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.

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.

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.

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.

The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins.

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.

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.

Discovery of a ZIP7 inhibitor from a Notch pathway screen.

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.

Zinc transporters belonging to the Cation Diffusion Facilitator (CDF) family have complementary roles in transporting zinc out of the cytosol.

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.

A Critical and Comparative Review of Fluorescent Tools for Live-Cell Imaging.

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.

Engineering of a Brighter Variant of the FusionRed Fluorescent Protein Using Lifetime Flow Cytometry and Structure-Guided Mutations.

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.

Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution.

Compartmentalized biochemical activities are essential to all cellular processes, but there is no generalizable method to visualize dynamic protein activities in living cells at a resolution commensurate with cellular compartmentalization. Here, we introduce a new class of fluorescent biosensors that detect biochemical activities in living cells at a resolution up to threefold better than the diffraction limit. These 'FLINC' biosensors use binding-induced changes in protein fluorescence dynamics to translate kinase activities or protein-protein interactions into changes in fluorescence fluctuations, which are quantifiable through stochastic optical fluctuation imaging. A protein kinase A (PKA) biosensor allowed us to resolve minute PKA activity microdomains on the plasma membranes of living cells and to uncover the role of clustered anchoring proteins in organizing these activity microdomains. Together, these findings suggest that biochemical activities of the cell are spatially organized into an activity architecture whose structural and functional characteristics can be revealed by these new biosensors.

A multicolor riboswitch-based platform for imaging of RNA in live mammalian cells.

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.

Inhibition of the Deubiquitinase Usp14 Diminishes Direct MHC Class I Antigen Presentation.

Infected or transformed cells must present peptides derived from endogenous proteins on MHC class I molecules to be recognized and targeted for elimination by Ag-specific cytotoxic T cells. In the first step of peptide generation, proteins are degraded by the proteasome. In this study, we investigated the role of the ubiquitin-specific protease 14 (Usp14), a proteasome-associated deubiquitinase, in direct Ag presentation using a ligand-stabilized model protein expressed as a self-antigen. Chemical inhibition of Usp14 diminished direct presentation of the model antigenic peptide, and the effect was especially pronounced when presentation was restricted to the defective ribosomal product (DRiP) form of the protein. Additionally, presentation specifically from DRiP Ags was diminished by expression of a catalytically inactive form of Usp14. Usp14 inhibition did not appreciably alter protein synthesis and only partially delayed protein degradation as measured by a slight increase in the half-life of the model protein when its degradation was induced. Taken together, these data indicate that functional Usp14 enhances direct Ag presentation, preferentially of DRiP-derived peptides, suggesting that the processing of DRiPs is in some ways different from other forms of Ag.

Thiolutin is a zinc chelator that inhibits the Rpn11 and other JAMM metalloproteases.

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.

Capzimin is a potent and specific inhibitor of proteasome isopeptidase Rpn11.

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.

A Multicolor Split-Fluorescent Protein Approach to Visualize Listeria Protein Secretion in Infection.

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.

Long-term live-cell imaging reveals new roles for Salmonella effector proteins SseG and SteA.

Salmonella Typhimurium is an intracellular bacterial pathogen that infects both epithelial cells and macrophages. Salmonella effector proteins, which are translocated into the host cell and manipulate host cell components, control the ability to replicate and/or survive in host cells. Due to the complexity and heterogeneity of Salmonella infections, there is growing recognition of the need for single-cell and live-cell imaging approaches to identify and characterize the diversity of cellular phenotypes and how they evolve over time. Here, we establish a pipeline for long-term (17 h) live-cell imaging of infected cells and subsequent image analysis methods. We apply this pipeline to track bacterial replication within the Salmonella-containing vacuole in epithelial cells, quantify vacuolar replication versus survival in macrophages and investigate the role of individual effector proteins in mediating these parameters. This approach revealed that dispersed bacteria can coalesce at later stages of infection, that the effector protein SseG influences the propensity for cytosolic hyper-replication in epithelial cells, and that while SteA only has a subtle effect on vacuolar replication in epithelial cells, it has a profound impact on infection parameters in immunocompetent macrophages, suggesting differential roles for effector proteins in different infection models.

Critical Comparison of FRET-Sensor Functionality in the Cytosol and Endoplasmic Reticulum and Implications for Quantification of Ions.

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 (Zn2+). 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.