UTX condensation underlies its tumour-suppressive activity.

UTX (also known as KDM6A) encodes a histone H3K27 demethylase and is an important tumour suppressor that is frequently mutated in human cancers1. However, as the demethylase activity of UTX is often dispensable for mediating tumour suppression and developmental regulation2-8, the underlying molecular activity of UTX remains unknown. Here we show that phase separation of UTX underlies its chromatin-regulatory activity in tumour suppression. A core intrinsically disordered region (cIDR) of UTX forms phase-separated liquid condensates, and cIDR loss caused by the most frequent cancer mutation of UTX is mainly responsible for abolishing tumour suppression. Deletion, mutagenesis and replacement assays of the intrinsically disordered region demonstrate a critical role of UTX condensation in tumour suppression and embryonic stem cell differentiation. As shown by reconstitution in vitro and engineered systems in cells, UTX recruits the histone methyltransferase MLL4 (also known as KMT2D) to the same condensates and enriches the H3K4 methylation activity of MLL4. Moreover, UTX regulates genome-wide histone modifications and high-order chromatin interactions in a condensation-dependent manner. We also found that UTY, the Y chromosome homologue of UTX with weaker tumour-suppressive activity, forms condensates with reduced molecular dynamics. These studies demonstrate a crucial biological function of liquid condensates with proper material states in enabling the tumour-suppressive activity of a chromatin regulator.

Multiplexed bioluminescence microscopy via phasor analysis.

Bioluminescence imaging with luciferase-luciferin pairs is a well-established technique for visualizing biological processes across tissues and whole organisms. Applications at the microscale, by contrast, have been hindered by a lack of detection platforms and easily resolved probes. We addressed this limitation by combining bioluminescence with phasor analysis, a method commonly used to distinguish spectrally similar fluorophores. We built a camera-based microscope equipped with special optical filters to directly assign phasor locations to unique luciferase-luciferin pairs. Six bioluminescent reporters were easily resolved in live cells, and the readouts were quantitative and instantaneous. Multiplexed imaging was also performed over extended time periods. Bioluminescent phasor further provided direct measures of resonance energy transfer in single cells, setting the stage for dynamic measures of cellular and molecular features. The merger of bioluminescence with phasor analysis fills a long-standing void in imaging capabilities, and will bolster future efforts to visualize biological events in real time and over multiple length scales.

Automated segmentation and tracking of mitochondria in live-cell time-lapse images.

Mitochondria display complex morphology and movements, which complicates their segmentation and tracking in time-lapse images. Here, we introduce Mitometer, an algorithm for fast, unbiased, and automated segmentation and tracking of mitochondria in live-cell two-dimensional and three-dimensional time-lapse images. Mitometer requires only the pixel size and the time between frames to identify mitochondrial motion and morphology, including fusion and fission events. The segmentation algorithm isolates individual mitochondria via a shape- and size-preserving background removal process. The tracking algorithm links mitochondria via differences in morphological features and displacement, followed by a gap-closing scheme. Using Mitometer, we show that mitochondria of triple-negative breast cancer cells are faster, more directional, and more elongated than those in their receptor-positive counterparts. Furthermore, we show that mitochondrial motility and morphology in breast cancer, but not in normal breast epithelia, correlate with metabolic activity. Mitometer is an unbiased and user-friendly tool that will help resolve fundamental questions regarding mitochondrial form and function.

Fluorescence lifetime: Beating the IRF and interpulse window.

Fluorescence lifetime imaging captures the spatial distribution of chemical species across cellular environments employing pulsed illumination confocal setups. However, quantitative interpretation of lifetime data continues to face critical challenges. For instance, fluorescent species with known in vitro excited-state lifetimes may split into multiple species with unique lifetimes when introduced into complex living environments. What is more, mixtures of species, which may be both endogenous and introduced into the sample, may exhibit 1) very similar lifetimes as well as 2) wide ranges of lifetimes including lifetimes shorter than the instrumental response function or whose duration may be long enough to be comparable to the interpulse window. By contrast, existing methods of analysis are optimized for well-separated and intermediate lifetimes. Here, we broaden the applicability of fluorescence lifetime analysis by simultaneously treating unknown mixtures of arbitrary lifetimes-outside the intermediate, Goldilocks, zone-for data drawn from a single confocal spot leveraging the tools of Bayesian nonparametrics (BNP). We benchmark our algorithm, termed BNP lifetime analysis, using a range of synthetic and experimental data. Moreover, we show that the BNP lifetime analysis method can distinguish and deduce lifetimes using photon counts as small as 500.

