Myhrvold and te Velthuis Labs
Multiplexed detection of influenza A virus mini viral RNAs using Cas13
Influenza A virus (IAV) can cause mild to severe respiratory illness in humans, affecting 9 million to 41 million people annually in the United States alone. During infection, IAV produces mini viral RNAs (mvRNAs) that trigger the innate immune response. The extent of the immune response plays a role in disease outcome. The timing and the degree of mvRNA production, along with how those factors influence the innate immune response, remains elusive. Furthermore, there are no sensitive, cost-effective, and specific methods to understand mvRNAs. Thus, I developed a Cas13-based detection assay to quantify mvRNAs in transfected and infected cells. I aim to use this method to study the temporal dynamics of mvRNA production and discover how the production of mvRNAs correlates with the innate immune response in tissue culture, animal models, and clinical samples. Additionally, I will create a multiplexed Cas13-based detection assay to evaluate different species of mvRNAs present in a single sample to gain a better insight into the production of various mvRNAs and disease outcomes. Together, these experiments will allow us to understand the interactions between mvRNAs and innate immunity and help us create models that will revolutionize viral diagnostics.
Avalos and Cohen Labs
A novel Yeast-Liquid-Hybrid (YLH) high-throughput method to uncover protein condensate constituents
The field of intracellular condensates is an emerging and growing research area. Liquid membranelles organelles have been found to be ubiquitous in cellular biology with functional roles in signaling, transcription, metabolism, etc. However, our ability to study such condensates in a high throughput manner is limited. Therefore, we sought to redesign and repurpose the classical Yeast-Two-Hybrid (Y2H) system into a new Yeast-Liquid-Hybrid (YLH) method to identify and study partitioning of proteins into phase-separated droplets. We have selected FUS1 and Tardigrade Intrinsically Disordered proteins to test and establish the system. The development of this method will allow for the discovery of interaction at a much larger scale than currently possible.
SERAYA JONES NELSON
Structural basis of dynamin regulation by amphiphysin during endocytosis
The extra- and intracellular eukaryotic membranes undergo constant membrane remodeling via concerted cycles of fission and fusion events, essential for many biological processes including nutrient uptake, receptor recycling, cell signaling and endocytosis. The proteins responsible for membrane remodeling include evolutionarily conserved guanosine triphosphatases (GTPases) belonging to the dynamin family. Dynamin is regulated by the integral accessory proteins, however, the mechanism, structural and functional basis by which these integral proteins interact with and regulate dynamin remains elusive. Amphiphysin, a Bin/amphiphysin/Rvs (BAR) domain protein mediates the invagination and fission steps of vesicles by sensing or facilitating membrane curvature and stimulating the GTPase activity of dynamin. Amphiphysin has been shown to regulate dynamin’s GTPase activity, a key requirement for fission, through an unknown mechanism. To understand this, I will elucidate the mechanisms by which amphiphysin co-operates with dynamin to promote fission in endocytosis. To do this, I intend to uncover the structure of full length amphiphysin in solution and in lipid tubules, which mimic their endogenous assembly on the neck of budding endocytic vesicles, using Cryogenic Electron Microscopy (cryo-EM). Additionally, I will conduct GTPase assays and fission assays to measure how amphiphysin modulates dynamin’s GTPase activity and measure the progressive appearance of high curvature fission products upon exposure to vesicles. By utilizing both structural and biophysical studies, the findings derived from this work will provide key insight into the spatio-temporal coordination of membrane fission during endocytosis. The insight obtained through this work will be instrumental in understanding amphiphysin’s regulation of dynamin to form endocytic vesicles involved in intracellular trafficking.