Oxygen (O₂) is essential for aerobic organisms to complete development. However, genetic mechanisms exist to promote development and avoid damage under low oxygen conditions. Elucidating how these mechanisms regulate development is important for understanding diseases, such as ischemia and tumor formation, which have a hypoxic component. We are studying the hypoxic response in Caenorhabditis elegans, a genetically tractable organism that uses simple diffusion for gas exchange with the environment. Previous research has suggested that at 5000ppm O₂, loss-of-function mutations in either the transcription factor hif-1, or an AMP-activated protein kinase (AMPK) subunit aak-2, prematurely arrest development. Given that these two mutants have similar phenotypic responses in severe hypoxia, we hypothesized that aak-2 and hif-1 act in the same genetic pathway to coordinate developmental arrest in hypoxia. To test this, we pharmacologically activated AMPK and performed epistasis analysis with genetic mutations. We found that activating AMPK by treatment with Metformin in a hif-1 mutant was not sufficient to sustain reproductive activity in 5000 ppm O₂. We also generated a strain that constitutively expresses HIF-1 in an aak-2 mutant background and found no difference in developmental arrest from aak-2 mutant animals with wild-type HIF-1 activity. We conclude that aak-2 and hif-1 act in distinct genetic pathways to promote development in hypoxia. Our results suggest that C. elegans has multiple, redundant pathways that coordinate development in hypoxic conditions.

Endophilin is an essential protein for membrane dynamics. It harbors a BAR (Bin–Amphiphysin–Rvs) domain, which acts as a basic modual for shaping membranes. Deletion of Endophilin impairs endocytosis, which leads to defects in cellular signaling. Despite the importance, how Endophilin is regulated remains elusive. Here, we propose that the bending activity of the Endophilin BAR domain is modulated by phosphorylation. We will use biophyiscal/biochemical approaches to investigate Endophilin-membrane interactions. Our result will provide new insights into molecular mechanisms for controling membrane dynamics. We use liposomes as an in vitro model. We follow the kinetics of Endophilin-membrane interactions using fluorescent probes. We identify key residues for Endophilin-membrane binding using site-directed mutagenesis. We believe this research will help us understand the mechanism for shaping cellular membranes, in particular, how Endophilin BAR domain bends membranes.

The vertebrate body forms through a process called embryogenesis. In my research, I focused on the establishment of body length during embryogenesis of the model organism zebrafish (Danio rerio). Under normal conditions, the cells of the developing body intercalate towards the back, or dorsal side, of the embryo, and extend longitudinally to grow the body axis. One observed defect in this process blocks cell intercalation, thus leading to underdeveloped bodies, which is known as a convergent extension phenotype. In my research, I studied the role of Microtubule-Associated Serine/Threonine kinase-Like (MASTL) RNA in body axis formation. Because of a known role for MASTL in regulating chromosome segregation, I hypothesized that overexpression of MASTL would interfere with normal cell function and lead to a convergent extension phenotype with a shortened body axis in the developing zebrafish embryo. To test this hypothesis, I injected embryos with different concentrations of RNA and allowed them to develop. Overexpression of MASTL shortened body lengths at all but the lowest concentration of RNA. I then chose a low but effective concentration of MASTL RNA, injected embryos and quantified embryo length. Finally, I performed RNA in situ hybridizations, a process by which labeled probes are used to localize specific RNA transcripts in embryos. Using a variety of labeled probes, I found in addition to shortened body length, the distribution of the prospective spinal cord cells and muscle cells were abnormal in MASTL-injected embryos. The results of these experiments clearly show that overexpression of MASTL leads to a convergent extension phenotype. To continue this research, I will look at whether or not MASTL affects body length formation through Protein Phosphatase 2a, a known target of MASTL. The results of my research will lead to a better understanding of the role MASTL in cell intercalation and vertebrate body elongation.

