Video-based pooled screening yields improved far-red genetically encoded voltage indicators.

Video-based screening of pooled libraries is a powerful approach for directed evolution of biosensors because it enables selection along multiple dimensions simultaneously from large libraries. Here we develop a screening platform, Photopick, which achieves precise phenotype-activated photoselection over a large field of view (2.3 × 2.3 mm, containing >103 cells, per shot). We used the Photopick platform to evolve archaerhodopsin-derived genetically encoded voltage indicators (GEVIs) with improved signal-to-noise ratio (QuasAr6a) and kinetics (QuasAr6b). These GEVIs gave improved signals in cultured neurons and in live mouse brains. By combining targeted in vivo optogenetic stimulation with high-precision voltage imaging, we characterized inhibitory synaptic coupling between individual cortical NDNF (neuron-derived neurotrophic factor) interneurons, and excitatory electrical synapses between individual hippocampal parvalbumin neurons. The QuasAr6 GEVIs are powerful tools for all-optical electrophysiology and the Photopick approach could be adapted to evolve a broad range of biosensors.

Alternative Splicing of FN (Fibronectin) Regulates the Composition of the Arterial Wall Under Low Flow.

OBJECTIVE: Exposure of the arterial endothelium to low and disturbed flow is a risk factor for the erosion and rupture of atherosclerotic plaques and aneurysms. Circulating and locally produced proteins are known to contribute to an altered composition of the extracellular matrix at the site of lesions, and to contribute to inflammatory processes within the lesions. We have previously shown that alternative splicing of FN (fibronectin) protects against flow-induced hemorrhage. However, the impact of alternative splicing of FN on extracellular matrix composition remains unknown. Approach and Results: Here, we perform quantitative proteomic analysis of the matrisome of murine carotid arteries in mice deficient in the production of FN splice isoforms containing alternative exons EIIIA and EIIIB (FN-EIIIAB null) after exposure to low and disturbed flow in vivo. We also examine serum-derived and endothelial-cell contributions to the matrisome in a simplified in vitro system. We found flow-induced differences in the carotid artery matrisome that were impaired in FN-EIIIAB null mice. One of the most interesting differences was reduced recruitment of FBLN1 (fibulin-1), abundant in blood and not locally produced in the intima. This defect was validated in our in vitro assay, where FBLN1 recruitment from serum was impaired by the absence of these alternatively spliced segments. CONCLUSIONS: Our results reveal the extent of the dynamic alterations in the matrisome in the acute response to low and disturbed flow and show how changes in the splicing of FN, a common response in vascular inflammation and remodeling, can affect matrix composition.

Bioelectrical domain walls in homogeneous tissues.

Electrical signaling in biology is typically associated with action potentials, transient spikes in membrane voltage that return to baseline. Hodgkin-Huxley and related conductance-based models of electrophysiology belong to a more general class of reaction-diffusion equations which could, in principle, support spontaneous emergence of patterns of membrane voltage which are stable in time but structured in space. Here we show theoretically and experimentally that homogeneous or nearly homogeneous tissues can undergo spontaneous spatial symmetry breaking through a purely electrophysiological mechanism, leading to formation of domains with different resting potentials separated by stable bioelectrical domain walls. Transitions from one resting potential to another can occur through long-range migration of these domain walls. We map bioelectrical domain wall motion using all-optical electrophysiology in an engineered cell line and in human induced pluripotent stem cell (iPSC)-derived myoblasts. Bioelectrical domain wall migration may occur during embryonic development and during physiological signaling processes in polarized tissues. These results demonstrate that nominally homogeneous tissues can undergo spontaneous bioelectrical symmetry breaking.

Alternative RNA splicing in the endothelium mediated in part by Rbfox2 regulates the arterial response to low flow.

