The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5

In yeast and mammals, the AAA ATPase Vps4p/SKD1 (for Vacuolar protein sorting 4/SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1) is required for the endosomal sorting of secretory and endocytic cargo. We identified a VPS4/SKD1 homolog in Arabidopsis thaliana, which localizes to the cytoplasm and to multivesicular endosomes. In addition, green fluorescent protein-SKD1 colocalizes on multivesicular bodies with fluorescent fusion protein endosomal Rab GTPases, such as ARA6/RabF1, RHA1/RabF2a, and ARA7/RabF2b, and with the endocytic marker FM4-64. The expression of SKD1E232Q, an ATPase-deficient version of SKD1, induces alterations in the endosomal system of tobacco (Nicotiana tabacum) Bright Yellow 2 cells and ultimately leads to cell death. The inducible expression ofSKD1E232Q in Arabidopsis resulted in enlarged endosomes with a reduced number of internal vesicles. In a yeast two-hybrid screen using Arabidopsis SKD1 as bait, we isolated a putative homolog of mammalian LYST-INTERACTING PROTEIN5 (LIP5)/SKD1 BINDING PROTEIN1 and yeast Vta1p (for Vps twenty associated 1 protein). Arabidopsis LIP5 acts as a positive regulator of SKD1 by increasing fourfold to fivefold its in vitro ATPase activity. We isolated a knockout homozygous Arabidopsis mutant line with a T-DNA insertion in LIP5. lip5 plants are viable and show no phenotypic alterations under normal growth conditions, suggesting that basal SKD1 ATPase activity is sufficient for plant development and growth.Facultad de Ciencias Agrarias y Forestale

