Patterson, Joseph R et al. (2011) International Worm Meeting "RNP granules: insights into their regulation and function."

Wood, Megan P, Patterson, Joseph R, Schisa, Jennifer A

[International Worm Meeting, 2011]

As sperm is depleted in Caenorhabditis elegans hermaphrodites, ovulation arrests and ribonucleoprotein (RNP) granules assemble in the arrested oocytes. RNP granules are also induced in non-arrested oocytes by environmental stresses such as heat shock, osmotic shock, and anoxia. The large RNP granules in oocytes are hypothesized to maintain mRNA stability and prevent precocious translation when fertilization is delayed or a stress is present. The assembly of RNP granules in response to extended meiotic arrest and stress is conserved in a related species, Caenorhabditis remanei. We have focused on two broad questions: 1) how is the assembly of RNP granules regulated? and 2) what is the consequence to an arrested oocyte if RNP granules fail to assemble normally? To gain insights into the mechanism for RNP granule assembly we first performed an ultrastructural analysis of the C. remanei germline. We were very surprised to see dramatic nuclear blebbing along the nuclear envelope of oocytes of unmated and heat stressed females. A combination of TEM, confocal analyses, and live imaging studies indicate the blebs appear to detach from the nucleus and may traffick to the cell cortex and assemble into annulate lamellae, in close proximity to RNP granules. Using RNAi, we further show that several nucleoporins are required for the assembly of RNP granules, and a disruption in RNP granule assembly coupled with a low frequency of nuclear blebbing in arrested oocytes negatively impacts embryonic viability. Our observations support a model where nuclear membrane blebbing is required to increase the trafficking of nucleoporins to the cell cortex in stressed or meiotically-arrested cells and to facilitate the recruitment of RNA and protein components of RNPs into large complexes. To further address the question of the function of RNP granules, we are performing additional fertility tests after preventing normal RNP granule assembly. We are using RNAi to knockdown genes that have recently been identified in our lab as required for normal RNP granule assembly, and performing mating assays to assess any consequences to embryonic viability. We expect these experiments to address part of our hypothesis for the function of RNP granules in meiotically arrested oocytes.

Wood, Megan et al. (2013) International Worm Meeting "Assembly of RNP granules in C. elegans oocytes promotes oocyte quality and is regulated by the cytoskeleton."

Gorman, Kevin, Schisa, Jennifer, Boag, Peter, Severance, Ashley, Davis, Gregory, Patterson, Joseph, Hollis, Angela, Wood, Megan

[International Worm Meeting, 2013]

In many animal species, oocytes arrest in meiosis until they are fertilized. It is well established that fertility diminishes as oocytes age. Our goal is to better understand the regulation and function of large ribonucleoprotein (RNP) granules that assemble in the germ lines of Caenorhabditis nematodes that are either stressed or in which ovulation is arrested. The RNP granules are hypothesized to maintain oocyte quality by regulating mRNA stability or translation in arrested or stressed oocytes (Jud et al., 2008). Their assembly is influenced by nuclear pore proteins, and we have hypothesized that nuclear blebs trafficking from the nuclear envelope to the cortex may promote the formation of the cortical RNP granules (Patterson et al., 2011). We have performed a targeted, functional RNAi screen to identify genes that are required for the assembly of RNP granules in arrested oocytes and identified 143 genes that are necessary. Among the gene classes of our positives are several cytoskeleton proteins including KCA-1 (kinesin cargo adaptor), several beta-tubulins, and WSP-1 (involved in actin polymerization). To gain insight into the mechanism of action of RNP granule regulators we are dissecting defined protein complexes; e.g. we are determining if KLC-1 (kinesin light chain) and UNC-116 (kinesin 1 heavy chain) which function with KCA-1 to position the meiotic spindle (Yang et al., 2005), also contribute to RNP granule assembly. The discovery of these novel regulators of RNP granule assembly allows for direct testing of our hypothesis for their function. When the normal assembly of RNP granules is prevented, we observe fertility is decreased, supporting the hypothesis that RNP granules maintain the quality of oocytes when fertilization is delayed. On-going studies are testing if RNA stability is diminished or translation of maternal mRNAs is de-repressed when RNP granule assembly is defective. These results have provided insight into novel regulators of RNP dynamics that likely apply to RNPs important for fertility and stress responses in many species.

Muneesh Tewari et al. (2001) International C. elegans Meeting "Searching for new proteins involved in dauer formation by two-hybrid protein interaction mapping."

