Larance, Mark et al. (2013) International Worm Meeting "Dissecting Ageing-Related Pathways by Studying Protein Changes after Calorie Restriction."

Lamond, Angus, Larance, Mark, Pourkarimi, Ehsan, Gartner, Anton

[International Worm Meeting, 2013]

Calorie restriction has been identified in many organisms to mediate increased life-span. Many pathways are involved in ageing including insulin/IGF-1 signalling, AMPK, autophagy, and TOR. In this study, we have used state-of-the-art quantitative proteome analysis of C. elegans, combined with follow-up genetics studies to identify the protein-level mechanisms that mediate the response to calorie restriction. We have recently established a method to enable stable isotope labelling with amino acids in cell culture (SILAC) for quantitative proteomic analysis in C. elegans. We have used advanced chromatography to fractionate C. elegans peptide digests to enhance sensitivity. To date, we have quantified the proteome-wide response in nematodes to short term calorie restriction. After characterising >4000 proteins across three biological replicates, we first identified those proteins changing significantly after calorie restriction. As a harsh threshold we required that proteins changed in abundance by more than two-fold and had a p-value <0.05. These cut-offs yielded 341 proteins significantly increased and 127 significantly decreased after calorie restriction for 16 h. We have also quantified many post-translational modifications detected in our unbiased dataset, including some that change significantly in response to calorie restriction. Gene ontology enrichment analysis based on biological processes showed that the up-regulated proteins were associated with nucleosome assembly, ageing, the response to heat, and macromolecular complex assembly. Conversely, the down-regulated proteins were associated with the biological processes of fatty acid metabolism, amino acid biosynthesis, cell division, and the positive regulation of growth. Of the 341 up-regulated proteins, 30 of the genes encoding these proteins have previously been shown to bind the transcription factor DAF-16, which plays a critical role in the increased longevity caused by calorie restriction. The mechanisms that regulate the remaining proteins remain to be elucidated. These datasets provide a comprehensive overview of the networks that mediate the response to calorie restriction and which may provide the associated increased longevity.

Emerson F et al. (2020) Elife "A memory of longevity."

Lee SS, Li CL, Emerson F

[Elife, 2020]

Worms with increased levels of the epigenetic mark H3K9me2 have a longer lifespan that can be passed down to future generations.

Wu Y et al. (2017) Curr Top Dev Biol "Regulation of Cell Polarity by PAR-1/MARK Kinase."

Wu Y, Griffin EE

[Curr Top Dev Biol, 2017]

PAR-1/MARK kinases are conserved serine/threonine kinases that are essential regulators of cell polarity. PAR-1/MARK kinases localize and function in opposition to the anterior PAR proteins to control the asymmetric distribution of factors in a wide variety polarized cells. In this review, we discuss the mechanisms that control the localization and activity of PAR-1/MARK kinases, including their antagonistic interactions with the anterior PAR proteins. We focus on the role PAR-1 plays in the asymmetric division of the Caenorhabditis elegans zygote, in the establishment of the anterior/posterior axis in the Drosophila oocyte and in the control of microtubule dynamics in mammalian neurons. In addition to conserved aspects of PAR-1 biology, we highlight the unique ways in which PAR-1 acts in these distinct cell types to orchestrate their polarization. Finally, we review the connections between disruptions in PAR-1/MARK function and Alzheimer's disease and cancer.

Benecke M et al. (1994) Worm Breeder's Gazette "DNA Fingerprinting in C. elegans - An Approach."

Epplen JT, Schierenberg E, Benecke M

[Worm Breeder's Gazette, 1994]

DNA fingerprinting in C. elegans - an approach Mark Beneckea, Jorg T. Epplenb and Einhard Schierenberga a Zoologisches Institut der Univeritat, 50923 Koln, Germany b Ruhr-Universitat, Ab1. fur Molekulare Humangenetik, 44780 Bochum, Germany

Butkevich E et al. (2016) Sci Rep "Phosphorylation of FEZ1 by Microtubule Affinity Regulating Kinases regulates its function in presynaptic protein trafficking."

