Isoform a4 of the vacuolar ATPase a subunit promotes 4T1-12B breast cancer cell-dependent tumor growth and metastasis in vivo.

The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump that governs the pH of various intracellular compartments and also functions at the plasma membrane in certain cell types, including cancer cells. Membrane targeting of the V-ATPase is controlled by isoforms of subunit a, and we have previously shown that isoforms a3 and a4 are important for the migration and invasion of several breast cancer cell lines in vitro. Using CRISPR-mediated genome editing to selectively disrupt each of the four a subunit isoforms, we also recently showed that a4 is critical to plasma membrane V-ATPase localization, as well as in vitro migration and invasion of 4T1-12B murine breast cancer cells. We now report that a4 is important for the growth of 4T1-12B tumors in vivo. We found that BALB/c mice bearing a4-/- 4T1-12B allografts had significantly smaller tumors than mice in the control group. In addition, we determined that a4-/- allografts showed dramatically reduced metastases to the lung and reduced luminescence intensity of metastases to bone relative to the control group. Taken together, these results suggest that the a4 isoform of the V-ATPase represents a novel potential therapeutic target to limit breast cancer growth and metastasis.

Isoform-specific gene disruptions reveal a role for the V-ATPase subunit a4 isoform in the invasiveness of 4T1-12B breast cancer cells.

The vacuolar H+-ATPase (V-ATPase) is an ATP-driven proton pump present in various intracellular membranes and at the plasma membrane of specialized cell types. Previous work has reported that plasma membrane V-ATPases are key players in breast cancer cell invasiveness. The two subunit a-isoforms known to target the V-ATPase to the plasma membrane are a3 and a4, and expression of a3 has been shown to correlate with plasma membrane localization of the V-ATPase in various invasive human breast cancer cell lines. Here we analyzed the role of subunit a-isoforms in the invasive mouse breast cancer cell line, 4T1-12B. Quantitation of mRNA levels for each isoform by quantitative RT-PCR revealed that a4 is the dominant isoform expressed in these cells. Using a CRISPR/Cas9-based approach to disrupt the genes encoding each of the four V-ATPase subunit a-isoforms, we found that ablation of only the a4-encoding gene significantly inhibits invasion and migration of 4T1-12B cells. Additionally, cells with disrupted a4 exhibited reduced V-ATPase expression at the leading edge, suggesting that the a4 isoform is primarily responsible for targeting the V-ATPase to the plasma membrane in 4T1-12B cells. These findings suggest that different subunit a-isoforms may direct V-ATPases to the plasma membrane of different invasive breast cancer cell lines. They further suggest that expression of V-ATPases at the cell surface is the primary factor that promotes an invasive cancer cell phenotype.

Glucose starvation increases V-ATPase assembly and activity in mammalian cells through AMP kinase and phosphatidylinositide 3-kinase/Akt signaling.

The vacuolar H+-ATPase (V-ATPase) is an ATP-driven proton pump involved in many cellular processes. An important mechanism by which V-ATPase activity is controlled is the reversible assembly of its two domains, namely the peripheral V1 domain and the integral V0 domain. Although reversible assembly is conserved across all eukaryotic organisms, the signaling pathways controlling it have not been fully characterized. Here, we identify glucose starvation as a novel regulator of V-ATPase assembly in mammalian cells. During acute glucose starvation, the V-ATPase undergoes a rapid and reversible increase in assembly and activity as measured by lysosomal acidification. Because the V-ATPase has recently been implicated in the activation of AMP kinase (AMPK), a critical cellular energy sensor that is also activated upon glucose starvation, we compared the time course of AMPK activation and V-ATPase assembly upon glucose starvation. We observe that AMPK activation precedes increased V-ATPase activity. Moreover, the starvation-induced increase in V-ATPase activity and assembly are prevented by the AMPK inhibitor dorsomorphin. These results suggest that increased assembly and activity of the V-ATPase upon glucose starvation are dependent upon AMPK. We also find that the PI3K/Akt pathway, which has previously been implicated in controlling V-ATPase assembly in mammalian cells, also plays a role in the starvation-induced increase in V-ATPase assembly and activity. These studies thus identify a novel stimulus of V-ATPase assembly and a novel signaling pathway involved in regulating this process. The possible function of starvation-induced increase in lysosomal V-ATPase activity is discussed.

