Light sheet imaging differs from other forms of microscopy by illuminating the entire plane of an ex vivo extracted brain sample and computationally compiling these planes post-hoc into a 3-dimensional image. This technique allows users to preserve the structural integrity and map biological units such as cells, axons, and genes onto a standardized brain atlas. Using the SHIELD clearing, preservation, and staining protocol, we prepared fluorescently labelled ex vivo whole, intact brains for light sheet imaging and used the ClearMap open source pipeline on the Hyak cluster operated by University of Washington-IT to overcome computational limitations. While ClearMap registers images and identifies cell counts per brain region, it lacks quality assurance metrics as well as figures for visual confirmation of preprocessing performance. To overcome these deficits, we have built a quality assurance package for ClearMap that includes registration validation and group-level quantification figures. In testing this package we analyzed multiple mouse brain samples exhibiting Fos expression patterns under varied conditions. Using this dataset we found that registration performance between expected and actual slices measured in F1 score averaged 0.7 in medial sections of the brain and we were able to visually inspect the alignment of subregions identified in a standardized brain atlas. Additionally, we created an interactive script to visualize heatmaps that indicate expression patterns for user-defined brain regions on an animal level. This work will enable rapid validation of collected data and third-party package performance while streamlining the analysis, interpretation, and visualization of results. In the future we intend to include useful statistics (one-way ANOVA, classification models, etc.) to provide additional functionality and further increase the speed and quality of data analysis.

The mechanistic target of rapamycin, mTOR, functions in the mTORC1 complex with another protein called raptor as a master regulator of eukaryotic cellular metabolism thereby regulating cell growth including from cancer, cell death including after stroke, inflammation, and aging. In a forward genetic screen for hypoxia resistant mutants, the Crowder lab recently identified a missense mutation in the daf-15 gene, which encodes C. elegans raptor. The mutation produces a heat-sensitive reduction of raptor function, hereafter referred to as daf-15(rf). At 20°C, daf-15(rf) is normally hypoxic sensitive, at 22°C very hypoxia resistant, and at 25°C incapable of normal development. Raptor negatively regulates autophagy, a mechanism for breakdown and recycling of proteins and organelles. Activation of autophagy has been found to promote hypoxic survival in C. elegans and higher organisms. Thus, we hypothesized that activation of autophagy was responsible for the hypoxia resistance of our daf-15(rf) mutant. To test this hypothesis, we first asked whether we could detect increased autophagy using fluorescently-tagged autophagy proteins at 22°C in daf-15(rf) but saw no effect compared to wild type. Next, we asked whether a C. elegans transcription factor, HLH-30, that promotes expression of autophagy proteins was activated by daf-15(rf) and found activation at 25°C but not at 22°C. Finally, we tested whether proteins essential for autophagy were also necessary for the hypoxia resistance of daf-15(rf). By generating double mutant strains, we showed that animals with daf-15(rf) but without essential autophagy proteins were still hypoxia resistant. Thus, we conclude that C. elegans raptor regulates hypoxic sensitivity by an autophagy-independent mechanism. These findings demonstrate that raptor can control hypoxic cellular injury by mechanisms distinct from autophagy. Such mechanisms, if identified, could be targeted for treatment of cancer, stroke, and other diseases where hypoxia plays a role.

Mitochondria are the main oxygen consumers in eukaryotic cells and as such are the primary organelles affected by oxygen deprivation, hypoxia. Hypoxia alters the size and shape of mitochondria, called the mitochondrial dynamics, but their role in hypoxic cell death is unknown. The Crowder lab has recently discovered that a mutation in the Mechanistic Target of Rapamycin Complex One (mTORC1) protein Raptor confers hypoxia resistance in the nematode C. elegans. mTORC1 is a master regulator of metabolism and is known to affect certain aspects of mitochondrial biology. Given these two facts, we tested the hypothesis that the hypoxia resistance of the C. elegans Raptor mutant is from alterations of mitochondrial dynamics. First, I showed that hypoxia induces small, rounded mitochondria in C. elegans caused from mitochondrial fission. Second consistent with the hypothesis, I showed that the mitochondria appear to have more normal morphology before and after hypoxia in the Raptor mutant. However, not consistent with the hypothesis, a C. elegans mutant with excess mitochondrial fission was not hypersensitive to hypoxia. Then combining the hyper fission mutant with the Raptor mutant did not diminish the hypoxia resistance produced by reduced Raptor function. Thus, our data demonstrates abrogating mitochondrial fission is not necessary for the hypoxia resistance produced by the Raptor mutant and leads us to reject our hypothesis. By exploring the interaction of mitochondrial fusion and fission with Raptor, we are beginning to understand how these important organelle and metabolic regulators combine to control hypoxic cell death.

