Nikonorova, Inna et al. (2021) International Worm Meeting "Deciphering the ciliary extracellular vesicle (EV) proteome"

Nikonorova, Inna, Cope, Alexander, Barr, Maureen, Power, Kaiden, Walsh, Jonathon, Wang, Juan, Shah, Premal, Akella, Jyothi

[International Worm Meeting, 2021]

Extracellular vesicles (EVs) are emerging as a universal means of cell-to-cell communication and hold great potential in diagnostics and regenerative therapies. However, the EV field lacks a fundamental understanding of biogenesis, cargo content, signaling, and target interactions. EVs that are transmitted by cilia represent a particular challenge due to small volume of the organelle. Here, we used our established C. elegans system to determine the composition and explore the function of ciliary EVs. We took advantage of the fact that C. elegans releases ciliary EVs from 21 male-specific neurons and 6 core IL2 neurons into environment and thus provides a great platform for discovery of evolutionarily conserved ciliary EV cargo. To collect ciliary EVs we developed a biochemical enrichment procedure based on buoyant density centrifugation and high-resolution fractionation. Using fluorescent-tagged EV cargo PKD-2::GFP and superresolution microscopy we tracked ciliary EVs in the collected fractions and identified two populations of PKD-2 carrying EVs that differ in their densities. Proteomic analysis of the PKD-2 EV-enriched fractions revealed 2,888 proteins of C. elegans EVome that likely originate from multiple tissues. Top candidates were validated via generation of transgenic or CRISPR reporters and visualization of EV release using super-resolution microscopy. This strategy revealed that the male reproductive system is a major source of non-ciliary EVs. To extract ciliary EV cargoes, we integrated our dataset with published transcriptomic data. We identified new ciliary EV cargo involved in nucleotide binding and RNA interference, suggesting that environmentally-released ciliary EVs may also carry nucleic acids. Our work serves as a springboard for discoveries in the EV field and will help shed light on the contribution of ciliary EVs to the pathophysiology of abnormal EV signaling, including ciliopathies, cancer, and neurodegenerative diseases.

Shen X et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Biomechanical analysis of C. elegans motility"

Arratia P E, Krajacic P, Lamitina T, Purohit P, Shen X

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Previous studies of C. elegans motility have focused mainly on simple quantitative assays, such as the ''thrashing'' assay. More recently, quantitative approaches have been used to study nematode kinematics. Such quantitative approaches could provide a new level of phenotypic resolution to the study of C. elegans motility. We recently developed a simple mathematical model to describe the C. elegans swim gait. This model provides estimates for the biomechanical properties of motility including force, power, tissue viscosity, and Young''s modulus. Here, we used high-speed video as well as image analysis and particle tracking methods to experimentally measure the forces imparted on the surrounding fluid by a swimming C. elegans. Analysis of the local forces (propulsive and lateral) over the wave period is computed by performing a simple force balance and by coupling the fluid velocity field with the nematode body postures. The propulsive force shows a sharp peak as the lateral force approaches zero. The magnitude of the peak propulsive force (i.e. thrust) and the average mechanical power of a swimming nematode in M9 solution are approximately 3.0 nN and 2.0 pW. By comparison, values of force and power estimated using theoretical models are approximately 3.5 nN and 5.0 pW. These data provide experimental validation for our model of motility and demonstrate that biomechanical phenotyping can provide a sensitive and quantitative phenotypic metric for the analysis of C. elegans motility.

Delia Garigan et al. (2001) International C. elegans Meeting "Characterization of a Short-Lived C. elegans Mutant"

Delia Garigan, Cynthia Kenyon

[International C. elegans Meeting, 2001]

Our lab screened 20,000 haploid genomes for long- and short-lived mutants and found at least ten short-lived mutants. We would like to know whether these animals are aging more rapidly than normal, or whether they are simply sick and unhealthy due to their mutations. Preliminary characterization of short-lived mutants reveals one candidate that appears healthy in early life but then dies early. We are using high-power microscopy and DNA microarray analysis to determine if this mutant has an accelerated progression of age-related tissue degeneration and biomarkers.

