Yeast Fex1p Is a Constitutively Expressed Fluoride Channel with Functional Asymmetry of Its Two Homologous Domains.

Fluoride is a ubiquitous environmental toxin with which all biological species must cope. A recently discovered family of fluoride export (FEX) proteins protects organisms from fluoride toxicity by removing it from the cell. We show here that FEX proteins in Saccharomyces cerevisiae function as ion channels that are selective for fluoride over chloride and that these proteins are constitutively expressed at the yeast plasma membrane. Continuous expression is in contrast to many other toxin exporters in yeast, and this, along with the fact that two nearly duplicate proteins are encoded in the yeast genome, suggests that the threat posed by fluoride ions is frequent and detrimental. Structurally, eukaryotic FEX proteins consist of two homologous four-transmembrane helix domains folded into an antiparallel dimer, where the orientation of the two domains is fixed by a single transmembrane linker helix. Using phylogenetic sequence conservation as a guide, we have identified several functionally important residues. There is substantial functional asymmetry in the effect of mutation at corresponding sites in the two domains. Specifically, mutations to residues in the C-terminal domain proved significantly more detrimental to function than did similar mutations in the N-terminal domain. Our data suggest particular residues that may be important to anion specificity, most notably the necessity of a positive charge near the end of TMH1 in the C-terminal domain. It is possible that a cationic charge at this location may create an electrostatic well for fluoride ions entering the channel from the cytoplasm.

Eukaryotic resistance to fluoride toxicity mediated by a widespread family of fluoride export proteins.

Fluorine is an abundant element and is toxic to organisms from bacteria to humans, but the mechanisms by which eukaryotes resist fluoride toxicity are unknown. The Escherichia coli gene crcB was recently shown to be regulated by a fluoride-responsive riboswitch, implicating it in fluoride response. There are >8,000 crcB homologs across all domains of life, indicating that it has an important role in biology. Here we demonstrate that eukaryotic homologs [renamed FEX (fluoride exporter)] function in fluoride export. FEX KOs in three eukaryotic model organisms, Neurospora crassa, Saccharomyces cerevisiae, and Candida albicans, are highly sensitized to fluoride (>200-fold) but not to other halides. Some of these KO strains are unable to grow in fluoride concentrations found in tap water. Using the radioactive isotope of fluoride, (18)F, we developed an assay to measure the intracellular fluoride concentration and show that the FEX deletion strains accumulate fluoride in excess of the external concentration, providing direct evidence of FEX function in fluoride efflux. In addition, they are more sensitive to lower pH in the presence of fluoride. These results demonstrate that eukaryotic FEX genes encode a previously unrecognized class of fluoride exporter necessary for survival in standard environmental conditions.

Loss-of-function mutations in Notch receptors in cutaneous and lung squamous cell carcinoma.

Squamous cell carcinomas (SCCs) are one of the most frequent forms of human malignancy, but, other than TP53 mutations, few causative somatic aberrations have been identified. We identified NOTCH1 or NOTCH2 mutations in ~75% of cutaneous SCCs and in a lesser fraction of lung SCCs, defining a spectrum for the most prevalent tumor suppressor specific to these epithelial malignancies. Notch receptors normally transduce signals in response to ligands on neighboring cells, regulating metazoan lineage selection and developmental patterning. Our findings therefore illustrate a central role for disruption of microenvironmental communication in cancer progression. NOTCH aberrations include frameshift and nonsense mutations leading to receptor truncations as well as point substitutions in key functional domains that abrogate signaling in cell-based assays. Oncogenic gain-of-function mutations in NOTCH1 commonly occur in human T-cell lymphoblastic leukemia/lymphoma and B-cell chronic lymphocytic leukemia. The bifunctional role of Notch in human cancer thus emphasizes the context dependency of signaling outcomes and suggests that targeted inhibition of the Notch pathway may induce squamous epithelial malignancies.

Macrophage mediated mesoscale brain mechanical homeostasis mechanically imaged via optical tweezers and Brillouin microscopy in vivo.

Tissues are active materials where epithelial turnover, immune surveillance, and remodeling of stromal cells such as macrophages all regulate form and function. Scattering modalities such as Brillouin microscopy (BM) can non-invasively access mechanical signatures at GHz. However, our traditional understanding of tissue material properties is derived mainly from modalities which probe mechanical properties at different frequencies. Thus, reconciling measurements amongst these modalities remains an active area. Here, we compare optical tweezer active microrheology (OT-AMR) and Brillouin microscopy (BM) to longitudinally map brain development in the larval zebrafish. We determine that each measurement is able to detect a mechanical signature linked to functional units of the brain. We demonstrate that the corrected BM-Longitudinal modulus using a density factor correlates well with OT-AMR storage modulus at lower frequencies. We also show that the brain tissue mechanical properties are dependent on both the neuronal architecture and the presence of macrophages. Moreover, the BM technique is able to delineate the contributions to mechanical properties of the macrophage from that due to colony stimulating factor 1 receptor (CSF1R) mediated stromal remodeling. Here, our data suggest that macrophage remodeling is instrumental in the maintenance of tissue mechanical homeostasis during development. Moreover, the strong agreement between the OT-AM and BM further demonstrates that scattering-based technique is sensitive to both large and minute structural modification in vivo.