Superoxide Release by Macrophages through NADPH Oxidase Activation Dominating Chemistry by Isoprene Secondary Organic Aerosols and Quinones to Cause Oxidative Damage on Membranes.

Oxidative stress mediated by reactive oxygen species (ROS) is a key process for adverse aerosol health effects. Secondary organic aerosols (SOA) account for a major fraction of fine particulate matter, and their inhalation and deposition into the respiratory tract causes the formation of ROS by chemical and cellular processes, but their relative contributions are hardly quantified and their link to oxidative stress remains uncertain. Here, we quantified cellular and chemical superoxide generation by 9,10-phenanthrenequinone (PQN) and isoprene SOA using a chemiluminescence assay combined with electron paramagnetic resonance spectroscopy as well as kinetic modeling. We also applied cellular imaging techniques to study the cellular mechanism of superoxide release and oxidative damage on cell membranes. We show that PQN and isoprene SOA activate NADPH oxidase in macrophages to release massive amounts of superoxide, overwhelming the superoxide formation by aqueous chemical reactions in the epithelial lining fluid. The activation dose for PQN is 2 orders of magnitude lower than that of isoprene SOA, suggesting that quinones are more toxic. While higher exposures trigger cellular antioxidant response elements, the released ROS induce oxidative damage to the cell membrane through lipid peroxidation. Such mechanistic and quantitative understandings provide a basis for further elucidation of adverse health effects and oxidative stress by fine particulate matter.

Number and molecular brightness analysis reveals Htt25Q protein aggregation upon the uptake of Htt97Q aggregates.

Number and molecular Brightness (N&B) analysis is a powerful method used to monitor protein aggregation in living cells. Here, we used the N&B method to characterize the unexpanded HTT protein oligomerization after the internalization of the mutant HTT (mHTT) which contains a CAG repeat extensions encoding for long polyglutamine (polyQ) proteins resulting in misfolding and aggregation. HEK cells expressing Htt25Q-mCherry proteins were infected with Htt97Q-EGFP aggregates, by cell to cell uptake, in cultured conditions resulting in an increasing population of dimers and tetramers compared to our controls. This study shows for the first time the impact of protein aggregation in the unexpanded Htt25Q-mCherry expressing cells that occurs from cell to cell transfer of the expanded Htt97Q-EGFP. These results signify the sporadic behavior of the polyQ inclusion that gives insight into the mechanism of protein dynamics as a consequence of secreted mHTT aggregates.

Caffeine and Cisplatin Effectively Targets the Metabolism of a Triple-Negative Breast Cancer Cell Line Assessed via Phasor-FLIM.

Triple-negative tumor cells, a malignant subtype of breast cancer, lack a biologically targeted therapy. Given its DNA repair inhibiting properties, caffeine has been shown to enhance the effectiveness of specific tumor chemotherapies. In this work, we have investigated the effects of caffeine, cisplatin, and a combination of the two as potential treatments in energy metabolism for three cell lines, triple-negative breast cancer (MDA-MB-231), estrogen-receptor lacking breast cancer (MCF7) and breast epithelial cells (MCF10A) using a sensitive label-free approach, phasor-fluorescence lifetime imaging microscopy (phasor-FLIM). We found that solely using caffeine to treat MDA-MB-231 shifts their metabolism towards respiratory-chain phosphorylation with a lower ratio of free to bound NADH, and a similar trend is seen in MCF7. However, MDA-MB-231 cells shifted to a higher ratio of free to bound NADH when cisplatin was added. The combination of cisplatin and caffeine together reduced the survival rate for MDA-MD231 and shifted their energy metabolism to a higher fraction of bound NADH indicative of oxidative phosphorylation. The FLIM and viability results of MCF10A cells demonstrate that the treatments targeted cancer cells over the normal breast tissue. The identification of energy metabolism alteration could open up strategies of improving chemotherapy for malignant breast cancer.

Label-Free Metabolic Classification of Single Cells in Droplets Using the Phasor Approach to Fluorescence Lifetime Imaging Microscopy.