Turfgrass research report 1996

Preemergence herbicide efficacy on crabgrass-1996 / J. Street and R. Stewart --Postemergence herbicide efficacy on crabgrass -1996 / J. Street and R. Stewart -- General turfgrass broadleaf weed control evaluation / W. Pound -- Nonselective herbicide evaluation / W. Pound -- Postemergent yellow nutsedge evaluation / W. Pound and R. Stewart -- Preemergent broadleaf weed control / W. Pound -- Control of ant mounds in turfgrass-1996 / D. J. Shetlar, H. D. Niemczyk and K. T. Power -- Control of hairy chinch bugs in turfgrass -1996 / D. J. Shetlar, H. D. Niemczyk and K. T. Power -- Preventive and curative insecticide applications for control of hairy chinch bugs in turfgrass - 1996 / D. J. Shetlar, H. D. Niemczyk and K. T. Power -- Quick kill of black cutworm larvae in bentgrass - 1996 / D. J. Shetlar, H. D. Niemczyk and K. T. Power -- Control of black cutworm, Agrotis ipsilon (Hufnagel), and sod webworms (Pyralidae: Crambinae) in creeping bentgrass, Agrostis palustris Hudson, with spinosad formulations, Columbus, Ohio, 1996 / D. J. Shetlar, H. D. Niemczyk and M. Belcher -- Control of black cutworm, Agrotis ipsilon (Hufnagel), and sod webworms (Pyralidae: Crambinae) in creeping bentgrass, Agrostis palustris Hudson, with observations on black turfgrass Ataenius adults, Ataenius spretulus (Haldeman), Columbus, Ohio, 1996 / D. J. Shetlar, H. D. Niemczyk and M. Belcher -- Control of black cutworm, Agrotis ipsilon (Hufnagel), sod webworms (Pyralidae: Crambinae), and black turfgrass Ataenius adults, Ataenius spretulus (Haldeman), in creeping bentgrass, Agrostis palustris Hudson, Columbus, Ohio, 1996 / D. J. Shetlar, H. D. Niemczyk and M. Belcher -- Control of bluegrass billbug, Sphenophorus parvulus Gyllenhal, larvae in lawn turf, Wooster, Ohio, 1996 / D. J. Shetlar, H. D. Niemczyk and M. Belcher -- Efficacy of spinosad applied prior to oviposition for control of bluegrass billbug larvae in turfgrass - 1996 / D. J. Shetlar, H. D. Niemczyk and K. T. Power -- Efficacy of insecticides applied prior to oviposition for control of bluegrass billbug larvae in turfgrass - 1996 / D. J. Shetlar, H. D. Niemczyk and K. T. Power -- Application of insecticides to kill black turfgrass Ataenius adults prior to egg laying on golf course fairways-1996 / D. J. Shetlar, H. D. Niemczyk and K. T. Power -- Preventive applications for control of Japanese beetle larvae in turfgrass - 1996 / D. J. Shetlar, H. D. Niemczyk and K. T. Power -- Influence of application date on the efficacy of insecticides applied for control of Japanese beetle larvae in turfgrass - 1996 / D. J. Shetlar, H. D. Niemczyk and K. T. Power -- Application of insecticides for preventive control of Japanese beetle larval populations in turfgrass - 1996 / D. J. Shetlar, H. D. Niemczyk and K. T. Power -- Application of Bacillus thuringiensis strain 'buibui' for control of white grubs in turfgrass -1996 / D. J. Shetlar, H. D. Niemczyk and K. T. Power -- Curative control of masked chafer larvae in turfgrass -1996 / D. J. Shetlar, H. D. Niemczyk and K. T. Power -- A field test of RH-0345 2SC and 2.5G at 2.0 lb.ai/ acre and Merit 75WP at 0.3 lb. ai/ acre for control of black cutworm larvae on golf course greens / H. D. Niemczyk -- Dollar spot control study - 1996 / K. Danneberger, J. Rimelspach, M. Boehm, and J. Taylor -- quality ratings for various fungicide treatments on creeping bentgrass putting green turf / K. Danneberger, J. Rimelspach, M. Boehm, and J. Taylor -- Brown patch control study on creeping bentgrass turf - 1996 / K. Danneberger, J. Rimelspach, M. Boehm, and J. Taylor -- Brown patch control on tall fescue / K. Danneberger, J. Rimelspach, M. Boehm, and J. Taylor -- Yellow tuft study-1996 / K. Danneberger, J. Rimelspach, M. Boehm, and J. Taylor -- Creeping bentgrass melting-out study / K. Danneberger, J. Taylor, R. Golembiewski, G. Bell, J. Rimelspach, and M. Boehm -- Kentucky bluegrass melting-out study / K. Danneberger, J. Rimelspach, M. Boehm and J. Taylor -- Red thread control study on perennial ryegrass / J. Rimelspach, K. Danneberger, and M. Boehm -- Red thread control study on Kentucky bluegrass / J. Rimelspach, K. Danneberger and M. Boehm -- Evaluation of fungicides for the control of red thread in Kentucky bluegrass, 1996 / J. Rimelspach, M. Boehm, K. Danneberger and J. Taylor -- Evaluation of fungicides for the control of red thread in perennial ryegrass, 1996 / J. Rimelspach, M. Boehm, K. Danneberger and J. Taylor -- Pink snow mold control study-1995-1996 / J. Rimelspach and K. Danneberger -- Primo & Sentinel applications on Poa annua quality / K. Danneberger, J. Taylor, R. Golembiewski, and G. Bell -- Nitrogen source, rate, and timing effect on Kentucky bluegrass -1996 / J. R. Street and R. M. Stewart -- Natural organic source evaluation on a Kentucky bluegrass-perennial ryegrass mixture-1996 / J. R. Street and R. M. Stewart -- Polymer-coated urea source and rate effect on Kentucky bluegrass / J. R. Street and R. M. Stewart -- Polymer-coated urea and IBDU fertilizer performance on Kentucky bluegrass / J. R. Street and R. M. Stewart -- 1993 NTEP bentgrass (fairway/tee) cultivar evaluation / J. A. Taylor -- 1993 NTEP fineleaf fescue cultivar evaluation / J. Taylor -- 1994 NTEP perennial ryegrass cultivar evaluation / J. Taylor -- 1995 Kentucky bluegrass (medium/high input) cultivar evaluation / J. Taylor -- 1995 NTEP Kentucky bluegrass (low input) cultivar evaluation / J. Taylor -- Controlling annual bluegrass and rough bluegrass in creeping bentgrass fairways: a nutritional approach / G. E. Bell, E. Odorizzi and T. K. Danneberger -- "Primo" growth regulator evaluation on creeping bentgrass / W. Pound and R. Stewart -- Influence of dollar spot on a blend of two creeping bentgrass cultivars / R. C. Golembiewski, T. K. Danneberger and P. M. Sweeney -- Dollar spot severity as influenced by Primo, creeping bentgrass cultivars, and nitrogen fertility / R. C. Golembiewski and T. K. Danneberger -- Identification of bulk samples of perennial ryegrass cultivars with RAPD Markers / P. M. Sweeney and T. K. Danneberge

