EM Lapoczka et al. (1999) International C. elegans Meeting "Altered Response Of Dauer Mutants To Sodium Azide Exposure And The Acquisition Of Thermotolerance."

EM Lapoczka, GE White, KE Stine

[International C. elegans Meeting, 1999]

We have demonstrated that exposure to low levels of sodium azide confers thermotolerance in C. elegans by a mechanism similar to the heat shock response (see Massie et al., this meeting). As a part of that study, we found that daf-21 (hsp90), a constitutive dauer forming mutant, acquired sodium azide induced thermotolerance when grown at the permissive temperature, but not at the restricted temperature, implying an important role for hsp90 in the worm's response to stress. Since the dauer state represents a significant response to external stresses, we wanted to determine whether those results reflected the importance of hsp90 in the response to stress, or was this a general characteristic of all dauers. We tested four different dauer mutants: daf-2 (m41, an insulin/IGF-I receptor), daf-7 (m62, a member of the TGF-b superfamily), daf-14 (m77, encodes dwarfin/MAD/DPC-4), and daf-21 (p673, hsp90). We demonstrated the acquisition of sodium azide induced thermolerance in all mutants tested at the permissive temperature; however, our initial studies failed to demonstrate the acquisition of thermotolerance in any mutants grown at the restricted temperature. We noted that dauers took longer to anesthetize with sodium azide (15-30 mins vs. <5 for controls); therefore, we increased the exposure from 1 to 2 hours. In addition, we lengthened the post heat shock recovery. This allowed us to demonstrate azide induced thermotolerance in all of these daf mutants, including daf-21. These results demonstrate that it is not the dauer state alone which would prevent a worm from obtaining sodium azide induced thermotolerance, and that hsp90 may not be a critical part of the worm's response to stress.

Kniss SL et al. (1998) Midwest Worm Meeting "A genetic analysis of C. elegans elongation using sma-1 suppressors"

Austin JA, Kniss SL

[Midwest Worm Meeting, 1998]

During morphogenesis, the <EM>C. elegans</EM> embryo elongates four-fold from a ball of cells into a long, thin worm. This elongation is dependent on circumferentially oriented actin bundles which form in the embryonic epidermal cells and are thought to generate the contractile force required for elongation. SMA-1 is a cytoskeletal protein which is homologous to <EM>Drosophila</EM> Beta-H-spectrin and is required for normal elongation: in <EM>sma-1</EM> mutants, the rate of elongation is decreased resulting in larvae that are shorter than wild-type at hatching. In many of the <EM>sma-1</EM> mutants it is been found that the organization of the epidermal actin bundles is altered (V. Praitis, personal communication). The <EM>sma-1</EM> alleles isolated in our lab vary in their effect on the rate of elongation suggesting that, in addition to its likely structural function, SMA-1 plays a role in regulation of elongation. To understand these functions of SMA-1, we have begun to isolate suppressors of <EM>sma-1</EM> mutants. We have designed a simple genetic screen that takes advantage of the inability of <EM>sma-1</EM> larvae to chemotax to food and have used this screen to isolate both dominant and recessive suppressors. We are currently examining the suppressed phenotypes and mapping the mutations. Characterization of our <EM>sma-1</EM> suppressors will begin to answer questions regarding the role of the cytoskeleton during morphogenesis and the function of SMA-1 in the regulation of elongation. In addition, we hope to understand the relationship between SMA-1's role in body elongation and its role in morphogenesis of the pharynx and excretory cell canal.

Jiang, Hongbing et al. (2017) International Worm Meeting "Identification of an evolutionarily conserved pathway essential for virus infection using Orsay virus infection of C. elegans."

