Chiaki Fujitake et al. (2005) International Worm Meeting "Enzymological and Functional Analysis of Ubiquitin C-terminal Hydrolases"

Kenji Takahashi, Yoshie Watanabe, Hideshi Inoue, Chiaki Fujitake

[International Worm Meeting, 2005]

Ubiquitin C-terminal hydrolases (UCHs) contribute to the ubiquitin (Ub) system by processing Ub precursors or by removing Ub from ubiquitinated peptides or small C-terminal Ub adducts, and are thought to play a central role for a number of cellular processes including cytoplasmic protein degradation, endocytosis, etc. However, the functions of UCHs are still uncertain. C. elegans has four genes coding for UCH; ubh-1~4. The ubh-1, ubh-2, and ubh-3 genes are predicted to be organized into an operon, and the encoded proteins are 32-38% and 34-38% identical to human UCH-L1 and UCH-L3, respectively, while UBH-4 is 47% identical to human UCH-L5 that is associated with 26S proteasome. Enzymological characterization: All the UBH proteins were expressed in E. coli as fusion proteins with a His-tag and purified. UBH-1, UBH-2, and UBH-3 showed C-terminal hydrolase activity toward C. elegans Ub and NEDD8 fused with HSV- and His-tags at the C-termini but not toward SUMO-1 fused with HSV- and His-tags. This substrate specificity is consistent with that of UCH-L3. UBH-4 showed significant but far less activity toward Ub and NEDD8. Expression patterns: We made ubh-1::GFP and ubh-4::GFP translational fusion constructs. In the case of ubh-1, GFP-reporter expression was observed in some neuronal cells including the HSN and DVB neurons. The expression pattern observed by immunostaining with anti-UBH-1 antibody was similar to that by the GFP-reporter analysis. The neuron-specific expression of UBH-1 is analogous to UCH-L1. On the other hand, ubh-4::GFP was observed to be expressed almost ubiquitously. Phenotype of ubh-1-deletion mutant: The mutant allele tm526 has a deletion in the 3 half of the ubh-1 gene so that the gene product is considered to have lost its function. By RT-PCR analysis, ubh-2 and ubh-3 are confirmed to be expressed in the deletion mutant ubh-1(tm526). The mutant animals have a slower rate of larval growth and have a smaller brood size (63%). Additionally, the defecation cycle of the knockout animals was longer (60 sec) than that of the wild-type animals (45 sec). Transgenic expression of ubh-1::GFP rescued the extended defecation cycle of the ubh-1(tm526) mutant animals. Conclusions: UBH-1, UBH-2, and UBH-3 are structurally and enzymatically related to human UCH-L1 and UCH-L3. Analogous to UCH-L1, mutations of which are linked to certain familial forms of Parkinsons disease, UBH-1 is important for neuronal functions.

Moore A et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Mitochondria, PI3k, and the hypoxia inducible factor: What is the connection?"

Golden A, Moore A

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

We have determined that RNAi of a subset of subunits involved in The Citric Acid Cycle (TCA) or the Electron Transport Chain (ETC) activates a novel mitochondria checkpoint during the early stages of C. elegans development. This arrest halts embryonic progression during the 2-4 cell stage of development by a mechanism involving PTEN/daf-18. Watanabe et al. demonstrated that nutrient deprivation triggers cell cycle arrest through activation of the PTEN/daf-18 mediated nutrient sensing pathway through its ability to dephosphorylate a subunit of the spindle assembly checkpoint (SAC) MDF-1 (1). We propose that mitochondria dysfunction might cause a similar arrest through activation of the PTEN pathway. In order to address the signaling molecules responsible for this cell cycle arrest upon TCA or ETC RNAi, we took advantage of the numerous mutants available in C. elegans to ask a series of genetic questions. We determined that mutants in the PI3k signaling pathway can suppress the embryonic arrest caused by RNAi of nuclear encoded mitochondria genes through the activation of at least one target, the hypoxia inducible factor (HIF). Though various components of this pathway have been shown to respond to mitochondria signals, our genetic data suggests that downstream activation of HIF by PI3k may play a significant role in suppression of the mitochondria RNAi induced embryonic checkpoint. In addition to defects in cell cycle progression, weak TCA or ETC RNAi leads to chromosomal and centrosomal defects. These phenotypes may mimic what occurs in mammalian cells in the initial stages of carcinogenesis due to defects in nuclear encoded mitochondrial genes. These are important findings given the numerous diseases such as certain cancers, neuropathies, and encephalopathies that are caused by defects in mitochondria proteins. Our results demonstrate that C. elegans can serve as a model system to study the early stages of cellular dysfunction in response to abnormalities in mitochondrial proteins. 1. Watanabe S, Yamamoto G, Kitagawa R; EMBO Vol 27 No 7 2008

Wiesenfahrt, T. et al. (2009) International Worm Meeting "Shaping the intestinal tube: Organization of the intermediate filament network by MAP-kinase BMK1/ERK5 homolog SMA-5."

