Reversible assembly and disassembly of V-ATPase during the lysosome regeneration cycle.

Regulation of the luminal pH of late endocytic compartments in continuously fed mammalian cells is poorly understood. Using normal rat kidney fibroblasts, we investigated the reversible assembly/disassembly of the proton pumping V-ATPase when endolysosomes are formed by kissing and fusion of late endosomes with lysosomes and during the subsequent reformation of lysosomes. We took advantage of previous work showing that sucrosomes formed by the uptake of sucrose are swollen endolysosomes from which lysosomes are reformed after uptake of invertase. Using confocal microscopy and subcellular fractionation of NRK cells stably expressing fluorescently tagged proteins, we found net recruitment of the V1 subcomplex during sucrosome formation and loss during lysosome reformation, with a similar time course to RAB7a loss. Addition of invertase did not alter mTORC1 signalling, suggesting that the regulation of reversible V-ATPase assembly/disassembly in continuously fed cells differs from that in cells subject to amino acid depletion/refeeding. Using live cell microscopy, we demonstrated recruitment of a fluorescently tagged V1 subunit during endolysosome formation and a dynamic equilibrium and rapid exchange between the cytosolic and membrane bound pools of this subunit. We conclude that reversible V-ATPase assembly/disassembly plays a key role in regulating endolysosomal/lysosomal pH in continuously fed cells.

The soluble epoxide hydrolase inhibitor GSK2256294 decreases the proportion of adipose pro-inflammatory T cells.

Adipose tissue contains a complex immune environment and is a central contributor to heightened systemic inflammation in obese persons. Epoxyeicosatrienoic acids (EETs) are lipid signaling molecules that decrease inflammation in obese animals, but their effect on inflammation in humans is unknown. The enzyme soluble epoxide hydrolase (sEH) hydrolyzes EETs to less active diols, and we hypothesized that pharmacologic sEH inhibition would decrease adipose inflammation in obese individuals. We treated obese prediabetic adults with the sEH inhibitor GSK2256294 versus placebo in a crossover design, collected subcutaneous abdominal adipose tissue via lipoaspiration and characterized the tissue T cell profile. Treatment with GSK2256294 decreased the percentage of pro-inflammatory T cells producing interferon-gamma (IFNγ), but not interleukin (IL)-17A, and decreased the amount of secreted tumor necrosis factor-alpha (TNFα). Understanding the contribution of the EET/sEH pathway to inflammation in obesity could lead to new strategies to modulate adipose and systemic inflammation.

Organelle tethering, pore formation and SNARE compensation in the late endocytic pathway.

To provide insights into the kiss-and-run and full fusion events resulting in endocytic delivery to lysosomes, we investigated conditions causing increased tethering and pore formation between late endocytic organelles in HeLa cells. Knockout of the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) VAMP7 and VAMP8 showed, by electron microscopy, the accumulation of tethered lysosome-associated membrane protein (LAMP)-carrier vesicles around multivesicular bodies, as well as the appearance of 'hourglass' profiles of late endocytic organelles attached by filamentous tethers, but did not prevent endocytic delivery to lysosomal hydrolases. Subsequent depletion of the SNARE YKT6 reduced this delivery, consistent with it compensating for the absence of VAMP7 and VAMP8. We also investigated filamentous tethering between multivesicular bodies and enlarged endolysosomes following depletion of charged multi-vesicular body protein 6 (CHMP6), and provide the first evidence that pore formation commences at the edge of tether arrays, with pore expansion required for full membrane fusion.

Wnt1 Promotes Cementum and Alveolar Bone Growth in a Time-Dependent Manner.