mTOR, the mechanistic target of rapamycin, is a serine/threonine kinase that regulates protein synthesis, cell growth, and metabolism in response to nutrients and energy in most eukaryotes. mTOR consists of two distinct complexes, mTORC1 and mTORC2. These complexes can be further divided into three components: mTORC1 consists of mTOR, Raptor, and mLST8, and mTORC2 consists of mTOR, Rictor, and mLST8. mTORC1 is critical in metazoan development and has been implicated in aging, cancer, diabetes, cardiovascular disease, and hypoxia. Previously, the Crowder lab conducted a mutant screen in C. elegans for hypoxia resistant mutations, and identified a missense reduction of function mutation in the daf-15 gene, the C.elegan ortholog of Raptor. A unique feature of this mutation is that the function of Raptor can be turned on and off by varying temperature. It has normal hypoxia resistance at 20 degrees, increased resistance between 21-22, and developmentally arrests at 25 degrees. I and the other authors made use of this conditional developmental arrest phenotype to screen for genetic suppressors. Using genetic mapping, sequencing, and complementation testing, we have identified multiple mutations in three different genes responsible for restoring Raptor function. One of the genes identified in this manner was rnf-126. Results show mutations in rnf-126 suppress the Raptor mutation. A null mutation in rnf-126 similarly suppressed the Raptor mutation. Previous work has implicated mammalian rnf-126 in degradation of the mTORC1 complex in cancer cells, suggesting that reduced levels of daf-15 may produce hypoxia resistance. We tested this hypothesis using auxin-mediated degradation of daf-15, finding that auxin-treated animals are hypoxia resistant. Current work by myself and others will further investigate how rnf-126 controls Raptor function and hypoxia sensitivity. Elaborating the function of this gene will define novel mechanisms whereby Raptor and mTORC1 controls metabolism, hypoxic injury, and development.

Proper chromosome segregation in mitosis relies on the correct attachment of kinetochores to the plus ends of microtubules. Kinetochores are protein complexes that assemble onto centromeres and bind microtubules. Microtubules are dynamic polymers of 𝛼- and 𝛽-tubulin subunits with an intrinsic structural polarity due to the repeated head-to-tail orientation of the heterodimer subunits in the lattice. This polarity results in a faster growing plus end and a slower growing minus end. Kinetochores are thought to initially bind to the microtubule lattice and then achieve plus end attachment by the action of plus end directed motor proteins or by the microtubule tip disassembling to the attachment point. While the plus end attachment is essential for mitotic fidelity, it remains unknown if the kinetochores themselves have an intrinsic polarity preference. Using total internal reflectance fluorescence microscopy, we have found that individual kinetochores assembled on centromeric DNA have a strong preference for binding the plus ends of stabilized microtubules in the absence of motor proteins and ATP or microtubule dynamics. Furthermore, using optical trapping we are able to measure the rupture forces of kinetochores on both ends of microtubules and have found that the observed preference for plus ends is matched by a greater binding strength at plus end tips. These results together give insight into how kinetochores could efficiently form plus end tip attachments and how they likely play a part in cell cycle regulation by using tension to sense a correct attachment. A better understanding of the specific mechanisms of kinetochore microtubule binding is valuable for understanding control of mitotic progression and could potentially inform more targeted anti-cancer therapies that focus specifically on dividing cells without impacting regular cell function.

