Sandra Encalada et al. (2001) International C. elegans Meeting "or358ts is involved in mitotic spindle orientation and positioning in the one-cell stage Caenorhabditis elegans embryo"

Rebecca Lyczak, Sandra Encalada, Bruce Bowerman

[International C. elegans Meeting, 2001]

The first mitotic division of the 1-cell stage C. elegans embryo is asymmetric, producing a smaller posterior daughter (P1), and a larger anterior daughter (AB), two cells born committed to distinct fates. During this first division, the mitotic spindle aligns along the longer anterior-posterior (a-p) axis of the zygote, and moves posteriorly during anaphase. This posterior displacement results in the first division being unequal. To understand how spindle orientation and positioning are regulated in the early worm embryo, we are characterizing the role or or358 ts, a temperature sensitive maternal-effect embryonic lethal mutant in which spindle positioning and orientation at the first division are defective. or358 ts was identified in a screen for temperature-sensitive embryonic lethal mutations. In this mutant, the first mitotic spindle sometimes fails to rotate fully and therefore does not always align with the a-p axis (its range is between 0-86 degrees relative to the a-p axis). The first spindle of these mutants then either remains transverse, or eventually flips to orient with the a-p axis. If the spindle of or358 ts mutant embryos aligns a-p, it is hyper-displaced posteriorly, resulting in larger than usual AB, and smaller than usual P1 daughter cells. Earlier defects of or358 ts can also be detected. These include reduced pseudocleavage, posteriorly hyper-displaced pro-nuclear meeting position, and failure of pro-nuclei to centrate after meeting. Preliminary data suggest that some or358 ts mutant embryos at the one-, two- and four-cell stages have microtubule nucleation defects, and in some embryonic cells microtubule nucleating centers appear dissociated from the nucleus. Mapping analyses of or358 ts positions it to linkage group (LG) I, near or to the left of lin-17 (-6.4 map units), and we are presently positionally cloning the wild-type gene. Although asymmetric cell divisions occur in a number of developmental contexts, the mechanisms regulating spindle positioning and orientation are not well understood. We think or358 ts should provide insights into the mechanisms that control these processes.

Sandra Encalada et al. (2002) European Worm Meeting "apo-5, a paternal gene involved in mitotic spindle orientation and positioning in the one-cell Caenorhabditis elegans embryo"

Bruce Bowerman, Rebecca Lyczak, Sandra Encalada

[European Worm Meeting, 2002]

During the first mitotic division of the one-cell C. elegans embryo, called P0, the mitotic spindle aligns along the longer anterior-posterior (a-p) axis and moves slightly posteriorly during anaphase, resulting in an asymmetric division that produces a smaller posterior daughter (P1), and a larger anterior daughter (AB). To understand how spindle positioning and orientation are regulated in the early worm embryo, we are characterizing apo-5, a temperature sensitive maternal- and paternal-effect embryonic lethal mutant with defects in the positioning and orientation of the first mitotic spindle along the a-p axis.

Sandra E Encalada et al. (2003) International Worm Meeting "A functional spindle-assembly checkpoint in early C. elegans embryos"

Sandra E Encalada, Rebecca Lyczak, Bruce A Bowerman

[International Worm Meeting, 2003]