The WNT/ß-catenin signaling pathway plays a central role in the biology of the periodontium, yet the function of specific extracellular WNT ligands remains poorly understood. By using a Wnt1-inducible transgenic mouse model targeting Col1a1-expressing alveolar osteoblasts, odontoblasts, and cementoblasts, we demonstrate that the WNT ligand WNT1 is a strong promoter of cementum and alveolar bone formation in vivo. We induced Wnt1 expression for 1, 3, or 9 wk in Wnt1Tg mice and analyzed them at the age of 6 wk and 12 wk. Micro-computed tomography (CT) analyses of the mandibles revealed a 1.8-fold increased bone volume after 1 and 3 wk of Wnt1 expression and a 3-fold increased bone volume after 9 wk of Wnt1 expression compared to controls. In addition, the alveolar ridges were higher in Wnt1Tg mice as compared to controls. Nondecalcified histology demonstrated increased acellular cementum thickness and cellular cementum volume after 3 and 9 wk of Wnt1 expression. However, 9 wk of Wnt1 expression was also associated with periodontal breakdown and ectopic mineralization of the pulp. The composition of this ectopic matrix was comparable to those of cellular cementum as demonstrated by quantitative backscattered electron imaging and immunohistochemistry for noncollagenous proteins. Our analyses of 52-wk-old mice after 9 wk of Wnt1 expression revealed that Wnt1 expression affects mandibular bone and growing incisors but not molar teeth, indicating that Wnt1 influences only growing tissues. To further investigate the effect of Wnt1 on cementoblasts, we stably transfected the cementoblast cell line (OCCM-30) with a vector expressing Wnt1-HA and performed proliferation as well as differentiation experiments. These experiments demonstrated that Wnt1 promotes proliferation but not differentiation of cementoblasts. Taken together, our findings identify, for the first time, Wnt1 as a critical regulator of alveolar bone and cementum formation, as well as provide important insights for harnessing the WNT signal pathway in regenerative dentistry.

Mechanism and evolution of the Zn-fingernail required for interaction of VARP with VPS29.

VARP and TBC1D5 are accessory/regulatory proteins of retromer-mediated retrograde trafficking from endosomes. Using an NMR/X-ray approach, we determined the structure of the complex between retromer subunit VPS29 and a 12 residue, four-cysteine/Zn++ microdomain, which we term a Zn-fingernail, two of which are present in VARP. Mutations that abolish VPS29:VARP binding inhibit trafficking from endosomes to the cell surface. We show that VARP and TBC1D5 bind the same site on VPS29 and can compete for binding VPS29 in vivo. The relative disposition of VPS29s in hetero-hexameric, membrane-attached, retromer arches indicates that VARP will prefer binding to assembled retromer coats through simultaneous binding of two VPS29s. The TBC1D5:VPS29 interaction is over one billion years old but the Zn-fingernail appears only in VARP homologues in the lineage directly giving rise to animals at which point the retromer/VARP/TBC1D5 regulatory network became fully established.

Fast and cloning-free CRISPR/Cas9-mediated genomic editing in mammalian cells.

CHoP-In (CRISPR/Cas9-mediated Homology-independent PCR-product integration) is a fast, non-homologous end-joining based, strategy for genomic editing in mammalian cells. There is no requirement for cloning in generation of the integration donor, instead the desired integration donor is produced as a polymerase chain reaction (PCR) product, flanked by the Cas9 recognition sequences of the target locus. When co-transfected with the cognate Cas9 and guide RNA, double strand breaks are introduced at the target genomic locus and at both ends of the PCR product. This allows incorporation into the genomic locus via hon-homologous end joining. The approach is versatile, allowing N-terminal, C-terminal or internal tag integration and gives predictable genomic integrations, as demonstrated for a selection of well characterised membrane trafficking proteins. The lack of donor vectors offers advantages over existing methods in terms of both speed and hands-on time. As such this approach will be a useful addition to the genome editing toolkit of those working in mammalian cell systems.

