Al-Hashimi, Hikmat et al. (2013) International Worm Meeting "EXC-2 and Maintenance of excretory canal tube structure."

Buechner, Matthew, Harte, Robyn, Al-Hashimi, Hikmat, Macdonald, Stuart, Hackett, Jenny

[International Worm Meeting, 2013]

Long, narrow, single-celled tubes are found in many tissues in our body, such as capillaries and nephrons. The excretory canal cell of C. elegans provides a model for investigating the formation and maintenance of narrow tubes. Mutations in a series of exc genes allow the excretory canals to swell into fluid-filled cysts. The position of all these genes has been narrowed, but a few remain to be cloned. We are currently using whole-genome sequencing methods to identify the sequence of exc-2. Preliminary data suggest that EXC-2 may act upstream of other EXC proteins that effect endosomal recycling. We are characterizing the potential role of EXC proteins in the endosomal transport pathway by monitoring several endosomal compartments via FRAP. Recovery of fluorescence of cytosolic molecules of labeled RAB-5 and RAB-11 are not affected by exc-2 mutation. A difference was seen, however, with RAB-5 and RAB-11 bound to their target compartments (the early and recycling endosomes, respectively). Puncta carrying marked RAB-5 recovered rapidly after bleaching. Puncta of RAB-11 molecules, exhibited a different behavior. In the wild-type background, bleached puncta recovered fluorescence fully within 180 seconds. When exc-2 was mutated, however, recovery was essentially halted, with no significant increase in fluorescence visible after 280 seconds. This result suggests that EXC-2 function is required for turnover of RAB-11 on the recycling endosome. We are now analyzing other subcellular markers such as EEA-1, RME-1, GRIP, GLO-1, and RAB-7 in order to gain a broader understanding of the role of EXC-2 and other EXC proteins in the endosomal recycling.

Al-Hashimi, Hikmat et al. (2015) International Worm Meeting "exc-2, a gene involved in maintaining tube structure of the excretory canals."

Al-Hashimi, Hikmat, Buechner, Matthew

[International Worm Meeting, 2015]

The excretory canals of C. elegans represent an attractive model for studying maintenance of seamless, narrow, single-cell biological tubes. Many genes have been identified that play a role in the maintenance of canal diameter.Mutation of the exc-2 gene leads to short (30-40% of wild-type length) canals containing multiple fluid-filled cysts. In order to identify the exc-2 gene, we carried out whole-genome sequencing for the allele rh90. The results showed a frameshift insertion in candidate gene ifa-4, which encodes intermediate filament A4. RNAi against ifa-4 in N2 worms causes formation of short, cystic canals similar to those of exc-2 mutant worms. In order to confirm the identity of the exc-2 gene, we performed a rescue assay and a complementation test between exc-2 and ifa-4. Surprisingly, injection of the ifa-4 sequence into exc-2 worms did not rescue the phenotype, and the two genes complemented each other, which indicates that exc-2 and ifa-4 are different genes. Sequencing of three additional alleles of exc-2 showed the same frameshift, as did sequencing of our wild-type strains! Galaxy Cloudmapping uses an older version of the genome (WS220); the latest version of Wormbase (WS247) has corrected this sequence error. We have now performed whole-genome sequencing of all 4 alleles of exc-2, two of which were outcrossed to Hawaiian wild-type worms to allow identification of the exc-2 mutation. We are now screening candidate genes via confirmation of mutations via Sanger sequencing, RNAi, and fosmid rescue in order to identify the exc-2 gene, which should have mutations in all 4 alleles.Our lab's previous work suggests that exc-2 acts genetically upstream of exc-9, exc-1, and exc-5. A FRAP analysis of mutants labeled with endosomal tags suggests that, while most endosomal compartments are unaffected, the turnover of RAB-11 on recycling endosomes requires wild-type EXC-2 function. EXC-2 may therefore participate in endosomal recycling.

Chiarelli, Travis et al. (2017) International Worm Meeting "Novel genes involved in excretory canal structure and maintenance."

