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Zn 2+ is an essential metal required by approximately 850 human transcription factors. How these proteins acquire their essential Zn 2+ cofactor and whether they are sensitive to changes in the labile Zn 2+ pool in cells remain open questions. Using ATAC-seq to profile regions of accessible chromatin coupled with transcription factor enrichment analysis, we examined how increases and decreases in the labile zinc pool affect chromatin accessibility and transcription factor enrichment. We found 685 transcription factor motifs were differentially enriched, corresponding to 507 unique transcription factors. The pattern of perturbation and the types of transcription factors were notably different at promoters versus intergenic regions, with zinc-finger transcription factors strongly enriched in intergenic regions in elevated Zn 2+ To test whether ATAC-seq and transcription factor enrichment analysis predictions correlate with changes in transcription factor binding, we used ChIP-qPCR to profile six p53 binding sites. We found that for five of the six targets, p53 binding correlates with the local accessibility determined by ATAC-seq. These results demonstrate that changes in labile zinc alter chromatin accessibility and transcription factor binding to DNA., (© 2024 Ocampo et al.)

Zinc (Zn 2+ ) plays roles in structure, catalysis, and signaling. The majority of cellular Zn 2+ is bound by proteins, but a fraction of total Zn 2+ exists in a labile form. Here, we present a protocol for measuring labile cytosolic Zn 2+ using an in situ calibration of a genetically encoded Förster resonance energy transfer (FRET) sensor. We describe steps for producing buffered Zn 2+ solutions for performing an imaging-based calibration and analyzing the imaging data generated to determine labile Zn 2+ concentration in single cells. For complete details on the use and execution of this protocol, please refer to Rakshit and Holtzen et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The exchangeable Zn 2+ 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 Zn 2+ 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 Zn 2+ in response to perturbation. For complete details on the use and execution of this protocol, please refer to Rakshit and Holtzen et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cells must replicate their genome quickly and accurately, and they require metabolites and cofactors to do so. Ionic zinc (Zn 2+ ) 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 Zn 2+ is required at the G1/S transition and during S phase for timely completion of S phase. A short pulse of Zn 2+ deficiency impairs DNA synthesis and increases markers of replication stress. These markers of replication stress are reversed upon resupply of Zn 2+ . Finally, we find that if Zn 2+ 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 Zn 2+ deficiency in S phase specifically induce replication stress, which causes downstream proliferation impairments in daughter cells., Competing Interests: Competing interests statement:The authors declare no competing interest.

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Amy E Palmer reports financial support was provided by National Institutes of Health., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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.

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., (© 2022. The Author(s).)

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.

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.Pten flox/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., (© 2022 Wiley Periodicals LLC.)

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.

Zinc (Zn 2+ ) is an essential metal in biology, and its bioavailability is highly regulated. Many cell types exhibit fluctuations in Zn 2+ that appear to play an important role in cellular function. However, the detailed molecular mechanisms by which Zn 2+ dynamics influence cell physiology remain enigmatic. Here, we use a combination of fluorescent biosensors and cell perturbations to define how changes in intracellular Zn 2+ impact kinase signaling pathways. By simultaneously monitoring Zn 2+ dynamics and kinase activity in individual cells, we quantify changes in labile Zn 2+ and directly correlate changes in Zn 2+ with ERK and Akt activity. Under our experimental conditions, Zn 2+ fluctuations are not toxic and do not activate stress-dependent kinase signaling. We demonstrate that while Zn 2+ 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 Zn 2+ fluctuations and correlate these fluctuations with signaling events in single cells to shed light on the role that Zn 2+ dynamics play in healthy cell signaling., Competing Interests: The authors declare no competing interest.

Zinc (Zn 2+ ) 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 Zn 2+ in cells. This review highlights recent developments fluorescent sensors to measure the labile Zn 2+ pool, chelators to manipulate Zn 2+ availability, and fluorescent tools and proteomics approaches for monitoring Zn 2+ -binding proteins in cells. Finally, we close with some highlights on the role of Zn 2+ in regulating cell function and in cell signaling., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Genetically encoded fluorescent sensors have been widely used to illuminate secretory vesicle dynamics and the vesicular lumen, including Zn 2+ 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 Zn 2+ 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 Zn 2+ levels, we developed CgA-Zn 2+ 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 Zn 2+ 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 Zn 2+ at the single-vesicle level, we were able to monitor Zn 2+ dynamics in populations of vesicles and detected high vesicular Zn 2+ 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 Zn 2+ from pM to nM ranges.

