ORCID Profile
0000-0001-6078-9312
Current Organisations
Flinders University
,
University of Wollongong
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Proteomics and Intermolecular Interactions (excl. Medical Proteomics) | Animal Neurobiology | Biochemistry and Cell Biology | Cell Neurochemistry
Publisher: Springer Science and Business Media LLC
Date: 28-08-2014
Abstract: In this protocol we describe the incorporation of bio-orthogonal amino acids as a versatile method for visualizing and identifying de novo-synthesized proteins in the roundworm Caenorhabditis elegans. This protocol contains directions on implementing three complementary types of analysis: 'click chemistry' followed by western blotting, click chemistry followed by immunofluorescence, and isobaric tags for relative and absolute quantification (iTRAQ) quantitative mass spectrometry. The detailed instructions provided herein enable researchers to investigate the de novo proteome, an analysis that is complicated by the fact that protein molecules are chemically identical to each other, regardless of the timing of their synthesis. Our protocol circumvents this limitation by identifying de novo-synthesized proteins via the incorporation of the chemically modifiable azidohomoalanine instead of the natural amino acid methionine in the nascent protein, followed by facilitating the visualization of the resulting labeled proteins in situ. It will therefore be an ideal tool for studying de novo protein synthesis in physiological and pathological processes including learning and memory. The protocol requires 10 d for worm growth, liquid culture and synchronization 1-2 d for bio-orthogonal labeling and, with regard to analysis, 3-4 d for western blotting, 5-6 d for immunofluorescence or ~3 weeks for mass spectrometry.
Publisher: Society for Neuroscience
Date: 23-06-2020
Publisher: Society for Neuroscience
Date: 14-01-2021
DOI: 10.1523/JNEUROSCI.1701-20.2020
Abstract: Sleep, a state of quiescence associated with growth and restorative processes, is conserved across species. Invertebrates including the nematode Caenorhabditis elegans exhibit sleep-like states during development, satiety, and stress. Here, we describe behavior and neural activity during sleep and awake states in adult C. elegans hermaphrodites using new microfluidic methods. We observed effects of fluid flow, oxygen, feeding, odors, and genetic perturbations on long-term sleep behavior over 12 h. We developed a closed-loop sleep detection system to automatically deliver chemical stimuli to assess sleep-dependent changes to evoked neural responses in in idual animals. Sleep increased the arousal threshold to aversive stimulation, yet the associated sensory neuron and first-layer interneuron responses were unchanged. This localizes adult sleep-dependent neuromodulation within interneurons presynaptic to the premotor interneurons, rather than afferent sensory circuits. However, sleep prolonged responses in appetitive chemosensory neurons, suggesting that sleep modulates responsiveness specifically across sensory systems rather than broadly d ing global circuit activity. SIGNIFICANCE STATEMENT Much is known about molecular mechanisms that facilitate sleep control. However, it is unclear how these pathways modulate neural circuit-level sensory processing or how misregulation of neural activity contributes to sleep disorders. The nematode Caenorhabditis elegans provides the ability to study neural circuitry with single-neuron resolution, and recent studies examined sleep states between developmental stages and when stressed. Here, we examine an additional form of spontaneous sleep in adult C. elegans at the behavioral and neural activity levels. Using a closed-loop system, we show that delayed behavioral responses to aversive chemical stimulation during sleep arise from sleep-dependent sensorimotor modulation localized presynaptic to the premotor circuit, rather than early sensory circuits.
Publisher: Springer Science and Business Media LLC
Date: 21-01-2016
DOI: 10.1038/SREP19582
Abstract: Proteins of the Homeodomain-Interacting Protein Kinase (HIPK) family regulate an array of processes in mammalian systems, such as the DNA damage response, cellular proliferation and apoptosis. The nematode Caenorhabditis elegans has a single HIPK homologue called HPK-1. Previous studies have implicated HPK-1 in longevity control and suggested that this protein may be regulated in a stress-dependent manner. Here we set out to expand these observations by investigating the role of HPK-1 in longevity and in the response to heat and oxidative stress. We find that levels of HPK-1 are regulated by heat stress and that HPK-1 contributes to survival following heat or oxidative stress. Additionally, we show that HPK-1 is required for normal longevity, with loss of HPK-1 function leading to a faster decline of physiological processes that reflect premature ageing. Through microarray analysis, we have found that HPK-1-regulated genes include those encoding proteins that serve important functions in stress responses such as Phase I and Phase II detoxification enzymes. Consistent with a role in longevity assurance, HPK-1 also regulates the expression of age-regulated genes. Lastly, we show that HPK-1 functions in the same pathway as DAF-16 to regulate longevity and reveal a new role for HPK-1 in development.