Involvement of a gut-retina axis in protection against dietary glycemia-induced age-related macular degeneration.

Age-related macular degeneration (AMD) is the major cause of blindness in developed nations. AMD is characterized by retinal pigmented epithelial (RPE) cell dysfunction and loss of photoreceptor cells. Epidemiologic studies indicate important contributions of dietary patterns to the risk for AMD, but the mechanisms relating diet to disease remain unclear. Here we investigate the effect on AMD of isocaloric diets that differ only in the type of dietary carbohydrate in a wild-type aged-mouse model. The consumption of a high-glycemia (HG) diet resulted in many AMD features (AMDf), including RPE hypopigmentation and atrophy, lipofuscin accumulation, and photoreceptor degeneration, whereas consumption of the lower-glycemia (LG) diet did not. Critically, switching from the HG to the LG diet late in life arrested or reversed AMDf. LG diets limited the accumulation of advanced glycation end products, long-chain polyunsaturated lipids, and their peroxidation end-products and increased C3-carnitine in retina, plasma, or urine. Untargeted metabolomics revealed microbial cometabolites, particularly serotonin, as protective against AMDf. Gut microbiota were responsive to diet, and we identified microbiota in the Clostridiales order as being associated with AMDf and the HG diet, whereas protection from AMDf was associated with the Bacteroidales order and the LG diet. Network analysis revealed a nexus of metabolites and microbiota that appear to act within a gut-retina axis to protect against diet- and age-induced AMDf. The findings indicate a functional interaction between dietary carbohydrates, the metabolome, including microbial cometabolites, and AMDf. Our studies suggest a simple dietary intervention that may be useful in patients to arrest AMD.

Regulation of V-ATPase activity.

V-ATPases are ATP-driven proton pumps present in both intracellular and cell surface membranes of eukaryotes that function in many normal and disease processes. V-ATPases are large, multi-subunit complexes composed of a peripheral domain (V1) that hydrolyzes ATP and a membrane integral domain (V0) that translocates protons. Because of the diversity of their functions, V-ATPase activity is controlled by a number of mechanisms. Regulated assembly of the V1 and V0 domains rapidly modulates V-ATPase activity in response to a variety of cues, including nutrient availability, growth factor stimulation and cellular differentiation. Considerable information has recently emerged concerning the cellular signaling pathways controlling regulated assembly. Acid secretion by epithelial cells in the kidney and epididymus is controlled by regulated trafficking of V-ATPases to the cell surface. Isoforms of subunit a of the V0 domain both control trafficking of V-ATPases to distinct cellular membranes and confer properties to the resultant complexes that help account for differences in pH between cellular compartments. Finally, differential expression of genes encoding V-ATPases subunits occurs in a number of contexts, including cancer.

Highly variable penetrance of abnormal phenotypes in embryonic lethal knockout mice.

Background: Identifying genes that are essential for mouse embryonic development and survival through term is a powerful and unbiased way to discover possible genetic determinants of human developmental disorders. Characterising the changes in mouse embryos that result from ablation of lethal genes is a necessary first step towards uncovering their role in normal embryonic development and establishing any correlates amongst human congenital abnormalities. Methods: Here we present results gathered to date in the Deciphering the Mechanisms of Developmental Disorders (DMDD) programme, cataloguing the morphological defects identified from comprehensive imaging of 220 homozygous mutant and 114 wild type embryos from 42 lethal and subviable lines, analysed at E14.5. Results: Virtually all mutant embryos show multiple abnormal phenotypes and amongst the 42 lines these affect most organ systems. Within each mutant line, the phenotypes of individual embryos form distinct but overlapping sets. Subcutaneous edema, malformations of the heart or great vessels, abnormalities in forebrain morphology and the musculature of the eyes are all prevalent phenotypes, as is loss or abnormal size of the hypoglossal nerve.Conclusions: Overall, the most striking finding is that no matter how profound the malformation, each phenotype shows highly variable penetrance within a mutant line. These findings have challenging implications for efforts to identify human disease correlates.