The development of fluorescent proteins (FPs) has revolutionized biological imaging. FusionRed, a monomeric red FP (RFP), is known for its low cytotoxicity and correct localization of target fusion proteins in mammalian cells but is limited in application by low fluorescence brightness. We report a brighter variant of FusionRed, "FR-MQV," which exhibits an extended fluorescence lifetime (2.8 ns), enhanced quantum yield (0.53), higher extinction coefficient (∼140 000 M -1 cm -1 ), increased radiative rate constant, and reduced nonradiative rate constant with respect to its precursor. The properties of FR-MQV derive from three mutations-M42Q, C159V, and the previously identified L175M. A structure-guided approach was used to identify and mutate candidate residues around the para-hydroxyphenyl and the acylimine sites of the chromophore. The C159V mutation was identified via lifetime-based flow cytometry screening of a library in which multiple residues adjacent to the para-hydroxyphenyl site of the chromophore were mutated. The M42Q mutation is located near the acylimine moiety of the chromophore and was discovered using site-directed mutagenesis guided by X-ray crystal structures. FR-MQV exhibits a 3.4-fold higher molecular brightness and a 5-fold increase in the cellular brightness in HeLa cells [based on fluorescence-activated cell sorting (FACS)] compared to FusionRed. It also retains the low cytotoxicity and high-fidelity localization of FusionRed, as demonstrated through assays in mammalian cells. These properties make FR-MQV a promising template for further engineering into a new family of RFPs.

The central dogma teaches us that DNA makes RNA, which in turn makes proteins, the main building blocks of the cell. But this over simplified linear transmission of information overlooks the vast majority of the genome produces RNAs that do not encode proteins and the myriad ways that RNA regulates cellular functions. Historically, one of the challenges in illuminating RNA biology has been the lack of tools for visualizing RNA in live cells. But clever approaches for exploiting RNA binding proteins, in vitro RNA evolution, and chemical biology have resulted in significant advances in RNA visualization tools in recent years. This review provides an overview of current tools for tagging RNA with fluorescent probes and tracking their dynamics, localization andfunction in live mammalian cells., Competing Interests: Declaration of Interests A.E.P. and E.B. are listed as inventors on a patent (U.S. application no. 16/526,835, Compositions and Methods for Tagging Ribonucleic Acids). The patent was filed July 31, 2019; it has not been awarded yet. One technology mentioned in the article (Riboglow) is mentioned in this patent., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Competing Interests: Declaration of competing interest None.

Zinc is the second most abundant transition metal in humans and an essential nutrient required for growth and development of newborns. During lactation, mammary epithelial cells differentiate into a secretory phenotype, uptake zinc from blood circulation, and export it into mother's milk. At the cellular level, many zinc-dependent cellular processes, such as transcription, metabolism of nutrients, and proliferation are involved in the differentiation of mammary epithelial cells. Using mouse mammary epithelial cells as a model system, we investigated the remodeling of zinc homeostasis during differentiation induced by treatment with the lactogenic hormones cortisol and prolactin. RNA-Seq at different stages of differentiation revealed changes in global gene expression, including genes encoding zinc-dependent proteins and regulators of zinc homeostasis. Increases in mRNA levels of three zinc homeostasis genes, Slc39a14 (ZIP14) and metallothioneins (MTs) I and II were induced by cortisol but not by prolactin. The cortisol-induced increase was partially mediated by the nuclear glucocorticoid receptor signaling pathway. An increase in the cytosolic labile Zn2+ pool was also detected in lactating mammary cells, consistent with upregulation of MTs. We found that the zinc transporter ZIP14 was important for the expression of a major milk protein, whey acid protein (WAP), as knockdown of ZIP14 dramatically decreased WAP mRNA levels. In summary, our study demonstrated remodeling of zinc homeostasis upon differentiation of mammary epithelial cells resulting in changes in cytosolic Zn2+ and differential expression of zinc homeostasis genes, and these changes are important for establishing the lactation phenotype.