Publisher: Elsevier BV
Date: 08-2020
Publisher: Springer Science and Business Media LLC
Date: 10-2017
DOI: 10.1038/NATURE24056
Publisher: Wiley
Date: 14-11-2014
DOI: 10.1111/ACEL.12285
Publisher: eLife Sciences Publications, Ltd
Date: 05-07-2017
DOI: 10.7554/ELIFE.28780
Abstract: A map of a neuronal circuit in a marine worm reveals how simple networks of neurons can control behavior.
Publisher: Springer Science and Business Media LLC
Date: 24-01-2014
DOI: 10.1007/S00018-014-1558-7
Abstract: Protein misfolding and aggregation as a consequence of impaired protein homeostasis (proteostasis) not only characterizes numerous age-related diseases but also the aging process itself. Functionally related to the aging process are, among others, ribosomal proteins, suggesting an intimate link between proteostasis and aging. We determined by iTRAQ quantitative proteomic analysis in C. elegans how the proteome changes with age and in response to heat shock. Levels of ribosomal proteins and mitochondrial chaperones were decreased in aged animals, supporting the notion that proteostasis is altered during aging. Mitochondrial enzymes of the tricarboxylic acid cycle and the electron transport chain were also reduced, consistent with an age-associated energy impairment. Moreover, we observed an age-associated decline in the heat shock response. In order to determine how protein synthesis is altered in aging and in response to heat shock, we complemented our global analysis by determining the de novo proteome. For that, we established a novel method that enables both the visualization and identification of de novo synthesized proteins, by incorporating the non-canonical methionine analogue, azidohomoalanine (AHA), into the nascent polypeptides, followed by reacting the azide group of AHA by ‘click chemistry’ with an alkyne-labeled tag. Our analysis of AHA-tagged peptides demonstrated that the decreased abundance of, for ex le, ribosomal proteins in aged animals is not solely due to degradation but also reflects a relative decrease in their synthesis. Interestingly, although the net rate of protein synthesis is reduced in aged animals, our analyses indicate that the synthesis of certain proteins such as the vitellogenins increases with age.
Publisher: Oxford University Press
Date: 08-01-2020
DOI: 10.1093/OXFORDHB/9780190860509.013.8
Abstract: Chronic pain is a significant public health problem, affecting 20–25% of the global population, and there is a clear need for more specific and effective therapeutics. To achieve this, a comprehensive understanding of the underlying mechanisms and molecular machinery driving pain-related diseases is required. The definition of pain as an “unpleasant sensory and emotional experience” associated with tissue injury is innately anthropomorphic, the emotional element being difficult to reconcile in nonhuman organisms. Even simple invertebrates are nevertheless capable of nociception, the neural processing of noxious stimuli. With the significant advantages of simpler nervous systems, experimental tractability, and a high level of conservation, they have a major role to play in advancing our understanding. This chapter reviews our current molecular- and circuit-level understanding of nociception in two of the most widely used invertebrate experimental models, the nematode Caenorhabditis elegans and the fly Drosophila melanogaster. In particular, it summarizes the molecules, cells, and circuits that contribute to nociception in response to erse noxious stimuli in these model organisms and the behavioral paradigms that we can harness to study them. The chapter discusses how mechanistic insights gained from these experimental systems can improve our understanding of pain in humans.
