Amir, Wafa et al. (2013) International Worm Meeting "Imaging the brain."

Kerr, Rex A., Swierczek, Nicholas, Amir, Wafa

[International Worm Meeting, 2013]

By coupling light-sheet microscopy with nuclear-localized GCaMP6 and nls-RFP for a reference channel, we can image essentially the entire brain at rates of up to 10 Hz. We find that the brain appears relatively quiet in animals that are not moving, and that only a small number of neurons show strong activity correlated with movement. However, without a robust method to identify neurons, the technique does not definitively determine which neurons are active. Nor do we know for certain which neurons are truly uninvolved and which merely have low activity levels or small calcium transients, though nls-GCaMP6 does robustly report calcium transients in gentle touch neurons. We are attempting both to use restricted expression patterns to simplify the identification problem, and to improve automated (or manual!) methods of identification, and will report on our progress in this regard.

Sapir, Amir et al. (2009) International Worm Meeting "Toward characterization of bacteria-to-nematodes HGT."

Sternberg, Paul, Sapir, Amir, Zaslaver, Alon, DeModena, John

[International Worm Meeting, 2009]

Horizontal gene transfer (HTG) is a fundamental process among unicellular organisms for acquiring new traits. Although initially thought to be extremely rare in metazoans, recent whole-genome sequencing projects reveal extensive gene transfer from prokaryotes to metazoans. This type of gene transfer is particularly relevant for symbiotic organisms that occupy new niches, where survival requires acquisition of new genes not previously present in the organism''s gene pool. For example, hemi-cellulose hydrolysis, induced by plant parasitic nematodes, is thought to have been acquired by the transfer of bacterial genes to the plant parasites'' bacteriovorous ancestors. In contrast to HGT between bacteria, the sequence of events leading to bacteria-to-nematodes HGT, as well as the molecular details of this process, remain elusive. So far, mechanistic studies of HGT in metazoans have been hindered by its rare occurrence, and the fact that symbiotic organisms are usually not suitable for long in-lab evolutionary studies. Our aim is to study HGT by combining the powerful genetics of E. coli (the donor) and C. elegans (the recipient). Specifically, we use the transfer of the unc-119 rescuing gene from E. coli to unc-119 mutant worms as an indicator for successful HGT. Rescued worms are examined to validate that gene transfer indeed happened, and these worms will be further analyzed to decipher the exact mechanism by which HGT occurred. We predict that the problem of identifying rare HGT events can be overcome by our experimental settings that involve growing multiple generations of worms in large numbers under specific selection. In a pilot experiment, we grew 4x106 worms per generation over 7 generations on E. coli carrying the unc-119 rescuing gene and validated that this approach is suitable for HGT studies. If HGT will not be identified, we will employ different conditions and genetic backgrounds that might increase HGT probabilities: i) Use of C. elegans mutants promoting bacterial propagation in the worm''s gut. ii) Exposing the cultured worms to various stress conditions. iii) Inducing HGT by unc-119 gene transposition in the E. coli donor. Ultimately, this system will serve as an empirical framework to elucidate the enigmatic process of bacteria-to-nematodes HGT.

Amir, Wafa et al. (2009) International Worm Meeting "Whole brain calcium imaging with plane illumination."

Amir, Wafa, Swierczek, Nicholas, Kerr, Rex A.

[International Worm Meeting, 2009]

Calcium imaging provides a non-invasive means of monitoring the activity of individual neurons in the worm. Ideally, hypotheses about the function of neural circuits could be verified by simultaneous observation of the activity of the neurons involved in the circuit. However, moving beyond single-neuron imaging has been challenging. The primary source of the difficulty is out-of-focus fluorescence; this is doubly-bad, as neurons out of the focal plane wash out the neurons of interest in the focal plane, and the out-of-focus calcium indicator molecules still photobleach as badly as always despite providing no useful information. To solve this problem, we are using plane illumination: the light source for fluorescent imaging is a laser brought in from the side and focused down to a sheet that is coincident with the focal plane of the microscope. This provides optical sectioning to approximately 6 microns, a 5-fold improvement over illuminating the entire nerve ring. We have built a microscope for plane illumination in C. elegans (PICE). This system couples the laser input to the objective through a bracket, and includes a piezo collar for rapid z-focusing. Using high speed EMCCD cameras, we can capture the nerve ring at a rate of ten volumes per second. We have demonstrated that the PICE system allows up to about two dozen neurons to be distinguished; full neuronal labeling still gives images which are difficult to analyze. We also show that the PICE method results in improved image quality and decreased photobleaching when performing volume scans as compared to traditional epi-illumination, and has minimal drawbacks when used for single-plane calcium imaging. The PICE system was originally designed for imaging where worms are glued on top of an agarose pad. Microfluidic devices have been used to provide greatly enhanced control of the worm''s spatial and chemical environment, but forming a light sheet inside such a device is nontrivial. We have devised several strategies to couple PICE with microfluidic devices and will describe the advantages of each and our progress towards implementing them.

Sapir, Amir (2015) International Worm Meeting "A novel circuit coordinating mevalonate pathway metabolism with mitochondrial stress ."