Mitochondria are essential organelles in eukaryotic cells and are most commonly known for their prominent role in energy production. In addition, mitochondria are involved in other important cellular processes such as lipid biosynthesis and apoptotic cell death. Mitochondria also have dynamic properties including fusion, division, and movement, which are all essential to maintain mitochondrial function. Given the plethora of essential cellular activities that require mitochondrial function, it is not surprising that defects in mitochondrial function are implicated in many different human diseases and disorders including diabetes and myopathies. Mitochondrial movement is important for the distribution of mitochondria, which is important in all cells, but especially important for asymmetric cells such as neurons. Transport is also required for mitochondrial fusion – the two organelles must move close enough to each other to fuse. Mitochondrial fusion in particular is required to maintain mitochondrial DNA and therefore, mitochondrial function. We are interested in the mechanism of mitochondrial movement and the connection between mitochondrial fusion and transport. Mitochondria move on microtubules using a complex of proteins. Miro 1 and Miro 2 are anchored to the mitochondrial outer membrane and interact with two adaptor proteins, TRAK1 and TRAK2. The TRAK proteins also interact with Kinesin, which binds to the microtubules and mediates microtubule-directed movement. Understanding the assembly and regulation of this complex and the stoichiometry of its components will provide insights into the mechanism of microtubule-directed mitochondrial movement. Our goal is to clone TRAK1 and TRAK2 complementary DNA (cDNA) into expression vectors for protein production in Escherichia coli cells and then to develop a purification protocol for TRAK1 and TRAK2. This will facilitate biochemical analysis of the mitochondrial motility complex.

 The vast majority of current protein-based drugs involve binding a therapeutic target with an exogenous molecule that neutralizes or blocks its function. A major drawback of this approach necessitates a stoichiometric number of binding sites be administered to the patient, resulting in many caveats such as high dosage, stability, and immunogenicity. Enzymes represent an underexplored avenue for protein therapeutics with vastly enhanced potency. Proteases are essential and ubiquitous enzymes that catalyze peptide bond cleavage. While there are multiple therapies available derived from naturally occurring proteases, currently there are no novel, engineered protease therapeutics. In this work, we seek to establish a highly specific and processive protease system with traits desirable for use as a protein therapy, such as high solubility and thermostability. For this purpose, we have chosen to engineer the small ubiquitin-like modifier (SUMO) protease. The target of this protease, SUMO, is a small protein important for cell cycle regulation in eukaryotes. The mechanism for SUMOylation is analogous to the ubiquitin regulation system; these small proteins are adducted to other molecules and serve as signals for degradation or cellular localization. Removal of these tags is essential to the regulatory process, and is mediated by the highly specific SUMO protease. This inherent specificity makes the SUMO protease an ideal engineering platform. Here, we take the first steps toward unlocking the therapeutic potential of the SUMO protease by enhancing the stability and solubility of the yeast homologue. To accomplish this, we re-engineered surface residues with the Rosetta software suite. Furthermore, we have developed a novel Förster resonance energy transfer assay to allow us to monitor protease activity in real time. In addition to laying a foundation for the creation of highly selective therapeutic proteases, our work has immediate implications for the production and purification of recombinant protein in a laboratory setting.

The lab I am involved with investigates the molecular events of neuromodulation, a form of neuronal chemical signaling that determines behavior, emotions, learning and memory. Neuromodulators are carried and released by dense core vesicles, a type of cellular vesicle which little is known about. Dr. Ailion's lab has performed genetic screens for C. elegans mutants that have altered dense core vesicle secretion and has isolated many unique mutants. During my time in Dr. Ailion's lab I focused on genetically mapping two of these mutants which were given the names yak90 and yak91. With yak90, I used a well-established mapping method where my mutant strain was crossed to marker strains which contained alleles causing obvious deformities on each chromosome. Generations later, I examined their offspring and was able to pinpoint one mutant phenotype that was not segregating according to Mendelian ratios, indicating linkage to chromosome IV. When a candidate gene was found near the linkage site, I did a complementation test and confirmed that yak90 contains a mutation in an already characterized gene, unc-31. With yak91, a new method was tested using a marker strain that contains alleles for multiple fluorescent tags instead of body deformities. This was necessary because yak91's mutation causes it to be "egl", or distended with mature eggs, and it was difficult to tell it apart from one of the marker phenotypes. The cross with the new strain was a success and after examining the progeny, linkage was found on chromosome IV. Another complementation test was performed, and yak91 was found to contain a mutation in ric-8. Total loss-of-function mutations in ric-8 are lethal so yak91 must contain a splice site or point mutation that significantly alters the function of the protein product. Both genes I identified play important roles in neuromodulation. 

