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The L-type Ca2+ channel (CaV1.2) is essential for cardiac excitation-contraction coupling. Alterations in CaV1.2 expression, localization, or function can underlie cardiac dysfunction found in aging and heart disease; thus the regulatory mechanisms that tune CaV1.2 expression to meet contractile demand are a crucial area of research. This dissertation work highlights the function of two proteins that regulate CaV1.2 trafficking: 14-3-3 and BIN1. We used a range of techniques including confocal imaging, proximity ligation assays, super-resolution imaging, In vivo echocardiography, and co-immunoprecipitation to explore the dynamics of this regulation. Our work elucidates a novel mechanism where 14-3-3 interacts with CaV1.2 in a phosphorylation-dependent manner to promote enhanced trafficking/recycling, clustering, and activity during β-adrenergic stimulation. We also report that a two-week treatment to restore youthful Bridging Integrator 1 (BIN1) levels in the hearts of 24-month-old mice rejuvenates cardiac function and substantially reverses the dysregulated endosomal recycling and disrupted CaV1.2 trafficking phenotype found in aging. These results increase our understanding of CaV1.2 regulation and provide a foundation for future work establishing a complete model of CaV1.2 trafficking.

Osteosarcoma (OS) is the most common primary bone malignancy in humans and canines, and in humans primarily affects younger patients 10-14 years of age. While considerable efforts have been put forth in new therapeutic approaches to this disease, the treatment and prognosis for OS has changed very little since the 1980s. Targeted therapeutics have made considerable progress in other cancer types, leveraging characteristics of cancer cells which differentiate them from that of normal healthy cells. In comparison to other cancers, OS is highly heterogeneic, and no single unifying driver mutation has yet been found. Development of a therapy which could overcome the high degree of heterogeneity amongst OS tumors could go a long way in improving the lives of OS patients. Targeting protein translation has been proposed as one mechanism by which to overcome tumor heterogeneity, and this dissertation focuses on studying a noncanonical role of core binding factor beta (CBFβ) as a regulator of protein translation, and elucidating whether this could represent a potential therapeutic target for OS. Utilizing a wide array of in vitro assays, we have been able to demonstrate that loss of CBFβ reduces protein expression of RUNX2 in a post-transcriptional manner, and this decrease in RUNX2 protein level is not fully explained by alterations in RUNX2 stability brought about by loss of its binding partner CBFβ. Additionally, we demonstrate that loss of CBFβ also causes a decrease in global protein translation, and confirmed an interaction between CBFβ and hnRNPK which has thus far only been observed in breast cancer cells. Importantly, this interaction with hnRNPK is said to be the mechanism by which CBFβ influences protein translation, and our results corroborate those observed in breast cancer cells and suggest CBFβ may also perform this role in OS. Reports of the interactions between CBFβ and hnRNPK or RUNX2 allude to mutual exclusivity in interaction, and with the transcriptional role of CBFβ accomplished via binding to RUNX proteins, and the translational role of CBFβ accomplished via binding to hnRNPK, it is entirely possible these two roles are antagonistic in some fashion. To investigate the relevance of certain CBFβ residues in terms of this translational role of CBFβ, and avoid confounding variables from the transcriptional role of CBFβ, we utilized point mutations to interrupt CBFβ-RUNX2 interaction. Using various in vitro assays, we validated key residues of CBFβ which are involved in its interaction with RUNX2, re-introduced this mutant form into CBFβ knockout cells, and measured alterations to RUNX2 interaction and nuclear shuttling. We confirmed that our mutant displays reduced binding to RUNX2, and drastically reduced nuclear shuttling. Lastly, we expanded our studies from RUNX2 to the entire genome and proteome. Encouraging data thus far had suggested CBFβ may play a role in protein translation, and necessary next steps were to assess which proteins CBFβ may be interacting with in performance of this role, and elucidate which proteins may be under the translational purview of CBFβ. Using immunoprecipitation mass spectrometry we identified numerous specific interactors of CBFβ, with high enrichment in pathway analysis terms associated with protein translation. Additionally, using two different methods we generated a list of proteins which may be under the translational purview of CBFβ, and found strong enrichment of numerous cancer-associated terms among this list. These studies establish that CBFβ participates in protein translation in OS, with many genes under its purview associated strongly with cancer in general, and OS specifically. This provides justification for future studies delving deeper into this novel role of CBFβ, and opens up another mechanism by which protein translation could be targeted therapeutically in OS.

AbstractThis dissertation investigates the role of the translation initiation machinery in the dynamics of stress granules (SGs). Stress granules are cytoplasmic, non-membrane bound organelles composed of mRNA transcripts, RNA-binding proteins, 40S ribosomal subunits, and eukaryotic initiation factors (eIFs). They form in response to environmental stress to promote cell survival and recovery. A major gap in knowledge addressed in this work is the need to measure real- time kinetics of SG assembly and disassembly. Furthermore, the involvement of translation initiation factors in SG assembly remains unclear. My research addresses these gaps by developing a system to monitor SG dynamics using a stable inducible Flp-In™ T-REx™-HeLa Cell Line system, enabling real-time tracking and modeling.Chapter 1 reviews the mechanisms of SG formation, their interplay with the translation initiation machinery, and their regulation under stress conditions. In Chapter 2, I describe the protocols developed to characterize the HeLa Fl-In cell line used throughout this study. Chapter 3 focuses on the role of the RNA Recognition Motif (RRM) of the initiation factor eIF4B in regulating SG assembly. Using single-cell live-imaging, I quantified the kinetics of SG formation and discovered that overexpression of GFP-tagged eIF4B with a point mutation in the RRM domain results in a decreased rate of SG formation, fewer total SGs, and a delayed stress response compared to wild type eIF4B. Furthermore, I demonstrated that eIF4B directly binds to G3BP1, a known SG nucleator, suggesting a mechanism by which eIF4B influences SG assembly. Chapter 4 examines the mechanisms of SG disassembly, a process poorly understood but linked to neurodegenerative disorders due to the inability to disassemble SGs, leaving cells in aconstant state of stress. I developed a method to measure the rate of SG disassemblyaccurately, addressing the challenge of focal drift in time-lapse imaging. Future work will explore overcoming focal drift using microfluidics devices or environmental chambers. Chapter 5 presents preliminary data on the interaction of GFP-tagged eIF4G with SGs. Unexpectedly, modest overexpression of eIF4G in HeLa cells results in hypersensitivity to oxidative stress, even though SGs still form. Proposed future experiments aim to confirm and extend understanding of this phenomenon.Overall, this dissertation provides novel insights into the dynamic interplay between translation initiation machinery and SGs, with potential therapeutic implications for targeting SG dynamics in diseases such as cancer and neurodegeneration.

During my dissertation in the lab of John Albeck, I have worked on various projects involving measurement of single-cell kinase signaling activity in response to stress using live-cell biosensors. This included optimizing and publishing methodology, the use of live-cell biosensors to measure cellular response to pro-inflammatory cytokines and oxidative stress, and the development of new biosensors. In Chapter 1, I introduce the methods we use to measure signal transduction in real time using fluorescent biosensors, which includes optimizations that I contributed to the lab. This protocol was accepted for publication as a chapter in Methods of Molecular Biology. In Chapter 2, with the help of my co-mentor Amir Zeki, I delve into the application of biosensors and show my finding that spatially coordinated ERK signaling (SPREADs) increased when airway epithelial cells are exposed to pro-inflammatory stimuli like those seen in common airway diseases. I then investigated the potential mechanisms of SPREADs and their suppression by hydrocortisone and metabolic stress that coincides with increased AMPK activity. Chapter 3 focuses on using live-cell microscopy to understand how the RNA-recognition motif of the translation protein eIF4B regulates the cell’s ability to form stress granules and suppress protein synthesis following treatment with sodium arsenate. Finally, in Chapter 4, I discuss my work developing, validating, and optimizing Far-Red FRET biosensors for ERK (REKAR67 and REKAR76) and AMPK (RAMPKAR2). Including the use of RAMPKAR2 to understand the single-cell dynamics of AMPK, ATP:ADP ratio, and glycolysis. This last chapter includes work from two different publications in preparation for submission.

Per- and polyfluoroalkyl substances (PFAS) are a class of compounds commonly used as surfactants in food contact materials and household items due to their hydrophobicity. However, their physicochemical properties render them highly resistant to degradation in the environment and human body. Thus, these chemicals are environmentally ubiquitous and have been found in the serum of 98% of U.S. citizens. Evidence implicates some well characterized PFAS in breast cell carcinogenesis and cancer progression. While PFAS-induced receptor-positive breast cancer progression has been widely investigated, PFAS-induced receptor-negative breast cancer has not, and molecular mechanisms remain unclear. Additionally, Generalized Read-Across of PFAS using cancer progression endpoints are lacking. This research analyzes and compares the effects of five PFAS of increasing alkyl chain length on triple negative breast cancer progression in vitro. Using the aggressive human cell line Hs578T the effects of five PFAS were analyzed across environmentally relevant doses. Progression was assessed through 2D cell proliferation assays and “2.5D” Matrigel migration and invasion assays. Results showed significant decreases in mean number of protrusions per cell and total colony formation per well with PFBA and PFDoA treatment. Significant decreases in colony formation were observed with PFDA treatment at all doses. The mean length of the longest protrusion per cell was largely unimpacted by PFAS, except for decreases by PFDoA. When viewing the data though read-across lenses, near significant trends were seen in protrusion counts with increasing alkyl chain length at 1 nM and 10 nM PFAS. The highest alkyl length significantly decreased colony formation in a pattern seen across 10 nM, 100 nM and 1000 nM doses. Cell proliferation was not impacted by PFAS treatment. These results will help elucidate whether triple negative breast cancer progression is related to dose and alkyl chain length, and whether short-chain PFAS exhibit increased progression as longer PFAS have been shown to promote.

