Iannacone, Michael et al. (2021) International Worm Meeting "Viral infection in C. elegans causes sleep, which is necessary for survival and energy maintenance"

van der Linden, Alexander, Um, Paul, Raizen, David, Iannacone, Michael, Fodor, Jeremy

[International Worm Meeting, 2021]

Cellular stressors including heat shock and ultraviolet radiation (UV) cause sleep in the nematode Caenorhabditis elegans. This stress-induced sleep is characterized by locomotor quiescence, cessation from feeding, an increased arousal threshold, and rapid reversibility. We have identified a novel stressor which causes sleep in C. elegans: the Orsay virus. The Orsay virus is the only known virus to infect C. elegans and is related to the nodavirus, which infects other invertebrates as well as vertebrates including mammals. The Orsay virus exclusively infects intestinal cells. Despite infection of non-neuronal cells, Orsay caused sleep through a similar neuronal mechanism as sleep caused by other stressors. We hypothesized the same genetic and neural circuitry which controls sleep in response to UV and sleep would also control sleep in response to Orsay. Similar to UV and heat, mutations which affected development of the ALA neuron and processing of neuropeptides reduced sleep in infected animals. Conversely, mutations which affected development of the RIS neuron (which is required for UV/heat sleep) did not affect sleep in response to Orsay. ALA-defective animals, which had reduced sleep, had decreased survival compared to wild-type animals. This effect on survival was not observed in the RIS-defective animals, which slept normally. Transgenic overexpression of somnogenic FLP-13 neuropeptides increased the survival of infected ALA-defective animals. The survival benefit of sleeping animals was not explained by a difference in viral replication levels or in the transcriptional intracellular pathogen response. Infection with Orsay caused a decrease in global ATP levels, and this decrease was more severe in ALA-defective animals. The decreased survival of virally-infected animals was accompanied by the proliferation of OP50 bacteria inside the gut of the animal, and in some cases beyond the intestines. Lifespan was extended in virus-infected animals when they were grown on UV-killed OP50 bacteria, suggesting that bacterial superinfection is partially responsible for virus-induced lethality. These findings suggest that sleep is beneficial for recovery from infection due to its effect on energetics, and this model presents a novel opportunity to explore the protective role of sleep in host-pathogen interactions.

Raizen, D.M. et al. (2019) International Worm Meeting "Sleep enhances survival during viral infection."

Raizen, D.M., Um, Paul, van der Linden, Alexander, Iannacone, Michael, Grubbs, Jeremy

[International Worm Meeting, 2019]

While it is widely appreciated that infection is associated with sleepiness and fatigue, the underlying mechanisms of infection-induced sleep and the benefit of this sleep has remained largely a mystery. Here, we used the Orsay virus model to explore these questions in C. elegans. The Orsay virus infects the intestinal cells yet has strong effects on behavior, indicating interactions between somatic and neural cells. Locomotion and feeding quiescence were increased in infected animals. Infection-induced sleep, like sleep in response to other stressors such UV light, required the neuropeptide processing enzyme EGL-21 as well as the ALA neuron. Surprisingly, our preliminary data suggests that the somnogenic neuron RIS, which is required for sleep in response to other stressors, was not required for virus-induced quiescence. Worms that lacked the ALA neuron (i.e. failed to sleep in response to infection), showed a 30% reduction in median lifespan compared to infected control animals. This survival benefit of sleep could not be explained by a difference in the viral load of sleeping vs. sleepless animals, nor could it be explained by differences in the transcriptional intracellular pathogen response. Sleepless infected animals had lower ATP levels than sleeping infected animals, suggesting that sleep benefits the animal at the level of energetics. This model presents an opportunity to understand the mechanism of sleepiness during infections and the protective role of sleep in host-pathogen interactions.

Francis McNally et al. (2007) International Worm Meeting "Restriction of meiotic spindle length by microtubule severing."