The spindle checkpoint acts during mitosis as a control mechanism to oversee the proper segregation of chromosomes. In C. elegans, previous observations indicate that treatment of early embryos with Nocodazole does not arrest mitosis. This has prompted the suggestion that spindle checkpoints do not act in early worm embryos. A spindle checkpoint component mdf-2 (mad2 homologue), has been shown to act in the C. elegans germline1. Furthermore, the MDF-2 protein is localized around DNA and in the central region of the mitotic spindle but its function during embryogenesis is not known1. To investigate the possibility that MDF-2 and other spindle checkpoint homologs influence cell cycle progression in the early embryo, we have examined requirements for these genes in embryos with delayed progression through mitosis. We became interested in cell cycle progression in the early embryo upon discovering that spindle assembly defective mutants exhibit delayed progression through mitosis. For example, we have identified one mutant called apo-5(or358ts) in which the first mitotic spindle is small and mispositioned. By carefully analyzing cell cycle progression using strains that express both histone and tubulin GFP fusion proteins, we have found that progression through mitosis is significantly delayed not only in apo-5(or358ts) mutant embryos but also in zyg-9, another mutant with spindle assembly defects. Strikingly, RNAi-mediated inactivation in apo-5(or358ts) mutants of C. elegans homologs of known components of the spindle-assembly checkpoint called mdf-1, mdf-2 and Cebub-1 (mad1, mad2, and bub1 homologs, respectively), and of mdf-1 in zyg-9 mutants restored more rapid progression through mitosis. Staining of one-cell apo-5 and zyg-9 mutant embryos with an antibody against BUB-1 shows persistence of this kinetochore protein at centrosomes through telophase, while in wildtype embryos BUB-1 is only detectable until late anaphase. These data suggests that the longer mitosis of apo-5 and zyg-9 embryos might depend on the prolongued persistence of BUB-1 during the latter stages of mitosis. Finally, Nocodazole treatment of one- and two-cell wildtype embryos also results in significant delays in the metaphase to anaphase transition, and these delays are rescued by mdf-1(RNAi). We conclude that a functional spindle checkpoint monitors chromosome capture and progression through mitosis in the early C. elegans embryo. 1 Kitagawa and Rose. 1999. Nature Cell Biology 1:514-521

James Carter et al. (2007) International Worm Meeting "Identification of Genes Required for P0 Spindle Alignment."

Encalada Sandra, Aleena Garner, James Carter, Bruce Bowerman

[International Worm Meeting, 2007]

After fertilization of the C. elegans oocyte, the fused maternal and paternal nuclei must rotate 90 degrees in order for the mitotic spindle to align correctly parallel to the anterior-posterior axis of the embryo. If this spindle rotation does not occur, P0 can instead divide perpendicular to the normal axis of division. This results in a failure of the embryo to hatch, or an embryonic lethal phenotype. In order to identify genes that are required for this event, we conducted a screen for temperature-sensitive embryonic lethal mutants. We collected several hundred mutants, and made nomarski (DIC) movies of the first mitotic division of all of the mutant lines. Twenty-one of these had some degree of failure in P0 spindle rotation. We used genetic complimentation and DNA sequencing to determine if any of these had mutations in genes that had previously been shown to be required for spindle rotation. We found seven alleles of zyg-9, (or624, or625, or628, or630, or634, or635, and or637) two alleles of tac-1, (or490 and or602) two alleles of tba-1, (or599 and or629) and one allele of dnc-1(or680) among this group. In addition, we have three mutants that map to chromosome I (or660, or491, and or358) and two alleles that map to Chromosome IV (or618 and or633). These mutants compliment genes known to be required for P0 spindle rotation. They are canditates to interact with the other genes known to be involved in setting up the spindle in the first mitotic division, or they may be new components of the mitotic spindle assembly. We will report on our efforts to further characterize the phenotypes of these mutants, in addition to 3-factor and SNP mapping of the remaining alleles to determine their genetic identity.

Gutierrez, Edgar et al. (2013) International Worm Meeting "Microfluidic devices for longitudinal imaging of gently immobilized worms and live imaging of early embryos during acute drug treatment."

Gutierrez, Edgar, Oegema, Karen, Green, Rebecca, Encalada, Sandra, Groisman, Alex

[International Worm Meeting, 2013]

A major challenge for studying dynamic developmental processes in C. elegans is long-term high-resolution imaging. We have used a micro-machined elastomer chip that is pushed against a cover glass by the application of vacuum to trap worms between the chip and the cover glass. The pushing force is widely adjustable by varying the level of vacuum, enabling a user-defined degree of immobilization for a variety of worm sizes. Loading the device is simple and minimizes the loss of worms. Worms can be easily recovered from the device by separating the elastomer chip from the substrate. A high level of vacuum is applied to completely immobilize worms during short intervals of high-resolution imaging; the vacuum is reduced between the imaging to allow worms to feed and develop. Upon reduction of the vacuum, the worms recover their normal behavior. We applied the device to image the vesicular transport in neurons of adult worms and the developmental time course of the gonad region, using DIC and confocal microscopy. We have performed longitudinal imaging over a 48 hour time interval, with the immobilization remaining fully functional and with worms developing from larvae to young adults. Small molecule inhibitors are a valuable tool for the analysis of fundamental cellular functions and an entry point for the development of therapeutic agents. We identified a gene whose inhibition renders the C. elegans eggshell permeable and built a microdevice for in situ worm dissection and high resolution imaging of embryos. The microdevice is made of hard plastics and has a rectangular well with an array of microwells on the bottom. After the dissection of worms, the fragile embryos are gently swept towards the microwell array, where they are protected from flow by the microwell walls. Permeable embryos are acutely exposed to drugs after the existing medium is aspirated and a medium with the drug is dispensed into the well with a pipette.