Sandoval, Luis, Leung, Christian, Wang, David, Chen, Kevin, Jiang, Hongbing

[International Worm Meeting, 2017]

Many fundamental biological discoveries have been made in <em abp="1258">C. elegans</em>. The discovery of Orsay virus has enabled studies of host-virus interactions in this model organism. To identify host factors critical for virus infection, we designed a forward genetic screen that utilizes a virally induced GFP reporter. Following chemical mutagenesis, two <em abp="1259">viro</em> (virus induced reporter off) mutants that failed to express GFP were mapped to <em abp="1260">sid-3</em>, a non-receptor tyrosine kinase, and <em abp="1261">B0280.13</em>, an orthologue of human WASP (Wiskott-Aldrich Syndrome Protein). Both mutants yielded 5-6 logs lower Orsay RNA levels than the parent strain, which approximates residual input Orsay levels, demonstrating the essential roles of these genes. However, Orsay virus replication initiated from an endogenous transgene bypasses the requirements for both SID-3 and B0280.13 suggesting that these two genes function at an early step in the virus lifecycle. Strikingly, the human ortholog of SID-3, activated CDC42 associated kinase (ACK1/TNK2), is capable of phosphorylating human WASP, suggesting that B0280.13 may be a substrate for SID-3 in <em abp="1262">C. elegans</em>. In support of this model, fluorescently tagged SID-3 and B0280.13 co-localized to the apical membrane of C. elegans intestinal cells. Further genetic ablation of TNK2 through CRISPR-Cas9 in two human cell lines, A549 (an adenocarcinomic human alveolar basal epithelial cell line) and Hap1 (a near haploid human cell line derived from myeloid leukemia patients), showed reduced infection by polio virus. Thus, an unbiased genetic screen in <em abp="1263">C. elegans</em> identified critical roles in virus infection for evolutionarily conserved genes in a known human pathway.

Christian Stigloher et al. (2009) European Worm Neurobiology Meeting "EM-TOMOGRAPHIC RECONSTRUCTION OF SYNAPTIC ULTRASTRUCTURE"

Christian Stigloher, Douglas Holmyard, Jean-Louis BESSEREAU, Mei ZHEN

[European Worm Neurobiology Meeting, 2009]

The specific functionality of a synapse is crucially linked to the spatial organization of its ultrastructural components. To perform electron microscopy (EM) on synapses in a close-to-native status, we froze living C. elegans under high pressure, which results in instant immobilization of the synaptic structures in amorphic ice. After processing, samples can be observed by classical imaging or used for immuno-EM analyses. We have previously shown that UNC-10/Rim is localized at the active zone, next to the dense projections which are the organizing centers of presynaptic specializations at C. elegans synapses. Genetic disruption of unc-10 causes a redistribution of membrane-contacting vesicles out of the active zone (Weimer et al., 2006). Our spatial analysis of the effect of loss of UNC-10/Rim function has up to now been hampered by the low resolution of classical EM imaging in the z-axis. In order to produce a seamless 3D model of synaptic structures, we are adapting EM-tomography techniques for the use in the C. elegans model. EM-tomography yields high resolution three dimensional ultrastructural images in samples with up to several hundred nm thickness. Specifically, we can visualize structures emanating from dense projections and contacting synaptic vesicles and filamentous connections between synaptic vesicles. With the resulting high resolution 3D reconstructions of synaptic ultrastructure and processing of the resulting datasets with image segmentation software, we will be able to compare wild-type and mutant organization in a comprehensive manner. In particular, this might provide a better insight into the function of UNC-10/Rim. Moreover, our approach can easily be applied to the study of other candidate synaptic proteins such as SYD-2.

Santella, Anthony et al. (2021) International Worm Meeting "An Electron Microscopy Pseudo Time Series of the C. elegans Embryo"

Bao, Zhirong, Kolotuev, Irina, Santella, Anthony

[International Worm Meeting, 2021]

Advances in Electron Microscopy (EM) bring about the possibility of temporal analysis using staged samples over time as well as offering opportunities to gain insights by synthesizing EM with other imaging modalities. We present a pseudo time series of C. elegans embryonic development, four volumes covering around 2 hours particularly rich in development between 320 and 475 minutes post first cleavage. This period encompasses neurulation, neural organogenesis and neuropil formation key events that build most of the major structures of the nervous system as well as critical events for many other organ systems. We correlate this EM data with florescence data spanning the first eight hours of embryogenesis. Every cell in the florescence data is identifiable via lineaging. To correlate these data sets we develop a novel computational method for alignment of identities between data sets in the challenging presence of spatial and temporal variation. This approach involves co-optimization of spatial alignment and the structure of labeled data based on a model of dynamic anatomy in the form of an adjacency graph with expected variation. This model captures both variable elements and consistent spatial proximity relationships. We identify every cell in three of the four time points. Identity results are accurate, ranging from 72 to 78 percent correct when assessed against a large set of manual annotations based on position and morphology. This is better than any previously reported results for identifying all cells in an organism based on position alone. The resulting single cell level annotation allows efficient navigation of this large EM data set. We use the sequence to probe the interactions over time between different components within the nerve ring elucidating the relationship between the temporal and spatial location of initial outgrowths into the ring and ultimate structure. We also examine the interaction between cells during the formation of the amphid dendrite structure providing insight into the timing and succession of events at the inter and intra cellular level. Our observations only scratch the surface of the details available in the data set. We will provide the EM data with single-cell level annotation as a resource for the community.