Feline Chronic Gingivostomatitis (FCGS) is a common oral inflammatory disease of cats. FCGS is associated with chronic viral infections, such as feline calicivirus (FCV) and feline foamy virus (FFV) and can be treated by full mouth extraction (FME) or mesenchymal stromal cell (MSC) therapy in cases where FME fails. We hypothesized that FCGS is characterized by a dysfunctional, exhausted, CD8+ T cells state that is characterized by decreased cytokine production, decreased proliferative potential, decreased cytotoxic activity and increased expression of inhibitory receptors. We further hypothesized that MSC therapy helps to reinvigorate exhausted CD8+ T cells. Transcriptomic analysis of peripheral blood CD8+ T cells indicate upregulation of genes and pathways associated with exhaustion in peripheral blood CD8+ T cells in cats with FCGS. Moreover, confocal immunofluorescence indicated a marked expansion of CTLA-4 positive T cells in the mandibular lymph nodes of cats with FCGS. Finally, MSC therapy suppressed CD8+ T cell activation and proliferation. By comparing the pre-treatment pathways of CD8+ T cell effector activity in successful and failed FME therapy, we identified IL12R, IL18R, and AKT1S1 as crucial for successful FME therapy, potentially linked with the Ras-MEK-ERK pathway, reflecting enhanced T cell signaling pathway activation. In summary, our study found CD8 T cell exhaustion in FCGS and identifies key molecular targets for FME and MSCs treatment.

Our immune system responds to extracellular cues to protect our health by coordinatingnecessary cell functions from cell motility to apoptosis. Engineering signaling pathwayscontrolling these processes is rapidly becoming a powerful approach for developing newbiomedical technology, with cancer immunotherapies serving as a prominent example. However,most current strategies rely on modifying or repurposing endogenous human signaling cascades.This can result in unintended crosstalk between the synthetic signaling pathways and thecomplex native signaling of human cells. Two-component systems (TCS) could address thischallenge and establish a scientific foundation for systematically improving and adding to thenatural abilities of cells to sense and respond to their environment in a robust yet flexiblemanner. With a newly paved avenue for engineering cell communication, researchers coulddevelop strategic signaling cascades in any human cell for their specific research interests.Importantly, TCS are absent in human cells, but present in all three domains of life, and offerdiverse yet simple and linear signaling pathways. In TCS, ligand binding modulates a histidinekinase (HK) sensor, a protein characteristically containing separate ligand binding and HKdomains. When activated, the HK transfers a phosphoryl group to the response regulator (RR),and the phosphorylated RR activates a specific downstream response through mechanismsranging from protein-to-protein interactions to transcription. In its inactive state, the HK acts as aphosphatase. These studies focus on a light-sensitive LOV TCS and a nitric oxide-sensitive HnoTCS as candidate model pathways for modifying mammalian cells. We demonstratedconstitutive activity with the LOV pathway and tested several modifications to achieve lightsensitivity. While some modifications affected pathway output, all tested pathways appeared tohave light-independent constitutive activity.

Recent reports of bovine embryonic stem cell (bESC) establishment and in vitro gametogenesis (IVG) in the mouse open the door to recapitulate oogenesis from bESCs in vitro, however little progress has been made toward this feat. Critical gaps remain in our understanding of bovine germ cell development, which will be necessary to establish a system that supports efficient in vitro germline formation as well as to assess the resulting cells once obtained. Additionally, progress in other models has shown the importance of pluripotent state on cellular capacity for in vitro germline induction. Therefore, the goal of this dissertation was to shed light on the requirements of livestock pluripotency and germline development to set the basis to establish reliable bovine in vitro oogenesis systems.We first examined the pluripotent state of bESC lines and explored the potential to stabilize these cells in intermediate states of pluripotency, due to the limitations of primed ESCs for germline induction. We found that regardless of the initial blastocyst stage that bESCs were derived from, cell lines exhibited similar transcriptional and functional characteristics of primed pluripotency. However, the demonstration that bESCs could be efficiently established from early blastocysts increases the pool of embryos that may be targeted for bESC derivation approaches. We also investigated alternative formative media for bESC establishment, but found that conditions that supported this state in other mammalian species did not translate to cattle. This highlights the unique signaling requirements of bovine pluripotency and the necessity of further studies to establish alternative conditions for bESCs with high germline competence.Specification of primordial germ cells (PGCs), the precursors of oocytes and sperm, occurs during early embryonic development. The first step of IVG is induction of ESCs into PGC-like cells. Despite wide interest in replicating this event in vitro, little was known about PGC specification in the cow. We identified founder PGCs in the posterior epiblast of bovine embryos at day 16, by unique co-expression of conserved early germline proteins SOX17, PRDM1, and TFAP2C, with absent SOX2. We found that PGCs had initiated migration by day 20, at which point they were distributed through the allantois and hindgut endoderm, but were not yet highly proliferative. Single-cell transcriptome analysis of bovine embryos at day 20-22 allowed us to capture a small population of bovine PGCs, and gave insights into early germline development, including acquisition of the germ cell program, repression of somatic marks, and initiation of epigenetic remodeling. Further, we identified candidate cell surface markers for sorting PGCs – or in vitro-induced counterparts – for future experiments. This work provides the earliest observation of PGCs in cattle and demonstrates a conserved program in bovine germline emergence with other species that form as an embryonic disc.We continued from studying PGC specification and migration onset in the embryo through the advanced development of these cells into oocytes in the fetal ovary. Expanding upon a report from day 50, we traced the bovine germline by single-cell RNA-sequencing at days 70, 90, and 120 of fetal ovarian development. This revealed the transcriptional profile of bovine germ cells from gonadal PGCs through germline commitment into oogonia, initiation of meiosis, arrest at prophase I, and development of primordial oocytes within follicles, as well as cell surface marker candidates for each of these stages. Further, we explored the molecular profile of the somatic niche of the ovary and the interactions of these cells with the germline, which gives evidence for signals that could support germ cell development in a culture dish.In conclusion, this dissertation provides detailed and novel insights into bovine pluripotency and germline establishment. The knowledge gained from these studies may help in the development of assisted reproductive technologies of livestock, humans, and endangered ungulates. Moreover, these studies highlight the cow as a valuable model of human reproductive development and offer important context of embryonic/fetal events that cannot be directly studied in humans.

Photosymbioses between cnidarian hosts and Symbiodiniaceae algae enable the success of coral reef ecosystems. Algae remain photosynthetically active in symbiosis capturing light energy to fix CO2, evolve O2, and produce photosynthates which are transferred to hosts as substrate for growth and metabolism. Host cells phagocytose free-living algae and sequester them in arrested phagosomes, termed “symbiosomes,” enabling control over symbionts’ metabolisms promoting the production of photosynthates while regulating symbiont population growth. The underlying cellular mechanisms are mostly unknown and only a V-type H+-ATPase (VHA)-dependent carbon-concentrating mechanism (CCM) is documented in corals. My thesis sought to identify whether the CCM is widespread in cnidarian photosymbioses, establish the CCM’s importance under various environmental conditions, and identify nitrogen transport mechanisms. Chapter 2 describes the cloning of a candidate coral NH3/NH4+ (total ammonia, Tamm) channel (ayRhp1) from Acropora yongei. Heterologous expression in Xenopus oocytes and flux assays determined ayRhp1 is a dual NH3/CO2 gas channel. Immunofluoresence Airyscan confocal microscopy (IACM) revealed ayRhp1 is present in the symbiosome membrane where, together with VHA, it concentrates Tamm in the symbiont’s microenvironment. Furthermore, ayRhp1 is preferentially present in the symbiosome membrane during the day; presumably to provide algae with Tamm to ensure photosynthate production during photosynthesis. Chapter 3 tested whether Cassiopea jellyfish, which host their symbiotic algae in motile cells called amebocytes, employ a CCM like that from coral host cells. Western blotting and AICM revealed that Cassiopea amebocytes express both VHA and carbonic anhydrase (CA) and that both proteins localized to the symbiosome membrane. Respirometry using VHA and CA inhibitors revealed significant reductions in O2 evolution rate, a proxy for photosynthetic activity, suggesting that host VHA and CA are functionally coupled in the CCM. Chapter 4 explored the prevalence of the CCM in the coral Stylophora pistillata after acclimation to various environmental light conditions. While VHA protein abundance correlated with increased irradiance, AICM found limited symbiosomal VHA and respirometry experiments did not detect functional evidence for CCMs. Instead, VHA was prominently observed in calcifying cells, where it may contribute to pH regulation for biomineralization. Together, my thesis advances our mechanistic understanding of nutrient exchange in cnidarian photosymbioses.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer, with von Hippel-Lindau (VHL) gene mutations prevalent in approximately 90% of cases. We have previously demonstrated synthetic lethality between VHL loss and the inhibition of Cyclin-Dependent Kinases (CDKs) in ccRCC, using the multi-CDK inhibitor Dinaciclib. Here, we extend these findings by investigating the specific role of CDK9 in synthetic lethality with VHL loss. CDK9 is a transcriptional CDK involved in the process of transcription elongation and splicing. Using three distinct and specific CDK9 inhibitors (AZD45753, KB0742 and NVP-2), we confirmed preferential toxicity towards VHL-deficient ccRCC cells that acted independently of the hypoxia-inducible factor (HIF) pathway, the main pathway regulated by VHL. An analysis of the current literature connecting inhibition of CDK9 to cancer cell death suggests that the prime mechanism of action is the reduction of CDK9’s target pro-survival proteins, MCL-1 or BFL-1. Interestingly, we found that BFL-1, not MCL-1, is the critical anti-apoptotic protein involved in the mechanism of synthetic lethality. The literature further suggested that CDK9 inhibition might cause immunogenic cell death (ICD). In vivo, immunocompetent and immunodeficient mouse studies using AZD4573 showed that CDK9 inhibition suppressed ccRCC tumor growth, although we did not observe significant changes in immune cell populations upon treatment in an immunocompetent mouse model. At the same time, CDK9 inhibition did not compromise immune system. Our results offer novel insights into the mechanism of CDK9 inhibition / VHL loss synthetic lethality in ccRCC and provide a compelling rationale for further development of CDK9 inhibitors as potential therapeutics in VHL-deficient ccRCC. The BFL-1 pathway, which is a part of the mechanism of AZD4573 action, represents a biomarker for drug sensitivity, paving the way for improved patient stratification and treatment outcomes.