Karen McNally, Francis McNally

[International Worm Meeting, 2007]

In animals, female meiotic spindles mediate the expulsion of chromosomes into polar bodies to generate a haploid egg. Time-lapse imaging of fluorescent protein fusions has revealed that wild-type C. elegans meiotic spindles undergo discrete length changes during each division. Meiosis I spindles, for example, maintain a constant metaphase length of 7.7 um for several minutes. Meiosis I spindles then shorten until they reach 58% of their starting length. During this initial shortening phase, the average fluorescence intensity of GFP-tubulin doubles (suggesting an inward sliding mechanism) and the spindles remain parallel to the cortex. After this initial shortening phase, spindles rotate perpendicular to the cortex, then initiate anaphase chromosome separation as they proceed through a second phase of spindle shortening. During the second shortening phase, the average fluorescence intensity of GFP tubulin decreases at poles while increasing in the midzone, suggesting a redistribution of microtubules within the spindle. At the end of the second shortening phase, MI spindles are 2.8 um long (38% of starting length). MEI-1 and MEI-2 are the C. elegans orthologs of the 2 subunits of a conserved microtubule-severing ATPase called katanin. MEI-1 and MEI-2 are localized on meiotic spindles and purified complexes of MEI-1 and MEI-2 sever microtubules in vitro, indicating that MEI-1/MEI-2 may regulate microtubule length within the meiotic spindle. We have analyzed a partial loss of function allele of mei-2 called ct98. mei-2(ct98) meiosis I spindles exhibit 4 defects. First, mei-2(ct98) MI spindles initially assembly with a longer than wild-type metaphase length of 9.6 um. Second, they do not rotate. Third, they do not undergo the second phase of spindle shortening although they undergo the first phase of shortening with wild-type velocity. Fourth, microtubules do not redistribute from the poles to the midzone. As a result of these defects, mei-2(ct98) spindles reach a final minimum length of 6.2 um (65% of starting length) and induce formation of larger than wild-type polar bodies. These results are consistent with a model in which microtubule severing reduces the average microtubule length during spindle assembly and during the second phase of spindle shortening. We are currently analyzing a collection of mei-1 misense alleles provided by Paul Mains to establish a relationship between in vitro microtubule-severing activity and in vivo spindle length control in order to address the perplexing question of why null alleles of mei-1 or mei-2 prevent assembly of bipolar meiotic spindles rather than causing assembly of extremely long meiotic spindles.

Anita U Rao et al. (2005) International Worm Meeting "Dissecting Heme Trafficking Pathways in C. elegans"

Iqbal Hamza, Anita U Rao, Melissa E Winn

[International Worm Meeting, 2005]

Heme serves as a cofactor for a number of proteins involved in key metabolic processes. In eukaryotes, heme synthesis occurs in the mitochondria by an evolutionarily conserved multi-step pathway. Hemes are hydrophobic and thus insoluble in the aqueous environment of the cell. Moreover, free heme is cytotoxic because of peroxidase activity. We therefore hypothesize that intracellular pathways exist for trafficking of heme from the site of synthesis in the mitochondria to various cellular destinations. However, identification of these heme transport pathways have been difficult because heme synthesis is regulated by multiple effectors and is tightly coordinated with apo-protein synthesis. We have previously shown that C. elegans and related helminths do not make heme albeit requiring exogenous heme for normal metabolic processes. Importantly, C. elegans show a biphasic response for heme; worms are growth-arrested at <sym14> 1um and <sym15> 800 uM heme. These results suggest that although worms are obligate heme auxotrophs they likely have all the pathways for heme utilization beyond the point of heme synthesis. To identify pathways for heme transport in C. elegans , we exploited their biphasic response to heme by screening for mutants that could survive heme toxicity. We screened 300,000 haploid genomes and isolated 13 mutants at 800 uM or 1000 uM heme in liquid axenic medium. These mutants can be preliminarily classified into two groups based on their growth in low and high heme, and their decreased sensitivity to gallium protoporphyrin (GaPP) a toxic heme analog. Mutants in the first group grow exceptionally well in low and high heme when compared to wild-type N2. These mutants also survive at 0.5 uM GaPP which normally causes growth arrest in N2 worms. Mutants in the second group do not differ greatly as compared to N2 in their ability to grow at low heme and 0.5 uM GaPP but they grow robustly at high heme. We are currently characterizing these mutants phenotypically and attempting to map the mutations by snip-SNPs.

