| 1. |
- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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| 2. |
- Chotard, Laëtitia, et al.
(författare)
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TBC-2 regulates RAB-5/RAB-7-mediated endosomal trafficking in Caenorhabditis elegans
- 2010
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Ingår i: Molecular Biology of the Cell. - : American Society for Cell Biology. - 1059-1524 .- 1939-4586. ; 21:13, s. 2285-2296
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Tidskriftsartikel (refereegranskat)abstract
- During endosome maturation the early endosomal Rab5 GTPase is replaced with the late endosomal Rab7 GTPase. It has been proposed that active Rab5 can recruit and activate Rab7, which in turn could inactivate and remove Rab5. However, many of the Rab5 and Rab7 regulators that mediate endosome maturation are not known. Here, we identify Caenorhabditis elegans TBC-2, a conserved putative Rab GTPase-activating protein (GAP), as a regulator of endosome to lysosome trafficking in several tissues. We show that tbc-2 mutant animals accumulate enormous RAB-7-positive late endosomes in the intestine containing refractile material. RAB-5, RAB-7, and components of the homotypic fusion and vacuole protein sorting (HOPS) complex, a RAB-7 effector/putative guanine nucleotide exchange factor (GEF), are required for the tbc-2(-) intestinal phenotype. Expression of activated RAB-5 Q78L in the intestine phenocopies the tbc-2(-) large late endosome phenotype in a RAB-7 and HOPS complex-dependent manner. TBC-2 requires the catalytic arginine-finger for function in vivo and displays the strongest GAP activity on RAB-5 in vitro. However, TBC-2 colocalizes primarily with RAB-7 on late endosomes and requires RAB-7 for membrane localization. Our data suggest that TBC-2 functions on late endosomes to inactivate RAB-5 during endosome maturation.
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| 3. |
- Elle, Ida C, et al.
(författare)
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Tissue- and paralogue-specific functions of acyl-CoA-binding proteins in lipid metabolism in Caenorhabditis elegans
- 2011
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 437:2, s. 231-241
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Tidskriftsartikel (refereegranskat)abstract
- ACBP (acyl-CoA-binding protein) is a small primarily cytosolic protein that binds acyl-CoA esters with high specificity and affinity. ACBP has been identified in all eukaryotic species, indicating that it performs a basal cellular function. However, differential tissue expression and the existence of several ACBP paralogues in many eukaryotic species indicate that these proteins serve distinct functions. The nematode Caenorhabditis elegans expresses seven ACBPs: four basal forms and three ACBP domain proteins. We find that each of these paralogues is capable of complementing the growth of ACBP-deficient yeast cells, and that they exhibit distinct temporal and tissue expression patterns in C. elegans. We have obtained loss-of-function mutants for six of these forms. All single mutants display relatively subtle phenotypes; however, we find that functional loss of ACBP-1 leads to reduced triacylglycerol (triglyceride) levels and aberrant lipid droplet morphology and number in the intestine. We also show that worms lacking ACBP-2 show a severe decrease in the β-oxidation of unsaturated fatty acids. A quadruple mutant, lacking all basal ACBPs, is slightly developmentally delayed, displays abnormal intestinal lipid storage, and increased β-oxidation. Collectively, the present results suggest that each of the ACBP paralogues serves a distinct function in C. elegans.