The broader theme of this dissertation is concerned with the molecular mechanismsemployed by neurons to regenerate dendrites after injury, with a special emphasis ondendrite injury detection. Much of the work provides evidence for a fundamental, yetnecessary, question: Does dendrite injury trigger calcium influx and, if so, how necessary isthis early calcium influx for repair? To answer this, I used Drosophila dendritic arborizationneurons of the larval peripheral nervous system as a model. Injuries were performed usingprecise two-photon laser-mediated removal of dendrites in live, intact larvae. Elevations incalcium were measured using the most advanced version of the genetically encoded calciumindicator GCaMP7f and morphological regeneration was assessed using membrane-boundfluorophores. The findings indicate that dendrite injury-induced elevations in calcium maybe cell type- and neurite type-specific. Importantly, shunting injury-induced calcium influxresulted in less, but not abolished, regeneration, suggesting that this early calcium plays asupportive role in dendrite regeneration. Mechanistically, I provided evidence for thepositive role of L-type VGCCs, IP3 signaling, and PKD activity on dendrite regeneration. Inaddition to my research endeavors, this thesis also describes my experience completing agraduate research internship with Eli Lilly and Company in Boston, MA. I then reflect on howthat opportunity made me a better scientist, and briefly discuss the literature on graduatestudent internships. Lastly, I detail the results of some final projects regarding the roles ofhistone deacetylases, protein kinase D, and the neuronal cytoskeleton, in addition to the useof hippocampal neuron culture to study dendrite regeneration in a system that providesunique characteristics distinct from fly sensory neurons.

Autophagy is a fundamental process of cellular quality control and maintenance that is used to protect cells during natural, healthy aging. Defects in autophagy are implicated in a host of pathologies of aging, notably neurodegenerative diseases. The Rubicon Homology (RH) proteins are a class of autophagy-regulating proteins defined by their RAB7A binding RH domains, consisting of the proteins Rubicon, Pacer, and PLEKHM1. These proteins bidirectionally regulate autophagy, as determined by the phosphorylation state of RAB7A.Here, I demonstrate a mitochondrial autophagy (mitophagy) specific form of regulation for RH family proteins. Activation of PINK1 and Parkin in response to mitochondrial damage initiates a response that includes phosphorylation of RAB7A at Ser72. Rubicon is a RAB7A binding negative regulator of autophagy. The structure of the Rubicon:RAB7A complex suggests that phosphorylation of RAB7A at Ser72 would block Rubicon binding. Indeed, in vitro phosphorylation of RAB7A by TBK1 abrogates Rubicon:RAB7A binding. Pacer, a positive regulator of autophagy, has an RH domain with a basic triad predicted to bind an introduced phosphate. Consistent with this, Pacer-RH binds to phosho-RAB7A but not to unphosphorylated RAB7A. In cells, mitochondrial depolarization reduces Rubicon:RAB7A colocalization whilst recruiting Pacer to phospho-RAB7A–positive puncta. Pacer knockout reduces Parkin mitophagy with little effect on bulk autophagy or Parkin-independent mitophagy. Rescue of Parkin-dependent mitophagy requires the intact pRAB7A phosphate-binding basic triad of Pacer. Together these structural and functional data support a model in which the TBK1-dependent phosphorylation of RAB7A serves as a switch, promoting mitophagy by relieving Rubicon inhibition and favoring Pacer activation.The stability of the Rubicon protein has recently been established as a natural regulator of autophagy flux in a variety of tissues (adipose, hepatic, cardiac, and neuronal), where cells will stabilize or degrade Rubicon as a means of inhibiting or promoting autophagy flux respectively. To develop a novel pharmacological inducer of autophagy, I sought to mimic this form of regulation by creating a small molecule heterobifunctional degrader of Rubicon that colocalizes Rubicon with the E3 ligase Cereblon, inducing ubiquitinylation and subsequent degradation.

In order to promote sustained growth and health, the cell must carefully balance its metabolic state, responding to nutrient shortages, acute injury, and altered environment to maintain energetic homeostasis. The machinations of this task are extremely complex, and require extensive sensors and signals to diagnose cellular nutrient state, identify specific issues, and maintain functional balance. Two organelles, the lysosome and the mitochondrion, are central to cellular metabolic processes and host a wide variety of signaling pathways that are used to assess energetic health and capacity. Their coregulation is necessary to support cellular health in instances of energetic stress, and the breakdown of their communication often leads to debilitating degenerative diseases. While much work has been done to understand how the signals and functions of these two organelles overlap during stress, it is less clear what functional changes are initiated by lysosomal signaling itself, and how these may alter mitochondrial function to promote survivability.Lysosomes are the primary catabolic site of the cell, where the autophagic and endocytic pathways converge to deliver cargo for degradation. By this mechanism, the lysosomal lumen reflects the nutrient status of the cellular environment and can use this information to influence the metabolic state of the cell. To this purpose, the lysosomal surface hosts the active state of the kinase mechanistic Target of Rapamycin (mTOR) as part of mTOR Complex I (mTORC1). Here, mTORC1 responds to the presence of insulin, growth factors, and nutrients to promote anabolic processes for cell growth. If any of these inputs are depleted, mTORC1 is rendered inactive and removed from the lysosome, switching the cell to catabolic programming. With this process, mTORC1 is central to maintaining metabolic and energetic homeostasis in the cell.Meanwhile, the mitochondria is the cellular site of bulk metabolite and energetic production. Robust mitochondrial function is required for the survival of high-energy tissues, such as the heart and brain, and is necessary for cellular growth and differentiation. Mitochondrial stress results in depleted energetic supply, toxic redox species, and in some cases, the trigger of apoptotic cell death. Defects in mitochondrial function are thus detrimental to cellular and organismal health. Previous work has established that many signals that reflect mitochondrial stress interact with mTORC1, either directly or by pathway convergence, to coordinate energetic status with whole-cell programming. In many models of mitochondrial disease, coordinated mTORC1 function is required to allow survivability.While we generally understand how mTORC1 interacts with and supports the objectives of mitochondrial stress pathways, what is less clear is how mTORC1 signaling influences mitochondrial function at baseline. Here, I study how acute inhibition of mTORC1 by pharmacological intervention alters mitochondrial function in the absence of induced stress, thereby shedding light on mitochondrial pathways that may be influenced by mTORC1 status. Then, I study how these alterations map to mitochondria who have been injured by depletion of the iron-sulfur cluster biogenesis protein Frataxin (FXN) to identify which pathways may be valuable for mTORC1-supported mitochondrial survival under stress.With functional assays, we identify impaired function of Complex III of the mitochondrial electron transport chain as a result of FXN loss, with a converse restoration when mTORC1 is inhibited. Proteomic analysis of mitochondria in these conditions reveal an alteration of redox homeostasis upon mTORC1 inhibition, irrespective of FXN loss. Further exacerbation of the system by FXN loss then exposes a potential role of mTORC1 signaling for modulation of CoQ biosynthesis machinery, which may represent a newly discovered branch of mTORC1 influence.

The correct function of organisms ranging from bacteria to humans critically depends on the cells’ ability to rapidly change its metabolism through dedicated signaling cascades that transduce signals present in the surrounding environment. Two examples of these signaling cascades: mechanistic target of rapamycin complex 1 (mTORC1) and cAMP-dependent protein kinase A (PKA) work in tangent with cellular processes like autophagy to maintain cellular homeostasis. The two-way regulation between mTORC1 and autophagy is clear but the relationship between autophagy and PKA is more complex. Here I will focus on this complex interaction and propose a model where autophagy is regulating the PKA holoenzyme but also becomes a signaling scaffold for AKAP11-bound PKA. Furthermore, I also talk about the implications of our findings in the context of schizophrenia and bipolar disorder.

Mycobacterium tuberculosis (Mtb) is a bacterial pathogen that establishes a pulmonary infection in the lung, and a quarter of the world’s population is estimated to be latently infected with Mtb. The genetics behind Mtb’s ability to survive in its primary niche, the phagosome of macrophages, is still under investigation. In chapter one, I introduce the history of Mtb forward genetics and discuss how laboratory techniques for forward genetics in Mtb have progressed over time. Then, I discuss mechanisms that are important for Mtb survival in the macrophage, including resistance against oxidative stress, nitrosative stress, and acidic pH. This chapter also reviews how Mtb acquires and metabolizes nutrients, specifically carbon and nitrogen sources.In the second chapter, I present how a prokaryotic organelle, an encapsulin nanocompartment, is important for defense against oxidative stress in Mtb. Encapsulin nanocompartments are a method of compartmentalization in bacteria and archaea that are comprised of a proteinaceous cell surrounding an enzymatic cargo protein. Much of the previous work on encapsulin nanocompartments has been characterizing their biochemical properties rather than elucidating the contribution of nanocompartments to bacterial physiology. We show that Mtb assembles encapsulin nanocompartments containing the peroxidase DyP, which can detoxify hydrogen peroxide. A mutant lacking the DyP nanocompartment in Mtb is attenuated at acidic pH in the presence of hydrogen peroxide, and the DyP nanocompartment mutant is susceptible to pyrazinamide in broth and a murine mouse model.Next, in chapter 3, the investigation of encapsulin nanocompartments is expanded to nontuberculosis mycobacteria, including Mycobacterium smegmatis (M. smeg) and Mycobacterium abscessus (M. abs). M. smeg and M. abs are predicted to encode the DyP nanocompartment in addition to a nanocompartment encoding a cysteine desulfurase (CyD). Genetic deletion nanocompartments in M. smeg and M. abs are largely unable to recapitulate the phenotypes observed in Mtb. However, M. smeg nanocompartment mutants have unique colony morphologies that are not observed in Mtb. Chapter 3 also investigates remaining hypotheses about Mtb nanocompartments, including the ability of Mtb DyP to use lipid peroxides as a substrate. We also explore the possibility that KatG, a catalase peroxidase in Mtb, acts redundantly with Mtb DyP in infection models.In chapter 4, random barcode-transposon site sequencing (RB-TnSeq), a technique for high-throughput forward genetics, is used to uncover the function of unknown genes in Mtb. RB-TnSeq is a pooled, transposon-mutant based method wherein each transposon contains a unique twenty nucleotide barcode, which bypasses steps of laborious protocols used for standard TnSeq screens. We exposed an RB-TnSeq library in Mtb to a chemical library of carbon sources, nitrogen sources, and stress compounds. From the genetic screen data for Mtb growth on carbon sources, we find that the Nuo complex, one of the three NADH dehydrogenases encoded by Mtb, is specifically required for growth on propionate and asparagine. Additionally, we explore the possibility of D- lactate to act as a carbon source for Mtb. Finally, we identify novel mutants that confer resistance to pretomanid, a recently approved tuberculosis antibiotic. Broadening our knowledge concerning Mtb genetics and how the bacterium survives in the face of immunological stressors in vivo is critical for furthering the development of novel bacterial therapeutics.