Grosche J, Erck C, Urlaub H, Nikolov M, Klopfenstein DR, Butkevich E, Chua JJ, Hartig W, Schmidt CF

[Sci Rep, 2016]

Adapters bind motor proteins to cargoes and therefore play essential roles in Kinesin-1 mediated intracellular transport. The regulatory mechanisms governing adapter functions and the spectrum of cargoes recognized by individual adapters remain poorly defined. Here, we show that cargoes transported by the Kinesin-1 adapter FEZ1 are enriched for presynaptic components and identify that specific phosphorylation of FEZ1 at its serine 58 regulatory site is mediated by microtubule affinity-regulating kinases (MARK/PAR-1). Loss of MARK/PAR-1 impairs axonal transport, with adapter and cargo abnormally co-aggregating in neuronal cell bodies and axons. Presynaptic specializations are markedly reduced and distorted in FEZ1 and MARK/PAR-1 mutants. Strikingly, abnormal co-aggregates of unphosphorylated FEZ1, Kinesin-1 and its putative cargoes are present in brains of transgenic mice modelling aspects of Alzheimer's disease, a neurodegenerative disorder exhibiting impaired axonal transport and altered MARK activity. Our findings suggest that perturbed FEZ1-mediated synaptic delivery of proteins arising from abnormal signalling potentially contributes to the process of neurodegeneration.

Pourkarimi, Ehsan et al. (2013) International Worm Meeting "Starving chromosome; reshaping the chromatin to survive."

Pourkarimi, Ehsan, Larance, Mark, Lamond, Angus, Gartner, Anton

[International Worm Meeting, 2013]

Caloric restriction is associated to increase in life span in many model systems including C. elegans. Previously many pathways have been identified to be involved in ageing, including Insulin/IGF-1 and TOR signalling. Loss of function mutation in DAF-2, a C. elegans insulin receptor, results in life span extension while depletion of the most down stream component of insulin signalling (IS), DAF-16, results in reduced longevity. In addition to increased lifespan, daf-2 mutants are resistant to stress responses such as heat, hypoxia and oxidative stress. Many of DAF-2/DAF-16 targets have been identified using; genetics approach, transcriptome analysis and chromatin immunoprecipitation. Besides IS, TOR regulates ageing and function as a master switch to regulates cell growth in response to nutrition availability. However despite the intensive research, the exact mechanism as to how cells response to starvation is not fully understood. We present the use of quantitative mass spectrometry of C. elegans to analyse organismal proteome changes upon starvation. We have recently developed a stable isotope labelling of amino acids in cell culture (SILAC) based technique for use in worms. Using SILAC we were able to detect more than 4000 proteins of which nearly 350 proteins were significantly up regulated while 127 were down regulated upon starvation. Gene ontology analysis has shown that majority of the up regulated proteins are associated to nucleosome assembly, ageing and stress response while a number of down regulated proteins are known to be involved in fatty acid and amino acid metabolism and cell division of which some are known to be a target of TOR. Nearly 10% of the up-regulated proteins that we have detected have previously been reported to be a target of daf-16. Interestingly, we have detected up regulation of some chromatin remodelers and histone variants. The up-regulation of some of the chromatin remodellers observed in our study has been confirmed using transgenic worms expressing these proteins tagged with GFP. A null mutant of one of the remodellers exhibits a dramatic reduction in life span. Currently we are analyzing the protein changes of worms depleted for DAF-2 and AMPK signalling.

Akay, Alper et al. (2015) International Worm Meeting "Proteomic analysis of the nuclear RNAi pathway identifies EMB-4/AQR in transposable element regulation."

