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A directed RNAi screen based on larval growth arrest reveals new modifiers of C. elegans insulin signaling
Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery.
Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery.
Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery.
Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery.
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 4, e34507- p.Article in journal (Refereed) Published
Abstract [en]

Genes regulating Caenorhabditis elegans insulin/IGF signaling (IIS) have largely been identified on the basis of their involvement in dauer development or longevity. A third IIS phenotype is the first larval stage (L1) diapause, which is also influenced by asna-1, a regulator of DAF-28/insulin secretion. We reasoned that new regulators of IIS strength might be identified in screens based on the L1 diapause and the asna-1 phenotype. Eighty-six genes were selected for analysis by virtue of their predicted interaction with ASNA-1 and screened for asna-1-like larval arrest. ykt-6, mrps-2, mrps-10 and mrpl-43 were identified as genes which, when inactivated, caused larval arrest without any associated feeding defects. Several tests indicated that IIS strength was weaker and that insulin secretion was defective in these animals. This study highlights the role of the Golgi network and the mitochondria in insulin secretion and provides a new list of genes that modulate IIS in C. elegans.

Place, publisher, year, edition, pages
2012. Vol. 7, no 4, e34507- p.
National Category
Endocrinology and Diabetes
Identifiers
URN: urn:nbn:se:umu:diva-57394DOI: 10.1371/journal.pone.0034507ISI: 000305338600033OAI: oai:DiVA.org:umu-57394DiVA: diva2:541338
Available from: 2012-07-17 Created: 2012-07-16 Last updated: 2017-12-07Bibliographically approved
In thesis
1. New modifiers of insulin signalling identified by interaction screens with ASNA-1 in C. elegans
Open this publication in new window or tab >>New modifiers of insulin signalling identified by interaction screens with ASNA-1 in C. elegans
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background: Insulin is a hormone released by the pancreatic beta cells in response to elevated levels of nutrients in the blood. Insulin triggers the uptake of glucose, fatty acids and amino acids into the liver, adipose tissue and muscles. Genes regulating insulin signalling are thus of vital importance for metabolic homeostasis and for preventing the development of diabetes. This thesis aims to identify new modifiers of insulin signalling, while carrying out functional studies of a homolog to human arsenite translocating ATPase, ASNA1. ASNA1 activates the insulin signalling pathway and promotes insulin secretion in mammalian cell lines and in Caenorhabditis elegans. A second aim is to better understand how ASNA1 and its interactors regulate sensitivity to the chemotherapeutic drug, cisplatin. Results: Regulators of insulin/IGF signalling (IIS) in C. elegans were identified based on the Larval arrest arrest aspect of the asna-1 depletion phenotype. Sixty-five genes were selected by virtue of their predicted interaction with ASNA-1 and screened for asna-1-like larval arrest upon inactivation of the genes . mrps-2, mrps-10, mrpl-43 encoding mitochondrial ribosomal protein subunits, and enpl-1 encoding an ER chaperone, GRP94 homolog were identified as the genes which when inactivated caused larval arrest without any associated feeding defects. IIS was weaker and insulin secretion was defective in these knockdown animals. ENPL-1 and ASNA-1 proteins interacted with one another both ex vivo and in vitro. ASNA-1 protein and mRNA level swere greatly reduced in enpl-1 mutants and enpl-1(-);asna-1(-) double-mutant worms displayed synthetic lethality. Overexpression of the insulins INS-4 and DAF-28 caused partial rescue of the germline phenotype of enpl-1 mutants, indicating that the phenotype of enpl-1 mutants was due at least in part to insufficient insulin levels. Studies of enpl-1 mutants also helped to understand the role of asna-1 in cisplatin sensitivity. The unfolded protein response (UPR) was induced in asna-1 and enpl-1 knockdown animals. enpl-1 mutants displayed higher sensitivity to cisplatin, when compared to asna-1 mutants and this correlated to higher UPR in enpl-1 knockdown animals. Pharmacological induction of the UPR in intrinsically cisplatin resistant wildtype worms also resulted in increased cisplatin sensitivity. This suggests that manipulation of ENPL-1 levels or of the UPR could enhance the anti-tumoral effects of cisplatin based cancer therapy. With a yeast two hybridscreen 27 putative physical interactors of ASNA-1 were identified. Amongst these candidate swas smn-1, which encodes survival of motor neuron protein homolog. RNAi knockdown of smn-1 caused a larval arrest phenotype similar to asna-1 depleted animals and smn-1 positively regulated IIS, like asna-1. Defects in IIS may be at the level of insulin release because neuropeptide secretion was impaired upon smn-1 knockdown. Further in vitro binding studies showed that SMN-1 and ASNA-1 interacted and inactivation of smn-1 in asna-1 mutants resulted in decreased viability. This implies that SMN-1 is another modifier of ASNA-1 and also a new component in IIS. Conclusion: With a directed RNAi screen and a yeast two hybrid screen several interactors of ASNA-1 that are also IIS modifiers were identified. ENPL-1 and SMN-1 are both involved in insulin release. We also found that induction of the UPR in enpl-1 and asna-1 mutants is a possible mechanism for increased sensitivity to cisplatin.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2012. 75 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1530
Keyword
Insulin, C. elegans, ASNA1, Cisplatin, GRP94, unfolded protein response, SMN1
National Category
Medical and Health Sciences
Research subject
Developmental Biology
Identifiers
urn:nbn:se:umu:diva-60949 (URN)978-91-7459-508-6 (ISBN)
Public defence
2012-12-10, Sal B, 9tr, Norrlands universitetssjukhus (NUS), Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2012-11-09 Created: 2012-11-05 Last updated: 2012-11-09Bibliographically approved
2. Insulin secretion and ASNA-1-dependent function of the endoplasmic reticulum in C. elegans
Open this publication in new window or tab >>Insulin secretion and ASNA-1-dependent function of the endoplasmic reticulum in C. elegans
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