Aging is the process of the decline of the physiological function of organs, which is thought to arise from both endogenous (i.e., cellular metabolism) and exogenous factors (i.e., air pollutants) that eventually lead to death. However, the exact spatiotemporal mechanisms that cause the decline in organisms are still unknown. Fortunately, extensive research has been done to elucidate how the decline happens and the biological cues that drive the aging process. A common convergence in the field of geroscience is the factors found in the circulatory system and their regenerative and, more commonly, deleterious effects. Pro-rejuvenating factors decline with age, and when replenished to youthful levels, they can restore function to the pathways that are dysregulated due to their depletion. Conversely, factors that have become deleterious due to deregulation can be reduced or inhibited to allow the organ's natural repair mechanisms to function to restore proper organ homeostasis. Both of these strategies may be used to reverse the age-associated decline of organ function and, ideally, extend both lifespan and healthspan, the time we live disease-free. However, considering the upregulation of many factors with age, and the even more numerous pathways disturbed by this upregulation, a more holistic approach would target these broader changes rather than merely attempting to restore, or more often, flood a single factor that decreases with age.Heterochronic blood exchange has emerged as a model to understand how the systemic milieu contributes to the aging process. Numerous individual factors have been discovered to contribute to the aging process when elevated, but the exact mechanism of how the old milieu exacerbates aging is largely unknown. By using a heterochronic blood exchange model, I showed an increase in senescent muscle stem cells in young mice after blood exchange with old mice. Moreover, after delivery of navitoclax, a common senolytic, to the old mouse prior to the blood exchange, the senescence transfer was no longer seen. This was verified using a second senolytic, dasatinib and quercetin. These results reinforce the idea that cellular senescence is not only a response to stress over a lifetime, but can be an immediate response from other senescent cells. Pharmacological approaches for targeting pathogenic cells, such as cancer or senescent, have the capacity to revolutionize biomedicine, but these cells resist apoptosis, and high doses of drugs also harm healthy cells in a patient. It would be very useful to simultaneously eliminate senescent and cancer cells because the senescent niche supports cancer progression, but drugs that target proliferating cells do not work against senescent cells. Approaches for energy starvation of cancer have not yet met with clinical success; moreover, senolytic attributes of this direction are largely unknown, and metabolic similarities between senescence and cancer are not well understood. Here I define the shared metabolic shifts in cancer and senescence, and develop a novel formulation of dichloroacetate, metformin, and 10-fold reduced dose of BCL-2 inhibitor that critically diminishes respiratory spare capacity for ATP synthesis only in the senescent and cancerous cells, allowing their simultaneous targeting without damage to healthy cells. In vivo, this three-drug formulation, DMA, is rejuvenative in old mice, suggesting a broadly available solution to counteract senescence, cancer, and aging effectively and simultaneously without deleterious side effects.

The enzyme rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes the majority of biological carbon fixation on Earth. Although the vast majority of rubiscos across the tree of life assemble as homo-oligomers, the globally predominant form I enzyme—found in plants, algae, and cyanobacteria—forms a unique hetero-oligomeric complex. The recent discovery of a homo-oligomeric sister group to form I rubisco (named form I′) has filled a key gap in our understanding of the enigmatic origins of the form I clade. However, to elucidate the series of molecular events leading to the evolution of form I rubisco, we must examine more distantly related sibling clades to contextualize the molecular features distinguishing form I and form I′ rubiscos. Here, we present a comparative structural study retracing the evolutionary history of rubisco that reveals a complex structural trajectory leading to the ultimate hetero-oligomerization of the form I clade. We structurally characterize the oligomeric states of deep-branching form Iα and I′′ rubiscos recently discovered from metagenomes, which represent key evolutionary intermediates preceding the form I clade. We further solve the structure of form I′′ rubisco, revealing the molecular determinants that likely primed the enzyme core for the transition from a homo-oligomer to a hetero-oligomer. Our findings yield new insight into the evolutionary trajectory underpinning the adoption and entrenchment of the prevalent assembly of form I rubisco, providing additional context when viewing the enzyme family through the broader lens of protein evolution.Proteins form oligomeric complexes with other proteins, enabling a diversity of biological functions. The acquisition of protein-protein interactions between binding partners is generally considered to be the primary driver of oligomerization. However, ligand binding is similarly capable of driving higher-order assembly, though how these interactions contribute to and constrain the evolution of protein complex formation is less well understood. Here, we demonstrate how protein oligomerization can emerge via ligand-protein interactions within an enzyme family. Through phylogenetic sampling of a unique clade of rubisco, we discover a range of oligomeric responses to ligand binding across the phylogeny and identify the structural features driving an observed dynamic shift in oligomeric state. We further identify the critical role of a metal cofactor in governing higher-order assembly. Our results describe the acquisition of a ligand-protein interaction that mediates oligomerization in rubisco, illustrating a means by which stochastic binding and entrenchment of a ligand enables an increase in complexity.

Membrane potential (Vm) is a critical biophysical property in electrically excitable and non-electrically excitable cells. Established methods for measuring Vm successfully report the large, rapid changes that trigger key signaling pathways in electrically excitable cells but lack the capabilities to record over extended time periods or reliably quantify smaller changes in Vm. Emerging evidence suggests that smaller fluctuations in membrane potential occurring over seconds to hours may have a regulatory role in fundamental physiological processes in non-electrically excitable cells, such as cell cycle progression. However, robust characterization of membrane potential dynamics and possible mechanisms of bioelectric control has been limited by insufficient techniques for quantifying Vm over these physiologically relevant timescales. Fluorescence lifetime imaging with VoltageFluor dyes (VF-FLIM) provides a new approach for making minimally invasive and high-throughput measurements of absolute membrane potential over extended timescales.This dissertation demonstrates the application of VF-FLIM to quantify membrane potential and track changes in non-electrically excitable cells. Chapter 1 introduces techniques for measuring membrane potential and summarizes evidence for membrane potential dynamics and bioelectric signaling during cell cycle progression. Chapter 2 presents initial work using VF-FLIM to identify differences in membrane potential across cell cycle phases, revealing a ~10 mV depolarization in G2/M phase cells compared to cells in G1 and S phase. Chapter 3 expands on this work and describes efforts towards monitoring real-time changes in Vm in live cells with development of a stable cell line expressing the sensor DHB-mVenus to track cell cycle progression, combined with a red-shifted VoltageFluor. Chapter 4 explores characterization of membrane potential dynamics induced by epidermal growth factor receptor signaling (EGFR), spanning four different EGFR ligands across two cell lines showing disparate responses to EGFR stimulation. A general protocol for VF-FLIM experiments in provided in Chapter 5, and Chapter 6 details essential considerations for data acquisition and analysis. Together, this work highlights the utility of VF-FLIM as an approach for optically recording Vm over extended time scales and offers new insights into membrane potential dynamics during cell cycle progression and epidermal growth factor receptor signaling in non-electrically excitable cells.

This dissertation aims to bring new perspective to our understanding of the growth, development, and evolution of the vestiture which adorns the wings of the Lepidoptera—butterflies and moths. This perspective is wrought from both advancing imaging techniques to observe the growth of these elaborate cell-secreted structures directly and through examining parallel trends in the scales and bristles of their sister order, the Trichoptera—caddisflies. Firstly, I develop methods to observe at high resolution the organization, arrangement, and dynamics of the cytoskeleton and cellular organelles through early scale development using commercially available live cell dyes. These protocols permit observing any timepoint of wing growth for any butterfly and moth hindwing, and any wing surfaces of insect species with transparent pupal cuticle. These techniques help confirm that scales grow through actin filament elongation and subsequent bundling into thick rods and allow us to observe nuclear divisions and lineages of sensory organ precursors across species. Secondly, I correlate patterns of microtubule and actin filament distribution across scale types throughout the lepidopteran phylogeny and make an association between reticulated microtubule networks and flattened scale morphologies. I then identify a transcription factor that seems to specify a cylindrical, non-flattened, scale shape fate and tie this into the evolution of transparency and specialized, elongated androconial scales. Using live-imaging of microtubule networks across normal and drug-perturbed development, I connect these cytoskeletal and transcription factor data to hypothesize that this gene initiates a cellular program to stabilize microtubules, preventing their transition to a reticulate network and producing cylindrical cell shapes and that this fate switch has been used in the evolution of transparent wing patches and scent-dispersing scales throughout lepidopteran evolution. Lastly, I develop tools, including a genome, to study a genus of long-horned caddisflies, Nectopsyche, that has independently evolved scales resembling those of butterflies and moths, and present phylogenetic and morphological data to contextualize this convergence with our current knowledge of macrochaete cytoskeletal organization and the evolution of wing patterns. Altogether, this document establishes a first step in producing an understanding of comparative growth dynamics of amphiesmenopteran macrochaetae with an eye to uncover general trends linking cytoskeletal structure and cellular morphology.