Decreasing sequencing costs have driven a rapid expansion of novel genotyping methods. One of these methods is the exploitation of restriction enzyme cut sites to generate genome-wide but reduced representation sequencing libraries (RRLs), alternatively termed genotyping by sequencing or restriction-site associated DNA sequencing. Without a reference genome, the resulting short sequence reads must be assembled de novo. There are many possible assembly programs, most not explicitly developed for RRL data, and we know little of their effectiveness. In this issue of Molecular Ecology Resources, LaCava et al. (2020) systematically evaluate six commonly used programs and two commonly varied parameters for complete and accurate assembly of RRLs, using simulated double digests of Homo sapiens and Arabidopsis thaliana genomes with varied mutation rates and types. The authors find substantial variation in performance across assembly programs. The most consistently high-performing assembler is infrequently used in their literature survey (CD-HIT; Li and Godzik, 2006), while several others fail to produce complete, accurate assemblies under many conditions. LaCava et al. additionally recommend best practices in parameter choice and evaluation of future assembly programs-advice that molecular ecologists working to assemble sequences of all kinds should take to heart., (© 2020 John Wiley & Sons Ltd.)

Ionic Zn 2+ has increasingly been recognized as an important neurotransmitter and signaling ion in glutamatergic neuron pathways. Intracellular Zn 2+ transiently increases as a result of neuronal excitation, and this Zn 2+ signal is essential for neuron plasticity, but the source and regulation of the signal is still unclear. In this study, we rigorously quantified Zn 2+ , Ca 2+ , and pH dynamics in dissociated mouse hippocampal neurons stimulated with bath application of high KCl or glutamate. While both stimulation methods yielded Zn 2+ signals, Ca 2+ influx, and acidification, glutamate stimulation induced more sustained high intracellular Ca 2+ and a larger increase in intracellular Zn 2+ . However, the stimulation-induced pH change was similar between conditions, indicating that a different cellular change is responsible for the stimulation-dependent difference in Zn 2+ signal. This work provides the first robust quantification of Zn 2+ dynamics in neurons using different methods of stimulation.

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., Competing Interests: ML, LD, JW, SJ, AP No competing interests declared, (© 2020, Lo et al.)

A key approach to investigating RNA species in live mammalian cells is the ability to label them with fluorescent tags and track their dynamics in the complex cellular environment. The growing appreciation for the diversity of RNAs in nature, especially the roles of small, non-coding RNAs for cell function, calls for development of orthogonal RNA tagging systems. We previously developed Riboglow, a new RNA tagging system that features modular elements and hence the possibility to customize features for each application of choice. Riboglow consists of an RNA tag that is genetically fused to the RNA of interest and a small molecule that binds the RNA tag and elicits a fluorescence light up signal. Here, we present an overview of the Riboglow platform and compare and contrast the system with existing RNA tagging systems. Two step by step protocols for implementation of RNA imaging with Riboglow in live mammalian cells are presented, with special emphasis on guidelines that drive choices for modular elements in the Riboglow platform. Such modular elements include the RNA tag sequence and size, the number of RNA tag repeats per tagged RNA, the fluorescent color of the probe, the identity of the chemical linker in the probe, and the concentration of the probe used in live cells. Together, Riboglow is a new RNA tagging platform that enables robust live cell imaging of RNA dynamics, and this detailed protocol and guidelines for implementation will enable broad usage of Riboglow., (© 2020 Elsevier Inc. All rights reserved.)