Our lab has recently developed computational methods for designing cage-like protein nanomaterials built through the self-assembly of multiple copies of two distinct protein components. This new technology holds great promise for a wide range of applications including targeted drug delivery, vaccine design, and biofuels. Moving towards these real-world applications, one desired but as of yet unrealized feature of our current designs is the ability to produce each protein component independently of one another and then trigger assembly by mixing the purified components in vitro. Obtaining this level of control over the assembly process requires that the individual protein components are stable and soluble in the absence of their designed binding partner. This property is not explicitly considered in our current design protocol. Here we present work aimed at addressing this problem by implementing a new step into our design protocol wherein we redesign the amino acid sequences of the proteins to increase the net positive charge on the surface of one protein subunit and the net negative charge on the surface of the other subunit. This supercharging approach should improve solubility by disfavoring aggregation between identical subunits of individually purified components while favoring the interactions between distinct subunits required for nanocage formation. To supercharge the proteins, we use the molecular modeling software package Rosetta to determine positions on the surface of each protein that are predicted to tolerate mutations to either positively or negatively charged residues. This information is used to automatically generate initial designs, which we then manually inspect and refine.

Proteins perform a vast array of functions within all living organisms. These functions are heavily dependent on the protein’s 3-D structure. Cellular stress can result in the loss of protein structure and can lead to a wide variety of problems, one being the formation of insoluble protein aggregates. Aggregates can inhibit proper protein function, disrupt cellular homeostasis, and are implicated in many diseases. There exists a family of proteins, known as the Small Heat Shock Proteins (sHSPs), which are thought to combat aggregate formation. They interact with misfolded and aggregate-prone proteins (clients) to delay the formation of insoluble aggregates. How sHSPs delay aggregation is not well understood. I seek to characterize interactions between sHSPs and their clients. A binding interaction between the sHSP αB crystallin and the model client Δ131Δ (a mutant of staphylococcal nuclease) has previously been characterized by NMR. My goal is to confirm this interaction and look for additional interactions between these two proteins through chemical cross-linking. Chemical cross-linking is a process that links two proteins that interact. One can use this method to detect which surfaces of proteins are involved in a protein-protein interaction. The chemical cross-linker I am using is incorporated as an unnatural amino acid (p-benzoyl-L-phenylalanine, or BpA) into the sHSP αB crystallin. Upon exposure to UV light, BpA can covalently cross-link to another protein if it comes into close contact with it. This approach can allow us to explore how sHSPs interact with the client Δ131Δ. By gaining an understanding of this specific sHSP-client interaction, I hope to provide insight into general mechanisms of how sHSPs interact with clients, a process that plays a critical role in cellular health.

Autophagy is the process in which eukaryotic cells selectively degrade proteins and organelles. Autophagy has been well documented in organisms ranging from yeast to mammalian cells, with demonstrated roles in response to starvation, aging, and stress. However, there has not been an effective method for measuring autophagy in C. elegans, despite C. elegans being a popular model organism for the study of disease and aging. We refined an assay that uses double-fluorescent protein tagged Atg-8/Lgg-1 to measure autophagy in both yeast as well as C. elegans. This assay reports an increase in autophagy in response to starvation in both model organisms. We then applied this to the study of nematodes containing a mutation in Rheb, an important protein in the upstream signaling cascade that regulates autophagy. Autophagy is damped by signaling through the TORC1 pathway. A mutagenesis screen revealed two different worm mutants (ENU9 and ENU10) which are predicted to hyper-activate TORC1 signaling. Using the aforementioned assay we found that ENU9 and 10 mutants have dramatically reduced levels of autophagy. This work has implications for further research in Alzheimer’s, Parkinson’s and related diseases associated with ageing and proteostasis.