Human PAH is characterized by a pattern of lipid-related insulin resistance.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a deadly disease of the small pulmonary vasculature with an increased prevalence of insulin resistance (IR). Insulin regulates both glucose and lipid homeostasis. We sought to quantify glucose- and lipid-related IR in human PAH, testing the hypothesis that lipoprotein indices are more sensitive indices of IR in PAH. METHODS: Oral glucose tolerance testing in PAH patients and triglyceride-matched (TG-matched) controls and proteomic, metabolomics, and lipoprotein analyses were performed in PAH and controls. Results were validated in an external cohort and in explanted human PAH lungs. RESULTS: PAH patients were similarly glucose intolerant or IR by glucose homeostasis metrics compared with control patients when matched for the metabolic syndrome. Using the insulin-sensitive lipoprotein index, TG/HDL ratio, PAH patients were more commonly IR than controls. Proteomic and metabolomic analysis demonstrated separation between PAH and controls, driven by differences in lipid species. We observed a significant increase in long-chain acylcarnitines, phosphatidylcholines, insulin metabolism-related proteins, and in oxidized LDL receptor 1 (OLR1) in PAH plasma in both a discovery and validation cohort. PAH patients had higher lipoprotein axis-related IR and lipoprotein-based inflammation scores compared with controls. PAH patient lung tissue showed enhanced OLR1 immunostaining within plexiform lesions and oxidized LDL accumulation within macrophages. CONCLUSIONS: IR in PAH is characterized by alterations in lipid and lipoprotein homeostasis axes, manifest by elevated TG/HDL ratio, and elevated circulating medium- and long-chain acylcarnitines and lipoproteins. Oxidized LDL and its receptor OLR1 may play a role in a proinflammatory phenotype in PAH. FUNDING: NIH DK096994, HL060906, UL1 RR024975-01, UL1 TR000445-06, DK020593, P01 HL108800-01A1, and UL1 TR002243; American Heart Association 13FTF16070002.

The Lysosome and Intracellular Signalling.

In addition to being the terminal degradative compartment of the cell's endocytic and autophagic pathways, the lysosome is a multifunctional signalling hub integrating the cell's response to nutrient status and growth factor/hormone signalling. The cytosolic surface of the limiting membrane of the lysosome is the site of activation of the multiprotein complex mammalian target of rapamycin complex 1 (mTORC1), which phosphorylates numerous cell growth-related substrates, including transcription factor EB (TFEB). Under conditions in which mTORC1 is inhibited including starvation, TFEB becomes dephosphorylated and translocates to the nucleus where it functions as a master regulator of lysosome biogenesis. The signalling role of lysosomes is not limited to this pathway. They act as an intracellular Ca2+ store, which can release Ca2+ into the cytosol for both local effects on membrane fusion and pleiotropic effects within the cell. The relationship and crosstalk between the lysosomal and endoplasmic reticulum (ER) Ca2+ stores play a role in shaping intracellular Ca2+ signalling. Lysosomes also perform other signalling functions, which are discussed. Current views of the lysosomal compartment recognize its dynamic nature. It includes endolysosomes, autolysosome and storage lysosomes that are constantly engaged in fusion/fission events and lysosome regeneration. How signalling is affected by individual lysosomal organelles being at different stages of these processes and/or at different sites within the cell is poorly understood, but is discussed.

The Rhodococcus equi virulence protein VapA disrupts endolysosome function and stimulates lysosome biogenesis.

Rhodococcus equi (R. equi) is an important pulmonary pathogen in foals that often leads to the death of the horse. The bacterium harbors a virulence plasmid that encodes numerous virulence-associated proteins (Vaps) including VapA that is essential for intracellular survival inside macrophages. However, little is known about the precise function of VapA. Here, we demonstrate that VapA causes perturbation to late endocytic organelles with swollen endolysosome organelles having reduced Cathepsin B activity and an accumulation of LBPA, LC3 and Rab7. The data are indicative of a loss of endolysosomal function, which leads cells to upregulate lysosome biogenesis to compensate for the loss of functional endolysosomes. Although there is a high degree of homology of the core region of VapA to other Vap proteins, only the highly conserved core region of VapA, and not VapD of VapG, gives the observed effects on endolysosomes. This is the first demonstration of how VapA works and implies that VapA aids R. equi survival by reducing the impact of lysosomes on phagocytosed bacteria.