Although human genetic studies have implicated many susceptible genes associated with plasma lipid levels, their physiological and molecular functions are not fully characterized. Here we demonstrate that orphan G protein-coupled receptor 146 (GPR146) promotes activity of hepatic sterol regulatory element binding protein 2 (SREBP2) through activation of the extracellular signal-regulated kinase (ERK) signaling pathway, thereby regulating hepatic very low-density lipoprotein (VLDL) secretion, and subsequently circulating low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG) levels. Remarkably, GPR146 deficiency reduces plasma cholesterol levels substantially in both wild-type and LDL receptor (LDLR)-deficient mice. Finally, aortic atherosclerotic lesions are reduced by 90% and 70%, respectively, in male and female LDLR-deficient mice upon GPR146 depletion. Taken together, these findings outline a regulatory role for the GPR146/ERK axis in systemic cholesterol metabolism and suggest that GPR146 inhibition could be an effective strategy to reduce plasma cholesterol levels and atherosclerosis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Zinc (Zn 2+ ) is an integral component of many proteins and has been shown to act in a regulatory capacity in different mammalian systems, including as a neurotransmitter in neurons throughout the brain. While Zn 2+ plays an important role in modulating neuronal potentiation and synaptic plasticity, little is known about the signaling mechanisms of this regulation. In dissociated rat hippocampal neuron cultures, we used fluorescent Zn 2+ sensors to rigorously define resting Zn 2+ levels and stimulation-dependent intracellular Zn 2+ dynamics, and we performed RNA-Seq to characterize Zn 2+ -dependent transcriptional effects upon stimulation. We found that relatively small changes in cytosolic Zn 2+ during stimulation altered expression levels of 931 genes, and these Zn 2+ dynamics induced transcription of many genes implicated in neurite expansion and synaptic growth. Additionally, while we were unable to verify the presence of synaptic Zn 2+ in these cultures, we did detect the synaptic vesicle Zn 2+ transporter ZnT3 and found it to be substantially upregulated by cytosolic Zn 2+ increases. These results provide the first global sequencing-based examination of Zn 2+ -dependent changes in transcription and identify genes that may mediate Zn 2+ -dependent processes and functions.

Genetically encoded Förster resonance energy transfer (FRET) sensors enable the visualization of ions, molecules, and processes in live cells. However, despite their widespread use, the molecular states that determine sensor performance are usually poorly understood, which limits efforts to improve them. We used dynamic light scattering (DLS) and time-resolved fluorescence anisotropy to uncover the sensing mechanism of ZifCV1.173, a Zn 2+ FRET sensor. We found that the dynamic range (DR) of ZifCV1.173 was dominated by the high FRET efficiency of the Zn 2+ -free state, in which the donor and acceptor fluorescent proteins were closely associated. Mutating the donor-acceptor interface revealed that the DR of ZifCV1.173 could be increased or decreased by promoting or disrupting the donor-acceptor interaction, respectively. Adapting the same mutations to a related sensor showed the same pattern of DR tuning, supporting our sensing mechanism and suggesting that DLS and time-resolved fluorescence anisotropy might be generally useful in the biophysical characterization of other FRET sensors.

The identification of activating mutations in NOTCH1 in 50% of T cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic transformation and in targeting the pathway. A phenotypic screen identified compounds that interfere with trafficking of Notch and induce apoptosis via an endoplasmic reticulum (ER) stress mechanism. Target identification approaches revealed a role for SLC39A7 (ZIP7), a zinc transport family member, in governing Notch trafficking and signaling. Generation and sequencing of a compound-resistant cell line identified a V430E mutation in ZIP7 that confers transferable resistance to the compound NVS-ZP7-4. NVS-ZP7-4 altered zinc in the ER, and an analog of the compound photoaffinity labeled ZIP7 in cells, suggesting a direct interaction between the compound and ZIP7. NVS-ZP7-4 is the first reported chemical tool to probe the impact of modulating ER zinc levels and investigate ZIP7 as a novel druggable node in the Notch pathway.

This study was to investigate the prevalence of and factors associated with inconsistent condom use with regular female sex partners (RPs) among heterosexual male STD patients (MSTDPs) in China. We interviewed 413 MSTDPs who had had sex with at least one RP and had received a diagnosis within the past week to six months. The prevalence of inconsistent condom use with RPs was 42.9% since STD diagnosis. Associated factors included perceptions related to condom use, regret about the decision of having unprotected sex with RP, and perceived personal partners' responsibility of using condoms.