The evolutionary transition to complex multicellularity in the animal stem lineage remains a profound mystery. To gain insights into this puzzle, I focused on choanoflagellates, the closest living relatives of animals. Despite their significance, understanding of the environmental factors influencing choanoflagellate evolution, life history, and behaviors remains limited. My doctoral research focused on the characterization of Choanoeca flexa, a recently discovered colonial choanoflagellate. This enigmatic microorganism is an example of how, by exploring the diversity of choanoflagellates, we can uncover novel biological phenomena that help us reconstruct the biology of animal progenitors and better understand the origins of animals.Chapter 1 reviews the history of C. flexa as an emerging model organism. C. flexa forms monolayered colonies capable of reversible inversions between flagella-in and flagella-out conformations, each with distinct multicellular behaviors. The colony inversion of C. flexa mirrors aspects of embryogenesis and behavioral escape responses in animals. Thus, this system offers a unique platform to investigate the evolutionary mechanisms underlying the origins of animal multicellular development and behavior. Additionally, its reliable presence in splash pools of the Caribbean island of Curaçao provides an opportunity to study its ecology, making C. flexa an appealing and promising model for eco- and evo-cell biology. For the work in Chapter 2, I collaborated with Nuria Ros-Rocher (Pasteur Institute) to characterize the life history of C. flexa and its environmental influences. We discovered that C. flexa exhibits ‘clonal-aggregative development’, forming colonies through clonal division, aggregation, or a combination of both. Field and laboratory investigations revealed that C. flexa multicellularity is entrained by extreme salinity fluctuations typical of splash pools undergoing evaporation-refilling cycles. Upon gradual evaporation, colonies dissociate into solitary desiccation-resistant cells, which reform colonies after rehydration. We proposed that C. flexa clonal-aggregative development may offer a rapid and versatile route to multicellularity, which can be particularly advantageous in ephemeral habitats like splash pools. This discovery blurs the line between clonal and aggregative multicellularity and expands our notion about the evolutionary trajectory leading to animals. Finally, in Chapter 3, I demonstrate how studying choanoflagellate multicellular behavior can shed light on ancient animal signaling pathways. Specifically, I explored nitric oxide (NO) signaling in choanoflagellates and found that C. flexa and two other species express genes for canonical animal NO signaling. In C. flexa, NO induces colony inversion, partially mediated by the secondary messenger cGMP, similar to animals. Moreover, the behavioral output of NO signaling in C. flexa – contraction and regulation of feeding and swimming – parallels the effect of NO in certain animal groups. This discovery suggests that NO/cGMP signaling predates animals and may have regulated similar behaviors in their ancestors. Together, these findings highlight the potential of choanoflagellates, particularly C. flexa, as a valuable system for unraveling the mysteries of multicellularity and animal evolution.

Translocase of outer mitochondrial membrane 40 (TOMM40, aka TOM40) is the major channel-forming subunit of the translocase of outer mitochondrial membrane (TOM) complex that regulate mitochondrial function by importing nuclear-encoded proteins. TOMM40 is located at mitochondria-endoplasmic reticulum contact sites (MERCs), which play a key role in cellular signaling, with known effects on mitochondria bioenergetics and lipid synthesis. Furthermore, we have used RNA-seq to assess global transcriptional response to statin exposure in immortalized lymphoblastoid cell lines (LCLs) derived from participants in a clinical trial who were treated with 40mg/day of simvastatin for 6 weeks. Our previous studies using this model system identified a number of novel genes affecting cholesterol metabolism and in vivo statin LDL (low-density lipoprotein) cholesterol response, including TOMM40. Statins are the drugs most commonly used for lowering plasma LDL cholesterol and cardiovascular disease risk. Although generally well tolerated, statins can induce myopathy, a major cause of non-adherence to treatment. Cellular studies have identified statin-induced myopathy to be associated with impaired mitochondrial function and morphology. In Chapter 2, we show that TOMM40 and TOMM22 play a key role in mediating statin-induced mitochondrial dysfunction as well as disruption of mitochondrial dynamics in skeletal myotubes. These findings suggest a novel mechanism by which adverse effects of statin treatment on mitochondrial dynamics that may contribute to the development of statin-induced myopathy are mediated by reduced TOMM40 and TOMM22 gene expression.Beyond the mitochondria, we explore the role of TOMM40 on regulation of lipid metabolism via MERCs. Fundamentally, the gene encoding TOMM40 is adjacent to that for APOE, a protein with central functions in lipid and lipoprotein transport. Recent GWAS studies have shown that a number of SNPs in linkage disequilibrium between these two genes are significantly associated with plasma lipid levels. While these associations have generally been attributed to APOE, no previous studies have addressed the possibility that TOMM40 has an independent effect on lipid metabolism. In Chapter 3, our investigation of molecular mechanisms underlying the effects of TOMM40 knockdown on lipid metabolism has revealed a number of novel findings, including a key role for the LXR transcription factor due both to transcriptional upregulation and increased activation by oxysterols that are generated by mitochondria-induced reactive oxygen species. Interestingly, the increased LXR activity also results in reduced intracellular cholesterol via both upregulation of the membrane cholesterol transporter ABCA1 and increased production and secretion of oxysterol-derived cholic acid. Finally, and importantly, we have shown using a number of experimental approaches that the LXR-mediated effects described above result from disruption of mitochondria-ER contact sites (MERCs), where TOMM40 plays a key role, and that lipid droplet contact sites are consequently shifted from the ER to mitochondria. Overall, these findings demonstrate TOMM40 plays a key role in connecting mitochondrial function and lipid metabolism.

Sexual reproduction relies on meiosis, a specialized cell division process that produces haploid gametes. Successful segregation of homologous chromosomes during meiosis requires the pairing of homologs, synapsis, and crossover recombination during meiotic prophase.Upon initiation of meiosis, in many organisms, chromosome ends establish connections with the nuclear envelope (NE) and interact with motor proteins that drive processive movements along the NE, facilitating homolog chromosome pairing and synapsis. In C. elegans, specialized chromosome regions known as “pairing centers” (PCs) serve this role. To understand the roles of PCs in C. elegans meiosis, I exploited a genetic screen and identified a novel protein required for homolog pairing and synapsis. Chapter 1 focuses on this newly discovered NE protein, MJL-1, which is essential for homolog pairing and the regulation of synapsis at the PCs. Chapter 2 describes my efforts to develop methodology for genome-wide profiling of chromosome-interacting proteins in the C. elegans germline, leading to new insights into chromosome organization during meiosis. Through this work, I have discovered correlations between the genome-wide distribution of chromosome axis proteins, active chromatin marks, and a protein required for meiotic double-strand breaks (DSBs), DSB-2, during meiosis in C. elegans.

Stem cell differentiation is key to plant development, which utilizes asymmetric cell divisions (ACD) to maintain stem cells, promote growth, and encourage cell diversity. Previous work in Arabidopsis thaliana elucidated some pathways controlling stem cell dynamics during morphogenesis. In the leaf epidermis, pre-mitotic, oriented nuclear migrations in meristemoids, a transient stem cell in Arabidopsis thaliana, allow creation of daughter cells with differing sizes, fates, and placements. Pre-division, the nucleus uses microtubules to migrate away from a polarity crescent that controls division orientation. Post-division, it is known that the nucleus orients towards the polarity crescent using F-actin, but precise mechanisms, including relevant nucleators, are unknown. Additionally, while behavior of the nucleus during ACDs in meristemoids has been characterized, cytoskeletal regulators linking the nucleus to F-actin remain unknown. Time-lapse imaging of Arabidopsis seedlings treated with an F-actin Arp2/3 complex inhibitor (CK-666) and a control compound (CK-689), revealed that Arp2/3 complex-mediated F-actin controls nuclear migration post-division. Novel regulators of nuclear migration were found by phenotypic characterization of stomatal formation in loss-of-function mutants in protein components linking the cytoskeleton to the nuclear envelope. We found three candidate proteins (CRWN1, CRWN3, and SINE1) decreased stomatal density, while one (SUN1) increased stomatal density. This suggests ACD frequency is altered in these mutants and identifies new factors to probe in future studies. These candidate proteins, SUN1, CRWN1, CRWN3, and SINE1, may control nuclear migration in Arabidopsis thaliana, alongside Arp2/3 complex-mediated F-actin. Determining mechanisms behind nuclear migration gives insight into how organelle positioning controls ACDs and stomatal development.

Stem cell biology has enabled the development of 3D organ-specific in vitro models that mimic aspects of in vivo tissue. The analysis of tissue cultures, particularly brain organoids, takes a high degree of coordination, measurement, and monitoring. Here, I have developed a novel microfluidic platform that automates the culture of individual organoids in isolated microenvironments at user-defined flow rates. This technology has enabled greater homeostatic regulation of culture's media through frequent, low-volume replenishment cycles. RNA sequencing (RNA-seq) analysis of automated cerebral cortex organoid cultures showed benefits in reducing glycolytic and endoplasmic reticulum stress compared to conventional in vitro cell cultures. For the first time, longitudinal metabolic profiles tracing glucose and lactate over 105 days of culture were achieved for six cerebral cortex organoid protocols.The microfluidic technology was advanced to integrate electrophysiology and imaging into a unified system for feedback-driven studies. An Internet of Things (IoT) architecture was developed to enable continuous, communicative interactions among sensing and actuation devices, achieving precisely timed control of biological experiments. Computer vision for fluid volume estimations of aspirated media was used as feedback to rectify deviations in microfluidic perfusion during media feeding/aspiration cycles. The system performed 7-day studies of mouse cerebral cortex organoids, comparing manual and automated protocols. The automated experimental samples maintained robust neural activity throughout the experiments; however, the system enabled hourly electrophysiology recordings that revealed dramatic temporal changes in neuron firing rates not observed in once-a-day recordings.