Protein homeostasis encompasses the creation, maintenance, and degradation of proteins, and it is essential for organisms to function properly. Yet, environmental stressors can disrupt protein homeostasis, which can lead to protein aggregation. Protein aggregation has been linked to serious aging diseases such as Huntington’s, Parkinson’s, and Alzheimer’s disease. In order to gain a better understanding of the pathways important for maintaining and disrupting protein homeostasis, we subject Caenorhabditis elegans to various environmental stressors that disrupt protein homeostasis. We have found that specific concentrations of hypoxia, or low oxygen conditions, induce an increased amount of protein aggregates in C. elegans compared to animals in room air, suggesting defects in protein homeostasis. In addition, the AMP-activated protein kinase (AMPK) seems to be crucial for allowing the formation of aggregates in hypoxia. AMPK mutants that are placed in hypoxia show a decrease in protein aggregation compared to wild-type animals. Furthermore, another environmental stressor, heat shock, also affects protein homeostasis. Heat shocking C. elegans prior to the hypoxic exposure, decreases the amount of aggregates compared to worms that did not experience heat shock. However, unlike hypoxia-induced protein aggregation, heat shock does not seem to require the presence of functional AMPK in order to influence protein aggregation.

My project is to reveal mechanisms that regulate the abundance and the distribution of dopamine receptors in C. elegans. The neurotransmitter dopamine carries out its modulatory activities though transmembrane dopamine receptors. These receptors that reside on plasma membranes bind extracellular dopamine and propagate activities through G-protein coupled signaling pathways. The abundance and location of dopamine receptors are highly dynamic, which permits dopamine to modulate brain activities in response to physical changes such as stress and ageing. Here, I propose to image fluorescently tagged dopamine receptors in living worms. I will use CRISPR/Cas9-mediated gene editing approaches to insert fluorescent tags into genes encoding dopamine receptors, which results transgenes located in near native gene environment (such as promoters and 3' UTR regions). To quantify the abundance of dopamine receptors on plasma membranes, I will tag a pH-sensitive green fluorescent protein (pHluorin) to extracellular domains of dopamine receptors. When dopamine receptors are in transporter vesicles, the pHluorin fluorescence is quenched because extracellular domains of dopamine receptors reside in the acidic vesicle lumen. Once dopamine receptors are delivered onto plasma membrane, the pHluorin fluorescence becomes visible as pH is neutralized. To monitor the total amount of dopamine receptors, I will introduce a pH-insensitive red fluorescent protein to the intracellular domain of dopamine receptors. The ratio between green and red fluorescence will indicate the surface fraction of dopamine receptors. As a control, I will conduct pharmacological and behavior assays to test whether the engineered dopamine receptors are functional in vivo. Results from this study will allow us to visualize how dopamine receptors are regulated to support brain functions.

Alberta’s Athabasca oil sands are one of the world’s largest petroleum reserves. Oil sands comprise sand, clay, and bitumen – a viscous form of petroleum which is more difficult to extract than conventional crude. Current bitumen extraction practices such as surface mining and steam-assisted gravity drainage are energy-intensive. This independent project investigated the potential of employing microbial biosurfactants to reduce the energy required for bitumen recovery in oil sands. Biosurfactants are employed in the industrial practice of microbial enhanced oil recovery (MEOR), which is viable in the conventional oil industry, but has not been applied to the Athabasca oil sands. In this study, a biosurfactant-producing bacterial strain was isolated from an environmental sample and its biosurfactant compound was characterized. Biofilm formation under simulated oil sands reservoir conditions was analyzed by scanning electron microscopy, and bitumen extraction was tested in a small-scale model. Potential microbial applications in oil sands extraction, upgrading, and bioremediation were explored in a literature review.