Wiesenfahrt, T., Leube, R., Husken, K., Gerhardus, H., Bossinger, O.

[International Worm Meeting, 2009]

Intermediate filaments (IFs) make up one of the three major fibrous cytoskeletal systems in metazoans. Numerous IF polypeptides are synthesized in cell type-specific combinations suggesting specialized functions. C. elegans carries great promise to elucidate the still unresolved mechanisms of IF assembly into complex networks and to determine IF function in a living organism. Intestinal IFs are abundant in the mechanically resilient endotube, a prominent feature of the C. elegans intestinal terminal web region. This IF-rich structure brings together all three cytoskeletal filaments that are integrated into a coherent entity by the apical junction thereby completely surrounding and stabilizing the intestinal lumen with its characteristic brush border. To identify factors that are responsible for the formation and positioning of this exquisite structure, IFB-2::CFP reporter strains were subjected to chemical mutagenesis. Among the resulting mutants we found one strain in which the IFB-2::CFP-labelled subapical IF network displays bubble-like protuberances, a phenotype reminiscent to the knock-down of IFC-2 by RNAi (Husken et al., 2008). By combined snp-mapping and RNAi-analyses the mutation was localized to the sma-5 gene (Watanabe et al., 2005). sma-5(kc1) encodes a protein that displays an amino acid exchange (R to H, pos. 125) in the catalytic domain. kc1 worms show a reduced body size, produce less progeny and have a shorter mean life span. Larval development of kc1 intestines occur regularly but considerable luminal alterations emerge later becoming manifest and most pronounced in adult worms. Multiple bubble-shaped invaginations protrude into the intestinal cells without affecting junctional integrity or apical domain morphology and composition. This phenotype suggests that the loss of sma-5 compromises intestinal stress resistance, probably by directly interfering with the phosphorylation state of the intestinal IF network.

Watanabe, Shigeki et al. (2011) International Worm Meeting "Three-dimensional molecular architecture of a cell using photo-activated localization microscopy and electron microscopy."

Chen, Pin-An, Richards, Jackson, Watanabe, Shigeki, Jorgensen, Erik, Frokjaer-Jensen, Christian, Hollopeter, Gunther, Hobson, Robert

[International Worm Meeting, 2011]

Mapping the molecular architecture of proteins within a cell is essential for understanding their function. Fluorescence microscopy has been widely used for this purpose. However, the diffraction limit of light limits this approach, since fluorophores that are within ~200 nm of each other cannot be resolved. Several microscopy techniques have been developed to break this limit, including photo-activated localization microscopy (PALM)[1] and related techniques (STORM[2] and fPALM[3]). PALM allows one to precisely pinpoint the location of a single molecule in a cell to within 10 nm. However, precise localization in a field of black is not useful. Where is that protein in the context of cellular structure? PALM images need to be overlaid on the structures visualized by electron microscopy. Therefore, a method to preserve fluorophores in tissues fixed and embedded in plastic needs to be developed. To preserve fluorescence in plastic, three conditions are required: hydration, limited oxidation from fixatives, and neutral pH. We found that use of 5% water, a less oxidative fixative (KMnO4), and hydrophilic plastic (GMA - pH8), were sufficient to preserve signals and morphology. To test the feasibility of fEM using PALM, we tagged histone, TOM-20, and liprin, with photo-convertible fluorescent proteins. We showed that histone, TOM-20, and liprin were successfully localized to the expected organelles - nucleus, mitochondrial membrane, and dense projection, respectively[4]. We also have mapped the a2d subunits of voltage-gated calcium channels in the nerve ring of C. elegans. We found the subunits to be exclusively localized to dense projections, supporting the previous localization pattern by immuno-EM[5]. This result suggests that the fEM is feasible for protein localization, and many proteins can be localized to their cellular structures if they can be tagged by fluorophores. References [1]Betzig, E. et al. Science 313, 1642-1645 (2006). [2]Rust, M.J., Bates, M. & Zhuang, X. Nat. Methods 3, 793-795 (2006). [3]Hess, S.T., Girirajan, T.P.K. & Mason, M.D. Biophys. J 91, 4258-4272 (2006). [4]Watanabe, S. et al. Nat. Methods 8, 80-84 (2011). [5]Gracheva, E.O., Hadwiger, G., Nonet, M.L. & Richmond, J.E. Neurosci. Lett 444, 137-142 (2008).

Watanabe, Shigeki et al. (2011) International Worm Meeting "Two endocytic pathways revealed by capturing channelrhodopsin induced synaptic transmission."