Small-molecule fluorescent probes are powerful and ubiquitous tools for measuring the concentration and distribution of analytes in living cells. However, accurate characterization of these analytes requires rigorous evaluation of cell-to-cell heterogeneity in fluorescence intensities and intracellular distribution of probes. In this study, we perform a parallel and systematic comparison of two small-molecule fluorescent vesicular Zn 2+ probes, FluoZin-3 AM and SpiroZin2, to evaluate each probe for measurement of vesicular Zn 2+ pools. Our results reveal that SpiroZin2 is a specific lysosomal vesicular Zn 2+ probe and affords uniform measurement of resting Zn 2+ levels at the single cell level with proper calibration. In contrast, FluoZin-3 AM produces highly variable fluorescence intensities and non-specifically localizes in the cytosol and multiple vesicular compartments. We further applied SpiroZin2 to lactating mouse mammary epithelial cells and detected a transient increase of lysosomal free Zn 2+ at 24-hour after lactation hormone treatment, which implies that lysosomes play a role in the regulation of Zn 2+ homeostasis during lactation. This study demonstrates the need for critical characterization of small-molecule fluorescent probes to define the concentration and localization of analytes in different cell populations, and reveals SpiroZin2 to be capable of reporting diverse perturbations to lysosomal Zn 2+ .

RNAs directly regulate a vast array of cellular processes, emphasizing the need for robust approaches to fluorescently label and track RNAs in living cells. Here, we develop an RNA imaging platform using the cobalamin riboswitch as an RNA tag and a series of probes containing cobalamin as a fluorescence quencher. This highly modular 'Riboglow' platform leverages different colored fluorescent dyes, linkers and riboswitch RNA tags to elicit fluorescence turn-on upon binding RNA. We demonstrate the ability of two different Riboglow probes to track mRNA and small noncoding RNA in live mammalian cells. A side-by-side comparison revealed that Riboglow outperformed the dye-binding aptamer Broccoli and performed on par with the gold standard RNA imaging system, the MS2-fluorescent protein system, while featuring a much smaller RNA tag. Together, the versatility of the Riboglow platform and ability to track diverse RNAs suggest broad applicability for a variety of imaging approaches.

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.

Listeria monocytogenes is an intracellular food-borne pathogen that has evolved to enter mammalian host cells, survive within them, spread from cell to cell, and disseminate throughout the body. A series of secreted virulence proteins from Listeria are responsible for manipulation of host-cell defense mechanisms and adaptation to the intracellular lifestyle. Identifying when and where these virulence proteins are located in live cells over the course of Listeria infection can provide valuable information on the roles these proteins play in defining the host-pathogen interface. These dynamics and protein levels may vary from cell to cell, as bacterial infection is a heterogeneous process both temporally and spatially. No assay to visualize virulence proteins over time in infection with Listeria or other Gram-positive bacteria has been developed. Therefore, we adapted a live, long-term tagging system to visualize a model Listeria protein by fluorescence microscopy on a single-cell level in infection. This system leverages split-fluorescent proteins, in which the last strand of a fluorescent protein (a 16-amino-acid peptide) is genetically fused to the virulence protein of interest. The remainder of the fluorescent protein is produced in the mammalian host cell. Both individual components are nonfluorescent and will bind together and reconstitute fluorescence upon virulence-protein secretion into the host cell. We demonstrate accumulation and distribution within the host cell of the model virulence protein InlC in infection over time. A modular expression platform for InlC visualization was developed. We visualized InlC by tagging it with red and green split-fluorescent proteins and compared usage of a strong constitutive promoter versus the endogenous promoter for InlC production. This split-fluorescent protein approach is versatile and may be used to investigate other Listeria virulence proteins for unique mechanistic insights in infection progression., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Zinc is an essential trace element that is required for the function of a large number of proteins. As these zinc-binding proteins are found within the cytosol and organelles, all eukaryotes require mechanisms to ensure that zinc is delivered to organelles, even under conditions of zinc deficiency. Although many zinc transporters belonging to the Cation Diffusion Facilitator (CDF) families have well characterized roles in transporting zinc into the lumens of intracellular compartments, relatively little is known about the mechanisms that maintain organelle zinc homeostasis. The fission yeast Schizosaccharomyces pombe is a useful model system to study organelle zinc homeostasis as it expresses three CDF family members that transport zinc out of the cytosol into intracellular compartments: Zhf1, Cis4, and Zrg17. Zhf1 transports zinc into the endoplasmic reticulum, and Cis4 and Zrg17 form a heterodimeric complex that transports zinc into the cis-Golgi. Here we have used the high and low affinity ZapCY zinc-responsive FRET sensors to examine cytosolic zinc levels in yeast mutants that lack each of these CDF proteins. We find that deletion of cis4 or zrg17 leads to higher levels of zinc accumulating in the cytosol under conditions of zinc deficiency, whereas deletion of zhf1 results in zinc accumulating in the cytosol when zinc is not limiting. We also show that the expression of cis4, zrg17, and zhf1 is independent of cellular zinc status. Taken together our results suggest that the Cis4/Zrg17 complex is necessary for zinc transport out of the cytosol under conditions of zinc-deficiency, while Zhf1 plays the dominant role in removing zinc from the cytosol when labile zinc is present. We propose that the properties and/or activities of individual CDF family members are fine-tuned to enable cells to control the flux of zinc out of the cytosol over a broad range of environmental zinc stress.

Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) are powerful tools for quantifying and visualizing analytes in living cells, and when targeted to organelles have the potential to define distribution of analytes in different parts of the cell. However, quantitative estimates of analyte distribution require rigorous and systematic analysis of sensor functionality in different locations. In this work, we establish methods to critically evaluate sensor performance in different organelles and carry out a side-by-side comparison of three different genetically encoded sensor platforms for quantifying cellular zinc ions (Zn 2+ ). Calibration conditions are optimized for high dynamic range and stable FRET signals. Using a combination of single-cell microscopy and a novel microfluidic platform capable of screening thousands of cells in a few hours, we observe differential performance of these sensors in the cytosol compared to the ER of HeLa cells, and identify the formation of oxidative oligomers of the sensors in the ER. Finally, we use new methodology to re-evaluate the binding parameters of these sensors both in the test tube and in living cells. Ultimately, we demonstrate that sensor responses can be affected by different cellular environments, and provide a framework for evaluating future generations of organelle-targeted sensors.

The bacterial pathogen Salmonella uses sophisticated type III secretion systems (T3SS) to translocate and deliver bacterial effector proteins into host cells to establish infection. Monitoring these important virulence determinants in the context of live infections is a key step in defining the dynamic interface between the host and pathogen. Here, we provide a modular labeling platform based on fluorescence complementation with split-GFP that permits facile tagging of new Salmonella effector proteins. We demonstrate enhancement of split-GFP complementation signals by manipulating the promoter or by multimerizing the fluorescent tag and visualize three effector proteins, SseF, SseG, and SlrP, that have never before been visualized over time during infection of live cells. Using this platform, we developed a methodology for visualizing effector proteins in primary macrophage cells for the first time and reveal distinct differences in the effector-defined intracellular niche between primary macrophage and commonly used HeLa and RAW cell lines.

Intracellular bacterial pathogens like Salmonella enterica use secretion systems, such as the Type III Secretion System, to deliver virulence factors into host cells in order to invade and colonize these cells. Salmonella virulence factors include a suite of effector proteins that remodel the host cell to facilitate bacterial internalization, replication, and evasion of host immune surveillance. A number of diverse and innovative approaches have been used to identify and characterize the role of effector proteins during infection. Recent techniques for studying infection using single cell and animal models have illuminated the contribution of individual effector proteins in infection. This review will highlight the techniques applied to study Salmonella effector proteins during infection. It will describe how different approaches have revealed mechanistic details for effectors in manipulating host cellular processes including: the dynamics of effector translocation into host cells, cytoskeleton reorganization, membrane trafficking, gene regulation, and autophagy.