Al-Hashimi, Hikmat, Chiarelli, Travis, Lundquist, Erik, Buechner, Matthew

[International Worm Meeting, 2017]

The excretory canal cell is the largest single epithelial cell within C. elegans, and its long fluid-filled hollow canals forms a useful model for understanding single-cell tubulogenesis. Our previous studies have identified a set of exc genes whose products regulate and maintain the narrow diameter of the canals during outgrowth. Some of these genes encode cytoskeletal proteins (including beta Hspectrin, formin, and intermediate filament proteins), and vesicular transport proteins (CRIP, IRG, and FGD proteins). The Miller lab has made available a list of genes highly enriched in various tissues of C. elegans (www.vanderbilt.edu/wormdoc/wormmap/WormViz.html), including genes enriched in the excretory canal cell. We have gratefully built on these results to search for additional genes that affect excretory canal structure, as well as genes that interact with known exc genes. We designed and are conducting a feeding RNAi screen with ~200 candidate genes strongly expressed in the canal cell, and which encode proteins with good potential to interact with EXC proteins, with the purpose of understanding the network of structural and trafficking required to build and maintain a narrow tube in a growing, moving animal. Since the excretory canal cell may be refractory to RNAi, as it has some neural characteristics (long processes following neural guidance cues during outgrowth), we crossed our chosen exc strains to an RNAi-sensitive strain (rrf-3) that carries an integrated construct marking the canal cell cytoplasm (Pvha-1::gfp). The selected exc mutant backgrounds represent several pathways of interest: cytoskeletal structure (exc-2), vesicular transport (exc-5 and exc-9) , RNA-binding (exc-7), and ion transport (exc-4), as well as animals with wild-type canals. At present, we have isolated and verified close to ten hits, some of which exacerbate canal defects, and excitingly, one which at least partially rescues the strongly cystic phenotype of the exc-5 mutant. These knockdowns offer insight into novel gene interactions as well as alternative pathways that have yet to be explored in regards to excretory canal maintenance. We are continuing the RNAi screen.

Kazatskaya, Anna et al. (2020) MicroPubl Biol

Philbrook, Alison, Sengupta, Piali, Amin-Wetzel, Niko, Kazatskaya, Anna, de Bono, Mario, Yuan, Lisa

[MicroPubl Biol, 2020]

A subset of sensory neurons in C. elegans contains compartmentalized sensory structures termed cilia at their distal dendritic ends (Ward et al. 1975; Perkins et al. 1986; Doroquez et al. 2014). Cilia present on different sensory neuron types are specialized both in morphology and function, and are generated and maintained via shared and cell-specific molecules and mechanisms (Perkins et al. 1986; Evans et al. 2006; Mukhopadhyay et al. 2007; Mukhopadhyay et al. 2008; Morsci and Barr 2011; Doroquez et al. 2014; Silva et al. 2017). The bilaterally symmetric pair of URX oxygen-sensing neurons in the C. elegans head (Figure 1A) is thought to be non-ciliated (Ward et al. 1975; Doroquez et al. 2014) but nevertheless exhibits intriguing morphological similarities with ciliated sensory neurons. URX dendrites extend to the nose where they terminate in large bulb-like complex structures (Ward et al. 1975; Doroquez et al. 2014; Cebul et al. 2020) (Figure 1A). These structures concentrate oxygen-sensing signaling molecules (Gross et al. 2014; Mclachlan et al. 2018) suggesting that similar to cilia, these structures are specialized for sensory functions. Microtubule growth events similar to those observed in ciliated sensory neurons were also reported at the distal dendritic regions of URX, implying the presence of a microtubule organizer such as a remodeled basal body (Harterink et al. 2018). Moreover, a subset of ciliary genes is expressed in URX (Kunitomo et al. 2005; Harterink et al. 2018; Mclachlan et al. 2018). We tested the hypothesis that URX dendrites contain cilia at their distal ends.

Wang W et al. (2009) Biochem Biophys Res Commun "Ced-9 inhibits Al-induced programmed cell death and promotes Al tolerance in tobacco."

Pan J, Bian H, Wang W, Shao H, Wang J, Chen H, Zheng K, Zhu M, Guo Y, Han N

[Biochem Biophys Res Commun, 2009]

Our previous data showed that apoptotic suppressors inhibit aluminum (Al)-induced programmed cell death (PCD) and promote Al tolerance in yeast cells, however, very little is known about the underlying mechanisms, especially in plants. Here, we show that the Caenorhabditis elegans apoptotic suppressor Ced-9, a Bcl-2 homologue, inhibited both the Al-induced PCD and Al-induced activity of caspase-like vacuolar processing enzyme (VPE), a crucial executioner of PCD, in tobacco. Furthermore, we show that Ced-9 significantly alleviated Al inhibition of root elongation, decreased Al accumulation in the root tip and greatly inhibited Al-induced gene expression in early response to Al, leading to enhancing the tolerance of tobacco plants to Al toxicity. Our data suggest that Ced-9 promotes Al tolerance in plants via inhibition of Al-induced PCD, indicating that conserved negative regulators of PCD are involved in integrated regulation of cell survival and Al-induced PCD by an unidentified mechanism.

Halic M et al. (2009) Mol Cell "22G-RNAs in transposon silencing and centromere function."