Sapir, Amir

[International Worm Meeting, 2015]

The mevalonate pathway catalyzes the synthesis of various metabolites fundamental for human health and disease including cholesterol and molecules essential for protein prenylation. Little is known, however, about regulation of the pathway beyond the well-characterized control of HMGR, the second enzyme of the pathway. We discovered an evolutionary conserved circuit that governs the activity of C. elegans HMGS-1, the first enzyme of the pathway, by ubiquitination and age-dependent sumoylation. Sumoylation is reversed by the spatiotemporal activity of a SUMO protease, ULP-4, that undergoes an intriguing cytosol-to-mitochondria translocation. Recently, I have found that this circuit is regulated by a mitochondrial stress response that induces the expression of both HMGS-1, and its SUMO protease ULP-4. The mitochondrial stress-activated transcription factor ATFS-1 governs HMGS-1 expression upon mitochondrial stress but, surprisingly, does not trigger ULP-4 expression. This discovery shows that a previously uncharacterized, ATFS-1-independent, mitochondrial stress pathway controls ULP-4 expression in order to regulate HMGS-1 activity and mevalonate pathway metabolism. My work bridges a conceptual gap between mitochondria function, mitochondria-related intercellular signaling pathways, and the regulation of the mevalonate pathway. Molecular understanding of this crosstalk has the potential for broad medical implications as mevalonate pathway dysregulation plays an important role in impaired cholesterol homeostasis and in tumorigenesis. .

Szitenberg, Amir et al. (2014) Evolutionary Biology of Caenorhabditis and Other Nematodes "THE EVOLUTION OF TYROSINE-RECOMBINASE ELEMENTS IN NEMATODA"

Blaxter, Mark L, Szitenberg, Amir, Lunt, David H, Koutsovoulos, Georgios

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2014]

Transposable elements can be categorised into DNA and RNA elements based on their mechanism of transposition. Tyrosine recombinase elements (YREs) are relatively rare and poorly understood, despite sharing characteristics with both DNA and RNA elements. Previously, the Nematoda have been reported to have a substantially different diversity of YREs compared to other animal phyla: the Dirs1-like YRE retrotransposon was encountered in most animal phyla but not in Nematoda, and a unique Pat1-like YRE retrotransposon has only been recorded from Nematoda. We explored the diversity of YREs in Nematoda by sampling broadly across the phylum and including 34 genomes representing the three classes within Nematoda. We developed a method to isolate and classify YREs based on both feature organization and phylogenetic relationships in an open and reproducible workflow. We also ensured that our phylogenetic approach to YRE classification identified truncated and degenerate elements, informatively increasing the number of elements sampled. We identified Dirs1-like elements (thought to be absent from Nematoda) in the nematode classes Enoplia and Dorylaimia indicating that nematode model species do not adequately represent the diversity of transposable elements in the phylum. Nematode Pat1- like elements were found to be a derived form of another PAT element that is present more widely in animals. Several sequence features used widely for the classification of YREs were found to be homoplasious, highlighting the need for a phylogenetically-based classification scheme. Nematode model species do not represent the diversity of transposable elements in the phylum.

Sapir, Amir et al. (2009) Worm Breeder's Gazette "Studying mechanisms of bacteria-to-metazoans horizontal gene transfer"

Zaslaver, Alon, Sternberg, Paul, DeModena, John, Sapir, Amir

[Worm Breeder's Gazette, 2009]

Amir Sapir et al. (2007) International Worm Meeting "AFF-1, a Novel FOS-1-regulated Fusogen Mediates Anchor-Cell fusion."

Anna P. Newman, Leonid V. Chernomordik, Benjamin Podbilewicz, Amir Sapir, Jaebok Choi, Clari Valansi, Evgenia Leikina, Ori Avinoam

[International Worm Meeting, 2007]

Cell fusion is required for fundamental biological processes like reproduction, organ formation, viral pathogenesis, and tumor progression. Little is known about the proteins that mediate cell fusion and the molecular mechanisms by which these fusogens exert their activity. Fusion between most C. elegans epithelial cells is mediated by EFF-1, the first developmental fusogen identified in metazoans. Fusion between the anchor-cell (AC) and the utse syncytium that establishes a continuous uterine-vulval tube proceeds normally in eff-1 mutants. This fusion, therefore, is facilitated by an additional unidentified fusogen. Using a forward genetic screen, we isolated mutations in the aff-1 gene in which the AC differentiates normally but fails to fuse with the utse cells. AFF-1 ectopic expression results in fusion of cells that normally do not fuse indicating that AFF-1 is sufficient to fuse cells. The fusogenic activity of AFF-1 was further confirmed by its ability to fuse heterologous Sf9 insect cells. AFF-1 and EFF-1 differ in their fusogenic activity and expression patterns but share eight conserved predicted disulfide bonds in their ectodomains including a putative TGF-beta-type-I-Receptor domain. Identification of this conserved domain is the first step in deciphering the molecular mechanism of developmental cell fusion. In addition we found that FOS-1, the Fos transcription factor ortholog that controls anchor-cell invasion during nematode development, is a specific activator of aff-1-mediated anchor-cell fusion. Thus, FOS-1 links cell invasion and fusion in a novel developmental cascade.