Jorgensen, Erik, Hollopeter, Gunther, Gu, Mingyu, Davis, Wayne, Watanabe, Shigeki, Liu, Qiang

[International Worm Meeting, 2011]

To sustain release of neurotransmitters at high rates of stimulation, synaptic vesicles need to be recycled locally at a synapse. Two major pathways for endocytosis were identified nearly 40 years ago: clathrin-scaffolds[1] and kiss-and-run[2]. Clathrin-mediated endocytosis is a slow process, which seems to take place distant from release sites. On the other hand, kiss-and-run occurs at release sites immediately after neurotransmission. The existence of either pathway at synapses has not been satisfactorily demonstrated[3]. Moreover, molecular mechanisms behind these pathways are poorly understood[3]. To address these issues, we need to observe synaptic vesicles as well as their associated proteins in action at nanoscale resolution. We developed two techniques that allow such an observation. First, we combined optogenetics with electron microscopy to induce synaptic transmission and capture rapid events at nanoscale resolution. Specifically, we expressed a variant of channelrhodopsin, ChIEF, in the C. elegans acetylcholine neurons, and stimulated them at various times before the high-pressure freezing. Using this technique, we identified two endocytic pathways that act at different times and in different parts of the synapse: a slow process distant from the dense projection and a fast process near the dense projection. Second, we developed a correlative super-resolution fluorescence microscopy and electron microscopy (fEM) imaging technique[4] to identify molecules that act in the processes. We found that the adaptor protein AP2 is localized to both identified endocytic sites, suggesting that AP2 may be functioning in both processes. By combining these two techniques, we are beginning to understand how endocytosis takes place at synapses. References: [1]Heuser, J.E. & Reese, T.S. Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cell Biol 57, 315-344 (1973). [2]Ceccarelli, B., Hurlbut, W.P. & Mauro, A. Depletion of vesicles from frog neuromuscular junctions by prolonged tetanic stimulation. J. Cell Biol 54, 30-38 (1972). [3]Royle, S.J. & Lagnado, L. Clathrin-mediated endocytosis at the synaptic terminal: bridging the gap between physiology and molecules. Traffic 11, 1489-1497 (2010). [4]Watanabe, S. et al. Protein localization in electron micrographs using fluorescence nanoscopy. Nat. Methods 8, 80-84 (2011).

Han, Ting et al. (2009) International Worm Meeting "26G RNAs regulate gene expression during spermatogenesis and larval development."

Chu, Diana, Kim, John, Fitzpatrick, Colin, Manoharan, Arun Prasad, Han, Ting

[International Worm Meeting, 2009]

Endogenous siRNAs (endo-siRNAs) fine tune gene expression in eukaryotes (1-4). Although endo-siRNAs were initially discovered in C. elegans (5), the biology of these small RNAs has not been studied in depth. Here we show that a class of endo-siRNAs is generated in the proliferating germline and regulates specific transcriptional programs during parental spermatogenesis and filial larval development. Using Solexa (Illumina) deep sequencing technology, we identified a unique class of endo-siRNAs that are 26nt in length and invariantly start with a G nucleotide (therefore, we name them 26G RNAs; 6). The 26G RNAs are highly enriched in purified germ cells (sperm and oocytes), depleted in the soma (glp-4(bn2) grown at 25 deg C), and antisense to mRNA transcripts. There are two distinct classes of 26G RNAs. Class I 26G RNAs peak in L4/young adult stages and tightly associate with the spermatogenesis transcriptional program. Class II 26G RNAs are maternally inherited, with highest expression in oocytes and embryos, and decline through the four larval stages. Importantly, by monitoring target transcript levels, we show that these two 26G RNA classes regulate non-overlapping sets of mRNA transcripts. Class I preferentially silences genes expressed during spermatogenesis and this target regulation is strongly coupled with 26G RNA levels. Class II 26G RNAs regulate maternal transcripts and the silencing effect perdures throughout larval development, indicating Class II 26G RNAs function as maternally deposited silencing factors for regulating zygotic gene expression. We further probed the genetic requirements for 26G RNA expression. In addition to several known eri pathway genes (eri-1, 3, 5, 7), an argonaute (ergo-1), and an RNA-dependent RNA polymerase (rrf-3) are required for 26G RNA expression. The fact that these mutants all exhibit enhanced exogenous RNAi suggests a competition between the 26G endo-siRNA pathway and exogenous RNAi for limiting factors. We are currently studying the biochemical mechanisms of 26G RNA biogenesis and target regulation. Take together, our findings indicate that 26G RNAs are generated by the endogenous RNAi machinery in the parental germline and regulate gene expression during paternal spermatogenesis and zygotic development. 1. Ghildiyal, M. et al., Science 320, 1077 (2008). 2. Okamura, K. et al., Nature 453, 803 (2008). 3. Tam, O. H. et al., Nature 453, 534 (2008). 4. Watanabe, T. et al., Nature 453, 539 (2008). 5. Ambros, V. et al., Curr Biol 13, 807 (2003). 6. Ruby, J. G. et al., Cell 127, 1193 (2006).