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| 4. |
- Goergen, Philip, 1986-
(författare)
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The Molecular Mechanism of Aggression and Feeding Behaviour in Drosophila melanogaster
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Obesity is a complex disorder which has become a growing health concern. Twin studies have demonstrated a strong genetic component to the development of obesity and genome wide association studies have identified several genetic loci associated with it. However, most of these loci are still poorly understood in a functional context. Interestingly, many of the hormones and neurobiological messengers responsible for regulating feeding behaviour and metabolism are also linked to controlling aggression, but it is still not understood how they interact to maintain metabolic homeostasis. In this thesis, the model organism Drosophila melanogaster was employed to dissect the molecular mechanisms of the genetic cascades regulating aggressive behaviour and metabolic homeostasis.In paper I and II, the role of transcription factor AP-2 (TfAP-2) and Tiwaz Twz, Drosophila homologues of two human obesity-linked genes were investigated in aggression and feeding behaviour. Paper I demonstrated that TfAP-2 and Twz genetically interact in octopaminergic neurons to modulate male aggression by controlling the expression of genes necessary for octopamine (fly analogue of noradrenaline) production and secretion. Moreover, it was revealed that octopamine in turn regulates aggression through the Drosophila cholecystokinin (CCK) satiation hormone homologue Drosulfakinin (Dsk). Paper II revealed that TfAP-2 and Twz also initiate feeding through regulation of octopamine poduction and secretion. Octopamine then induces Dsk expression leading to inhibition of feeding.Paper III established that the activity of the small GTPase Ras-related C3 botulinum toxin substrate 2 (Rac2) is required in Drosophila for the proper regulation of metabolic homeostasis, as well as overt behaviours. Rac2 mutants were starvation susceptible, had less lipids and exhibited disrupted feeding behaviour. Moreover, they displayed aberrant aggression and courtship behaviour towards conspecifics.Paper IV studied Protein kinase D (PKD), the homologue of a third obesity-linked gene PRKD1, and another kinase Stretchin-Mlck (Strn-Mlck). Reducing PKD transcript levels in the insulin producing cells led to flies with increased starvation susceptibility, decreased levels of lipids and diminished insulin signalling compared to controls. Reduced Strn-Mlck expression resulted in a starvation phenotype and slight reduction in insulin signalling and lipid content. These findings imply a function for PKD and Strn-Mlck in insulin release.
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| 5. |
- Korta, Dorota Z, et al.
(författare)
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S6K links cell fate, cell cycle and nutrient response in C. elegans germline stem/progenitor cells.
- 2012
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Ingår i: Development. - : The Company of Biologists. - 0950-1991 .- 1477-9129. ; 139:5, s. 859-870
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Tidskriftsartikel (refereegranskat)abstract
- Coupling of stem/progenitor cell proliferation and differentiation to organismal physiological demands ensures the proper growth and homeostasis of tissues. However, in vivo mechanisms underlying this control are poorly characterized. We investigated the role of ribosomal protein S6 kinase (S6K) at the intersection of nutrition and the establishment of a stem/progenitor cell population using the C. elegans germ line as a model. We find that rsks-1 (which encodes the worm homolog of mammalian p70S6K) is required germline-autonomously for proper establishment of the germline progenitor pool. In the germ line, rsks-1 promotes cell cycle progression and inhibits larval progenitor differentiation, promotes growth of adult tumors and requires a conserved TOR phosphorylation site. Loss of rsks-1 and ife-1 (eIF4E) together reduces the germline progenitor pool more severely than either single mutant and similarly to reducing the activity of let-363 (TOR) or daf-15 (RAPTOR). Moreover, rsks-1 acts in parallel with the glp-1 (Notch) and daf-2 (insulin-IGF receptor) pathways, and does not share the same genetic dependencies with its role in lifespan control. We show that overall dietary restriction and amino acid deprivation cause germline defects similar to a subset of rsks-1 mutant phenotypes. Consistent with a link between diet and germline proliferation via rsks-1, loss of rsks-1 renders the germ line largely insensitive to the effects of dietary restriction. Our studies establish the C. elegans germ line as an in vivo model to understand TOR-S6K signaling in proliferation and differentiation and suggest that this pathway is a key nutrient-responsive regulator of germline progenitors.
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| 6. |
- Nilsson, Lars, et al.