Organ specific microenvironmental MR1 expression in cutaneous melanoma.

The microenvironment is an important regulator of intertumoral trafficking and activity of immune cells. Understanding how the immune system can be tailored to maintain anti-tumor killing responses in metastatic disease remains an important goal. Thus, immune mediated eradication of metastasis requires the consideration of organ specific microenvironmental cues. Using a xenograft model of melanoma metastasis in adult zebrafish, we perturbed the dynamic balance between the infiltrating immune cells in the metastatic setting using a suite of different transgenic zebrafish. We employed intravital imaging coupled with metabolism imaging (FLIM) to visualize and map the organ specific metabolism with near simultaneity in multiple metastatic lesions. Of all the MHC complexes examined for brain and skeletal metastases, we determined that there is an organ specific expression of mhc1uba (human ortholog, MR1) for both the melanoma cells and the resident and infiltrating immune cells. Specifically, immune clusters did not express mhc1uba in brain metastatic lesions in immune competent fish. Finally, the differential immune response drove organ specific metabolism where tumor glycolysis was increased in brain metastases compared to skeletal and parental lines as measured using fluorescence lifetime imaging microscopy (FLIM). As MR1 belongs to the MHC class I molecules and is a target of immunotherapeutic drugs, we believe that our data presents an opportunity to understand the relationship between organ specific tumor metabolism and drug efficacy in the metastatic setting.

YAP localization mediates mechanical adaptation of human cancer cells during extravasation in vivo.

Biophysical profiling of primary tumors has revealed that individual tumor cells fall along a highly heterogeneous continuum of mechanical phenotypes. One idea is that a subset of tumor cells is "softer" to facilitate detachment and escape from the primary site, a step required to initiate metastasis. However, it has also been postulated that cells must be able to deform and generate sufficient force to exit into distant sites. Here, we aimed to dissect the mechanical changes that occur during extravasation and organ colonization. Using multiplexed methods of intravital microscopy and optical tweezer based active microrheology, we obtained longitudinal images and mechanical profiles of cells during organ colonization in vivo. We determined that cells were softer, more liquid like upon exit of the vasculature but stiffened and became more solid like once in the new organ microenvironment. We also determined that a YAP mediated mechanogenotype influenced the global dissemination in our in vivo and in vitro models and that reducing mechanical heterogeneity could reduce extravasation. Moreover, our high throughput analysis of mechanical phenotypes of patient samples revealed that this mechanics was in part regulated by the external hydrodynamic forces that the cancer cells experienced within capillary mimetics. Our findings indicate that disseminated cancer cells can keep mutating with a continuum landscape of mechano-phenotypes, governed by the YAP-mediated mechanosensing of hydrodynamic flow.

Genome-Wide Identification of Genes Involved in General Acid Stress and Fluoride Toxicity in Saccharomyces cerevisiae.

Hydrofluoric acid elicits cell cycle arrest through a mechanism that has long been presumed to be linked with the high affinity of fluoride to metals. However, we have recently found that the acid stress from fluoride exposure is sufficient to elicit many of the hallmark phenotypes of fluoride toxicity. Here we report the systematic screening of genes involved in fluoride resistance and general acid resistance using a genome deletion library in Saccharomyces cerevisiae. We compare these to a variety of acids - 2,4-dinitrophenol, FCCP, hydrochloric acid, and sulfuric acid - none of which has a high metal affinity. Pathways involved in endocytosis, vesicle trafficking, pH maintenance, and vacuolar function are of particular importance to fluoride tolerance. The majority of genes conferring resistance to fluoride stress also enhanced resistance to general acid toxicity. Genes whose expression regulate Golgi-mediated vesicle transport were specific to fluoride resistance, and may be linked with fluoride-metal interactions. These results support the notion that acidity is an important and underappreciated principle underlying the mechanisms of fluoride toxicity.

Independent regulation of age associated fat accumulation and longevity.