Xu M et al. (2010) Biology of the C. elegans Male, Madison, WI "Digital Alignment of Electron Microscopic Images for Nervous System Reconstruction"

Xu M, Hood G, Bloniarz A, Emmons S, Wetzel A

[Biology of the C. elegans Male, Madison, WI, 2010]

It is important to have electron microscopic images aligned before nervous system reconstruction. Alignment corrects the translation, rotation, magnification and distortion that occur during E.M. acquisition. A well-aligned E.M. dataset facilitates reconstruction using our program ELEGANCE, making it easier to identify neuron profiles and trace their progress through the EM stack. Moreover, alignment smooths the neuron process through sections to get better and more accurate maps. Alignment between high power series and low power series makes it possible to create a global coordinate system for neuron reconstruction across the whole worm. Alignment software created at the Pittsburgh Supercomputing Center is being used in our male nervous system reconstruction project. This software, which utilizes parallel computing algorithms, corrects misalignments including image warping. This program is installed on the Albert Einstein College of Medicine's Rock version computer cluster server with 65 nodes and 520 processors. We are currently using it to montage and align our new EM dataset of the male head, which when completed will consist of tens of thousands electron micrographs necessary to reconstruct the male anterior nervous system across 5000 serial sections. The software can also be applied to our already completed reconstruction of the male posterior nervous system to improve the accuracy of neuron maps.

Santos S I C O et al. (2008) European Worm Meeting "Ultra-fast optics: a novel tool to study C. elegans"

Mathew M, Santos S I C O

[European Worm Meeting, 2008]

In the last decade nonlinear techniques using ultra-fast optics has emerged. as a novel tool for biological investigation. We present the use of these. techniques in studying C. elegans viz. Two Photon Microscopy (TPEFM),. Harmonic Generation Microscopy (SHGM & THGM) and Ultra-fast laser Induced. Nanosurgery.. Nonlinear imaging techniques are superior to commonly used techniques with. respect to penetration depth, photobleaching and signal to noise ratio,. owing to the use of very high intensity low average power ultra-short near. infra red (NIR) pulses.. In the last few years these high intensity NIR pulses have also been used. to precisely target, cut and manipulate sub-cellular structures with sub-. micrometer resolution. This offers the possibility of in vivo surgery. without collateral damage.

Yukinobu Arata et al. (2007) International Worm Meeting "Quantitative measurement of the timing of cell division in C. elegans embryo."

Hitoshi Sawa, Yukinobu ARATA

[International Worm Meeting, 2007]

During animal development, cell cycle progression seems to be regulated tightly. In this study, we quantitatively measured cell volume and cell cycle duration. We found that cell cycle duration was in inverse proportion to the 0.26 (~1/4) th power of cell volume in the AB and MS lineages and the 0.34 (~1/3) th power of cell volume in the C lineage, but was independent of cell volume in the E and D lineages. These findings indicate that cells divide in size-dependent and -independent manners during the development, and suggest that cells possess unidentified mechanisms to regulate cell cycle progression, depending on cell volume. In addition, we also observed a relationship between cell division timing and cell position in the embryo, and showed that most cell divisions occur semi-synchronously throughout the embryo; anteriorly-positioned cells tended to divide slightly earlier than those in the posterior. This phenomenon seems to correspond to the mitotic wave that has been reported in Drosophila</I>, Xenopus</I> and zebrafish development. Is cell division timing affected by cell division in adjacent cells? We changed the relative positions of cells in embryos cultured in vitro</I>. Although most cells divide in the same order as in the embryo in egg shell, the division order of E cell daughters was earlier. This result suggests that cell division timing is affected by the embryonic microenvironment. Given these findings, we propose that cell cycle progression is regulated by size- and position-dependent mechanisms in the C. elegans</I> embryo.

Itskovits, Eyal et al. (2015) International Worm Meeting "A new multi-worm tracker reveals unprecedented insights on worm chemotaxis."

Itskovits, Eyal, Zaslaver, Alon

[International Worm Meeting, 2015]

We have developed a novel Multi Worm Tracker (MWT) that is capable of tracking over a hundred worms simultaneously. The tracker employs a machine learning classification approach to identify the behaving worms. The system produces a long informative track for each individual worm, and generally maintains tracking despite frequent animal collision events. This MWT provides unprecedented statistical power revealing subtle, yet significant, behavioral features. Here we present results that challenge the prevalent "biased random walk" strategy of worms' chemotaxis. Moreover, we readily obtain data with satisfactory statistical significance following a single chemotaxis assay. Our system includes a suite of solutions for acquisition, tracking, and statistical analyses via a friendly user interface that is easy to operate with minimal programming skills.

Evans, K.S. et al. (2017) International Worm Meeting "Leveraging the power of Caenorhabditis elegans to dissect quantitative traits across a diverse collection of drug responses."

Andersen, E.C., Evans, K.S., Cook, D.E., Brady, S.C.

