Wong, K, Aalto, A.P., Pagliuso, D, Pasquinelli, A.E., Chen, J.S., Schreiner, W.P.
[
International Worm Meeting,
2017]
Outside of the laboratory in the natural world, organisms are subject to various environmental insults that, if not properly addressed, can have lethal consequences. Organisms employ a carefully orchestrated and conserved Heat Shock Response (HSR) in order to cope with the potentially lethal effects of increased temperature. While the transcriptional response to HS is well-established, post-transcriptional control mechanisms involved in the HSR are yet to be fully investigated. We are particularly interested in the role of non-coding RNAs (ncRNAs) in modulating gene expression in response to heat stress. This focus stems from our discovery of nearly one hundred novel long ncRNAs (lncRNAs) induced by heat shock in C. elegans. Additionally, we have found several miRNAs to be differentially regulated by heat stress. While changes in miRNA expression and a functional role for some of these miRNAs in mitigating the HSR have been previously published, their targets are yet to be established. We aim to determine how HS induces the expression of long and short ncRNAs and how they might contribute to the HSR. Preliminary evidence shows that Heat Shock Factor 1 (HSF-1) is required for the expression of some novel lncRNAs, indicating that this transcription factor directs the production of ncRNAs as well as protein-coding mRNAs. Additionally, we find that the levels of some lncRNAs continue to be elevated long after the heat shock episode, raising the possibility that these RNAs provide resistance to future exposures to heat stress. Overall, these studies have revealed that heat shock causes extensive remodeling of the ncRNA landscape, providing new candidates for regulating the HSR.
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).