Abbhirami Rajagopal et al. (2005) International Worm Meeting "Genome-wide analysis of genes involved in regulation of heme homeostasis in C. elegans"

Iqbal Hamza, Abbhirami Rajagopal, Michael Krause

[International Worm Meeting, 2005]

Heme is a prosthetic group of proteins that perform wide variety of cellular functions. Heme synthesis occurs in the mitochondria via a multistep pathway, which is highly conserved across eukaryotes. We have shown that C. elegans lacks the ability to make heme, albeit containing hemoproteins for various metabolic processes. Furthermore, our results indicate that worms utilize heme from their diet for incorporation into hemoproteins. Worms grown in liquid axenic CeHR medium achieved optimal growth at 20 uM hemin but failed to grow beyond the early L4 stage at concentrations <sym14> 1 uM hemin; worms were growth-arrested at L2/L3 stage at concentrations <sym15> 800 uM hemin. Thus, heme requirement in worms appears to be biphasic. We rationalized that worms must have the ability to regulate heme transport and coordinate transport with hemoprotein synthesis because hemes are hydrophobic macrocycles and free heme can cause cell injury due to its peroxidase activity. To understand the molecular mechanisms of heme homeostasis in eukaryotes, an Affymetrix C. elegans genome array analysis was performed using RNA samples extracted from worms that were grown in CeHR medium supplemented with 4, 20 and 500 uM hemin. Of the 22,627 probe sets, a total of 886 genes revealed changes in mRNA expression in response to heme. Amongst these genes were 145 genes with fold-changes of two or greater, including putative transporters, transcription factors, and known hemoproteins such as cytochrome P450s. Expression profiles for these genes are currently being validated by real-time PCR analysis. Based on the results from RNA quantitations, sequence motif searches, and secondary structure predictions, we will perform RNA interference analysis on specific candidate genes to directly examine the role of these genes in organismic heme homeostasis.

Hsin-ya Yang et al. (2002) West Coast Worm Meeting "Spindle Movements during Female Meiosis"

Hsin-ya Yang, Francis J McNally, Karen L McNally

[West Coast Worm Meeting, 2002]

During female meiosis, a haploid chromosome number is achieved by sequentially segregating half of the homologous chromosomes, then half of the sister chromatids into polar bodies. Both meiotic segregation events require positioning a microtubule-based spindle at the cell cortex in an orientation perpendicular to the cortex. In many metazoans, this final spindle position is achieved after the meiotic spindle first translocates through the cytoplasm to the cortex and subsequently rotates to the perpendicular orientation. These spindle movements are critical for proper segregation of chromosomes into polar bodies, yet the molecular basis of these movements is largely unknown. We are using C. elegans as a model system to determine the molecules and mechanisms that drive these spindle movements. Meiotic spindle movements were observed by time-lapse imaging of intact, anesthetized worms expressing GFP-beta tubulin or GFP-histone H2B. Forty to fifty minutes elapsed between the start of meiosis at nuclear envelope breakdown and the end of meiosis marked by formation of the female pronucleus. After nuclear envelope breakdown, an 8 um long, anastral spindle assembled several um from the cell cortex. The spindle then translocated to the cortex with an average velocity of 1.5 um/min. The spindle initially adopted an orientation roughly parallel to the cell cortex and slid along the cortex for an average of 8.8 um before the metaphase-anaphase transition. At the metaphase-anaphase transition, the 8 um spindle first shortened to 6 um in length, then rotated perpendicular to the cortex. After rotation, separation of homologous chromosomes occurred as the spindle continued to shorten to a final length of 2.5 um. As the first polar body formed, the short post-anaphase spindle narrowed and lengthened into a midbody. Meiosis II proceeded in a similar fashion. To test whether microtubules are required for meiotic spindle movements, GFP-histone time lapse imaging was conducted on tubulin (RNAi) worms. Translocation of meiotic bivalents to the cortex was completely blocked in these worms indicating that microtubules are required for translocation of bivalent chromosomes to the cortex. The spindle checkpoint was apparently not activated by this treatment because homologs separated with normal timing. Diakinesis nuclei were also mispositioned relative to the oocyte cortex in tubulin (RNAi) worms, raising the possibility that the failure in bivalent translocation was an indirect effect of starting out too far from the cortex. Zyg-9 null worms, however, also had mispositioned diakinesis nuclei but meiotic bivalents translocated to the cortex after maturation (9/9 worms). In addition, in worms null for mei-1, a microtubule regulator, bivalents failed to translocate to the cortex in 50% of matured oocytes (4/6 worms). These results indicate that microtubules are directly involved in translocation of bivalent chromosomes to the cell cortex. To test the role of F-actin in spindle movements, time-lapse imaging was conducted on worms treated with latrunculin A. All aspects of meiosis I spindle translocation and rotation were normal in latrunculin-treated worms but polar bodies did not form and the segregated chromosomes collapsed back together at the end of anaphase I. In addition, the narrowing of the post-anaphase spindle to a midbody was slow and incomplete. These results indicate that meiotic spindle translocation and rotation are less dependent on F-actin than is polar body formation. Further studies will concentrate on the motors and microtubule dynamics regulators involved in meiotic spindle translocation.