The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5

In yeast and mammals, the AAA ATPase Vps4p/SKD1 (for Vacuolar protein sorting 4/SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1) is required for the endosomal sorting of secretory and endocytic cargo. We identified a VPS4/SKD1 homolog in Arabidopsis thaliana, which localizes to the cytoplasm and to multivesicular endosomes. In addition, green fluorescent protein-SKD1 colocalizes on multivesicular bodies with fluorescent fusion protein endosomal Rab GTPases, such as ARA6/RabF1, RHA1/RabF2a, and ARA7/RabF2b, and with the endocytic marker FM4-64. The expression of SKD1E232Q, an ATPase-deficient version of SKD1, induces alterations in the endosomal system of tobacco (Nicotiana tabacum) Bright Yellow 2 cells and ultimately leads to cell death. The inducible expression ofSKD1E232Q in Arabidopsis resulted in enlarged endosomes with a reduced number of internal vesicles. In a yeast two-hybrid screen using Arabidopsis SKD1 as bait, we isolated a putative homolog of mammalian LYST-INTERACTING PROTEIN5 (LIP5)/SKD1 BINDING PROTEIN1 and yeast Vta1p (for Vps twenty associated 1 protein). Arabidopsis LIP5 acts as a positive regulator of SKD1 by increasing fourfold to fivefold its in vitro ATPase activity. We isolated a knockout homozygous Arabidopsis mutant line with a T-DNA insertion in LIP5. lip5 plants are viable and show no phenotypic alterations under normal growth conditions, suggesting that basal SKD1 ATPase activity is sufficient for plant development and growth.Facultad de Ciencias Agrarias y Forestale

Sphingosine kinases and their metabolites modulate endolysosomal trafficking in photoreceptors

Alterations in sphingosine kinase activity change the degradation rates of Rhodopsin and the transient receptor potential (TRP) channel by lysosomes and can result in retinal degeneration

Regulation of human intestinal T-cell responses by type 1 interferon-STAT1 signaling is disrupted in inflammatory bowel disease

This work was supported by a research fellowship grant from the Crohn’s and Colitis in Childhood Research Association (CICRA) and a small project grant from Crohn’s and Colitis UK (CCUK). We would like to acknowledge Professor Ian Sanderson, who helped with the initial design of this work, and provided important support throughout. We would also like to thank Dr Gary Warne for his advice and assistance in the use of the sorting by flow cytometry. We would also like to thank Dr Raj Lahiri and Professor Graham Foster for the kind gift of the primers for the ISGs (2’5’ OAS and MxA)

IL-2(high) tissue-resident T cells in the human liver: Sentinels for hepatotropic infection

The liver provides a tolerogenic immune niche exploited by several highly prevalent pathogens as well as by primary and metastatic tumors. We have sampled healthy and hepatitis B virus (HBV)-infected human livers to probe for a subset of T cells specialized to overcome local constraints and mediate immunity. We characterize a population of T-bet(lo)Eomes(lo)Blimp-1(hi)Hobit(lo) T cells found within the intrahepatic but not the circulating memory CD8 T cell pool expressing liver-homing/retention markers (CD69(+)CD103(+) CXCR6(+)CXCR3(+)). These tissue-resident memory T cells (TRM) are preferentially expanded in patients with partial immune control of HBV infection and can remain in the liver after the resolution of infection, including compartmentalized responses against epitopes within all major HBV proteins. Sequential IL-15 or antigen exposure followed by TGFβ induces liver-adapted TRM, including their signature high expression of exhaustion markers PD-1 and CD39. We suggest that these inhibitory molecules, together with paradoxically robust, rapid, cell-autonomous IL-2 and IFNγ production, equip liver CD8 TRM to survive while exerting local noncytolytic hepatic immunosurveillance

LGR5 Is a Negative Regulator of Tumourigenicity, Antagonizes Wnt Signalling and Regulates Cell Adhesion in Colorectal Cancer Cell Lines