Hall DH et al. (1995) International C. elegans Meeting "ULTRASTRUCTURE OF CELL DEATH IN MEC-4 & DEG-1"

Gu GQ, Gong L, Hall DH, Driscoll MA, Chalfie M, Garcia-Anoveros J

[International C. elegans Meeting, 1995]

Dying cells have been examined by EM to determine the cellular pathology caused by dominant toxic alleles of two "degenerin" genes, mec-4 and deg-1. These genes encode ion channel subunits that are similar to amiloride-sensitive Na+ channels in vertebrates. Equivalent change in a single amino acid in either subunit causes cell death with a similar morphology: cells swell and vacuolate. The cause of death is unknown, but aberrant channel gating may cause osmotic imbalance. The first indication of damage seen by EM in DIC-staged L1 larvae is infolding of the plasma membrane. Later, cytoplasmic vacuoles form and the cell swells. More slowly, the nucleus invaginates and chromatin aggregates. In distinction to programmed cell death, these degenerating cells are not quickly engulfed by a neighbor, but can linger as swollen ghosts after degradation of cytoplasmic components. In a mec-4(d) transgenic strain carrying many copies of the mutant gene, a low level of membrane defects were seen by EM in non-touch cells and hypodermis, but no extra deaths were observed. In deg-1(d) u38, similar defects were noted in the tail in several muscle cells. This muscle phenotype is consistent with the pattern of deg-1 expression which includes muscle expression. Thus even low levels of gene expression may result in local cell pathology.

Stephen D Fields et al. (2003) International Worm Meeting "Identification and characterization of functional synapses in cultured C. elegans neurons."

Jim Rand, Kiely M Grundahl, Stephen D Fields

[International Worm Meeting, 2003]

Establishing C. elegans neurons in primary culture has been refined to the point that tissue culture now provides one more option for studying cellular processses. There is not solid physiological or structural evidence, however, verifying that functional synapses are actually formed in culture. We use several approaches in this study to confirm the presence of functioning synapses. Cells were isolated (following the method of Christensen et al., 2002) from several worm strains expressing neuron-specific GFP. For electron microscopy (EM), cultures enriched for neurons were obtained by fluorescence-activated cell sorting of cells from a strain with an unc-41-promoter driven GFP that is expressed in early embryos. For fluorescence studies, cells were cultured from a strain expressing a GFP-tagged synaptogyrin (SNG-1) fusion protein (Nonet, 1999). Cultured, fluorescent cells from these strains exhibited one to several processes with periodic varicosities along the length of the process. SNG-1::GFP was expressed in a punctate pattern corresponding to the varicosities in these processes. Immunofluorescence staining of neurons from the SNG-1::GFP reporter strain shows that synaptic proteins such as UNC-41, UNC-13, CHA-1 and SNT-1 as well as the vesicular proteins VMAT and UNC-17 all colocalize with SNG-1::GFP. FM4-64, a styryl dye that fluoresces upon binding to membranes, is also endocytosed and then exocytosed in a Ca2+-dependent manner at these GFP-expressing varicosities, although it appears that not all processes actively take up the FM4-64. In addition, transmission EM reveals that cationized ferritin is also taken up at neuronal varicosities and that structures that appear to be active zones are present near clusters of synaptic vesicles. Taken together, these results indicate that the varicosities of cultured neurons seem to have all the machinery present to form synapses and that these synapses actually function in at least a rudimentary fashion. Scanning EM of neuronal processes is currently being used to reveal the cytoskeletal organization of potential active zones in "opened" varicosities. We are also beginning to employ the technique of immunogold labelling EM of neurons in culture, since chemical fixations that are conducive to immunolabelling of whole worms usually yield poor ultrastructure of neurons.