Amir Sapir et al. (2008) Development & Evolution Meeting "EFF-1 and AFF-1, Founders of a Family of Cell-Cell Fusion Proteins in Nematodes"

Meital Oren-Suissa, Amir Sapir, Ori Avinoam, Clari Valansi, Benjamin Podbilewicz

[Development & Evolution Meeting, 2008]

Cell fusion is essential for sexual reproduction and organ development. Yet, despite many years of research, little is known about the mechanism of cell-cell fusion. Our molecular genetic studies of developmental cell fusion in C. elegans have led to the discovery of the first family of proteins that mediate cell fusion (the FF family of fusogens). These fusogens, EFF-1 and AFF-1, are type I membrane proteins that are essential for cell fusion and can fuse cells when ectopically expressed on the membranes of C. elegans and heterologous insect or mammalian cells. EFF-1 is a developmental fusogen that drives most epithelial fusion events in C. elegans. AFF-1 is responsible for the last step in the life of the anchor-cell: its fusion to the utse syncytium forming the worm hymen. AFF-1 is necessary to fuse the anchor-cell, hyp5, a few pharyngeal muscles, the vulA and vulD rings of the vulva, and the lateral seam cells. We suggest that there is a structural domain shared between AFF-1, EFF-1 and TGF-beta-type-I-receptors. Furthermore, FOS-1 controls AFF-1-mediated developmental anchor-cell fusion. We will discuss how the FF proteins fuse cells via an universal intermediate in which the outer leaflets of the plasma membranes merge before the inner leaflets (hemifusion). The FF proteins are the first genuine cell fusogens because they are: (1) Essential for mesodermal and ectodermal cell-cell fusion. (2) Expressed on the surface of both fusing cells at the time of cell fusion. (3) Sufficient to fuse cells that normally do not fuse in vivo and in tissue culture cells. Understanding the mechanism of cell fusion mediated by EFF-1 and AFF-1 may facilitate the identification of other fusogens required for fertilization and somatic fusion in different organisms.

Amir Sapir et al. (2005) International Worm Meeting "Fuse to reproduce: exploring putative roles for EFF-1 in the reproductive system"

Gidi Shemer, Nirit Assaf, Benjamin Podbilewicz, Amir Sapir

[International Worm Meeting, 2005]

Fusion of cells as a way to mix their internal content is used during development at different stages and tissues of multicellular animals. Fundamental biological processes like fertilization and muscle formation are dependant on fusion events that are tightly regulated spatially and temporally. While detailed description of the morphological changes of cells undergoing fusion was established, the molecular mechanism that mediates this process remains elusive. Recently, the necessary and sufficient role of EFF-1 (Epithelial Fusion Failure-1) as mediator of epidermal cell fusion in C. elegans was demonstrated. In addition to epidermal fusion, fusion events in other tissues are required for normal development. In the reproductive system sperm-egg fusion as well as fusion of somatic uterine and vulval cells is necessary for successful reproduction. This study examines the possible role of EFF-1 as a mediator of cell fusion events in the reproductive system. In order to do so, the morphology and function of the reproductive system has been analyzed in eff-1 mutant worms. Morphological analysis revealed that lack of eff-1 leads to abnormal development of the gonad. Specifically, we detected abnormal gonad migration concomitant with germ line syncytium mislocalization to the proximal side of the gonad arm. In addition cell fusion failure has been detected in toroidal cells of the vulva and uterus but not between the AC and the uterine seam cells. Sterility assays in different alleles of eff-1 revealed that between 10-80% of eff-1 hermaphrodites are sterile. Analyzing an allelic series of eff-1 revealed that there is positive correlation between allelic strength and the percentage of sterility. Crossing eff-1 mutant hermaphrodites with wild-type males resulted in partial rescue of their sterility, leading to the development of outcross and self progeny. The ability of wild type sperm to induce fertilization between eff-1 mutant gametes hints for a nonautonomous function of EFF-1 in the germ line which is essential for normal fertilization. Detailed characterization of eff-1 mutant effects in the reproductive system can shed light on the importance of fusion events for successful reproduction. We are currently trying to discriminate between the somatic and germ line roles of EFF-1 affecting reproduction.

Amir W et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Optimizing multi-neuronal calcium imaging"

Kerr R A, Amir W

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

We are imaging calcium indicators in multiple neurons simultaneously by illuminating with a light sheet (selective plane illumination microscopy) and recording with fast EM-CCD cameras while scanning through the worm's brain. This approach allows us to record from sparse subsets of neurons (~10-20) in the head for up to several minutes at 5-10 Hz. However, these imaging conditions pose challenges that are less acute in single-neuron, single-plane imaging: increased photobleaching, increased sensitivity to motion artifacts, a range of expression levels, and conditions that are less extensively optimized for each neuron. Thus, we have been evaluating calcium indicators and analysis strategies to obtain high-quality calcium imaging data from multiple neurons. Our progress in this endeavor, and perhaps some interesting neural activity, will be described.