Characterization of single cell metabolism is imperative for understanding subcellular functional and biochemical changes associated with healthy tissue development and the progression of numerous diseases. However, single-cell analysis often requires the use of fluorescent tags and cell lysis followed by genomic profiling to identify the cellular heterogeneity. Identifying individual cells in a noninvasive and label-free manner is crucial for the detection of energy metabolism which will discriminate cell types and most importantly critical for maintaining cell viability for further analysis. Here, we have developed a robust assay using the droplet microfluidic technology together with the phasor approach to fluorescence lifetime imaging microscopy to study cell heterogeneity within and among the leukemia cell lines (K-562 and Jurkat). We have extended these techniques to characterize metabolic differences between proliferating and quiescent cells-a critical step toward label-free single cancer cell dormancy research. The result suggests a droplet-based noninvasive and label-free method to distinguish individual cells based on their metabolic states, which could be used as an upstream phenotypic platform to correlate with genomic statistics. © 2018 International Society for Advancement of Cytometry.

Quantitative image mean squared displacement (iMSD) analysis of the dynamics of profilin 1 at the membrane of live cells.

Image mean square displacement analysis (iMSD) is a method allowing the mapping of diffusion dynamics of molecules in living cells. However, it can also be used to obtain quantitative information on the diffusion processes of fluorescently labelled molecules and how their diffusion dynamics change when the cell environment is modified. In this paper, we describe the use of iMSD to obtain quantitative data of the diffusion dynamics of a small cytoskeletal protein, profilin 1 (pfn1), at the membrane of live cells and how its diffusion is perturbed when the cells are treated with Cytochalasin D and/or the interactions of pfn1 are modified when its actin and polyphosphoinositide binding sites are mutated (pfn1-R88A). Using total internal reflection fluorescence microscopy images, we obtained data on isotropic and confined diffusion coefficients, the proportion of cell areas where isotropic diffusion is the major diffusion mode compared to the confined diffusion mode, the size of the confinement zones and the size of the domains of dynamic partitioning of pfn1. Using these quantitative data, we could demonstrate a decreased isotropic diffusion coefficient for the cells treated with Cytochalasin D and for the pfn1-R88A mutant. We could also see changes in the modes of diffusion between the different conditions and changes in the size of the zones of pfn1 confinements for the pfn1 treated with Cytochalasin D. All of this information was acquired in only a few minutes of imaging per cell and without the need to record thousands of single molecule trajectories.

Wnt signaling directs a metabolic program of glycolysis and angiogenesis in colon cancer.

Much of the mechanism by which Wnt signaling drives proliferation during oncogenesis is attributed to its regulation of the cell cycle. Here, we show how Wnt/ß-catenin signaling directs another hallmark of tumorigenesis, namely Warburg metabolism. Using biochemical assays and fluorescence lifetime imaging microscopy (FLIM) to probe metabolism in vitro and in living tumors, we observe that interference with Wnt signaling in colon cancer cells reduces glycolytic metabolism and results in small, poorly perfused tumors. We identify pyruvate dehydrogenase kinase 1 (PDK1) as an important direct target within a larger gene program for metabolism. PDK1 inhibits pyruvate flux to mitochondrial respiration and a rescue of its expression in Wnt-inhibited cancer cells rescues glycolysis as well as vessel growth in the tumor microenvironment. Thus, we identify an important mechanism by which Wnt-driven Warburg metabolism directs the use of glucose for cancer cell proliferation and links it to vessel delivery of oxygen and nutrients.

Absence of REV3L promotes p53-regulated cancer cell metabolism in cisplatin-treated lung carcinoma cells.

Lung cancer is one of the deadliest cancers in the world because of chemo-resistance to the commonly used cisplatin-based treatments. The use of low fidelity DNA polymerases in the translesional synthesis (TLS) DNA damage response pathway that repairs lesions caused by cisplatin also presents a mutational carcinogenic burden on cells that needs to be regulated by the tumor suppressor protein p53. However, there is much debate over the roles of the reversionless 3-like (REV3L) protein responsible for TLS and p53 in regulating cancer cell metabolism. In this study, the fluorescence lifetime of the metabolic coenzyme NADH reveals that the absence of REV3L can promote the p53-mediated upregulation of oxidative phosphorylation in cisplatin-treated H1299 lung carcinoma cells and increases cancer cell sensitivity to this platinum-based chemotherapy. These results demonstrate a previously unrecognized relationship between p53 and REV3L in cancer cell metabolism and may lead to improvements in chemotherapy treatment plans that reduce cisplatin resistance in lung cancer.

Spatial Characterization of Bioenergetics and Metabolism of Primordial to Preovulatory Follicles in Whole Ex Vivo Murine Ovary.