Muneesh Tewari, Patrick Hu, Marc Vidal, Gary Ruvkun, Svetlana Busiguina

[International C. elegans Meeting, 2001]

This project aims to identify new proteins involved in dauer formation by large-scale yeast two-hybrid screens using all the known dauer gene products as baits. Open reading frames encoding each of 34 proteins implicated in dauer formation (daORFs) were cloned into yeast two-hybrid bait (DB-daORFs) and prey (AD-daORFs) vectors by Gateway recombinational cloning. First all pairwise DB-daORF/AD-daORF combinations were tested. This matrix experiment recapitulated an interaction between the DAF-3 (an ortholog of SMAD4) and DAF-5 (a relative of the SNO oncoprotein; Garth Patterson, personal communication) gene products. Individual yeast two-hybrid screens of a C. elegans AD-cDNA library were then carried out with each DB-daORF. This large-scale mapping experiment identified a total of 58 putative interactors. Among others, an interaction between AGE-1 (an ortholog of PI-3-kinase) and the worm ortholog of the p85 adaptor protein was detected. Most of the interactors identified correspond to previously uncharacterized gene products. They will be subjected to functional analysis by systematic RNA-mediated interference to test their involvement in dauer formation.

King KV et al. (1998) Midwest Worm Meeting "DAF-8 is a SMAD protein required in amphid chemosensory neurons to promote growth."

King KV, Riddle DL, Estevez AOZ

[Midwest Worm Meeting, 1998]

Non-dauer development in C. elegans involves activation of a signal transduction pathway analogous to Transforming Growth Factor-beta (TGF-beta) signaling pathways in other organisms. Previous genetic analysis indicates that daf-8 and daf-14 are partially redundant positive regulators of non-dauer development, whereas daf-3 promotes dauer formation. The daf-8 (King et al., unpublished), daf-3 (Patterson et al., 1997) and daf-14 (J. Thomas, personal communication) genes each encode SMAD proteins. SMAD proteins mediate transcriptional regulation of TGF-beta-responsive genes in response to phosphorylation by TGF-beta receptors. Mutations in daf-8 result in constitutive dauer larva formation, and some embryonic and larval lethality. One mutation truncates the protein in the putative DNA binding domain and eight other daf-8 mutations change residues in the putative transcriptional activation domain. Expression of a daf-8::GFP fusion gene is restricted to one pair of amphid chemosensory neurons in the head and is down-regulated in dauer larvae. This implies that dauer formation is sensitive to the levels of daf-8. This limited expression pattern suggests that DAF-8s function in regulating dauer formation may be different from DAF-14.

Teixeira-Castro, A. et al. (2009) International Worm Meeting "Ataxin-3 protein context and cell-specific factors modulate polyQ-mediated neuronal aggregation in a C. elegans model of Machado-Joseph disease."

Morimoto, R., Maciel, P., Teixeira-Castro, A.

[International Worm Meeting, 2009]

Understanding the basis for neuronal subtype-specific protein aggregation is of central importance for several human neurodegenerative diseases, including Machado-Joseph disease. In this study, we developed a novel Ataxin-3 (ATXN3) pathogenesis model in Caenorhabditis elegans and examined the aggregation profile of human ATXN3 by performing FRAP analysis, in live neuronal cells. We found that full-length ATXN3 aggregates only at high Q-length, not found in human patients, whereas C-terminal ATXN3 causes aggregation and neurotoxicity at a threshold length of 75 glutamines. Analysis of specific neurons in C. elegans, reveals that the ventral nerve cord motor neurons are highly affected. Interestingly, certain sensory neurons of the head contain aggregated foci only when the polyQ-stretch is expressed within ATXN3 protein flanking sequences. Moreover, co-expression of full-length human pathological ATXN3 (below aggregation threshold) with an aggregated species capable of initiating the nucleation events, aggravates the aggregation phenotype and new ATXN3-polyQ co-aggregates are formed also in the sensory neurons of these animals, which are not affected when the two species are expressed alone. These results provide direct evidence that protein context and cell-specific factors are major modifiers of polyQ pathogenesis.

McGehee, Annette et al. (2011) International Worm Meeting "The DAF-7/TGF-b signaling pathway regulates the abundance of the glutamate receptor GLR-1 in the ventral nerve cord."

McGehee, Annette, Juo, Peter

[International Worm Meeting, 2011]