Microbial methane utilization (also known as methanotrophy) stands out as an exceptional form of chemoorganotrophy, which requires a set of unique cellular machineries (membrane compartments and metabolic pathways) fully dedicated for energy recovery from the simplest organic compounds (methane or methanol) and reconstruction of all cellular parts from a single carbon unit. Despite significant progress in understanding fundamentals of methanotrophy, several critical elements remain unresolved. The most critical knowledge gaps include the lack of mechanistic understanding of methane oxidation, such as the source of electrons for the copper dependent methane monooxygenase (pMMO), the plasticity of metabolic pathways, and their dynamic interplay modulated by metal availability. These elements need to be thoroughly understood before they can be established as a chassis organism for biomanufacturing. To solve these mysteries, the research in this thesis is presented in three chapters, each dedicated to different cellular and molecular aspects of the methane metabolism using Methylotuvimicrobium alcaliphilum 20ZR as a model methanotrophic organism. The first chapter of my thesis is focused on characterization of methane oxidation machinery in the strain 20ZR at a cellular level. Exploration into this machinery was performed by a series of biochemical studies with strains that lack different elements of the methane oxidation network, such as methanol dehydrogenase (MDH), formate dehydrogenase (FDH), and downregulation/ expression of particulate methane monooxygenase (pMMO) proteins. This is followed by the second chapter of the thesis, which investigates cellular function of the surface layer proteins (SLPs). I performed comparative genetic analysis, knockout studies and a series of phenotyping tests complemented by -omics studies to gain insights into the SLP function. The data revealed that the surface layer protein matrix not only stabilizes the cell envelope at suboptimal conditions, but also contributes to metal scavenging.The last chapter of my thesis is dedicated to evaluating the feasibility of utilizing SLPs from methanotrophic bacteria for biomanufacturing. I developed an array of genetic tools to functionalize the surface layer proteins and use them as a platform to display heterologous proteins and peptides. The identification and utilization of surface layer proteins in methanotrophic bacteria introduces a novel approach for biomining.

Desmoplakin (DSP) is a critical protein component of desmosomes, specialized cell structures that function as intercellular junctions and are essential for maintaining the structural integrity of cardiac muscle cells. Mutations or loss of DSP are linked to arrhythmogenic right ventricular cardiomyopathy (ARVC), a genetic cardiac disease characterized by ventricular arrhythmias, fibrofatty myocardial replacement, and sudden cardiac death. ARVC patients with DSP mutations often show a unique form of cardiomyopathy associated with myocarditis, further complicating the clinical presentation of this disease.In my studies, I have investigated the therapeutic potential of restoring Connexin-43 (Cx-43), a key gap junction protein, through gene therapy in a novel mouse model of myocarditis-driven DSP cardiomyopathy. Utilizing a cardiac-specific DSP heterozygous mice (DSP Het) invoked with inflammatory stress, I have assessed the effects of adeno-associated virus (AAV9) mediated Cx-43 gene therapy on electrical, structural and cardiac function. I hypothesized that restoring Cx-43 levels will reduce myocardial inflammation indirectly by stabilizing DSP levels, delaying the progression of ARVC, and improving the electrical and mechanical integrity of the heart.My thesis studies have shown that Cx-43 restoration stabilizes electrical, mechanical, and structural function of the heart, and improves the levels of desmosomal proteins, particularly DSP. Electrocardiogram (ECG) and magnetic resonance imaging (MRI) analyses showed notable improvements in electrical conduction and cardiac function, respectively, in the treated compared untreated (diseased) group. Histological examinations revealed that treated mice showed no signs of fat or fibrosis, indicating a structural improvement. In conclusion, these findings suggest that Cx-43 gene therapy mitigates the adverse effects of inflammatory mediated DSP deficiency, providing a promising therapeutic strategy for myocarditis driven ARVC in patients with DSP mutations. This research advances our understanding of the molecular mechanisms linking DSP deficiency to myocarditis, highlighting the potential for developing targeted therapies for ARVC patients with DSP mutation.

Approximately 30% of epilepsy patients experience intractable epilepsy, where traditional pharmacological therapies fail to provide seizure relief. Although challenging to sustain, patients often rely on the high-fat, low-carbohydrate ketogenic diet (KD) as a viable alternative for managing seizures. Despite its clinical success, the exact anti-epileptic mechanisms of the KD, remain unclear. Mitochondria have emerged as central players in this mechanism, with studies highlighting them as both targets through which the KD protects against seizures and key regulators of neuronal health and excitability. This research centers on leveraging human cerebral organoids (COs) and CRISPR-Cas9 genome editing to develop an in vitro epilepsy model. Utilizing advanced lattice light-sheet microscopy (LLSM), this study captures live 4D (x, y, z, time) morphological and dynamic data of mitochondria in COs, allowing for a comprehensive analysis of mitochondrial network behavior and its response to KD treatment. The study aims to elucidate the mechanisms through which the KD modulates mitochondrial structure and function, potentially guiding the development of novel therapeutics for drug-resistant epilepsy. By integrating cutting-edge in vitro human neural tissue models, CRISPR-Cas9 genome editing, and innovative live tissue 4D microscopy techniques, this thesis seeks to advance our understanding of the KD's anti-seizure mechanisms and contribute to the broader field of epilepsy research.

Faithful cell division is crucial for all eukaryotic cells to ensure that daughter cells receive thecorrect number of chromosomes. Errors in chromosome segregation during meiosis in germcells can result in aneuploid gametes, potentially leading to infertility, genetic disorders likeDown Syndrome, and miscarriages. Similarly, chromosome segregation errors during mitosiscan produce aneuploid somatic cells, which are often associated with cancer. Although theregulation of these two types of cell division is vital, the mechanisms that ensure theiraccuracy are still actively being studied. Among the proteins identified as key regulators ofboth mitotic and meiotic fidelity in various species, including Caenorhabditis elegans (C.elegans), is PCH-2. PCH-2 is a conserved AAA+ ATPase required for timely coordination ofthe meiotic prophase events pairing, synapsis and recombination, but the mechanism for howit accomplishes this coordination is currently unknown. Additionally, PCH-2 has been shownto promote crossover formation and affect their distribution. Here I use C. elegans as a modelsystem to investigate the conserved meiotic role of PCH-2.In Chapter 1, I begin my investigation of PCH-2’s conserved role by observing it’s localizationto meiotic chromosomes in the mnT12 background, where two chromosomes, X and IV, arefused. In order to fully understand how defects in chromosomal rearrangements can affectPCH-2’s localization, I also studied its localization in heterozygous inversions, mIn1/+ andhIn1/+. I find that PCH-2 localization is extended in the chromosomal fusion backgroundmnT12, but not in mIn1 heterozygotes and hIn1 heterozygotes. I also find that the extensionof PCH-2 in mnT12 worms results in the promotion of crossover formation, providing anotherexample of how PCH-2 can affect crossover formation.In Chapter 2, I demonstrate that PCH-2 regulates the number and distribution of crossoversby opposing their formation. This antagonistic action has distinct effects at various stages ofmeiotic prophase. I show that in the early stages, PCH-2 restricts the conversion of doublestrand breaks (DSBs) into crossovers, thereby limiting crossover events at the initial sites ofDSB formation and homolog interactions. And then later in meiotic prophase, PCH-2 reducesthe number of crossover-eligible intermediates, aiding in the reinforcement and designation ofthese intermediates, and ultimately ensuring crossover assurance. Additionally, I show thatPCH-2 exerts this control through the meiotic HORMAD protein, HIM-3. The data I presentstrongly support a model where PCH-2's conserved function is to remodel meiotic HORMADsthroughout meiotic prophase, destabilizing crossover-eligible precursors, coordinating meioticrecombination with synapsis, and contributing to the progressive execution of meioticrecombination to ensure precise crossover regulation.Finally in Chapter 3, I test PCH-2’s role on modulating synapsis. Specifically by observinghow the loading and unloading of a central region component of the synaptonemal complex(SC) is affected in the absence of PCH-2. I show that loss of PCH-2 does not affect how thisprotein – SYP-3, is loading onto chromosomes during meiotic prophase.Added together, my work establishes a crucial framework for understanding the role of PCH-2in regulating the number and distribution of crossovers (Chapter 1 and Chapter 2), a functionI propose is conserved across species. While specific mechanisms may differ amongsystems (Chapter 3), I suggest that PCH-2 plays a key role in remodeling meiotic HORMADsthroughout meiotic prophase to destabilize crossover-eligible precursors. This processcoordinates meiotic recombination with synapsis, contributing to the stepwise progression ofrecombination and ensuring crossover assurance, interference, and homeostasis.

Changes to chromatin structure, including both epigenetic modifications and structural variants, are recurrent in many cancer types and can be the primary tumor driver. These variants are difficult to detect through traditional short-read sequencing and often remain under-characterized, especially in relation to their functional impacts on the transcriptome. With recent improvements in long-read sequencing, it has become possible to detect these alterations with a single sequencing run with greater accuracy and context than before. I have developed new software methods for that identify chromatin modifications with greater accuracy from long-read DNA sequencing and applied these methods to identify chromatin accessibility and transcriptomic alterations resulting from the perturbation of chromatin remodelers in Saccharomyces cerevisiae as a model system. I have also developed a software pipeline to integrate detection of gene fusions, SNVs, mid-sized insertions and deletions, and alternative splicing from long-read RNA-seq, resulting in a comprehensive characterization of functional alterations in cancer-relevant genes. I apply these methods to three cell lines derived from patients with osteosarcoma, a tumor that is driven by poorly characterized and difficult to detect structural variants. I identify multiple functional alterations in these tumors with potential therapeutic implications. This work provides a more complete understanding of the many layers of gene regulation in cancer cells.