Publisher: Public Library of Science (PLoS)
Date: 16-12-2016
Publisher: EMBO
Date: 2016
Publisher: Wiley
Date: 31-01-2020
Publisher: Frontiers Media SA
Date: 2013
Publisher: Life Science Alliance, LLC
Date: 23-06-2022
Abstract: Sexual dimorphism occurs where different sexes of the same species display differences in characteristics not limited to reproduction. For the nematode Caenorhabditis elegans , in which the complete neuroanatomy has been solved for both hermaphrodites and males, sexually dimorphic features have been observed both in terms of the number of neurons and in synaptic connectivity. In addition, male behaviours, such as food-leaving to prioritise searching for mates, have been attributed to neuropeptides released from sex-shared or sex-specific neurons. In this study, we show that the lury-1 neuropeptide gene shows a sexually dimorphic expression pattern being expressed in pharyngeal neurons in both sexes but displaying additional expression in tail neurons only in the male. We also show that lury-1 mutant animals show sex differences in feeding behaviours, with pharyngeal pumping elevated in hermaphrodites but reduced in males. LURY-1 also modulates male mating efficiency, influencing motor events during contact with a hermaphrodite. Our findings indicate sex-specific roles of this peptide in feeding and reproduction in C. elegans , providing further insight into neuromodulatory control of sexually dimorphic behaviours.
Publisher: Wiley
Date: 02-03-2022
DOI: 10.1111/JNC.15591
Abstract: The aggregation of proteins into inclusions or plaques is a prominent hallmark of a erse range of pathologies including neurodegenerative diseases. The quantification of such inclusions in Caenorhabditis elegans models of aggregation is usually achieved by fluorescence microscopy or other techniques involving biochemical fractionation of worm lysates. Here, we describe a simple and rapid flow cytometry‐based approach that allows fluorescently tagged inclusions to be enumerated in whole worm lysate in a quantitative and unbiased fashion. We demonstrate that this technique is applicable to multiple C. elegans models of aggregation and importantly, can be used to monitor the dynamics of inclusion formation in response to heat shock and during ageing. This includes the characterisation of physicochemical properties of inclusions, such as their apparent size, which may reveal how aggregate formation is distinct in different tissues or at different stages of pathology or ageing. This new method can be used as a powerful technique for the medium‐ to high‐throughput quantification of inclusions in future studies of genetic or chemical modulators of aggregation in C. elegans . image
Publisher: Frontiers Media SA
Date: 04-11-2020
Publisher: American Society for Microbiology
Date: 07-2012
DOI: 10.1128/JVI.06636-11
Abstract: Egress of wrapped virus (WV) to the cell periphery following vaccinia virus (VACV) replication is dependent on interactions with the microtubule motor complex kinesin-1 and is mediated by the viral envelope protein A36. Here we report that ectromelia virus (ECTV), a related orthopoxvirus and the causative agent of mousepox, encodes an A36 homologue (ECTV-Mos-142) that is highly conserved despite a large truncation at the C terminus. Deleting the ECTV A36R gene leads to a reduction in the number of extracellular viruses formed and to a reduced plaque size, consistent with a role in microtubule transport. We also observed a complete loss of virus-associated actin comets, another phenotype dependent on A36 expression during VACV infection. ECTV ΔA36R was severely attenuated when used to infect the normally susceptible BALB/c mouse strain. ECTV ΔA36R replication and spread from the draining lymph nodes to the liver and spleen were significantly reduced in BALB/c mice and in Rag-1-deficient mice, which lack T and B lymphocytes. The dramatic reduction in ECTV ΔA36R titers early during the course of infection was not associated with an augmented immune response. Taken together, these findings demonstrate the critical role that subcellular transport pathways play not only in orthopoxvirus infection in an in vitro context but also during orthopoxvirus pathogenesis in a natural host. Furthermore, despite the attenuation of the mutant virus, we found that infection nonetheless induced protective immunity in mice, suggesting that orthopoxvirus vectors with A36 deletions may be considered another safe vaccine alternative.