Endolysosomes Are the Principal Intracellular Sites of Acid Hydrolase Activity.

The endocytic delivery of macromolecules from the mammalian cell surface for degradation by lysosomal acid hydrolases requires traffic through early endosomes to late endosomes followed by transient (kissing) or complete fusions between late endosomes and lysosomes. Transient or complete fusion results in the formation of endolysosomes, which are hybrid organelles from which lysosomes are re-formed. We have used synthetic membrane-permeable cathepsin substrates, which liberate fluorescent reporters upon proteolytic cleavage, as well as acid phosphatase cytochemistry to identify which endocytic compartments are acid hydrolase active. We found that endolysosomes are the principal organelles in which acid hydrolase substrates are cleaved. Endolysosomes also accumulated acidotropic probes and could be distinguished from terminal storage lysosomes, which were acid hydrolase inactive and did not accumulate acidotropic probes. Using live-cell microscopy, we have demonstrated that fusion events, which form endolysosomes, precede the onset of acid hydrolase activity. By means of sucrose and invertase uptake experiments, we have also shown that acid-hydrolase-active endolysosomes and acid-hydrolase-inactive, terminal storage lysosomes exist in dynamic equilibrium. We conclude that the terminal endocytic compartment is composed of acid-hydrolase-active, acidic endolysosomes and acid hydrolase-inactive, non-acidic, terminal storage lysosomes, which are linked and function in a lysosome regeneration cycle.

[Bone and adipose tissue formation]. / Knochen- und Fettgewebebildung.

Leptin has been described to have a crucial role in bone homeostasis by systemic as well as local action. Systemically, leptin seems to inhibit bone formation controlled by a feedback loop including osteocalcin and insulin. Even though the action seems to be bone site specific, as well as gender- and time-dependent, the results showing the interaction of these three factors are in part still inconsistent. In this article the complex effects of leptin, insulin, and osteocalcin on bone and fat metabolism are summarized.

A Genetic Screen Identifies a Critical Role for the WDR81-WDR91 Complex in the Trafficking and Degradation of Tetherin.

Tetherin (BST2/CD317) is a viral restriction factor that anchors enveloped viruses to host cells and limits viral spread. The HIV-1 Vpu accessory protein counteracts tetherin by decreasing its cell surface expression and targeting it for ubiquitin-dependent endolysosomal degradation. Although the Vpu-mediated downregulation of tetherin has been extensively studied, the molecular details are not completely elucidated. We therefore used a forward genetic screen in human haploid KBM7 cells to identify novel genes required for tetherin trafficking. Our screen identified WDR81 as a novel gene required for tetherin trafficking and degradation in both the presence and absence of Vpu. WDR81 is a BEACH-domain containing protein that is also required for the degradation of EGF-stimulated epidermal growth factor receptor (EGFR) and functions in a complex with the WDR91 protein. In the absence of WDR81 the endolysosomal compartment appears swollen, with enlarged early and late endosomes and reduced delivery of endocytosed dextran to cathepsin-active lysosomes. Our data suggest a role for the WDR81-WDR91 complex in the fusion of endolysosomal compartments and the absence of WDR81 leads to impaired receptor trafficking and degradation.

Rsk2, the Kinase Mutated in Coffin-Lowry Syndrome, Controls Cementum Formation.