KA Swan et al. (1999) International C. elegans Meeting "or191ts and or182ts are Embryonic Lethal, Temperature-Sensitive Mutations that Disrupt Spindle Rotation and Cell Cycle Timing, Respectively, in Two-Cell Stage C. elegans Embryos."

DR Hamill, BA Bowerman, KA Swan

[International C. elegans Meeting, 1999]

In order to understand the processes governing the establishment of asymmetry within the early C. elegans embryo, our lab has begun an investigation of several conditional mutants isolated in a screen for temperature sensetive, embryonic lethal mutations (see abstract by D. Hamill). Two of these mutations, or191ts and or182ts disrupt different aspects of asymmetry normally observed at the two-cell stage. The or191ts mutation results in random spindle orientation within the two-cell stage blastomeres AB and P 1 , which normally divide transversely and longitudinally, respectively. In addition to dividing along different axes, the cell cycle times of AB and P 1 are unequal in wild-type embryos, with AB dividing slightly ahead of P 1 . A second mutation we have identified increases this asymmetry. The mutation or182ts , delays the cell cycle timing of both AB and P 1 . The AB cell cycle time, however, is increased only slightly, while the time of the P 1 cell cycle is doubled. or191ts maps to the center of chromosome V while or182ts maps to the far left arm of chomosome III. Phenotypic analysis of these mutants and progress on identifying the affected genes will be presented. For information concerning the analysis of other mutants affecting similar processes see posters presented by Danielle Hamill and Sandra Encalada in our laboratory.

Greiner, Erin et al. (2015) International Worm Meeting "Modulation of Transthyretin Degradation Alters Proteotoxicity in Caenorhabditis elegans Models of Degenerative Disease."

Encalada, Sandra, Paulsson, Johan, Dillin, Andrew, Greiner, Erin, Genereux, Joseph, Kelly, Jeffery, Porras-Gonzalez, Diana

[International Worm Meeting, 2015]

The transthyretin (TTR) amyloid diseases, including Familial Amyloid Polyneuropathy (FAP), are the most common autosomal-dominantly inherited systemic amyloidoses, wherein mutant TTR leads to peripheral and autonomic nervous system degeneration, cardiac dysfunction, or both. The tetrameric TTR protein is primarily secreted by the liver and undergoes dissociation and partial monomer denaturation, followed by extracellular TTR aggregation onto distal postmitotic tissues such as the neurons and heart. However, the mechanism(s) of this cell non-autonomous TTR proteotoxicity have not been elucidated in any model organism. To dissect the mechanism(s) of TTR secretion, degradation, and age-dependent aggregation on TTR cell non-autonomous proteotoxicity, we generated and characterized C. elegans transgenic lines expressing human WT TTR, or the destabilized TTR variants V30M or D18G expressed in the body wall muscle under the unc-54 promoter. We find that expressing WT TTR and V30M TTR in the muscle significantly reduced the mean lifespan and increased age-dependent paralysis in these strains. To scrutinize TTR toxicity mechanisms, we are using TTR conformational probes (small molecules and antibodies) to monitor the changes in the levels of TTR conformers generated (i.e., tetramer, misfolded oligomers) throughout the lifespan of these strains in order to formulate TTR structure-proteotoxicity relationships. Using a fluorogenic probe that selectively binds and reacts with TTR tetramers, we show that TTR tetramers are secreted from the muscle in WT TTR and V30M TTR strains and are taken up by coelomocytes, six macrophage-like cells in the body cavity where endocytosis and degradation usually takes place. Genetic ablation of coelomocytes appears to increase V30M TTR tetramer levels in the body cavity, suggesting that TTR degradation occurs in these cells. Moreover, coelomocyte ablation significantly exacerbated V30M TTR proteotoxicity resulting in increased sterility and reduced viability, suggesting that modulation of V30M TTR degradation influences its toxicity. Characterization of these models points toward protein degradation as a relevant factor for modulating toxicity in degenerative diseases of postmitotic tissues.