Halic M, Moazed D

[Mol Cell, 2009]

Three recent papers (Gu et al., 2009; Claycomb et al., 2009; van Wolfswinkel et al., 2009) provide evidence that links a new class of small RNAs and Argonaute-associated complexes to centromere function and genome surveillance.

Page KE et al. (2012) Metallomics "Aluminium exposure disrupts elemental homeostasis in Caenorhabditis elegans."

McCrohan CR, Page KE, White KN, Killilea DW, Lithgow GJ

[Metallomics, 2012]

Aluminium (Al) is highly abundant in the environment and can elicit a variety of toxic responses in biological systems. Here we characterize the effects of Al on Caenorhabditis elegans by identifying phenotypic abnormalities and disruption in whole-body metal homeostasis (metallostasis) following Al exposure in food. Widespread changes to the elemental content of adult nematodes were observed when chronically exposed to Al from the first larval stage (L1). Specifically, we saw increased barium, chromium, copper and iron content, and a reduction in calcium levels. Lifespan was decreased in worms exposed to low levels of Al, but unexpectedly increased when the Al concentration reached higher levels (4.8 mM). This bi-phasic phenotype was only observed when Al exposure occurred during development, as lifespan was unaffected by Al exposure during adulthood. Lower levels of Al slowed C. elegans developmental progression, and reduced hermaphrodite self-fertility and adult body size. Significant developmental delay was observed even when Al exposure was restricted to embryogenesis. Similar changes in Al have been noted in association with Al toxicity in humans and other mammals, suggesting that C. elegans may be of use as a model for understanding the mechanisms of Al toxicity in mammalian systems.

Page K E et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Genes Affecting Aluminium Toxicity in Caenorhabditis elegans"

Page K E, White K N, Lithgow G J, McCrohan C R

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Aluminium (Al) is a highly abundant crustal metal with known toxic effects in multiple biological systems. Using the nematode C. elegans we have found a set of genetic modulators of Al toxicity. C. elegans is widely used for toxicity and aging studies due to its small size, large progeny numbers, short lifespan, and simple methods of genetic manipulation. This makes C. elegans a good model to study the toxicity mechanism of Al in animals. The exposures were carried out on agar plates, with Al in the form of Al(NO3)3, mixed with a concentrated Escherichia coli food source. Here we show that a selection of genes involved in the mechanisms of Al toxicity and/or metabolism have potential relevance in the metallostasis (metal homeostasis) of other metals, or are key regulators of known stress resistance pathways. Al negatively affects C. elegans developmental progression from 3 microM Al, fertility at 30 microM Al, and body size from 1.9 mM Al. The developmental delay phenotype caused by Al exposure can be passed on to the un-exposed next generation. At 4.8mM exposures Al affects the levels of Al and other elements in C. elegans; shown using ICP-OES. We are also investigating the effect of Al exposure to the course of normal aging in C. elegans.

Chavez-Perez, Carlos et al. (2021) MicroPubl Biol

Keenan, Brendan T, Raizen, David M, Jafari, Niusha, Rohacek, Alex M, Chavez-Perez, Carlos

[MicroPubl Biol, 2021]

Like other animals, the nematode C. elegans exhibits reduced movement and sleep in response to sickness, which can be induced by exposure to high temperatures (Hill et al. 2014; Nelson et al. 2014) ultraviolet light (DeBardeleben et al. 2017), and other stressful exposures (Hill et al. 2014; Goetting et al. 2020). This response has been termed Stress/Sickness-Induced Sleep (SIS) (Hill et al. 2014; Trojanowski and Raizen 2016). Exposure to the stressor leads to quiescence in part via release of the cytokine Epidermal Growth Factor (EGF) (Hill et al. 2014; Konietzka et al. 2020), which is encoded by the gene lin-3 (Hill and Sternberg 1992). EGF activates the ALA and RIS neurons, which then release their respective neuropeptides to effect reduced movement and behavioral quiescence (Konietzka et al. 2020).

Yeon, Jihye et al. (2021) MicroPubl Biol

Yeon, Jihye, Sengupta, Piali, Takeishi, Asuka

[MicroPubl Biol, 2021]

Neuronal networks can achieve similar outputs via distinct underlying circuit mechanisms (Beverly et al., 2011; Marder et al., 2015; Saideman et al., 2007; Trojanowski et al., 2014; Wang et al., 2019). This degeneracy allows networks to maintain robustness without compromising functional flexibility (Cropper et al., 2016; Edelman and Gally, 2001). Since the contribution of degenerate neuronal pathways is likely to be revealed under defined genetic or environmental conditions, it is challenging to identify and describe the contributions of such pathways to neuronal circuit function.