Publisher: Oxford University Press
Date: 05-10-2017
DOI: 10.1093/OXFORDHB/9780190456757.013.5
Abstract: The nematode Caenorhabditis elegans is among the most intensely studied animals in modern experimental biology. In particular, because of its amenability to classical and molecular genetics, its simple and compact nervous system, and its transparency to optogenetic recording and manipulation, C. elegans has been widely used to investigate how in idual gene products act in the context of neuronal circuits to generate behavior. C. elegans is the first and at present the only animal whose neuronal connectome has been characterized at the level of in idual neurons and synapses, and the wiring of this connectome shows surprising parallels with the micro- and macro-level structures of larger brains. This chapter reviews our current molecular- and circuit-level understanding of behavior in C. elegans. In particular, we discuss mechanisms underlying the processing of sensory information, the generation of specific motor outputs, and the control of behavioral states.
Publisher: Informa UK Limited
Date: 21-09-2013
DOI: 10.4161/CIB.25288
Publisher: Springer Science and Business Media LLC
Date: 20-11-2014
Publisher: Cold Spring Harbor Laboratory
Date: 31-08-2021
DOI: 10.1101/2021.08.29.458141
Abstract: The aggregation of proteins into inclusions or plaques is a prominent hallmark of a erse range of pathologies including neurodegenerative diseases. The quantification of such inclusions in Caenorhabditis elegans models of aggregation is usually achieved by fluorescence microscopy or other techniques involving biochemical fractionation of worm lysates. Here, we describe a simple and rapid flow cytometry-based approach that allows fluorescently-tagged inclusions to be enumerated in whole worm lysate in a quantitative and unbiased fashion. We demonstrate that this technique is applicable to multiple C. elegans models of aggregation and importantly, can be used to monitor the dynamics of inclusion formation in response to heat shock and during aging. This includes the characterisation of physicochemical properties of inclusions, such as their size, which may reveal how aggregate formation is distinct in different tissues or at different stages of pathology or aging. This new method can be used as a powerful technique for the medium- to high-throughput quantification of inclusions in future studies of genetic or chemical modulators of aggregation in C. elegans .
Publisher: Springer Science and Business Media LLC
Date: 05-06-2014
DOI: 10.1038/SREP05185
Publisher: The Company of Biologists
Date: 2013
Abstract: Protein with tau-like repeats (PTL-1) is the sole Caenorhabditis elegans homolog of tau and MAP2, which are members of the mammalian family of microtubule-associated proteins (MAPs). In mammalian neurons, tau and MAP2 are segregated, with tau being mainly localised to the axon and MAP2 mainly to the dendrite. In particular, tau plays a crucial role in pathology, as elevated levels lead to the formation of tau aggregates in many neurodegenerative conditions including Alzheimer's disease. We used PTL-1 in C. elegans to model the biological functions of a tau-like protein without the complication of functional redundancy which is observed among the mammalian MAPs. Our findings indicate that PTL-1 is important for the maintenance of neuronal health as animals age, as well as in the regulation of whole organism lifespan. In addition, gene dosage of PTL-1 is critical, as variations from wild-type levels are detrimental. We also observed that human tau is unable to robustly compensate for loss of PTL-1, although phenotypes observed in tau transgenic worms are dependent on the presence of endogenous PTL-1. Our data suggest that some of the effects of tau pathology may result from the loss of physiological tau function, and not solely from a toxic gain-of-function due to accumulation of tau.
Publisher: Springer Science and Business Media LLC
Date: 02-07-2020
Abstract: Lesioning studies have provided important insight into the functions of brain regions in humans and other animals. In the nematode Caenorhabditis elegans , with a small nervous system of 302 identified neurons, it is possible to generate lesions with single cell resolution and infer the roles of in idual neurons in behaviour. Here we present a dataset of ~300 video recordings representing the locomotor behaviour of animals carrying single-cell ablations of 5 different motorneurons. Each file includes a raw video of approximately 27,000 frames each frame has also been segmented to yield the position, contour, and body curvature of the tracked animal. These recordings can be further analysed using publicly-available software to extract features relevant to behavioural phenotypes. This dataset therefore represents a useful resource for probing the neural basis of behaviour in C. elegans , a resource we hope to augment in the future with ablation recordings for additional neurons.
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 06-2022
End Date: 06-2025
Amount: $554,000.00
Funder: Australian Research Council
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