Low and disturbed blood flow drives the progression of arterial diseases including atherosclerosis and aneurysms. The endothelial response to flow and its interactions with recruited platelets and leukocytes determine disease progression. Here, we report widespread changes in alternative splicing of pre-mRNA in the flow-activated murine arterial endothelium in vivo. Alternative splicing was suppressed by depletion of platelets and macrophages recruited to the arterial endothelium under low and disturbed flow. Binding motifs for the Rbfox-family are enriched adjacent to many of the regulated exons. Endothelial deletion of Rbfox2, the only family member expressed in arterial endothelium, suppresses a subset of the changes in transcription and RNA splicing induced by low flow. Our data reveal an alternative splicing program activated by Rbfox2 in the endothelium on recruitment of platelets and macrophages and demonstrate its relevance in transcriptional responses during flow-driven vascular inflammation.

Deformability of Tumor Cells versus Blood Cells.

The potential for circulating tumor cells (CTCs) to elucidate the process of cancer metastasis and inform clinical decision-making has made their isolation of great importance. However, CTCs are rare in the blood, and universal properties with which to identify them remain elusive. As technological advancements have made single-cell deformability measurements increasingly routine, the assessment of physical distinctions between tumor cells and blood cells may provide insight into the feasibility of deformability-based methods for identifying CTCs in patient blood. To this end, we present an initial study assessing deformability differences between tumor cells and blood cells, indicated by the length of time required for them to pass through a microfluidic constriction. Here, we demonstrate that deformability changes in tumor cells that have undergone phenotypic shifts are small compared to differences between tumor cell lines and blood cells. Additionally, in a syngeneic mouse tumor model, cells that are able to exit a tumor and enter circulation are not required to be more deformable than the cells that were first injected into the mouse. However, a limited study of metastatic prostate cancer patients provides evidence that some CTCs may be more mechanically similar to blood cells than to typical tumor cell lines.

Tumor angiogenesis in the absence of fibronectin or its cognate integrin receptors.

Binding of α5ß1 and αvß3/ß5 integrin receptors on the endothelium to their fibronectin substrate in the extracellular matrix has been targeted as a possible means of blocking tumor angiogenesis and tumor growth. However, clinical trials of blocking antibodies and peptides have been disappointing despite promising preclinical results, leading to questions about the mechanism of the inhibitors and the reasons for their failure. Here, using tissue-specific and inducible genetics to delete the α5 and αv receptors in the endothelium or their fibronectin substrate, either in the endothelium or globally, we show that both are dispensable for tumor growth, in transplanted tumors as well as spontaneous and angiogenesis-dependent RIP-Tag-driven pancreatic adenocarcinomas. In the nearly complete absence of fibronectin, no differences in vascular density or the deposition of basement membrane laminins, ColIV, Nid1, Nid2, or the TGFß binding matrix proteins, fibrillin-1 and -2, could be observed. Our results reveal that fibronectin and the endothelial fibronectin receptor subunits, α5 and αv, are dispensable for tumor angiogenesis, suggesting that the inhibition of angiogenesis induced by antibodies or small molecules may occur through a dominant negative effect, rather than a simple functional block.

Platelets guide the formation of early metastatic niches.

During metastasis, host cells are recruited to disseminated tumor cells to form specialized microenvironments ("niches") that promote metastatic progression, but the mechanisms guiding the assembly of these niches are largely unknown. Tumor cells may autonomously recruit host cells or, alternatively, host cell-to-host cell interactions may guide the formation of these prometastatic microenvironments. Here, we show that platelet-derived rather than tumor cell-derived signals are required for the rapid recruitment of granulocytes to tumor cells to form "early metastatic niches." Granulocyte recruitment relies on the secretion of CXCL5 and CXCL7 chemokines by platelets upon contact with tumor cells. Blockade of the CXCL5/7 receptor CXCR2, or transient depletion of either platelets or granulocytes prevents the formation of early metastatic niches and significantly reduces metastatic seeding and progression. Thus, platelets recruit granulocytes and guide the formation of early metastatic niches, which are crucial for metastasis.