The regulation of glutamate receptor (GluR) abundance and localization can be controlled by activity, and has been implicated in learning and memory. Thus, understanding the cell biological mechanisms that regulate GluRs is of significant interest. We use quantitative fluorescence microscopy to study genes and mechanisms involved in regulating the localization and abundance of GFP-tagged GluR GLR-1 (GLR-1::GFP) at synapses. GLR-1 is an AMPA-type GluR that is expressed in ventral nerve cord (VNC) interneurons where it localizes to sensory-interneuron and interneuron-interneuron synapses (1-3). Here we describe a novel role for the DAF-7/TGF-b signaling pathway in regulating the abundance of GLR-1 in the VNC. TGF-b signaling regulates multiple cell biological and developmental processes in many organisms, including several aspects of neuronal development. In C. elegans, the DAF-7/TGF-b pathway has been well studied as a regulator of dauer formation (4). We used temperature-sensitive (ts) mutants to show that multiple components of the DAF-7/TGF-b signaling pathway are involved in regulating GLR-1 levels in the adult VNC. Specifically, we found that the abundance of GLR-1::GFP increases significantly in daf-7 (TGF-b/ligand), daf-1 (type I receptor), daf-8 (Smad), and daf-14 (Smad) ts mutants compared to wild type animals. DAF-3 is a Smad whose transcriptional activity is antagonized by DAF-8 (5). We found that mutation of daf-3 suppresses the increase in GLR-1::GFP observed in daf-8 mutants, suggesting that the effect of DAF-8 on GLR-1 is mediated through DAF-3. The effect on GLR-1::GFP is specific for the DAF-7/TGF-b signaling pathway because animals with mutations in sma-6, a type I receptor that acts in a parallel TGF-b signaling pathway to regulate body size, showed no increase in the abundance of GLR-1::GFP in the VNC. We also found no change in the distribution of a presynaptic marker, GFP-tagged synaptobrevin, in daf-8 or daf-14 mutant animals suggesting that the number of presynaptic inputs is likely not affected by the DAF-7/TGF-b pathway. These results identify a novel function for the DAF-7/TGF-b signaling pathway in regulating the abundance of GLR-1 in the VNC, and suggest that an extracellular factor (TGF-b) is required to maintain GluR levels in the mature nervous system. (1) Hart et al. (1995). Nature 378:82-85. (2) Maricq et al. (1995). Nature 378:78-81. (3) Rongo et al. (1998). Cell 94:751-759. (4) Patterson and Padgett (2000). TIGS 16:27-33. (5) Patterson et al. (1997) Genes & Dev 11:2679-2690.

Tao Liu et al. (2002) East Coast Worm Meeting "Identifying genes regulated by the TGF-beta mediated dauer pathway in Caenorhabditis elegans using cDNA microarrays"

John Wang, Garth I Patterson, Tao Liu, Stuart Kim

[East Coast Worm Meeting, 2002]

Dauer formation in Caenorhabditis elegans is regulated by several environmental stimuli, including food, pheromone and temperature. Three parallel pathways, including a transforming-growth-factor--like pathway, regulate dauer formation. Members of the TGF- superfamily play critical roles in cell growth and differentiation, and in embryogenesis. In C. elegans these pathways are essential regulators of dauer formation, body-size determination, male copulatory structures, and axonal guidance, but the precise pathways remain unclear. Repeat experiments suggest that the dauer TGF- pathway functions in neurons to control the dauer decision (Inove & Thomas (2000) Dev Biol 217: 192). We would like to understand the output of this pathway in neurons, and how the neurons signal other cells in the body to control dauer formation. We used microarray analysis of 17,871 genes to determine changes in gene expression caused by TGF- pathway mutations. In these experiments, 734 genes were repressed, and 499 genes were induced at least 2.145-fold at 99% confidence level. Two hundred eighty-seven genes were regulated more than 4-fold and 54 genes were regulated more than ten-fold. More than 90% of the genes identified in this analysis have not been reported previously as dauer-regulated genes. The identified genes reveal that the TGF- pathway affects many aspects of cell physiology including cell growth, protection, cytoskeletal organization and signaling. Regulated genes include several types of transcription factors, as well as cell surface and cytoplasmic signal transducers. Identified targets possibly linked to TGF-'s effects on cell growth include the large group of ribosomal protein and aromatic amino acid permeases, as well as the translation elongation factor EF-2 protein. The TGF--mediated genes with protective function include genes such as superoxide dismutase and many dauer-induced cytochrome P450's, which may serve to protect the worms from toxin, or to control signaling by modifying steroid hormones. Among the signaling molecules identified as targets, daf-12, one of the key genes in dauer formation signaling pathways, was induced; whereas daf-2, the gene that regulates dauer formation with daf-12, was repressed. Dauer-induced genes and dauer-repressed genes have different expression patterns in a gene expression map having 48 mountains which contain sets of highly correlated genes (See Kim et al., (2001) Science (2001) 2087-2092). We are using RNAi to suppress some of these regulated genes to test their function in dauer formation (see abstract by Pan & Patterson). We are also using computational analysis to identify putative transcriptional regulatory elements in these regulated genes (see abstract by Jani & Patterson). We will discuss what we have learned about dauer formation from our microarray analysis, and our plans to use further experiments to sort direct targets of TGF- signaling from indirect targets.