The ribosomal S6 kinase RSK2 is essential for osteoblast function, and inactivating mutations of RSK2 cause osteopenia in humans with Coffin-Lowry syndrome (CLS). Alveolar bone loss and premature tooth exfoliation are also consistently reported symptoms in CLS patients; however, the pathophysiologic mechanisms are unclear. Therefore, aiming to identify the functional relevance of Rsk2 for tooth development, we analyzed Rsk2-deficient mice. Here, we show that Rsk2 is a critical regulator of cementoblast function. Immunohistochemistry, histology, micro-computed tomography imaging, quantitative backscattered electron imaging, and in vitro assays revealed that Rsk2 is activated in cementoblasts and is necessary for proper acellular cementum formation. Cementum hypoplasia that is observed in Rsk2-deficient mice causes detachment and disorganization of the periodontal ligament and was associated with significant alveolar bone loss with age. Moreover, Rsk2-deficient mice display hypomineralization of cellular cementum with accumulation of nonmineralized cementoid. In agreement, treatment of the cementoblast cell line OCCM-30 with a Rsk inhibitor reduces formation of mineralization nodules and decreases the expression of cementum markers. Western blot analyses based on antibodies against Rsk1, Rsk2, and an activated form of the 2 kinases confirmed that Rsk2 is expressed and activated in differentiating OCCM-30 cells. To discriminate between periodontal bone loss and systemic bone loss, we additionally crossed Rsk2-deficient mice with transgenic mice overexpressing the osteoanabolic transcription factor Fra1. Fra1 overexpression clearly increases systemic bone volume in Rsk2-deficient mice but does not protect from alveolar bone loss. Our results indicate that cell autonomous cementum defects are causing early tooth loss in CLS patients. Moreover, we identify Rsk2 as a nonredundant regulator of cementum homeostasis, alveolar bone maintenance, and periodontal health, with all these features being independent of Rsk2 function in systemic bone formation.

Proteomics characterization of exosome cargo.

Characterization of exosomal cargo is of significant interest because this cargo can provide clues to exosome biogenesis, targeting, and cellular effects and may be a source of biomarkers for disease diagnosis, prognosis and response to treatment. With recent improvements in proteomics technologies, both qualitative and quantitative characterization of exosomal proteins is possible. Here we provide a brief review of exosome proteomics studies and provide detailed protocols for global qualitative, global quantitative, and targeted quantitative analysis of exosomal proteins. In addition, we provide an example application of a standard global quantitative analysis followed by validation via a targeted quantitative analysis of urine exosome samples from human patients. Advantages and limitations of each method are discussed as well as future directions for exosome proteomics analysis.

Using problem solving therapy to treat veterans with subsyndromal depression: a pilot study.

OBJECTIVE: We conducted a pilot study comparing problem solving therapy for primary care (PST-PC) to a dietary education control condition in middle-aged and older veterans with symptoms of emotional distress and subsyndromal depression. METHODS: This was a two-site study at the VA Pittsburgh Healthcare System and Philadelphia VA Medical Center. Participants included veterans >50 years of age referred from primary care clinics who were eligible if they obtained a pre-screen score >11 on the Centers for Epidemiologic Studies Depression (CES-D) scale. Exclusions were a DSM-IV Major Depressive Episode within the past year, active substance abuse/dependence within 1 month, current antidepressant therapy, and a Mini mental status exam score <24. Participants were randomized to receive one of two interventions--either PST-PC or an attention control condition consisting of dietary education (DIET)--each consisting of six to eight sessions within a 4-month period. RESULTS: Of 45 individuals randomized, 23 (11 PST-PC and 12 DIET) completed treatment. Using regression models in completers that examined outcomes at end of treatment while controlling for baseline scores, there were significant differences between treatment groups in SF-36 mental health component scores but not in depressive symptoms (as assessed with either the 17-item Hamilton Rating Scale for Depression or the Beck Depression Inventory), social problem solving skills, or physical health status (SF-36 physical health component score). CONCLUSIONS: These pilot study findings suggest that a six-to-eight session version of PST-PC may lead to improvements in mental health functioning in primary care veterans with subsyndromal depressive symptoms.