A bipolar spindle functions to segregate chromosomes during cell division in all eukaryotes, yet spindle size and microtubule organization vary dramatically across different species and cell types. Targeting protein for Xklp2 (TPX2) is one candidate factor for modulating spindle architecture through its roles in branching microtubule nucleation, activation of the mitotic kinase Aurora A, and association with the kinesin-5 (Eg5) motor. Xenopus egg extracts provide an ideal system to study the role of TPX2 in defining spindle architecture since in vitro reactions combining metaphase-arrested extracts and sperm nuclei recapitulate meiotic spindle assembly and can be biochemically manipulated. Here, we characterize a conserved nuclear localization sequence (NLS) motif, 123-KKLK-126, in X. laevis TPX2, which regulates astral microtubule formation and spindle pole morphology in Xenopus egg extracts. Addition of recombinant TPX2 with this sequence mutated to AALA dramatically increased spontaneous formation of microtubule asters and recruitment of phosphorylated Aurora A, Eg5 and pericentrin to meiotic spindle poles. TPX2 is frequently overexpressed in aggressive human cancers, and it is not well understood how overexpression of TPX2 affects spindle assembly and function. We analyzed the spindle morphology of human cell lines and showed that cells over-expressing the oncogene MYC and TPX2 possess shorter spindles with increased TPX2 localization at spindle poles. These results indicate that TPX2 alters spindle length and morphology in cancer cells, which may contribute their ability to divide despite MYC-induced mitotic stress and are consistent with previous observations in Xenopus egg extract showing that high levels of TPX2 shrink meiotic spindles. We propose that TPX2 is a lynchpin spindle assembly factor that contributes to the recruitment and activity of microtubule nucleation factors, regulating spindle size and generating distinct spindle architectures.

Rickettsia parkeri are obligate intracellular, arthropod-borne pathogens that are classified within the spotted fever group (SFG) of Rickettsia species. R. parkeri are capable of causing eschar-associated spotted fever rickettsiosis in humans. SFG Rickettsia infection of humans occurs when a human is bitten by an infected tick. In the human host, Rickettsia invade host cells, escape from vacuoles, and undergo two modes of actin-based motility mediated by either the effectors, RickA or Sca2. RickA-motility occurs during early timepoints of infection (5 hrs) and alone, is sufficient to polymerize bundled actin filaments against the bacterial surface. The forces generated by actin polymerization against the bacterial surface propels bacteria in the host cytosol. Sca2-motility has been implicated in cell-to-cell spread, a process that allows bacteria to infect nearby cells without being exposed to the extracellular environment via short protrusions of the host plasma membranes. The role of RickA-motility has been unclear. Using live-cell imaging of sca2::Tn mutant R. parkeri, which only undergo RickA-motility, we discovered a role for RickA-motility in cell-to-cell spread. Indeed, bacteria undergoing RickA-motility were observed to collide against host plasma membranes to form long protrusions that extended into and were engulfed by neighboring recipient cells. We compared the dynamics of RickA-spread to Sca2-spread and discovered that RickA-spread involves longer protrusions, longer time, and larger fluctuations in length during periods of oscillation, suggesting that RickA-motility and Sca2-motility contribute to two mechanistically different forms of cell-to-cell spread. We lastly assessed the role of RickA and Sca2 in pathogenesis during infection of Ifnar1-/-; Ifngr1-/- double knock out (DKO) mice, which can succumb to infection with wild-type R. parkeri and form eschars following intradermal inoculation. We discovered that RickA is involved in eschar formation while Sca2 contributes to larger foci of infection in skin, as well as dissemination from the skin into the internal organs. Overall, our results suggest that Rickettsia undergo two different forms of cell-to-cell spread during their infectious lifecycle and that these forms of spread contribute to different processes in pathogenesis. Further investigations on the molecular mechanisms of RickA-spread and Sca2-spread will improve our understanding of how bacteria can highjack host processes to support their infectious lifecycle; and continued studies of R. parkeri using the Ifnar1-/-; Ifngr1-/- DKO mice will expand our knowledge of rickettsial pathobiology.

One of the largest obstacles in modern drug discovery is that a significant portion (>90%) of the proteome is considered “undruggable”, in that these proteins lack a characterized, functional binding pocket or “ligandable hotspot” which small molecules can bind to and modulate the protein’s function for therapeutic benefit. To overcome such disease-causing proteins, targeted protein degradation (TPD) strategies have arisen, where the cell’s endogenous degradation machinery is hijacked to ubiquitinate and degrade the classically undruggable protein. Molecular glue degraders serve as a promising modality to achieve TPD. These are monovalent compounds that induce the proximity of a target protein with a component of the ubiquitin proteasome system to degrade the protein of interest. The systematic and modular synthesis of such small molecules, however, has not been possible due to a lack of known rational chemical design principles for converting protein-targeting ligands into molecular glue degraders. This dissertation discusses the elucidation of novel design principles for the synthesis of covalent molecular glue degraders through the discovery of a transplantable chemical moiety that converts protein-targeting ligands into monovalent degraders of their corresponding targets. We first discovered a covalent handle which, when appended to the CDK4/6 inhibitor ribociclib, resulted in the degradation of CDK4 in a proteasome-dependent manner. Structural optimization of the initial covalent handle led to the synthesis of a but-2-ene-1,4-dione (fumarate) moiety that induced a more potent dose-responsive degradation of CDK4. Utilizing quantitative chemoproteomic platforms to profile global cysteine reactivity throughout the proteome, we identified that the RING-family E3 ligase RNF126 was binding covalently to the monovalent CDK4 degraders. Transplanting this covalent fumarate handle onto various inhibitors and protein-targeting ligands allowed for the degradation of protein targets BCR-ABL and c-ABL, PDE5, SMARCA2/4, LRRK2, BRD4, BTK, HDAC1/3, and AR and AR-V7. We have identified a first-in-class minimal covalent chemical handle that can convert protein targeting ligands into molecular glue degraders of their corresponding targets across diverse chemical and protein classes.

Lipid droplets (LDs) are endoplasmic reticulum-derived neutral lipid storage organelles that consist of a core of neutral lipids bounded by a phospholipid monolayer decorated with associated proteins. The canonical role of LDs is to function as a lipid storage depot, and the accumulation of enlarged LDs is the pathological hallmark of numerous prevalent metabolic diseases, such as obesity and metabolic dysfunction-associated steatotic liver disease (MASLD). Recent findings also implicate LDs in a growing number of surprising cellular roles such as preventing lipotoxicity in neurons, increasing the bioactivation of hydrophobic drugs, and suppressing bacterial invasion as part of a protective innate immune response. Despite these important cellular functions, many details surrounding LD biogenesis and breakdown are incompletely understood. Furthermore, while it is appreciated that the regulation and function of LDs must drastically change under starvation, overnutrition, inflammation, and other altered environments, the pathways that govern these responses remain mostly unexplored.To address this gap in knowledge, we performed a series of CRISPR-Cas9 screens under different metabolic states to uncover mechanisms of hepatic neutral lipid flux. Clustering of chemical-genetic interactions identified CLIC-like chloride channel 1 (CLCC1) as a critical regulator of neutral lipid storage and secretion. Loss of CLCC1 resulted in the buildup of large LDs in hepatoma cells and knockout in mice caused liver steatosis. Remarkably, the LDs are in the lumen of the ER and exhibit properties of lipoproteins, indicating a profound shift in neutral lipid flux. Finally, remote homology searches identified a domain in CLCC1 that is homologous to yeast Brl1p and Brr6p, factors that promote the fusion of the inner and outer nuclear envelopes during nuclear pore complex assembly. Loss of CLCC1 lead to extensive nuclear membrane herniations, consistent with impaired nuclear pore complex assembly. Thus, we identify CLCC1 as the human Brl1p/Brr6p homolog and propose that CLCC1-mediated membrane remodeling promotes hepatic neutral lipid flux and nuclear pore complex assembly.One of the other novel genes from these screens is the interleukin IL32, a secreted pro-inflammatory cytokine that the Olzmann Lab unexpectedly identified as an LD protein using proximity labeling proteomics. Our data demonstrate that endogenous IL32 localizes to a subset of LDs following the induction of host defense signaling pathways, suggesting that IL32 may be a key host factor that acts to regulate LDs during an immune response. These findings not only lay a groundwork for elucidating the IL32-mediated mechanisms of LD-bacteria interactions, creating a foundation for an understanding of how targeting lipid storage can be exploited to halt infectious disease pathogenesis, but also highlight the importance of LD heterogeneity in a variety of cellular functions. Together, these studies provide an extensive compendium of lipid storage modulators and provide rich mechanistic insights into lipid droplet flux and LD heterogeneity in the liver.

The high turgor pressure across the plasma membrane of yeasts creates a requirement for substantial forces, produced by polymerization of an actin filament network, for clathrin-mediated endocytosis (CME). Endocytic internalization is impeded in the absence of fimbrin, an actin filament crosslinking protein called Sac6 in budding yeast. In the first chapter of this dissertation, I use live-cell microscopy to gain new insights into the role of actin filament crosslinking proteins in force generation. Quantitative measurement of the numbers of fimbrin and transgelin molecules at sites of CME reveals an interplay between recruitment of these two actin crosslinking proteins. Manipulation of turgor pressure shows that sites with more fimbrin are more effective at internalization under high load. Super-resolved microscopy of actin patches in fixed yeast reveals that sites with more fimbrin also have a higher density of actin filaments, implicating crosslinking proteins in assisting in force generation by the actin network during CME under increased load.In Chapter 2, I use simulations of an experimentally constrained, agent-based mathematical model of CME to recapitulate the result that endocytic networks with more double-bound fimbrin internalize the plasma membrane against elevated turgor pressure more effectively. Networks with large numbers of crosslinks also have more growing actin filament barbed ends at the plasma membrane, where the addition of new actin monomers contributes to force generation and vesicle internalization. These results provide a richer understanding of the crucial role played by actin filament crosslinking proteins during actin network force generation, highlighting the contribution of these proteins to the self-organization and force generation of the actin filament network.The myosin-Is, Myo3 and Myo5 in budding yeast, are also implicated in force generation and assist in assembly of the actin network during CME. The myosin-Is consist of a motor domain, a membrane binding tail homology 1 (TH1) domain, and an Src homology 3 (SH3) domain that works in concert with a central acidic (CA) region to activate the Arp2/3 complex and promote branched actin assembly. In the third chapter of this dissertation, I examine the ability of Myo5 domain mutant proteins to complement each other in diploid budding yeast. Through a growth assay and live cell microscopy, I reveal that the force generating motor domain and NPF activity from the SH3 domain of Myo5 each must be coupled to the membrane binding TH1 domain for successful progression of CME. However, motor and NPF activity are modular and separable, providing interesting insight into the function of the essential myosin-I in actin network assembly and force generation during budding yeast CME.