[International Worm Meeting, 2017]

Within natural populations, most phenotypes are determined by the contributions of multiple genetic loci and/or interactions with the environment. Additionally, genetic factors can act independently and/or interact to control trait differences across populations. Studies of yeast and Arabidopsis found that the majority of phenotypic variation is attributable to independent additive quantitative trait loci (QTL). By contrast, studies of Drosophila and humans suggest that most traits are controlled by interacting genetic loci. Here, we leverage the genetic power of a panel of 265 recombinant inbred advanced intercross lines (RIAILs) of Caenorhabditis elegans, derived from the Bristol strain (N2) and a wild isolate from Hawaii (CB4856), to reconcile this debate in a highly powered statistical study. For this panel of strains, we use a high-throughput fitness assay to analyze strain responses to 16 different conditions, including heavy metals, antineoplastic drugs, pesticides, and neuroactive compounds. Using linkage mapping, we identify a diverse set of genomic regions that underlie differences in responses to these compounds. We will present the relative contributions of additive QTL and higher-order genetic interactions across various growth parameters in responses to these 16 drugs. Additionally, we identified four genomic regions that impact responses to multiple classes of compounds. We generated chromosome-substitution strains (CSSs) and near-isogenic lines (NILs) to experimentally validate three of these "QTL hotspots". For many conditions, these strains recapitulate the phenotypic effect predicted using the recombinant inbred lines, validating the power of our efforts. Furthermore, the discovery of these QTL hotspots may be indicative of pleiotropic loci that control responses to multiple conditions.

Chriske, Austin et al. (2017) International Worm Meeting "Analysis of nutritional supplement effects on Caenorhabditis elegans lifespan."

Metzger, Brandon, Crossgrove, Kirsten, Chriske, Austin

[International Worm Meeting, 2017]

The molecular basis for the regulation of aging has been extensively characterized in Caenorhabditis elegans. We are using C. elegans to test the effects of nutritional supplements on lifespan. We have observed lifespan extension with purified grape seed extract and the commercial supplement OPC Synergy registered (Standard Process Inc.) using manual lifespan assays. We are currently using C. elegans mutants to try to isolate the genetic pathways involved in lifespan extension by OPC Synergy. We have also assembled a lifespan machine with six scanners (Stroustrup et al. 2013. Nature Methods, 10: 665-673) and will discuss how it compares to manual assays, both in terms of results and ease of use. Our goal is to use the higher statistical power of lifespan machine experiments to perform high throughput screening of nutritional supplements for efficacy and pathway analysis.

Kim, Do-Young et al. (2015) International Worm Meeting "Label-free imaging of C. elegans muscle structure using sum-frequency generation microcopy."

Kim, Hyunmin, Kim, Do-Young, Hahm, Jeong-Hoon, Kim, Kyuhyung, Moon, Daewon, Joo, Kyung-Il

[International Worm Meeting, 2015]

Label-free optical method provides a high contrast live imaging and non-invasive monitoring of biological processes. Recently, second-harmonic generation (SHG) imaging microscopy based on even-order nonlinear optical process has been developed to visualize skeletal muscle structures in multiple tissues in vivo without fluorescent labeling or immunostaining (Pfeffer et al,. 2011). Sum-frequency generation (SFG) is second order nonlinear optical process and requires the nonlinear optical wavelength up-conversion with two different frequency photons, which is achieved by doubling of single photon frequency in SHG. Thus, the SFG can provide more detailed chemical information such as the molecular vibration and dynamics. Here, we showed that the SFG visualizes the myosin filament structure of C. elegans muscle using a SFG/TPF (two photon fluorescence) dual mode imaging microscope. We further analyzed the observed SFG signal as a function of time overlap, power dependence, and pulse polarization at the body wall or pharyngeal muscle of wild type animals. We next examined previous identified mutant animals such as dim-1(ra102) and unc-89 (e1460) and observed their disorganized muscle structures as previously reported (Timothy et al., 2012). However, while we observed decreased muscle density in unc-89, we did not detect any difference in SFG signals of dim-1 or unc-89 mutants in the extent of the pulse overlapping or changes of power/polarization compared to those of wild type animals, indicating that the chemical property of muscle fibers is not altered in those mutants. Additionally, we compared the SFG signals with GFP-induced TPF signals as function of aging and found that the SFG signals were decreased faster than GFP signals as animals are getting old. Finally, using SFG microscopy, we are now trying to see muscle structures of uncharacterized mutants of which genes are known to be implicated in human muscular dystrophy.