Carmona, Javier et al. (2017) International Worm Meeting "2D worm tracking line-confocal microscope with virtual reality."

Wang, Charles, Carmona, Javier, Baldo, Anthony, Madruga, Blake, Mendoza, Steve, Jin, Suying, Arisaka, Katsushi, Liu, Larry, Thatcher, Joseph

[International Worm Meeting, 2017]

Calcium dynamic imaging and free motion tracking coupled with external stimulations allow for in depth analysis of C. elegans behavior. We have developed an integrated platform for monitoring and controlling C. elegans under a variety of external stimulations, including thermal, electrical, and photo stimuli. This innovative platform combines rapid volumetric (20 volume/s) diffraction limited dual line-confocal microscopy (0.5 um x 1 um x 5 um voxel) to determine the neural pathways different external stimuli induce, while tracking worm's two dimensional motion. Never before has dynamic signal propagation, from neuron to neuron, been observed for C. elegans in free motion at such high volume scanning rate. External stimuli are computer controlled with < 10 ms resolution for precise spatio-temporal synchronization with free motion behavior and whole-brain calcium dynamics. Physical linear and circular thermal gradients were implemented using customized temperature plates with thermal fluctuations of less than 0.05 deg C. In addition, thermal stimulation was applied via a 1490 nm infrared laser to create virtual temperature conditions, synchronized with head motion. Infrared laser stimulation allows C. elegans' thermoreceptor (AFD neuron) to perceive temperature fluctuations exclusively in the time domain, thereby allowing for the complete virtual manipulation of the nematode's thermal environment. Electrical responses were induced using a technique that involves applying a linear or spatially alternating electrical field through a gelatin sample with fields ranging from 4 to 14 V/cm. Photon stimulation was implemented using a 405 nm laser with intensities ranging from 0 to 10 mW/mm^2. Volumetric Calcium imaging of QW1217 has also allowed for the complete mapping of the neurons responsible for each of the aforementioned stimuli. The microscope and software accommodate multiple simultaneous stimuli applications, such as electrical and photon simulations. Also tested were the neural pathway differences between infrared and photo avoidance behavior due to their very similar behavioral responses.

Consortium Wormbase (2000) Worm Breeder's Gazette "Update on the C. elegans database WormBase"

Consortium Wormbase

[Worm Breeder's Gazette, 2000]

