Franco, Joy A., Pruitt, Beth L., Wang, Lingxin, Kuhl, Ellen, Wang, Lucy M., Das, Alakananda, Goodman, Miriam B., Chapman, Dail
[
International Worm Meeting,
2021]
The sense of touch is made possible by neurons that use ion channels to convert mechanical stimuli into electrical signals. Many other proteins affect mechanosensation, either through direct force transfer by acting as physical tethers, or indirectly by modulating cell stiffness and morphology, protein trafficking, and channel positioning. Subcellular localization of mechanosensory ion channels is especially important for localized force sensation, since channels further away from the point of stimulation are recruited as stimulus intensity and speed increase. Here we examine the micro-environment of ion channels to investigate the molecular basis of channel positioning, leveraging the well-characterized MEC-4 mechanoelectrical transduction channel in C. elegans touch receptor neurons (TRNs). We show that MEC-4 channels localize to punctae distributed along TRN neurites in vivo but not in cultured TRNs in vitro, suggesting that in vivo channel localization depends on factors that are absent in vitro. Both time-lapse imaging and fluorescence recovery after photobleaching shows that most in vivo MEC-4 puncta are immobile over several minutes, suggesting that they are anchored to stable structures. To identify these structures, we screened mutants affecting plasma membrane proteins, cytoskeletal proteins, and extracellular matrix (ECM) proteins to investigate what factors regulate the positioning and stability of the MEC-4 puncta. In doing so, we discovered that the ECM protein nidogen regulates MEC-4 puncta distribution and both behavioral and electrical responses to touch. Nidogen associates with laminin networks in basement membranes and we show that both nidogen and laminin co-localize with MEC-4 puncta, in a manner that depends upon another ECM protein, MEC-1. To learn more about the physiological significance of channel puncta, we modeled mechanical strain distribution along a neurite in silico and found that physical attachment of the neurite to the ECM at puncta generates localized regions of increased strain. We speculate that this amplifies and focuses the mechanical stimulus for ion channel opening. Future experiments will determine whether these ECM proteins physically anchor the MEC-4 channels, accounting for the immobility of the MEC-4 puncta, and whether such a physical tether is responsible for ion channel opening in response to a mechanical stimulus.
[
European Worm Meeting,
2006]
Ellen Nollen, Tjakko van Ham & Ronald H. A. Plasterk. Aggregation of misfolded proteins occurs in various age-related neurodegenerative disorders, including Parkinsons, Alzheimers, and Huntingtons disease. To understand how cells protect themselves against misfolded proteins, we search for genes that enhance or prevent protein aggregation. C. elegans strains expressing polyglutamine stretches fused to YFP with visible, age-dependend protein aggregation are used as a genetic model. Using a genome-wide RNAi screen, we have previously identified 186 genes that, when knocked down, cause premature protein aggregation. These genes include genes involved in protein synthesis, folding, degradation and RNA synthesis and processing. 1. Conversely, we performed a forward mutagenesis screen to identify genes that, when mutated, suppress age-dependent polyglutamine aggregation. For one suppressor mutant, in which aggregation is suppressed by more than 75%, we have now identified the responsible mutation. This mutation is a missense mutation in a gene encoding a protein of unknown function that is highly conserved between C. elegans and humans. Knock-down by RNAi of the same gene in wild-type worms yielded a similar reduction in aggregation, suggesting a loss-of-function mutation. We are currently further characterizing this mutant and the remaining suppressor mutants. In addition, to establish whether the genes we have identified are specific for polyglutamine aggregation or whether they comprise of a general protein homeostatic buffer, we have developed a worm model for aggregation of alpha synuclein, which occurs in Parkinson''s disease. Altogether our results will provide insight into cellular protection against misfolded proteins and yield targets for therapy against protein misfolding diseases.. 1Nollen E.A.A., Garcia S.M., van Haaften G., Kim S., Chavez A., Morimoto R.I., Plasterk R.H. (2004) Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation. Proc. Natl. Acad. Sci. U.S.A. 101(17):6403-8.