(författare)
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Caenorhabditis elegans Numb Inhibits Endocytic Recycling by Binding TAT-1 Aminophospholipid Translocase
- 2011
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Ingår i: Traffic. - Malden : Wiley-Blackwell. - 1398-9219 .- 1600-0854. ; 12:12, s. 1839-1849
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Tidskriftsartikel (refereegranskat)abstract
- Numb regulates endocytosis in many metazoans, but the mechanism by which it functions is not completely understood. Here we report that the Caenorhabditis ele-gans Numb ortholog, NUM-1A, a regulator of endocytic recycling, binds the C isoform of transbilayer amphipath transporter-1 (TAT-1), a P4 family adenosine triphosphatase and putative aminophospholipid translocase that is required for proper endocytic trafficking. We demonstrate that TAT-1 is differentially spliced during development and that TAT-1C-specific splicing occurs in the intestine where NUM-1A is known to function. NUM-1A and TAT-1C colocalize in vivo. We have mapped the binding site to an NXXF motif in TAT-1C. This motif is not required for TAT-1C function but is required for NUM-1A's ability to inhibit recycling. We demonstrate that num-1A and tat-1 defects are both suppressed by the loss of the activity of PSSY-1, a phosphatidylserine (PS) synthase. PS is mislocalized in intestinal cells with defects in tat-1 or num-1A function. We propose that NUM-1A inhibits recycling by inhibiting TAT-1C's ability to translocate PS across the membranes of recycling endosomes.
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| 7. |
- Stenvall, Jörgen, et al.
(författare)
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Selenoprotein TRXR-1 and GSR-1 are essential for removal of old cuticle during molting in Caenorhabditis elegans
- 2011
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 108:3, s. 1064-1069
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Tidskriftsartikel (refereegranskat)abstract
- Selenoproteins, in particular thioredoxin reductase, have been implicated in countering oxidative damage occurring during aging but the molecular functions of these proteins have not been extensively investigated in different animal models. Here we demonstrate that TRXR-1 thioredoxin reductase, the sole selenoprotein in Caenorhabditis elegans, does not protect against acute oxidative stress but functions instead together with GSR-1 glutathione reductase to promote the removal of old cuticle during molting. We show that the oxidation state of disulfide groups in the cuticle is tightly regulated during the molting cycle, and that when trxr-1 and gsr-1 function is reduced, disulfide groups in the cuticle remain oxidized. A selenocysteine-to-cysteine TRXR-1 mutant fails to rescue molting defects. Furthermore, worms lacking SELB-1, the C. elegans homolog of Escherichia coli SelB or mammalian EFsec, a translation elongation factor known to be specific for selenocysteine in E. coli, fail to incorporate selenocysteine, and display the same phenotype as those lacking trxr-1. Thus, TRXR-1 function in the reduction of old cuticle is strictly selenocysteine dependent in the nematode. Exogenously supplied reduced glutathione reduces disulfide groups in the cuticle and induces apolysis, the separation of old and new cuticle, strongly suggesting that molting involves the regulated reduction of cuticle components driven by TRXR-1 and GSR-1. Using dauer larvae, we demonstrate that aged worms have a decreased capacity to molt, and decreased expression of GSR-1. Together, our results establish a function for the selenoprotein TRXR-1 and GSR-1 in the removal of old cuticle from the surface of epidermal cells.
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| 8. |
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| 9. |
- Tuck, Simon
(författare)
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The control of cell growth and body size in Caenorhabditis elegans
- 2014
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 321:1, s. 71-76
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Forskningsöversikt (refereegranskat)abstract
- One of the most important ways in which animal species vary is in their size. Individuals of the largest animal ever thought to have lived, the blue whale (Balaenoptera musculus), can reach a weight of 190 t and a length of over 30 m. At the other extreme, among the smallest multicellular animals are males of the parasitic wasp, Dicopomorpha echmepterygis, which even as adults are just 140 mu m in length. In terms of volume, these species differ by more than 14 orders of magnitude. Since size has such profound effects on an organism's ecology, anatomy and physiology, an important task for evolutionary biology and ecology is to account for why organisms grow to their characteristic sizes. Equally, a full description of an organism's development must include an explanation of how its growth and body size are regulated. Here I review research on how these processes are controlled in the nematode, Caenorhabditis elegans. Analyses of small and long mutants have revealed that in the worm, DBL-1, a ligand in the TGF beta superfamily family, promotes growth in a dose-dependent manner. DBL-1 signaling affects body size by stimulating the growth of syncytial hypodermal cells rather than controlling cell division. Signals from chemosensory neurons and from the gonad also modulate body size, in part, independently of DBL-1-mediated signaling. Organismal size and morphology is heavily influenced by the cuticle, which acts as the exoskeleton. Finally, I summarize research on several genes that appear to regulate body size by cell autonomously regulating cell growth throughout the worm.
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