Fra-2/AP-1 controls adipocyte differentiation and survival by regulating PPARγ and hypoxia.

Adipocyte cell number is a crucial factor for controlling of body weight and metabolic function. The regulation of adipocyte numbers in the adult organism is not fully understood but is considered to depend on the homeostasis of cell differentiation and apoptosis. Herein, we show that targeted deletion of the activator protein (AP-1)-related transcription factor Fra-2 in adipocytes in vivo (Fra-2(Δadip) mice) induces a high-turnover phenotype with increased differentiation and apoptosis of adipocytes, leading to a decrease in body weight and fat pad mass. Importantly, adipocyte cell numbers were significantly reduced in Fra-2(Δadip) mice. At the molecular level, Fra-2 directly binds to the PPARγ2 promoter and represses PPARγ2 expression. Deletion of Fra-2 leads to increased PPARγ2 expression and adipocyte differentiation as well as increased adipocyte apoptosis through upregulation of hypoxia-inducible factors (HIFs). These findings suggest that Fra-2 is an important checkpoint to control adipocyte turnover. Therefore, inhibition of Fra-2 may emerge as a useful strategy to increase adipocyte turnover and to reduce adipocyte numbers and fat mass in the body.

Reconstruction of surface potential from Kelvin probe force microscopy images.

We present an algorithm for reconstructing a sample surface potential from its Kelvin probe force microscopy (KPFM) image. The measured KPFM image is a weighted average of the surface potential underneath the tip apex due to the long-range electrostatic forces. We model the KPFM measurement by a linear shift-invariant system where the impulse response is the point spread function (PSF). By calculating the PSF of the KPFM probe (tip+cantilever) and using the measured noise statistics, we deconvolve the measured KPFM image to obtain the surface potential of the sample.The reconstruction algorithm is applied to measurements of CdS-PbS nanorods measured in amplitude modulation KPFM (AM-KPFM) and to graphene layers measured in frequency modulation KPFM (FM-KPFM). We show that in the AM-KPFM measurements the averaging effect is substantial, whereas in the FM-KPFM measurements the averaging effect is negligible.

Aldosterone deficiency prevents high-fat-feeding-induced hyperglycaemia and adipocyte dysfunction in mice.

AIMS/HYPOTHESIS: Obesity is associated with aldosterone excess, hypertension and the metabolic syndrome, but the relative contribution of aldosterone to obesity-related complications is debated. We previously demonstrated that aldosterone impairs insulin secretion, and that genetic aldosterone deficiency increases glucose-stimulated insulin secretion in vivo. We hypothesised that elimination of endogenous aldosterone would prevent obesity-induced insulin resistance and hyperglycaemia. METHODS: Wild-type and aldosterone synthase-deficient (As (-/-)) mice were fed a high-fat (HF) or normal chow diet for 12 weeks. We assessed insulin sensitivity and insulin secretion using clamp methodology and circulating plasma adipokines, and examined adipose tissue via histology. RESULTS: HF diet induced weight gain similarly in the two groups, but As (-/-) mice were protected from blood glucose elevation. HF diet impaired insulin sensitivity similarly in As (-/-) and wild-type mice, assessed by hyperinsulinaemic-euglycaemic clamps. Fasting and glucose-stimulated insulin were higher in HF-fed As (-/-) mice than in wild-type controls. Although there was no difference in insulin sensitivity during HF feeding in As (-/-) mice compared with wild-type controls, fat mass, adipocyte size and adiponectin increased, while adipose macrophage infiltration decreased. HF feeding significantly increased hepatic steatosis and triacylglycerol content in wild-type mice, which was attenuated in aldosterone-deficient mice. CONCLUSIONS/INTERPRETATION: These studies demonstrate that obesity induces insulin resistance independently of aldosterone and adipose tissue inflammation, and suggest a novel role for aldosterone in promoting obesity-induced beta cell dysfunction, hepatic steatosis and adipose tissue inflammation.