Activation of Kappa Opioid Receptors (KOR)- either from a stress-evoked release of the endogenous dynorphin neuropeptide or pharmacologically- produces analgesic effects, aversive stress responses, and amplifies behaviors related to drug addiction. Influence on these specific behaviors can be attenuated through naloxone precipitated fentanyl withdrawal to model extreme distress. This was replicated by surgically implanting osmotic minipumps filled with fentanyl in mice for a 7-day period. Saline was utilized as a control against mice pretreated with norBNI (a long-lasting KOR antagonist) versus mice who only received fentanyl in order to determine if there was an effect on behavioral response following the precipitated withdrawal. Once the pumps were removed and fentanyl was eliminated from the system, mice underwent a 2-day spontaneous withdrawal phase prior to pairing 1 mg/kg naloxone injections with the presentation of almond extract. An observed aversion response to the almond odorant would exhibit a conditioned stimulus. The pairing of these components would then associate the negative feelings from withdrawal with introduced extract. The odorant aversion evident in fentanyl-treated mice was significantly reduced by pretreatment with the KOR antagonist norBNI, suggesting that the aversion was mediated by the release of endogenous dynorphin. With the continuation of this experiment, I would expect to see an increase in stress resilience as the KOR system becomes blocked with the administration of an antagonist.

The Kappa Opioid Receptor (KOR) is one of four opioid receptors found in the body and has an endogenous ligand known as Dynorphin. Binding of Dynorphin to this receptor has been shown to mediate some responses to stress, increase addiction risk, affect learning behaviors, and can provide analgesic effects. I am interested in learning how stress-induced activation of the dynorphin-KOR system affects addiction risk by identifying where in the brain dynorphin acts and how KOR activation affects brain function. I studied "where" by doing injections into specific regions in which Dynorphin is expressed and observing the mechanism of activated KOR. As a part of my project, I was tasked with performing stereotaxic viral infections of two different viruses in order to further study this system and its pathways. To allow for selective targeting of KOR expressing cells, the mice used have a gene for Cre Recombinase placed after the KOR promoter. This creates an environment where all neurons with expressed KOR also have Cre Recombinase in the cytoplasm. From here, I inject double-floxed inverse orientation viral vectors into specific KOR-expressing regions of the brain. The reversed virus is able to find its way into many cells but is only able to be inverted and properly expressed in cells containing Cre Recombinase. Functions of the active virus may differ but there are two primary examples used in my projects. The first is p38 CRISPR, which uses CRISPR-Cas9 technology to knockout the p38 mitogen-activated protein kinase gene, a protein kinase expressed after KOR activation. The second is a reactive oxygen species sensor, as ROS are associated with depalmitoylation and subsequent deactivation of the receptor. The use of a stereotax with this technology allows for precise targeting of different brain regions depending on the location relative to landmark sutures on the mouse skull.

Mitosis is the fundamental biological process that ensures equal separation of genetic material during cell division. Microtubules and their associated structures in the mitotic spindle execute this partitioning by carefully aligning and separating duplicated chromosomes. Despite intrinsically variable growth rates across microtubules and stochastic assembly and disassembly phases, chromosome-bound microtubules exhibit highly coordinated behavior that drive mitosis. The basis for this high degree of synchronization is currently unknown. Previously, we used a novel dual laser trap assay to show that microtubule pairs growing in vitro are coordinated by mechanical coupling (Leeds et al. 2023). A simple model incorporating both force-dependent pausing and growth speed heterogeneity explains the measured coordination of microtubule pairs. Our findings illustrate how microtubule growth may be synchronized during mitosis and provide a basis for modeling multiple microtubules in a bundle. In this project, we expand on the techniques we used with the dual optical laser setup and combine them with a cutting laser to induce disassembly in our microtubules. Studying the coordination of shortening microtubules encompasses a broader spectrum of microtubule dynamics and sheds light on other aspects of microtubule regulatory mechanisms. We can then extend our model to include the degree by which mechanical coupling can coordinate microtubules in disassembly in addition to growth. Bundles of multiple microtubules are in mixed states of shortening and growth while executing the coordinated motion necessary to drive mitosis, so understanding how mechanical coupling affects disassembling microtubules gives insight into the complete picture of the mechanisms behind their synchronous motion essential for life.