Thiolutin is a disulfide-containing antibiotic and anti-angiogenic compound produced by Streptomyces. Its biological targets are not known. We show that reduced thiolutin is a zinc chelator that inhibits the JAB1/MPN/Mov34 (JAMM) domain-containing metalloprotease Rpn11, a deubiquitinating enzyme of the 19S proteasome. Thiolutin also inhibits the JAMM metalloproteases Csn5, the deneddylase of the COP9 signalosome; AMSH, which regulates ubiquitin-dependent sorting of cell-surface receptors; and BRCC36, a K63-specific deubiquitinase of the BRCC36-containing isopeptidase complex and the BRCA1-BRCA2-containing complex. We provide evidence that other dithiolopyrrolones also function as inhibitors of JAMM metalloproteases.

Ca 2+ and Zn 2+ dynamics have been identified as important drivers of physiological processes. In order for these dynamics to encode function, the cell must have sensors that transduce changes in metal concentration to specific downstream actions. Here we compare and contrast the native metal sensors: calmodulin (CaM), the quintessential Ca 2+ sensor and metal-responsive transcription factor 1 (MTF1), a candidate Zn 2+ sensor. While CaM recognizes and modulates the activity of hundreds of proteins through allosteric interactions, MTF1 recognizes a single DNA motif that is distributed throughout the genome regulating the transcription of many target genes. We examine how the different inorganic chemistries of these two metal ions may shape these different mechanisms transducing metal ion concentration into changing physiologic activity. In addition to native metal sensors, scientists have engineered sensors to spy on the dynamic changes of metals in cells. The inorganic chemistry of the metals shapes the possibilities in the design strategies of engineered sensors. We examine how different strategies to tune the affinities of engineered sensors mirror the strategies nature developed to sense both Ca 2+ and Zn 2+ in cells., (© 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The proteasome is a vital cellular machine that maintains protein homeostasis, which is of particular importance in multiple myeloma and possibly other cancers. Targeting of proteasome 20S peptidase activity with bortezomib and carfilzomib has been widely used to treat myeloma. However, not all patients respond to these compounds, and those who do eventually suffer relapse. Therefore, there is an urgent and unmet need to develop new drugs that target proteostasis through different mechanisms. We identified quinoline-8-thiol (8TQ) as a first-in-class inhibitor of the proteasome 19S subunit Rpn11. A derivative of 8TQ, capzimin, shows >5-fold selectivity for Rpn11 over the related JAMM proteases and >2 logs selectivity over several other metalloenzymes. Capzimin stabilized proteasome substrates, induced an unfolded protein response, and blocked proliferation of cancer cells, including those resistant to bortezomib. Proteomic analysis revealed that capzimin stabilized a subset of polyubiquitinated substrates. Identification of capzimin offers an alternative path to develop proteasome inhibitors for cancer therapy.

Fluorescent tools have revolutionized our ability to probe biological dynamics, particularly at the cellular level. Fluorescent sensors have been developed on several platforms, utilizing either small-molecule dyes or fluorescent proteins, to monitor proteins, RNA, DNA, small molecules, and even cellular properties, such as pH and membrane potential. We briefly summarize the impressive history of tool development for these various applications and then discuss the most recent noteworthy developments in more detail. Particular emphasis is placed on tools suitable for single-cell analysis and especially live-cell imaging applications. Finally, we discuss prominent areas of need in future fluorescent tool development-specifically, advancing our capability to analyze and integrate the plethora of high-content data generated by fluorescence imaging.

Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Fluorescent biosensors are important measurement tools for in vivo quantification of pH, concentrations of metal ions and other analytes, and physical parameters such as membrane potential. Both the development of these sensors and their implementation in examining cellular heterogeneity requires technology for measuring and sorting cells based on the fluorescence levels before and after chemical or physical perturbations. We developed a droplet microfluidic platform for the screening and separation of cell populations on the basis of the in vivo response of expressed fluorescence-based biosensors after addition of an exogenous analyte. We demonstrate the capability to resolve the responses of two genetically encoded Zn 2+ sensors at a range of time points spanning several seconds and subsequently sort a mixed-cell population of varying ratios with high accuracy.