Previous work characterizing ovarian bioenergetics has defined follicular metabolism by measuring metabolic by-products in culture media. However, culture conditions perturb the native state of the follicle, and these methods do not distinguish between metabolism occurring within oocytes or granulosa cells. We applied the phasor approach to fluorescence lifetime imaging microscopy (phasor FLIM) at 740-nm two-photon excitation to examine the spatial distribution of free and protein-bound nicotinamide adenine dinucleotide hydride (NADH) during primordial through preovulatory stages of follicular development in fresh ex vivo murine neonatal and gonadotropin stimulated prepubertal ovaries. We obtained subcellular resolution phasor FLIM images of primordial through primary follicles and quantified the free/bound NADH ratio (relative NADH/NAD+) separately for oocyte nucleus and oocyte cytoplasm. We found that dynamic changes in oocyte nucleus free/bound NADH paralleled the developmental maturation of primordial to primary follicles. Immunohistochemistry of NAD+-dependent deacetylase SIRTUIN 1 (SIRT1) in neonatal ovary revealed that increasing SIRT1 expression in oocyte nuclei was inversely related to decreasing free/bound NADH during the primordial to primary follicle transition. We characterized oocyte metabolism at these early stages to be NADH producing (glycolysis/Krebs). We extended the results of prior studies to show that cumulus and mural granulosa cell metabolism in secondary through preovulatory follicles is mainly NADH producing (glycolysis/Krebs cycle), while oocyte metabolism is mainly NADH consuming (oxidative phosphorylation). Taken together, our data characterize dynamic changes in free/bound NADH and SIRT1 expression during early follicular development and confirm results from previous studies defining antral and preovulatory follicle metabolism in culture.

Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus.

UNLABELLED: Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. IMPORTANCE: The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry.

Millisecond spatiotemporal dynamics of FRET biosensors by the pair correlation function and the phasor approach to FLIM.

Here we present a fluctuation-based approach to biosensor Förster resonance energy transfer (FRET) detection that can measure the molecular flow and signaling activity of proteins in live cells. By simultaneous use of the phasor approach to fluorescence lifetime imaging microscopy (FLIM) and cross-pair correlation function (pCF) analysis along a line scanned in milliseconds, we detect the spatial localization of Rho GTPase activity (biosensor FRET signal) as well as the diffusive route adopted by this active population. In particular we find, for Rac1 and RhoA, distinct gradients of activation (FLIM-FRET) and a molecular flow pattern (pCF analysis) that explains the observed polarized GTPase activity. This multiplexed approach to biosensor FRET detection serves as a unique tool for dissection of the mechanism(s) by which key signaling proteins are spatially and temporally coordinated.

Modes of diffusion of cholera toxin bound to GM1 on live cell membrane by image mean square displacement analysis.

The image-mean square displacement technique applies the calculation of the mean square displacement commonly used in single-molecule tracking to images without resolving single particles. The image-mean square displacement plot obtained is similar to the mean square displacement plot obtained using the single-particle tracking technique. This plot is then used to reconstruct the protein diffusion law and to identify whether the labeled molecules are undergoing pure isotropic, restricted, corralled, transiently confined, or directed diffusion. In our study total internal reflection fluorescence microscopy images were taken of Cholera toxin subunit B (CtxB) membrane-labeled NIH 3T3 mouse fibroblasts and MDA 231 MB cells. We found a population of CTxB undergoing purely isotropic diffusion and one displaying restricted diffusion with corral sizes ranging from 150 to ∼1800 nm. We show that the diffusion rate of CTxB bound to GM1 is independent of the size of the confinement, suggesting that the mechanism of confinement is different from the mechanism controlling the diffusion rate of CtxB. We highlight the potential effect of continuous illumination on the diffusion mode of CTxB. We also show that aggregation of CTxB/GM1 in large complexes occurs and that these aggregates tend to have slower diffusion rates.

Eisosomes are dynamic plasma membrane domains showing pil1-lsp1 heteroligomer binding equilibrium.