Irene Wacker et al. (2005) International Worm Meeting "Exploring axon guidance mutants at the nanostructural level by serial block-face scanning electron microscopy"

Irene Wacker, Harald Hutter, Winfried Denk

[International Worm Meeting, 2005]

To address certain questions of neuronal development, in particular the process of neuronal circuit formation, spatial resolution at the EM level is essential. Almost 30 years ago this was demonstrated in "The Mind of the Worm" (White et al. 1986)1, an electron microscopical (EM) reconstruction of the nervous system of C. elegans based on ultrathin serial sections. This work provided the reference for C. elegans neuroanatomy down to the level of individual synaptic connections. Since then very few 3D reconstructions of worms have been published, obviously due to the immense effort in time and labor it takes to accomplish such a task. A recent development at our institute offers a novel solution to the problem of reliable large scale serial sectioning for electron microscopy. A scanning electron microscope was equipped with an ultramicrotome inside the vacuum chamber, which allows to image the block-face of a sample (Denk and Horstmann 2004)2. Several hundred sections of a sample can be produced and imaged reliably avoiding tedious collection of serial sections by hand and simplifying alignment of sections. We are testing whether this machine would make EM analysis of some of our axon guidance mutants feasible, in particular addressing questions of synapse formation in mutants with axonal outgrowth defects. Block-face imaging precludes poststaining of sections and we have to find a way to introduce sufficient contrast during fixation. Currently we are optimizing fixation protocols on mixed populations of worms using high pressure freezing and freeze substitution. We will present first data stacks and discuss the potential use of the method.1 White et al. (1986) Phil. Trans. R. Soc. Lond. B314, 1-3402 Denk W, Horstmann, H (2004) PLoS Biol 2 (11): e329

Jane SD et al. (1997) International C. elegans Meeting "OPTICAL IMAGING OF VIABLE, IDENTIFIABLE NEURONES IN THE ANTERIOR NERVE RING OF CAENORHABDITIS ELEGANS."

Franks CJ, Holden-Dye L, Jane SD, Chad JE, Walker RJ, Wallach LP

[International C. elegans Meeting, 1997]

Here we demonstrate the feasibility of using confocal calcium imaging in identifiable neurones as a refined phenotypic analysis for C.elegans. Experiments were performed on the anterior region of a transgenic strain of C.elegans expressing green fluorescent protein (GFP) in cholinergic neurones. Nematodes were transected at the level of the pharyngeal-intestinal valve. The semi-intact preparation was placed in a chamber on a confocal microscope stage containing modified Dent's saline. In the first series of experiments, dual confocal imaging of GFP (ex.488;em.>520>560) and propidium iodide (ex.568; em. >585) was used to assess the viability of the neurones. GFP fluorescence was observed in neurones within the nerve ring. There was no co-localisation of labelling following incubation with the membrane impermeant nucleic acid intercalalting dye, propidium iodide (15 !M). Subsequent addition of 1% TritonX-100 resulted in a loss of the GFP signal from neurones and a concomitant appearance of propidium iodide staining. Further confirmation of neuronal viability was obtained in a second series of experiments by loading with the acetoxy-methyl ester of calcium orange (10 !M). Differential fluorescence loading was observed in the nerve ring (ex.488;em>585) which was co-localised with GFP fluorescence, implying neuronal distribution. Following wash-off of the loading solution, the calcium orange fluorescence was stable for up to 30 mins, and addition of the calcium mobilising drug ryanodine (10 !M) produced an increased fluorescence within neurones (n=3 preparations; 60% increase in fluorescence). Addition of ionomycin (5 !M) elicited a further increase in pixel intensity indicating that the dye (Kd185nM) was not fully saturated even in the presence of ryanodine. From these data, we infer that in this semi-intact preparation, the neurones can be visualised, have intact cell membranes and maintain Ca++ homeostasis. We thank James Rand and Dennis Frisby for the gift of unc-17:GFP transgenic C.elegans

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.