Ayesha N Shajahan et al. (2001) International C. elegans Meeting "Regulation of endocytosis of albumin in the intestinal epithelial cells of C.elegans by TGFbeta-like signaling"

Ayesha N Shajahan, Shahid S Siddiqui

[International C. elegans Meeting, 2001]

In C. elegans , TGF b -like signaling pathways regulate a range of developmental processes such as dauer formation (Estevez et al., 1993), body size growth and male tail formation (Morita et al., 1999; Suzuki et al., 1999), and axon guidance (Colavita et al., 1998). Recently, Siddiqui et al. have shown that daf-4 (TGF b -like-RII) and daf-1 (TGF b -like-RI) (at 25 o C) mutants show poor uptake of FITC-BSA (fluorescein-5-isothiocyanate isomer- bovine serum albumin) in the intestinal cells when compared to wild-type animals. Our aim is to identify the genes in the TGF b -like pathways that affect endocytosis of albumin in the intestinal epithelial cells. We will examine the uptake of FITC-BSA in the mutants of the TGF b -like pathway in comparison to its uptake in wild type. daf-4::gfp (Patterson et al., 1997) and daf-1::gfp (Gunther et al., 2000) are expressed in the intestinal cells. Expression and regulation of other TGF b -like receptors, Smad proteins and the downstream trancription factors in the TGF b -like signaling pathway will be examined in the intestinal cells of wild type and various daf mutants, in the presence and absence of albumin, by immunofluorescence and immunoprecipitation assays. This approach will determine the role of TGF b -like signaling in endocytosis of albumin, and also uncover any possible cross talk between such pathways in the process.

Andreia Teixeira-Castro et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "In vivo dynamics of human ataxin-3 aggregation and neurotoxicity in C. elegans neuronal cells is modulated by Q-length and age"

Patricia Maciel, Richard Morimoto, Andreia Teixeira-Castro

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Machado-Joseph disease, like other polyglutamine (polyQ) diseases, is a late onset neurological disorder characterized by the appearance of misfolded protein species, aggregates, neuronal dysfunction and cell death. Although the mechanism(s) underlying the formation of ataxin-3 (AT3) neuronal inclusions are poorly understood, it is becoming increasingly evident that proteolysis of full-length AT3 is a biological relevant event in the disease since it occurs and affects aggregation both in vitro and in vivo. In this study, we developed a new model for AT3 pathogenesis in Caenorhabditis elegans, in which we observed that expression of the full-length human pathogenic AT3 alone did not cause aggregation in live neuronal cells. In contrast, expression of a C-terminal fragment of mutant AT3 resulted in protein aggregation, suggesting that the aggregation-prone fragment was behaving as seed capable of initiating the nucleation events. Moreover, we studied the dynamics of the sequestration process of full-length pathogenic and wild-type AT3 into polyQ aggregates and observed that this process occurs in an age-dependent manner and that there is a tight correlation between aggregation and neuronal toxicity onset. We are currently using this model to address the molecular mechanisms of the ageing-dependence of the aggregation and neurological phenotypes, which could provide clues to the late onset of the human disease.

Trombley, Alicia et al. (2015) International Worm Meeting "Membrane Remodeling in the C. elegans Germ Line."

Trombley, Alicia, Schmidt, Kevin, Wood, Megan, Schisa, Jennifer, Davis, Michael

[International Worm Meeting, 2015]

In many animal species, oocytes arrest in meiosis until they are fertilized. As oocytes age, changes occur in their cytoplasm, and fertility diminishes. Our goal is to better understand the regulation and function of ribonucleoprotein (RNP) and membrane remodeling in the oocytes of Caenorhabditis nematodes that are either arrested in meiosis or exposed to stress. The assembly of large RNP granules is hypothesized to maintain oocyte quality by regulating mRNA stability or translation (Jud et al., 2008). Increased blebbing of nuclear membranes also occurs in these oocytes, along with the assembly of annulate lamellae (Patterson et al., 2011). The remodeling of nuclear membranes into blebs in meiotically-arrested or heat-stressed oocytes correlates with the remodeling of RNPs and the formation of annulate lamellae at the oocyte cortex. Our current experiments are leveraging the discovery of large stacks of annulate lamellae in cgh-1 and ifet-1 oocytes. We are assessing the extent of nuclear blebbing in these, and related CGH-1 complex mutants, and so far have discovered increased blebbing in a subset of the mutants. We are also using TEM and the SP12::GFP strain to analyze membranes at the oocyte cortex and throughout the germ line. In addition to stacks of aligned membranes visualized by TEM in the mutant germ lines, we also observe large patches of ER membrane in oocytes and in the distal core. In combination with analyses of RNP granules, our results will address the hypothesis that nuclear blebbing initiates the formation of AL at the oocyte cortex, which in turn promotes the assembly of RNP granules.