Eisosomes are plasma membrane domains concentrating lipids, transporters, and signaling molecules. In the budding yeast Saccharomyces cerevisiae, these domains are structured by scaffolds composed mainly by two cytoplasmic proteins Pil1 and Lsp1. Eisosomes are immobile domains, have relatively uniform size, and encompass thousands of units of the core proteins Pil1 and Lsp1. In this work we used fluorescence fluctuation analytical methods to determine the dynamics of eisosome core proteins at different subcellular locations. Using a combination of scanning techniques with autocorrelation analysis, we show that Pil1 and Lsp1 cytoplasmic pools freely diffuse whereas an eisosome-associated fraction of these proteins exhibits slow dynamics that fit with a binding-unbinding equilibrium. Number and brightness analysis shows that the eisosome-associated fraction is oligomeric, while cytoplasmic pools have lower aggregation states. Fluorescence lifetime imaging results indicate that Pil1 and Lsp1 directly interact in the cytoplasm and within the eisosomes. These results support a model where Pil1-Lsp1 heterodimers are the minimal eisosomes building blocks. Moreover, individual-eisosome fluorescence fluctuation analysis shows that eisosomes in the same cell are not equal domains: while roughly half of them are mostly static, the other half is actively exchanging core protein subunits.

A mutation associated with centronuclear myopathy enhances the size and stability of dynamin 2 complexes in cells.

BACKGROUND: Dynamin 2 (Dyn2) is a ~100kDa GTPase that assembles around the necks of nascent endocytic and Golgi vesicles and catalyzes membrane scission. Mutations in Dyn2 that cause centronuclear myopathy (CNM) have been shown to stabilize Dyn2 polymers against GTP-dependent disassembly in vitro. Precisely timed regulation of assembly and disassembly is believed to be critical for Dyn2 function in membrane vesiculation, and the CNM mutations interfere with this regulation by shifting the equilibrium toward the assembled state. METHODS: In this study we use two fluorescence fluctuation spectroscopy (FFS) approaches to show that a CNM mutant form of Dyn2 also has a greater propensity to self-assemble in the cytosol and on the plasma membrane of living cells. RESULTS: Results obtained using brightness analysis indicate that unassembled wild-type Dyn2 is predominantly tetrameric in the cytosol, although different oligomeric species are observed, depending on the concentration of expressed protein. In contrast, an R369W mutant identified in CNM patients forms higher-order oligomers at concentrations above 1µM. Investigation of Dyn2-R369W by Total Internal Reflection Fluorescence (TIRF) FFS reveals that this mutant forms larger and more stable clathrin-containing structures on the plasma membrane than wild-type Dyn2. CONCLUSIONS AND GENERAL SIGNIFICANCE: These observations may explain defects in membrane trafficking reported in CNM patient cells and in heterologous systems expressing CNM-associated Dyn2 mutants.

The Ebola virus matrix protein penetrates into the plasma membrane: a key step in viral protein 40 (VP40) oligomerization and viral egress.

Ebola, a fatal virus in humans and non-human primates, has no Food and Drug Administration-approved vaccines or therapeutics. The virus from the Filoviridae family causes hemorrhagic fever, which rapidly progresses and in some cases has a fatality rate near 90%. The Ebola genome encodes seven genes, the most abundantly expressed of which is viral protein 40 (VP40), the major Ebola matrix protein that regulates assembly and egress of the virus. It is well established that VP40 assembles on the inner leaflet of the plasma membrane; however, the mechanistic details of plasma membrane association by VP40 are not well understood. In this study, we used an array of biophysical experiments and cellular assays along with mutagenesis of VP40 to investigate the role of membrane penetration in VP40 assembly and egress. Here we demonstrate that VP40 is able to penetrate specifically into the plasma membrane through an interface enriched in hydrophobic residues in its C-terminal domain. Mutagenesis of this hydrophobic region consisting of Leu(213), Ile(293), Leu(295), and Val(298) demonstrated that membrane penetration is critical to plasma membrane localization, VP40 oligomerization, and viral particle egress. Taken together, VP40 membrane penetration is an important step in the plasma membrane localization of the matrix protein where oligomerization and budding are defective in the absence of key hydrophobic interactions with the membrane.

Scanning image correlation spectroscopy.

Molecular interactions are at the origin of life. How molecules get at different locations in the cell and how they locate their partners is a major and partially unresolved question in biology that is paramount to signaling. Spatio-temporal correlations of fluctuating fluorescently tagged molecules reveal how they move, interact, and bind in the different cellular compartments. Methods based on fluctuations represent a remarkable technical advancement in biological imaging. Here we discuss image analysis methods based on spatial and temporal correlation of fluctuations, raster image correlation spectroscopy, number and brightness, and spatial cross-correlations that give us information about how individual molecules move in cells and interact with partners at the single molecule level. These methods can be implemented with a standard laser scanning microscope and produce a cellular level spatio-temporal map of molecular interactions.