WormBase (www.wormbase.org) is an international consortium of biologists and computer scientists dedicated to providing the research community with accurate, current, accessible information concerning the genetics, genomics and biology of C. elegans and some related nematodes. WormBase builds upon the existing ACeDB database of the C. elegans genome by providing curation from the literature, an expanded range of content and a user friendly web interface. The team that developed and maintained ACeDB (Richard Durbin, Jean Thierry-Mieg) remains an important part of WormBase. Lincoln Stein and colleagues at Cold Spring Harbor are leading the effort to develop the user interface, including visualization tools for the genome and genetic map. Teams at Sanger Centre (led by Richard Durbin) and the Genome Sequencing Center at Washington University, St. Louis (led by John Spieth) continue to curate the genomic sequence. Jean and Danielle Thierry-Mieg at NCBI spearhead importation of large-scale data sets from other projects. Paul Sternberg and colleagues at Caltech will curate new data including cell function in development, behavior and physiology, gene expression at a cellular level; and gene interactions. Paul Sternberg assumes overall responsibility for WormBase, and is delighted to hear feedback of any sort. WormBase has recently received major funding from the National Human Genome Research Institute at the US National Institutes of Health, and also receives support from the National Library of Medicine/NCBI and the British Medical Research Council. WormBase is an expansion of existing efforts, and as such continues to need you help and feedback. Even with the increased scope and funding, all past contributors to ACeDB remain involved. The Caenorhabditis Genetics Center (Jonathan Hodgkin and Sylvia Martinelli) collaborate with WormBase to curate the genetic map and related topics. Ian Hope and colleagues continue to supply expression data to WormBase. Leon Avery will continue his superb website and serves as one advisor to WormBase. While the major means of access to WormBase is via the world wide web, downloadable versions of WormBase as well as the acedb software engine will continue to be available.

Rachel McMullan et al. (2006) European Worm Meeting "Rho is a Pre-Synaptic Activator of Neurotransmitter Release at Pre-Existing Synapses in C.elegans"

Rachel McMullan, Emma Hiley, Paul Morrison, Stephen J. Nurrish

[European Worm Meeting, 2006]

Rachel McMullan, Emma Hiley, Paul Morrison and Stephen J. Nurrish. Rho GTPases have important roles in neuronal development but their function in adult neurons is less well understood. We demonstrate that pre-synaptic changes in Rho activity at C.elegans neuromuscular junctions can radically change animal behaviour via modulation of two separate pathways. In one, pre-synaptic Rho increases acetylcholine (ACh) release by stimulating the accumulation of diacylglycerol (DAG) and the DAG-binding protein UNC-13 at sites of neurotransmitter release; this pathway requires binding of Rho to the DAG kinase DGK-1. A second DGK-1-independent mechanism is revealed by the ability of a Rho inhibitor (C3 transferase) to decrease levels of release even in the absence of DGK-1; this pathway is independent of UNC-13 accumulation at release sites. We do not detect any Rho induced changes in neuronal morphology or synapse number, thus Rho facilitates synaptic transmission by a novel mechanism. Surprisingly, many commonly available human RhoA constructs contain an uncharacterised mutation that severely reduces binding of RhoA to DAG kinase. Thus a role for RhoA in controlling DAG levels has not been previously appreciated.

Minniti AN et al. (1995) International C. elegans Meeting "THE HERMAPHRODITE-INITIATED SPERMIOGENESIS PATHWAY."

Minniti AN, Nance JF, Sadler C, Ward S

[International C. elegans Meeting, 1995]

During spermiogenesis, round nonmotile spermatids are rapidly transformed into asymmetrical crawling spermatozoa. In addition to spe-8 and spe-12, we found two new genes, spe-27 and spe-29, that when mutated, disrupt spermiogenesis in an identical manner: mutant hermaphrodites are self-sterile, while mutant males are fertile. Sperm from both sexes activate abnormally in vitro, suggesting that these gene products are expressed in both sperm, but only hermaphrodites require them for activation. The hermaphrodite's spermatids, however, can be activated by mating. This phenotype has been explained by a model that has two distinct pathways of activation, one for males and one for hermaphrodites (Shakes and Ward, 1989). The four genes are needed only for the hermaphrodite pathway. spe-27 and spe-12 have been cloned. Their mRNA is found exclusively in the germline during spermatogenesis. Their predicted protein sequences show no similarities to known proteins. We are currently cloning spe-29. To further understand how these genes act during spermiogenesis we are determining the subcellular localization of their gene products. In addition, suppressor analysis is under way (see abstract by Paul Muhlrad and Samuel Ward) to look for other genes in this pathway and for interactions between these genes.