Regulation of V-ATPase assembly and function of V-ATPases in tumor cell invasiveness.

V-ATPases are ATP-driven proton pumps that function within both intracellular compartments and the plasma membrane in a wide array of normal physiological and pathophysiological processes. V-ATPases are composed of a peripheral V(1) domain that hydrolyzes ATP and an integral V(0) domain that transports protons. Regulated assembly of the V-ATPase represents an important mechanism of regulating V-ATPase activity in response to a number of environmental cues. Our laboratory has demonstrated that glucose-dependent assembly of the V-ATPase complex in yeast is controlled by the Ras/cAMP/PKA pathway. By contrast, increased assembly of the V-ATPase during dendritic cell maturation involves the PI-3 kinase and mTORC1 pathways. Recently, we have shown that amino acids regulate V-ATPase assembly in mammalian cells, possibly as a means to maintain adequate levels of amino acids upon nutrient starvation. V-ATPases have also been implicated in cancer cell survival and invasion. V-ATPases are targeted to different cellular membranes by isoforms of subunit a, with a3 targeting V-ATPases to the plasma membrane of osteoclasts. We have shown that highly invasive human breast cancer cell lines express higher levels of the a3 isoform than poorly invasive lines and that knockdown of a3 reduces both expression of V-ATPases at the plasma membrane and in vitro invasion of breast tumor cells. Moreover, overexpression of a3 in a non-invasive breast epithelial line increases both plasma membrane V-ATPases and in vitro invasion. Finally, specific ablation of plasma membrane V-ATPases in highly invasive human breast cancer cells using either an antibody or small molecule approach inhibits both in vitro invasion and migration. These results suggest that plasma membrane and a3-containing V-ATPases represent a novel and important target in the development of therapeutics to limit breast cancer metastasis. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Deciphering the mechanisms of developmental disorders: phenotype analysis of embryos from mutant mouse lines.

The Deciphering the Mechanisms of Developmental Disorders (DMDD) consortium is a research programme set up to identify genes in the mouse, which if mutated (or knocked-out) result in embryonic lethality when homozygous, and initiate the study of why disruption of their function has such profound effects on embryo development and survival. The project uses a combination of comprehensive high resolution 3D imaging and tissue histology to identify abnormalities in embryo and placental structures of embryonic lethal lines. The image data we have collected and the phenotypes scored are freely available through the project website (http://dmdd.org.uk). In this article we describe the web interface to the images that allows the embryo data to be viewed at full resolution in different planes, discuss how to search the database for a phenotype, and our approach to organising the data for an embryo and a mutant line so it is easy to comprehend and intuitive to navigate.

Recent Insights into the Structure, Regulation, and Function of the V-ATPases.

The vacuolar (H(+))-ATPases (V-ATPases) are ATP-dependent proton pumps that acidify intracellular compartments and are also present at the plasma membrane. They function in such processes as membrane traffic, protein degradation, virus and toxin entry, bone resorption, pH homeostasis, and tumor cell invasion. V-ATPases are large multisubunit complexes, composed of an ATP-hydrolytic domain (V1) and a proton translocation domain (V0), and operate by a rotary mechanism. This review focuses on recent insights into their structure and mechanism, the mechanisms that regulate V-ATPase activity (particularly regulated assembly and trafficking), and the role of V-ATPases in processes such as cell signaling and cancer. These developments have highlighted the potential of V-ATPases as a therapeutic target in a variety of human diseases.

The surface chemistry of metal-organic frameworks.

Metal-organic frameworks (MOFs) have received particular attention over the last 20 years as a result of their attractive properties offering potential applications in a number of areas. Typically, these characteristics are tuned by functionalisation of the bulk of the MOF material itself. This Feature Article focuses instead on modification of MOF particles at their surfaces only, which can also offer control over the bulk properties of the material. The differing surface modification techniques available to the synthetic chemist will be discussed, with a focus on the effect of surface modification of MOFs on their fundamental properties and application in adsorption, catalysis, drug delivery and other areas.