Larance, Mark, Bradshaw, Charles, Miska, Eric A., Di Domenico, Tomas, Akay, Alper, Lamond, Angus I., Allen, George, Medhi, Ragini

[International Worm Meeting, 2015]

Endogenous small RNA pathways can induce transcriptional gene silencing in C. elegans. piRNAs bound to the PRG-1 PIWI protein recognise their target by sequence complementarity and lead to the recruitment of RNA-dependent RNA polymerases (RdRPs). RdRPs, in turn, generate secondary siRNAs that are antisense to the target RNA and are 22 nt long with a 5' G (22G-RNAs). Other endogenous small RNA pathways such as the 26G-RNA pathway use similar amplification steps leading to 22G-RNA synthesis. In the C. elegans germline, the amplified 22G-RNAs are loaded into the argonaute protein HRDE-1 which localises to the nucleus initiating the nuclear RNAi pathway. The nuclear RNAi pathway leads to the transcriptional silencing of target genes and requires multiple chromatin factors. Silenced genes accumulate the repressive chromatin mark H3K9me3 and, remarkably, silencing of genes can last for several generations (1-6). The majority of the data linking small RNA mediated transcriptional and heritable silencing is based on evidence from genetic experiments. In order to better understand the mechanism of transcriptional and heritable silencing, it is essential to understand how key proteins in this silencing pathway function. We identified the intron binding protein EMB-4/AQR as an interaction partner of HRDE-1 in our immunoprecipitation experiments coupled to SILAC (7) based quantitative proteomics. Similar to hrde-1, emb-4/AQR is required for piRNA-mediated transgene silencing and the recently described 22G-RNA spreading (8) is inhibited in emb-4/AQR mutant animals. RNA-seq experiments revealed that emb-4/AQR is required for the repression of a subset of piRNA targets including transposable elements. In addition, some transposable elements which are de-silenced in emb-4/AQR mutants are not targeted by the piRNA pathway, indicating a more global role for emb-4/AQR in transposable element regulation. We are currently elucidating the molecular mechanism by which emb-4/AQR communicates with the nuclear RNAi machinery. 1. Ashe A et al. Cell. 150(1):88. (2012) 2. Buckley BA et al. Nature. 489(7416):447. (2012) 3. Luteijn MJ et al. EMBO J. 31(16):3422. (2012) 4. Shirayama M et al. Cell. 150(1):65. (2012) 5. Rechavi O et al. Cell. 158(2):277. (2014) 6. Han T et al. Proc Natl Acad Sci USA. 106(44):18674. (2009) 7. Larance M et al. Nat Meth. 8(10):849. (2011) 8. Sapetschnig A et al., PLoS Genet. 11(3):e1005078. (2015).

Skourti-Stathaki K et al. (2013) Mol Cell "Histone 3 s10 phosphorylation: "caught in the R loop!"."

Skourti-Stathaki K, Proudfoot NJ

[Mol Cell, 2013]

In this issue of Molecular Cell, Castellano-Pozo etal. (2013) describe a connection between R loop structures and histone 3 S10 phosphorylation (H3S10P), a mark of chromatin compaction. Their results constitute asignificant advance in our understanding of the role of R loops in genomic instability.

Bohdanowicz M et al. (2010) Curr Biol "Vesicular traffic: a Rab SANDwich."

Bohdanowicz M, Grinstein S

[Curr Biol, 2010]

The small GTPases Rab5 and Rab7 mark temporally distinct but sequentially connected stages in phagosome maturation, but the mechanism underlying the transition between these stages has been unclear. Recent studies in Caenorhabditis elegans have now uncovered a new protein complex that connects Rab5 to Rab7.

Hope IA (2001) Trends Genet "Broadcast interference - functional genomics."

Hope IA

[Trends Genet, 2001]

Four recent papers mark a major shift in functional genomic analysis for multicellular organisms. RNA-mediated interference was applied to inactivate individual genes systematically on a genomic scale. These studies subjected a third of the genes in the genome of Caenorhabditis elegans to reverse genetic analysis.