Age-dependent changes in metabolism can manifest as cellular lipid accumulation, but how this accumulation is regulated or impacts longevity is poorly understood. We find that Saccharomyces cerevisiae accumulate lipid droplets (LDs) during aging. We also find that over-expressing BNA2, the first Biosynthesis of NAD+ (kynurenine) pathway gene, reduces LD accumulation during aging and extends lifespan. Mechanistically, this LD accumulation during aging is not linked to NAD+ levels, but is anti-correlated with metabolites of the shikimate and aromatic amino acid biosynthesis (SA) pathways (upstream of BNA2), which produce tryptophan (the Bna2p substrate). We provide evidence that over-expressed BNA2 skews glycolytic flux from LDs towards the SA-BNA pathways, effectively reducing LDs. Importantly, we find that accumulation of LDs does not shorten lifespan, but does protect aged cells against stress. Our findings reveal how lipid accumulation impacts longevity, and how aging cell metabolism can be rewired to modulate lipid accumulation independently from longevity.

Fluoride export (FEX) proteins from fungi, plants and animals are 'single barreled' channels containing one functional and one vestigial ion pore.

The fluoride export protein (FEX) in yeast and other fungi provides tolerance to fluoride (F-), an environmentally ubiquitous anion. FEX efficiently eliminates intracellular fluoride that otherwise would accumulate at toxic concentrations. The FEX homolog in bacteria, Fluc, is a 'double-barreled' channel formed by dimerization of two identical or similar subunits. FEX in yeast and other eukaryotes is a monomer resulting from covalent fusion of the two subunits. As a result, both potential fluoride pores are created from different parts of the same protein. Here we identify FEX proteins from two multicellular eukaryotes, a plant Arabidopsis thaliana and an animal Amphimedon queenslandica, by demonstrating significant fluoride tolerance when these proteins are heterologously expressed in the yeast Saccharomyces cerevisiae. Residues important for eukaryotic FEX function were determined by phylogenetic sequence alignment and functional analysis using a yeast growth assay. Key residues of the fluoride channel are conserved in only one of the two potential fluoride-transporting pores. FEX activity is abolished upon mutation of residues in this conserved pore, suggesting that only one of the pores is functional. The same topology is conserved for the newly identified FEX proteins from plant and animal. These data suggest that FEX family of fluoride channels in eukaryotes are 'single-barreled' transporters containing one functional pore and a second non-functional vestigial remnant of a homologous gene fusion event.

Host phenotype characteristics and MC1R in relation to early-onset basal cell carcinoma.

Basal cell carcinoma (BCC) incidence is increasing, particularly among adults under the age of 40 years. Pigment-related characteristics are associated with BCC in older populations, but epidemiologic studies among younger individuals and analyses of phenotype-genotype interactions are limited. We examined self-reported phenotypes and melanocortin 1 receptor gene (MC1R) variants in relation to early-onset BCC. BCC cases (n=377) and controls with benign skin conditions (n=390) under the age of 40 years were identified through Yale's Dermatopathology database. Factors most strongly associated with early-onset BCC were skin reaction to first summer sun for 1 hour (severe sunburn vs. tan odds ratio (OR)=12.27, 95% confidence interval (CI)=4.08-36.94) and skin color (very fair vs. olive OR=11.06, 95% CI=5.90-20.74). Individuals with two or more MC1R non-synonymous variants were 3.59 times (95% CI=2.37-5.43) more likely to have BCC than those without non-synonymous variants. All host characteristics and MC1R were more strongly associated with multiple BCC case status (37% of cases) than a single BCC case status. MC1R, number of moles, skin reaction to first summer sun for 1 hour, and hair and skin color were independently associated with BCC. BCC risk conferred by MC1R tended to be stronger among those with darker pigment phenotypes, traditionally considered to be at low risk of skin cancer.

Correlation of Global MicroRNA Expression With Basal Cell Carcinoma Subtype.

Basal cell carcinomas (BCCs) are the most common cancers in the United States. The histologic appearance distinguishes several subtypes, each of which can have a different biologic behavior. In this study, global miRNA expression was quantified by high-throughput sequencing in nodular BCCs, a subtype that is slow growing, and infiltrative BCCs, aggressive tumors that extend through the dermis and invade structures such as cutaneous nerves. Principal components analysis correctly classified seven of eight infiltrative tumors on the basis of miRNA expression. The remaining tumor, on pathology review, contained a mixture of nodular and infiltrative elements. Nodular tumors did not cluster tightly, likely reflecting broader histopathologic diversity in this class, but trended toward forming a group separate from infiltrative BCCs. Quantitative polymerase chain reaction assays were developed for six of the miRNAs that showed significant differences between the BCC subtypes, and five of these six were validated in a replication set of four infiltrative and three nodular tumors. The expression level of miR-183, a miRNA that inhibits invasion and metastasis in several types of malignancies, was consistently lower in infiltrative than nodular tumors and could be one element underlying the difference in invasiveness. These results represent the first miRNA profiling study in BCCs and demonstrate that miRNA gene expression may be involved in tumor pathogenesis and particularly in determining the aggressiveness of these malignancies.