Determination of 35 cell surface antigen levels in malignant pleural effusions identifies CD24 as a marker of disseminated tumor cells.

Many targets have been identified in solid tumors for antibody therapy but it is less clear what surface antigens may be most commonly expressed on disseminated tumor cells. Using malignant pleural effusions as a source of disseminated tumor cells, we compared a panel of 35 antigens for their cancer specificity, antigen abundance and functional significance. These antigens have been previously implicated in cancer metastasis and fall into four categories: (i) cancer stem cell, (ii) epithelial-mesenchymal transition, (iii) metastatic signature of in vivo selection and (iv) tyrosine kinase receptors. We determined the antigen density of all 35 antigens on the cell surface by flow cytometry, which ranges from 3 × 10(3) -7 × 10(6) copies per cell. Comparison between the malignant and benign pleural effusions enabled us to determine the antigens specific for cancer. We further chose six antigens and examined the correlation between their expression levels and tumor formation in immunocompromised mice. We concluded that CD24 is one of the few antigens that could simultaneously meet all three criteria of an ideal target. It was specifically and abundantly expressed in malignant pleural effusions; CD24(high) tumor cells formed tumors in mice at a faster rate than CD24(low) tumor cells, and shRNA-mediated knockdown of CD24 in HT29 cells confirmed a functional requirement for CD24 in the colonization of the lung. Concomitant consideration of antigen abundance, specificity and functional importance can help identify potentially useful markers for disseminated tumor cells.

Direct signaling between platelets and cancer cells induces an epithelial-mesenchymal-like transition and promotes metastasis.

Interactions of cancer cells with the primary tumor microenvironment are important determinants of cancer progression toward metastasis but it is unknown whether additional prometastatic signals are provided during the intravascular transit to the site of metastasis. Here, we show that platelet-tumor cell interactions are sufficient to prime tumor cells for subsequent metastasis. Platelet-derived TGFß and direct platelet-tumor cell contacts synergistically activate the TGFß/Smad and NF-κB pathways in cancer cells, resulting in their transition to an invasive mesenchymal-like phenotype and enhanced metastasis in vivo. Inhibition of NF-κB signaling in cancer cells or ablation of TGFß1 expression solely in platelets protects against lung metastasis in vivo. Thus, cancer cells rely on platelet-derived signals outside of the primary tumor for efficient metastasis.

GPR56 Regulates VEGF production and angiogenesis during melanoma progression.

Angiogenesis is a critical step during cancer progression. The VEGF is a major stimulator for angiogenesis and is predominantly contributed by cancer cells in tumors. Inhibition of the VEGF signaling pathway has shown promising therapeutic benefits for cancer patients, but adaptive tumor responses are often observed, indicating the need for further understanding of VEGF regulation. We report that a novel G protein-coupled receptor, GPR56, inhibits VEGF production from the melanoma cell lines and impedes melanoma angiogenesis and growth, through the serine threonine proline-rich segment in its N-terminus and a signaling pathway involving protein kinase Cα. We also present evidence that the two fragments of GPR56, which are generated by autocatalyzed cleavage, played distinct roles in regulating VEGF production and melanoma progression. Finally, consistent with its suppressive roles in melanoma progression, the expression levels of GPR56 are inversely correlated with the malignancy of melanomas in human subjects. We propose that components of the GPR56-mediated signaling pathway may serve as new targets for antiangiogenic treatment of melanoma.

GPR56 is essential for testis development and male fertility in mice.

Testis development is critical for male fertility and continuation of the mammalian species. Essential structural components of testes are seminiferous tubules, which are lined by Sertoli cells and provide nutrients and physical protection for the maturation of sperm. Seminiferous tubule formation is initiated in embryos as testis cords and relies on their remodeling for maturation during development. Recently, three-dimensional image analyses showed that testis cords in different parts of embryonic gonads undergo distinct remodeling processes. How this asymmetric remodeling is regulated has not been investigated. We report here that the absence of an adhesion G protein-coupled receptor, GPR56, leads to partial disruption of seminiferous tubules and reduced fertility in male mice. The defects appear to originate asymmetrically in embryonic gonads, but subsequent to the initial establishment of testis cords, suggesting that GPR56 might act to establish a spatial and/or temporal cue for asymmetric cord remodeling during male gonad development.

