc availability by host cells. Changes in host cell gene expression suggest infection increases cytosolic zinc while simultaneously limiting zinc within the phagosome. Using a genetically encoded sensor, we demonstrate that cytosolic labile zinc increases 45-fold at 12 h postinfection. Further, manipulation of zinc in the medium alters bacterial clearance and replication, with zinc depletion inhibiting both processes. Comparing the transcriptomic changes to published data on infection of C57BL/6 macrophages revealed notable differences in metal regulation and the global immune response. Our results reveal that 129S6 macrophages represent a distinct model system compared to C57BL/6 macrophages. Further, our results indicate that manipulation of zinc at the host-pathogen interface is more nuanced than that of iron or copper. The 129S6 macrophages leverage intricate means of manipulating zinc availability and distribution to limit the pathogen's access to zinc, while simultaneously ensuring sufficient zinc to support the immune response.

Mycobacterium avium subsp. hominissuis (MAH) Microaggregate induction of host innate immunity is linked to biofilm formation.

Bacterial aggregation is a strategy employed by many pathogens to establish infection. Mycobacterium avium subsp. hominissuis (MAH) undergoes a phenotypic change, microaggregation, when exposed to the respiratory epithelium. We therefore compared how non-aggregated bacteria, or planktonic, and microaggregated MAH can establish lung infections by evaluating mucosal epithelial cell and phagocytic cell responses. It was determined that human mucosal lung epithelial cells recognition of MAH occurs through toll-like receptors 1 and 2. MAPK 1/3 is phosphorylated at 30 min post infection, and active at the transcriptional level 2 h post infection for both phenotypes. Microaggregate infected BEAS-2B cells up-regulated CCL5, IL-1ß, and TNF-α cDNA, while planktonic infected cells only up-regulated IL-1ß cDNA at 2 h post infection. Microaggregates are associated with increased uptake by macrophages after 1 h compared to planktonic bacteria (8.83% vs. 5.00%, P < 0.05). In addition, the microaggregate phenotype, when internalized by macrophages, had reduced growth compared to planktonic bacteria, which increased when the host cells were exposed to microaggregate supernatant, obtained from the incubation of MAH with HEp-2 cells. Moreover, microaggregate supernatant stimulated biofilm formation by planktonic and microaggregated bacteria. Microaggregate supernatant also induces the production of both pro- and anti-inflammatory cytokines, which was suppressed following MAH infection. The results suggest that epithelial recognition occurs during MAH infection, and the microaggregate phenotype stimulates an inflammatory response. The initial bacterial interaction with the mucosal epithelium and development of the microaggregate phenotype has a role in pathogenesis, allowing for more robust biofilm formation and infection establishment.

Direct Conjugation of NEDD8 to the N-Terminus of a Model Protein Can Induce Degradation.

While the role of ubiquitin in protein degradation is well established, the role of other ubiquitin-like proteins (UBLs) in protein degradation is less clear. Neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8) is the UBL with the highest level of amino acids identified when compared to ubiquitin. Here we tested if the N-terminal addition of NEDD8 to a protein of interest could lead to degradation. Mutation of critical glycine residues required for normal NEDD8 processing resulted in a non-cleavable fusion protein that was rapidly degraded within the cells by both the proteasome and autophagy. Both degradation pathways were dependent on a functional ubiquitin-conjugation system as treatment with MLN7243 increased levels of non-cleavable NEDD8-GFP. The degradation of non-cleavable, N-terminal NEDD8-GFP was not due to a failure of GFP folding as different NEDD8-GFP constructs with differing abilities to fold and fluoresce were similarly degraded. Though the fusion of NEDD8 to a protein resulted in degradation, treatment of cells with MLN4924, an inhibitor of the E1 activating enzyme for NEDD8, failed to prevent degradation of other destabilized substrates. Taken together these data suggest that under certain conditions, such as the model system described here, the covalent linkage of NEDD8 to a protein substrate may result in the target proteins degradation.

Zn2+ influx activates ERK and Akt signaling pathways.

Zinc (Zn2+) is an essential metal in biology, and its bioavailability is highly regulated. Many cell types exhibit fluctuations in Zn2+ that appear to play an important role in cellular function. However, the detailed molecular mechanisms by which Zn2+ dynamics influence cell physiology remain enigmatic. Here, we use a combination of fluorescent biosensors and cell perturbations to define how changes in intracellular Zn2+ impact kinase signaling pathways. By simultaneously monitoring Zn2+ dynamics and kinase activity in individual cells, we quantify changes in labile Zn2+ and directly correlate changes in Zn2+ with ERK and Akt activity. Under our experimental conditions, Zn2+ fluctuations are not toxic and do not activate stress-dependent kinase signaling. We demonstrate that while Zn2+ can nonspecifically inhibit phosphatases leading to sustained kinase activation, ERK and Akt are predominantly activated via upstream signaling and through a common node via Ras. We provide a framework for quantification of Zn2+ fluctuations and correlate these fluctuations with signaling events in single cells to shed light on the role that Zn2+ dynamics play in healthy cell signaling.

Tools and techniques for illuminating the cell biology of zinc.

Zinc (Zn2+) is an essential micronutrient that is required for a wide variety of cellular processes. Tools and methods have been instrumental in revealing the myriad roles of Zn2+ in cells. This review highlights recent developments fluorescent sensors to measure the labile Zn2+ pool, chelators to manipulate Zn2+ availability, and fluorescent tools and proteomics approaches for monitoring Zn2+-binding proteins in cells. Finally, we close with some highlights on the role of Zn2+ in regulating cell function and in cell signaling.

Systematic Comparison of Vesicular Targeting Signals Leads to the Development of Genetically Encoded Vesicular Fluorescent Zn2+ and pH Sensors.

Genetically encoded fluorescent sensors have been widely used to illuminate secretory vesicle dynamics and the vesicular lumen, including Zn2+ and pH, in living cells. However, vesicular sensors have a tendency to mislocalize and are susceptible to the acidic intraluminal pH. In this study, we performed a systematic comparison of five different vesicular proteins to target the fluorescent protein mCherry and a Zn2+ Förster resonance energy transfer (FRET) sensor to secretory vesicles. We found that motifs derived from vesicular cargo proteins, including chromogranin A (CgA), target vesicular puncta with greater efficacy than transmembrane proteins. To characterize vesicular Zn2+ levels, we developed CgA-Zn2+ FRET sensor fusions with existing sensors ZapCY1 and eCALWY-4 and characterized subcellular localization and the influence of pH on sensor performance. We simultaneously monitored Zn2+ and pH in individual secretory vesicles by leveraging the acceptor fluorescent protein as a pH sensor and found that pH influenced FRET measurements in situ. While unable to characterize vesicular Zn2+ at the single-vesicle level, we were able to monitor Zn2+ dynamics in populations of vesicles and detected high vesicular Zn2+ in MIN6 cells compared to lower levels in the prostate cancer cell line LnCaP. The combination of CgA-ZapCY1 and CgA-eCALWY-4 allows for measurement of Zn2+ from pM to nM ranges.