BACKGROUND: LGR5 (Leucine-rich repeat-containing G-protein coupled receptor 5) is the most established marker for intestinal stem cells. Mouse models show that LGR5+ cells are the cells of origin of intestinal cancer, and LGR5 expression is elevated in human colorectal cancers, however very little is known about LGR5 function or its contribution to the stem cell phenotype and to colorectal cancer. PRINCIPAL FINDINGS: We have modulated the expression of LGR5 by RNAi (inhibitory RNAs) or overexpression in colorectal cancer cell lines. Paradoxically, ablation of LGR5 induces increased invasion and anchorage-independent growth, and enhances tumourigenicity in xenografts experiments. Conversely, overexpression of LGR5 augments cell adhesion, reduces clonogenicity and attenuates tumourigenicity. Expression profiling revealed enhanced wnt signalling and upregulation of EMT genes upon knockdown of LGR5, with opposite changes in LGR5 overexpressing cells. These findings suggest that LGR5 is important in restricting stem cells to their niche, and that loss of LGR5 concomitant with activated wnt signalling may contribute to the invasive phenotype of colorectal carcinomas

The Sodium/Proton Exchanger NHE8 Regulates Late Endosomal Morphology and Function

The pH and lumenal environment of intracellular organelles is considered essential for protein sorting and trafficking through the cell. We provide the first evidence that a mammalian sodium (potassium)/proton exchanger, NHE8, plays a key role in the control of protein trafficking and endosome morphology

Enhanced Transferrin Receptor Expression by Proinflammatory Cytokines in Enterocytes as a Means for Local Delivery of Drugs to Inflamed Gut Mucosa

Therapeutic intervention in inflammatory bowel diseases (IBDs) is often associated with adverse effects related to drug distribution into non-diseased tissues, a situation which attracts a rational design of a targeted treatment confined to the inflamed mucosa. Upon activation of immune cells, transferrin receptor (TfR) expression increases at their surface. Because TfR is expressed in all cell types we hypothesized that its cell surface levels are regulated also in enterocytes. We, therefore, compared TfR expression in healthy and inflamed human colonic mucosa, as well as healthy and inflamed colonic mucosa of the DNBS-induced rat model. TfR expression was elevated in the colonic mucosa of IBD patients in both the basolateral and apical membranes of the enterocytes. Increased TfR expression was also observed in colonocytes of the induced colitis rats. To explore the underlying mechanism CaCo-2 cells were treated with various proinflammatory cytokines, which increased both TfR expression and transferrin cellular uptake in a mechanism that did not involve hyper proliferation. These findings were then exploited for the design of targetable carrier towards inflamed regions of the colon. Anti-TfR antibodies were conjugated to nano-liposomes. As expected, iron-starved Caco-2 cells internalized anti-TfR immunoliposomes better than controls. Ex vivo binding studies to inflamed mucosa showed that the anti-TfR immunoliposomes accumulated significantly better in the mucosa of DNBS-induced rats than the accumulation of non-specific immunoliposomes. It is concluded that targeting mucosal inflammation can be accomplished by nano-liposomes decorated with anti-TfR due to inflammation-dependent, apical, elevated expression of the receptor

The amyloid precursor protein controls PIKfyve function

While the Amyloid Precursor Protein (APP) plays a central role in Alzheimer's disease, its cellular function still remains largely unclear. It was our goal to establish APP function which will provide insights into APP's implication in Alzheimer's disease. Using our recently developed proteo-liposome assay we established the interactome of APP's intracellular domain (known as AICD), thereby identifying novel APP interactors that provide mechanistic insights into APP function. By combining biochemical, cell biological and genetic approaches we validated the functional significance of one of these novel interactors. Here we show that APP binds the PIKfyve complex, an essential kinase for the synthesis of the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate. This signalling lipid plays a crucial role in endosomal homeostasis and receptor sorting. Loss of PIKfyve function by mutation causes profound neurodegeneration in mammals. Using C. elegans genetics we demonstrate that APP functionally cooperates with PIKfyve in vivo. This regulation is required for maintaining endosomal and neuronal function. Our findings establish an unexpected role for APP in the regulation of endosomal phosphoinositide metabolism with dramatic consequences for endosomal biology and important implications for our understanding of Alzheimer's disease