GPR56 plays varying roles in endogenous cancer progression.

GPR56, a non-classical adhesion receptor, was previously reported to suppress tumor growth and metastasis in xenograft models using human melanoma cell lines. To understand whether GPR56 plays similar roles in the development of endogenous tumors, we analyzed cancer progression in Gpr56 (-/-) mice using a variety of transgenic cancer models. Our results showed that GPR56 suppressed prostate cancer progression in the TRAMP model on a mixed genetic background, similar to its roles in progression of melanoma xenografts. However, its roles in other cancer types appeared to be complex. It had marginal effects on tumor onset of mammary tumors in the MMTV-PyMT model, but had no effects on subsequent tumor progression in either the MMTV-PyMT mice or the melanoma model, Ink4a/Arf (-/-) tyr-Hras. These results indicate diverse roles of GPR56 in cancer progression and provide the first genetic evidence for the involvement of an adhesion GPCR in endogenous cancer development.

GPR56, an atypical G protein-coupled receptor, binds tissue transglutaminase, TG2, and inhibits melanoma tumor growth and metastasis.

The survival and growth of tumor cells in a foreign environment is considered a rate-limiting step during metastasis. To identify genes that may be essential for this process, we isolated highly metastatic variants from a poorly metastatic human melanoma cell line and performed expression analyses of metastases and primary tumors from these cells. GPR56 is among the genes markedly down-regulated in the metastatic variants. We show that overexpression of GPR56 suppresses tumor growth and metastasis, whereas reduced expression of GPR56 enhances tumor progression. Levels of GPR56 do not correlate with growth rate in vitro, suggesting that GPR56 may mediate growth suppression by interaction with a component in the tumor microenvironment in vivo. We show that GPR56 binds specifically to tissue transglutaminase, TG2, a widespread component of tissue and tumor stroma previously implicated as an inhibitor of tumor progression. We discuss the mechanisms whereby GPR56-TG2 interactions may suppress tumor growth and metastasis.

GPVI-deficient mice lack collagen responses and are protected against experimentally induced pulmonary thromboembolism.

Platelet glycoprotein VI (GPVI) is now considered to be a major player in platelet-collagen adhesive interactions leading to thrombus formation. GPVI blockade, or its depletion, has been shown in mice to result in complete protection against arterial thrombosis, without significant prolongation of bleeding time. GPVI may therefore represent a useful antithrombotic target. In order to reaffirm the role of GPVI in platelet-collagen interactions, we developed GPVI(null) mice by targeted disruption methodology. GPVI(null) mice platelets failed to respond to a high dose of fibrillar collagen, or convulxin, a GPVI agonist, but showed a normal response to other agonists such as ADP, PMA and arachidonic acid. We report, for the first time, that a proportion of GPVI(null) mice is protected against lethal thromboembolism, induced by the infusion of a mixture of collagen and epinephrine. Greater than 55% of GPVI(null) mice survived the challenge, whereas the maximal survival from the other genotypes was 17% (n=18 per genotype). Washed platelets obtained from GPVI(null) mice showed >90% reduction in adhesion to fibrillar collagen under static conditions. Platelet adhesion to collagen under dynamic conditions using a high shear rate (2600 s(-1)) was dramatically reduced using blood from GPVI(null) mice, while platelets from wild-type and heterozygous animals showed a similar amount of adhesion. Animals from each genotype had essentially similar tail bleeding time, suggesting that a complete deficiency of GPVI, at least in mice, does not result in an enhanced bleeding tendency. These observations clearly establish that blockade of GPVI may attenuate platelet-collagen interactions without adversely affecting the bleeding time.