ASNA1 is a well-conserved ATPase involved in a wide range of functions, including cisplatin resistance, growth control, insulin secretion and targeting of tail-anchored (TA) proteins to membranes. It is a positive regulator of insulin secretion both in the roundworm Caenorhabditis elegans and in humans. Insulin secretion and downstream insulin/IGF signalling (IIS) stands at the heart of many human pathologies, such as diabetes, Alzheimer’s disease and cancer. A better understanding of IIS may therefore prove vital for treatment and cure of these diseases. This thesis aims to further investigate the function of asna-1, and to identify new regulators of IIS based on the asna-1 phenotype in C. elegans.

Worms lacking ASNA-1 arrest growth in the first larval stage, L1, with reduced insulin secretion. The L1 arrest represents the strongest of the IIS phenotypes in worms. Most regulators of the insulin pathway have been identified in screens for other IIS phenotypes, influencing lifespan or the dauer diapause. Therefore, new regulators could be found by screening for genes which, when inactivated, cause an asna-1-like L1 arrest. Using bioinformatic approaches, a set of 143 putative asna-1 interactors were identified, based on their predicted or confirmed interaction with asna-1 in various organisms. Depletion of the Golgi SNARE homologue YKT-6 or the mitochondrial translocase homologue TOMM-40 caused asna-1-like larval arrests. Using several criteria, including genetic suppression by daf-16/Foxo, it was established that YKT-6 and TOMM-40 are positive regulators of IIS. Both proteins were also required for normal DAF-28/insulin secretion.

Further investigation of TOMM-40 identified it as a ubiquitously expressed mitochondrial translocase in C. elegans: It localized to mitochondrial membranes and was required for importing a tagged mitochondrial reporter across mitochondrial membranes. Depletion of TOMM-40 caused a collapse of the proton gradient across the inner mitochondrial membrane and triggered the mitochondrial unfolded protein response (UPR). Worms with defective mitochondria failed to grow normally in presence of food, but this growth defect was suppressed by daf-16(mgDf50). In addition, tomm-40(RNAi) led to DAF-16/FOXO activation, an effect that was suppressed by over expression of DAF-28/insulin. Taken together, these findings support a model whereby signals of food availability are conveyed through respiring mitochondria to promote DAF-28/insulin secretion, which in turn promotes growth.

Biochemical studies have identified ASNA-1 as a chaperone that targets a subset of newly synthesized TA proteins to a receptor at the endoplasmic reticulum (ER) membrane. However, these findings have not been tested in vivo in a metazoan model. A reporter-based system to analyse TA protein targeting into the ER in live animals using confocal microscopy was set up. A model asna-1-dependent TA protein, Y38F2AR.9/SEC-61β, required functional ASNA-1 for correct targeting to the ER. Conversely, a model asna-1-independent TA protein, CYTB5.1/cytochrome B5, did not. This phenotype was shared with the predicted asna-1 receptor homologue, wrb-1. Consistently, WRB-1 was found to localize to the ER. However, other wrb-1 mutant phenotypes only partially overlap with those of asna-1 mutants, suggesting that ASNA-1 is either partially independent of WRB-1 for TA protein targeting or that ASNA-1 has additional functions besides its role in TA protein targeting.

Confocal microscopy also indicated that the ER morphology was aberrant in asna-1 and wrb-1 mutants. ER UPR was elevated in the asna-1 mutants, as indicated by the upregulation of an hsp-4/BiP reporter. Transmission and immuno-electron microscopy of these mutants revealed a swollen ER lumen, which is another hallmark of ER stress. High levels of autophagy in asna-1 animals and the presence of ER-containing autophagosomes in both asna-1 and wrb-1 mutants indicated a stress-induced remodelling of the ER membrane in these two mutants. In addition, both mutants had normal mitochondrial morphology, but showed severe effects on Golgi compartment morphology. Hypothetically, all these phenotypes could be due to defects in the signal recognition particle (SRP) pathway. This is because Y38F2AR.9/SEC-61β is both a TA protein and a component of the SEC-61 translocon. However, both Golgi and ER morphology was normal in Y38F2AR.9/sec-61β(tm1986) mutant animals, suggesting that the organellar defects seen in asna-1 and wrb-1 were due to a TA protein-dependent mechanism rather than an SRP-dependent mechanism. In addition, asna-1 mutants displayed numerous protein aggregates, consistent with a proposed role for ASNA-1 in shielding aggregation-prone TA protein membrane anchors from the hydrophilic environment of the cytosol.

In conclusion, YKT-6 and TOMM-40 are positive regulators of IIS and DAF-28/insulin secretion, implicating roles for Golgi and mitochondria in IIS. DAF-28 is a metabolically regulated insulin in C. elegans, since its secretion depends on active mitochondria. Mutants for asna-1 and its predicted receptor wrb-1 show severe defects in ER and Golgi morphology. These defects may occur because TA protein targeting in asna-1 and wrb-1 mutants is defective, which is also demonstrated here in the first analysis of this process in live animals.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2014. 80 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1634
National Category
Cell and Molecular Biology
Research subject
cellforskning; Genetics
Identifiers
urn:nbn:se:umu:diva-85906 (URN)978-91-7601-004-4 (ISBN)
Public defence
2014-03-14, Hörsal Betula, by 6M, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2014-02-21 Created: 2014-02-13 Last updated: 2014-02-21Bibliographically approved

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