Hermann GJ et al. (2005) Mol Biol Cell "Genetic analysis of lysosomal trafficking in Caenorhabditis elegans."

Grant BD, Rabbitts BM, Fonarev P, Priess JR, Hieb CA, Kershner AM, Schroeder LK, Hermann GJ

[Mol Biol Cell, 2005]

Monitoring Editor: Sandra Schmid The intestinal cells of Caenorhabditis elegans embryos contain prominent, birefringent gut granules that we show are lysosome-related organelles. Gut granules are labeled by lysosomal markers, and their formation is disrupted in embryos depleted of AP-3 subunits, VPS-16, and VPS-41. We define a class of glo mutants (gut granule loss) that are defective in gut granule biogenesis. We show that the glo-1 gene encodes a predicted Rab GTPase that localizes to lysosome-related gut granules in the intestine, and that glo-4 encodes a possible GLO-1 guanine nucleotide exchange factor. These and other glo genes are homologous to genes implicated in the biogenesis of specialized, lysosome-related organelles such as melansomes in mammals and pigment granules in Drosophila. The glo mutants thus provide a simple model system for the analysis of lysosome-related organelle biogenesis in animal cells.

Rose S et al. (2007) Mol Biol Cell "Caenorhabditis elegans intersectin: a synaptic protein regulating neurotransmission."

Di Fiore PP, Rose S, Krag C, Tsushima H, Schultz A, Salcini AE, Malabarba MG

[Mol Biol Cell, 2007]

Monitoring Editor: Sandra Schmid Intersectin is a multifunctional protein that interacts with components of the endocytic and exocytic pathways and is also involved in the control of actin dynamics. Drosophila intersectin is required for viability, synaptic development and synaptic vesicle recycling. Here, we report the characterization of intersectin function in C. elegans. Nematode intersectin (ITSN-1) is expressed in the nervous system, and it is enriched in presynaptic regions. The C. elegans intersectin gene (itsn-1) is nonessential for viability. In addition, itsn-1-null worms do not display any evident phenotype, under physiological conditions. However, they display aldicarb-hypersensitivity, compatible with a negative regulatory role of ITSN-1 on neurotransmission. ITSN-1 physically interacts with dynamin and EHS-1, two proteins involved in synaptic vesicle recycling. We have previously shown that EHS-1 is a positive modulator of synaptic vesicle recycling in the nematode, likely through modulation of dynamin or dynamin-controlled pathways. Here, we show that ITSN-1 and EHS-1 have opposite effects on aldicarb sensitivity, and on dynamin-dependent phenotypes. Thus, the sum of our results identifies dynamin, or a dynamin-controlled pathway, as a potential target for the negative regulatory role of ITSN-1.

Mullen GP et al. (2006) Mol Biol Cell "The Caenorhabditis elegans snf-11 gene encodes a sodium-dependent GABA transporter required for clearance of synaptic GABA."

Quick MW, Moulder G, Rand JB, Fields SD, Saxena P, Mullen GP, McManus JR, Mathews EA, Barstead RJ

[Mol Biol Cell, 2006]

Monitoring Editor: Sandra Schmid Sodium-dependent neurotransmitter transporters participate in the clearance and/or recycling of neurotransmitters from synaptic clefts. The snf-11 gene in C. elegans encodes a protein of high similarity to mammalian GABA transporters (GATs). We show here that snf-11 encodes a functional GABA transporter; SNF-11-mediated GABA transport is Na(+)- and Cl(-)-dependent, has an EC50 of 168 microM, and is blocked by the GAT1 inhibitor SKF89976A. The SNF-11 protein is expressed in seven GABAergic neurons, several additional neurons in the head and retrovesicular ganglion, and three groups of muscle cells. Therefore, all GABAergic synapses are associated with either presynaptic or post-synaptic (or both) expression of SNF-11. Although a snf-11 null mutation has no obvious effects on GABAergic behaviors, it leads to resistance to inhibitors of acetylcholinesterase. In vivo, a snf-11 null mutation blocks GABA uptake in at least a subset of GABAergic cells; in a cell culture system, all GABA uptake is abolished by the snf-11 mutation. We conclude that GABA transport activity is not essential for normal GABAergic function in C. elegans; and the localization of SNF-11 is consistent with a GABA clearance function rather than recycling.