To date, neurodegenerative diseases remain with lackluster treatment options. Diseases such as Alzheimer’s are defined based on proteinopathic inclusions, and these inclusions have been the targets of most therapeutic development. The premise that simply addressing these inclusions will be sufficient for therapeutics is poorly supported. Here, I attempt to build an evidence base using selective vulnerability as a substrate to work towards more biologically informed disease-modifying therapeutics. First, I examine the physiological underpinnings of a potent risk factor for dementia and other age-related diseases – social isolation. In humans, social isolation has well-established epidemiological associations with dementia, cardiovascular disease, immune system dysfunction, metabolic disorders, and mental health disorders. Mice exposed to long-term social isolation are studied across several organ systems. Associations with the stressor are detected in renal structure and blood-brain-barrier dysfunction; however, I find that mice are a poor model of social isolation.Next, I examine a potential mechanism underlying why neurons with tau proteinopathic inclusions die. The best correlate of functional decline in neurodegeneration is neuron loss, so focusing on processes of neuron loss might confer better therapeutics. Here, I find that the Ras GTPase, Rhes, is dysregulated in neurons harboring tau proteinopathic inclusions in sporadic tauopathies. This study reinforces in vitro and in vivo work done in models of familial tauopathy demonstrating that a farnesyltransferase inhibitor may suppress neurodegeneration in tauopathies. I then approach selective vulnerability by focusing on the neuromodulatory subcortical system. Parts of the neuromodulatory subcortical system are affected early in Alzheimer’s disease and feature a wide gradient of selective vulnerability and resilience. Here, I find pathways related to cholesterol homeostasis and stress hormone signaling to the noradrenergic locus coeruleus that differentiate it from the more resilient substantia nigra. I close by focusing on the epistemological frameworks used to study neurodegenerative disease and comment on how a focus on selective vulnerability may drive significant paradigm changes in the field. Overall, this dissertation provides evidence of pathophysiology that may drive neurodegenerative diseases and adverse outcomes of stress disorders.

Skeletal muscle cellular function relies on mitochondrial oxidative phosphorylation and maintenance of the mitochondrial reticulum through naturally occurring fusion and fission. A key molecule in oxidative phosphorylation is lactate, a product of glycolysis formed under aerobic conditions and a major energy source, gluconeogenic precursor, and a signaling molecule. With age progression, mitochondrial function decreases in part due to elevated oxidative damage and increased mitochondrial fission, resulting in the inability to metabolize different energy substrates or “metabolic inflexibility”. Damage caused by oxidative stress, such as lipid peroxidation, leads to altered mitochondrial dynamics, oxidative cell death, and an increased risk of age-related diseases such as sarcopenia. Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme capable of repairing peroxidized membranes and mice lacking Prdx6 exhibit sarcopenia-like phenotypes suggesting an age-related effect of Prdx6. The purpose of this dissertation was to understand the effects of aging on lactate metabolism and mitochondrial dysfunction while studying the role of oxidative stress as a potential driver for age-related changes to mitochondrial metabolism. I hypothesized that aging impairs lactate metabolism in humans while promoting mitochondrial dysfunction in skeletal muscle-derived cells and that oxidative stress derived from age-related reductions in mitochondrial Prdx6 is a primary driver of this dysfunction. The effect of age on the Postprandial Lactate Shuttle and aging-related metabolic inflexibility and mitochondrial dysfunction was revealed. These findings collectively contribute to our understanding of the mechanisms underlying age-related mitochondrial dysfunction, emphasizing the critical role of Prdx6 in preserving mitochondrial integrity in skeletal muscle. These insights pave the way for future research and interventions aimed at improving mitochondrial health in the aging population. Here, I discuss how aging affects skeletal muscle, lactate metabolism, and mitochondrial function, and how oxidative stress is a primary driver of such effects in skeletal muscle.

Organisms must learn from their environment and keep these memories for the long term to ensure their survival. If an adverse event is not remembered, the animal may not outlive it a second time. Thus, understanding how an adverse stimulus is encoded is especially important. In this dissertation, we find that C. elegans have a 2-hour critical period for memory consolidation, where sleep and the OSM-9 TRPV-like cation channel are required to successfully form a long-lasting memory. Specifically, OSM-9 may function in integrating old and new information associated to starvation. In addition, we find a new behavioral marker for swimming that is maintained post swim-based repetitive olfactory conditioning. The neural correlates of this proprioceptive memory can be observed in animals up to 2 hours post-conditioning and is formed independently of aversive olfactory memory. We found that animals integrate past and present environmental stimuli to form a new motor plan, which biologically functions to accelerate crawl-to-swim transition. Neural correlates of starvation associated changes in reversal motor pattern also show a change in conditioned animals in comparison to naïve animals, however these changes are not maintained. This specificity in gait selection and procedural memory can be further probed to understand how different types of experiences are organized in the brain.

Macular degenerations impact millions across the globe, with this number predicted to grow due to lack of effective therapies to combat the disease. The complex etiology of macular degenerations makes their developing treatments difficult. This thesis seeks to explore the underlying mechanisms and how they contribute to the disease in three ways. First, I show how ceramide accumulation drives the release of extracellular vesicles from the retinal pigment epithelium. These extracellular vesicles contain pro-inflammatory cargo and are predominantly released from the apical surface of the retinal pigment epithelium, towards the retina. This disrupts communication in the retina and recruits harmful microglia into the subretinal space. Second, I show that the retinal pigment epithelium are uniquely mechanosensitive to their environment, such that “drusen-in-a-dish” models, where I use biologically inert silica beads, can drive mitochondrial fragmentation in retinal pigment epithelium and disrupt retinal pigment epithelium homeostasis. These can be extrapolated towards mechanisms of disease in age-related macular degeneration. Finally, I show that an Food and Drug Administration-approved drug for osteoporosis and Paget disease, zoledronic acid, can be repurposed to reduce ceramide levels within the retinal pigment epithelium. And by reducing ceramide levels, many of the disruptions to retinal pigment epithelium function can be ameliorated and improve vision in mouse models of inherited macular degenerations. Together, these findings can be used for a better understanding of macular degenerations and hopefully pave the way for a curative intervention.

When a cell divides, it builds the mitotic spindle, a micron-scale bipolar structure made of dynamic microtubules that must attach to chromosomes and segregate them evenly into two daughter cells. Segregation relies on microtubule-generated force to capture and move chromosomes, yet how this force contributes to a cell’s decision-making during division has been challenging to discern. In my thesis work, I have addressed how biophysical cues at the kinetochore contribute to two key decisions during mitosis: first, whether to hold on to chromosome attachments or let go and second, when to stop building the spindle and divide. The kinetochore is the macromolecular interface that connects chromosomes to spindle microtubules. For accurate chromosome segregation, sister kinetochores need to become bioriented or attach to opposite spindle poles. Biorientation occurs by a constant feedback loop, destabilizing incorrect attachments while reinforcing correct ones. How the kinetochore distinguishes between correct and incorrect attachments is not clear. In this work, I address the long-standing hypothesis that tension at the kinetochore dictates which attachments to maintain. Using live imaging to monitor outcomes, I directly perturb force on kinetochores globally by chromokinesin overexpression and locally by individual chromosome arm ablation. Together with experiments enriching for attachment errors, this work demonstrates that elevated force at the kinetochore promotes attachment stabilization, and this effect is not chromosome-agnostic but leads to impaired error correction on long chromosomes. In parallel with local error correction decisions, the cell must globally prevent anaphase until all chromosomes are attached correctly. The spindle assembly checkpoint (SAC) generates a diffusible signal at unattached kinetochores to prevent premature segregation. The precise cues that trigger SAC satisfaction at an individual kinetochore and how the cell measures and integrates these signals is not well understood. Here, I use laser ablation to generate unpaired, sisterless kinetochores and monitor anaphase entry timing. I find a progressive, titratable delay in mitosis with increasing number of unpaired kinetochores, suggesting that without a sister kinetochore or a discrete opposing force, attachments may not be sufficiently stable to satisfy the SAC with normal dynamics. Still, these unpaired kinetochores are insufficient to prevent anaphase entirely, suggesting either that the SAC does satisfy eventually or that the cell cannot detect this low level of SAC signal. Altogether, I find that altering the physical landscape of the kinetochore by perturbing opposing force on it, either by manipulating chromokinesins or removing its sister, alters two critical pathways: error correction and the SAC. I find that force directly impacts the fidelity of cell division by signaling the error correction machinery and may alter dynamics of SAC satisfaction either directly or indirectly.

Liver cancer remains a leading cause of cancer-related death in part due to the shortage of effective therapies, and MYC overexpression defines an aggressive and difficult to treat subset of liver cancer cases. Given MYC’s ability to reprogram cellular metabolism and the liver’s role in systemic metabolism, we hypothesized that MYC could induce tumor-specific metabolic dependencies that could be targeted to slow tumor growth. We used mass-spectrometry to identify alanine depletion as a conserved metabolic signature of both mouse and human liver tumors. In vitro cell culture and high-content microscopy experiments showed that alanine could promote the proliferation and survival of MYC-overexpressing liver cancer lines in low nutrient conditions. Mechanistically, we found that MYC driven liver cancer cell lines and tumors specifically express the alanine enzyme GPT2, and loss-of-function RNA interference studies confirmed that GPT2 was required for the ability of liver cancer lines to use alanine to proliferate in vitro. We then used a conditional GPT2 knockout mouse to show that GPT2 played a necessary role in MYC-driven liver tumorigenesis in vivo. Isotope tracing and metabolomics were used to identify the TCA cycle, amino acid synthesis, and nucleotide synthesis as metabolic pathways that were fueled downstream of alanine. Finally, we found that L-Cycloserine, an orally bioavailable compound that inhibits GPT2, was efficacious at slowing liver tumor growth in both mouse and human liver cancer models. Thus, we identify a targetable metabolic pathway that MYC-driven liver tumors repurpose to fuel their survival.