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Investigating the Supracellular Processes Underlying Emergent Material Phase Properties During Embryonic Skin Morphogenesis
No full textDuring embryonic development, vertebrate tissues are sculpted by the coordinated behaviors of cells.Vertebrate organs are complex, typically requiring the orchestrated dynamics of hundreds to thousands of cells. Yet, due to their functional significance, the mechanisms underlying organ formation are robust.This implies the need for mechanisms to constrain critical processes during embryogenesis. While much of developmental biology has focused on the aspects of self-organization that relate to patterns of chemicals and molecules across a tissue and their relationship to gene regulatory networks, less attention has been given to the physical mechanisms at play. In particular,it is poorly understood how, in complex tissues, cells self-organize in a robust manner. This line of inquiry has been expounded recently in a classic system for studying periodic pattern formation: the embryonic chicken skin. The formation of feather follicle primordia was shown to be mechanically driven by the developing dermis. Cellular pulling forces, balanced by tissue stiffness, generate periodically spaced multicellular aggregates of dermal progenitors. As the aggregates form, they compress epidermal cells, triggering molecular changes that initiate the feather follicle primordium gene expression program. Yet, two questions remained. How do cells coordinate and tune their mechanical forces across a field of cells in order to generate the correct pattern? Once the dermal condensate forms, what role do molecular signals from the epidermis have in three-dimensionally sculpting the feather follicle primordium? This thesis addresses these questions by examining tissue morphogenesis through the lens of regulatory processes that occur at the multicellular length scale. In Chapter Two, we develop an assay to reconstitute the initiation of follicle patterning ex vivo. We show that contractile cells rearrange and align the extracellular matrix (ECM). Reciprocal interactions between the cells and ECM, mediated by calcium signaling, progressively align the cell-ECM layer. This exchange transforms a mechanically unlinked collective of dermal cells into a continuum with coherent, long-range order. Combining theory with experiment, we show that this ordered cell-ECM layer behaves as an active contractile fluid that spontaneously forms regular patterns. In Chapter Three, we examine how molecular signals—BMPs and FGFs—enable transformation from a flat dermal condensate to a feather follicle primordium with three-dimensional architecture. By analyzing cellular and molecular patterns as the feather follicle primordium forms and matures, we show that distinct multicellular domains emerge during budding that spatially correlate with BMP and FGF activity. By reconstituting these BMP and FGF domains ex vivo, we show that FGF promotes solidification whereas BMP retains fluidity but enhances contractility. Furthermore, we show that a biphasic supracellular complex is sufficient to drive tissue budding, resulting in symmetry breaking in a new spatial dimension. Together, these results present a paradigm for how supracellular-scale material properties generate robust changes in tissue patterning and architecture
Dissecting the Roles of Cancer Cell Lipid Metabolism in Tumor Progression and Immune Evasion
No full textLipids, along with nucleic acids, carbohydrates, and proteins, are essential biomolecules.These diverse hydrophobic metabolites are composed of fatty acids and include sterols, phospholipids, and glycerides. Lipids can be broken down to fuel energy demands, form the structural basis of all membranes, and can both act as signaling molecules themselves and regulate signaling by impacting membrane fluidity and receptor stability. Given the diverse and essential functions of lipids for normal cell function, it is no surprise that cancer cells leverage lipid metabolic pathways to fuel uncontrolled growth and proliferation and evade cell death. Indeed, enhanced lipid availability has been implicated in tumor initiation, growth,and metastasis, and metabolic genes involved in lipid uptake and synthesis are downstream of oncogenic alterations.Though excess lipids are known to correlate with disease progression, the precise enzymatic players required for cancer cell survival under distinct stressors have not been systematically studied. In this body of work, we designed and implemented loss of function CRISPR-Cas9 genetic screens to identify lipid metabolic dependencies in cancer cells growing under lipid peroxidation or immune pressure. Cancer cells rewire their metabolism and rely on endogenous antioxidants to mitigate lethal oxidative damage to lipids. However, the metabolic processes which modulate the response to lipid peroxidation are poorly defined. Using genetic screens, we compared metabolic genes essential for proliferation upon inhibition of cystine uptake or glutathione peroxidase-4 (GPX4), two pathways reported to mitigate lipid reactive oxygen species (ROS). Interestingly, very few genes were commonly required under both conditions, suggesting that cystine limitation and GPX4 inhibition may impair proliferation via distinct mechanisms. Our screens also identified tetrahydrobiopterin (BH4) biosynthesis as an essential metabolic pathway upon GPX4 inhibition. Mechanistically, BH4 is a potent radical-trapping antioxidant that protects lipid membranes from autoxidation, alone and in synergy with Vitamin-E. Dihydrofolate reductase (DHFR) catalyzes the regeneration of BH4 and its inhibition by methotrexate synergizes with GPX4 inhibition. In sum, we identify the mechanism by which BH4 acts as an endogenous antioxidant and provides a compendium of metabolic modifiers of lipid peroxidation. This work is summarized in Chapter 2. Despite the critical functions of lipid metabolism in membrane physiology, signaling, and energy production, how specific lipids contribute to tumorigenesis is incompletely understood. Here, using functional genomics and lipidomic approaches, we identified de novo sphingolipid synthesis as an essential pathway for cancer immune evasion. Synthesis of sphingolipids is surprisingly dispensable for cancer proliferation in culture or in immunodeficient mice but required for tumor growth in multiple syngeneic models. Blocking sphingolipid production in cancer cells enhances the anti-proliferative effects of natural killer (NK) and CD8+T cells via interferon gamma (IFNγ) signaling. Mechanistically, glycosphingolipids impact IFNγ receptor subunit 1 (Ifngr1) localization, mediating IFNγ-induced growth arrest and proinflammatory signaling. Finally, high expression of sphingolipid synthesis genes correlates with poor survival in cancer patients and increasing tumor sphingolipids impairs immune surveillance. Altogether, we show that glycosphingolipids are both necessary and limiting metabolites for cancer immune evasion. This work is summarized in Chapter 3. Overall, this thesis defines metabolic pathways that are required for cell survival during ferroptotic stress and immune surveillance. Using a combination of in vitro and in vivo functional genetic screens in multiple cell lines, were port on a compendium of genes regulating cancer cell sensitivity to lipid ROS and immune pressure. Though we hone in on the GCH1-BH4-DHFR and glycosphingolipid-Ifngr1 axes, future work may unravel the molecular mechanisms underlying the many genetic dependencies identified by our screening strategies. Ultimately, we anticipate that the findings reported here will advance the understanding of how lipid metabolism in cancer cells impacts disease progression and hope it will contribute to the development and improvement of therapeutics
Using Small Molecule Tools to Study ATPase Mechanoenzymes
No full textThe dysregulation of AAA (ATPases associated with diverse cellular activities) mechanoenzymes has been linked to diseases, and chemical inhibitors and activators can be powerful tools to probe mechanisms and test therapeutic hypotheses. However,the structural conservation across the AAA protein family makes designing selective chemical inhibitors challenging. Additionally, unlike chemical inhibitors that can stabilizea single conformational state of an enzyme, activator binding must be permissive to different conformational states needed for enzyme function, and we do not know how AAA proteins can be activated by small molecules. My thesis work covers the development of a chemical genetics approach to inhibit AAA proteins, starting from atriazolopyridine-based fragment that binds the AAA domain of the microtubule severing protein katanin, and the identification of a druggable site for chemical activators in valosin-containing protein (VCP)/p97, a AAA unfoldase whose loss of function has been linked to protein aggregation-based disorders. For the chemical genetics approach, we designed ASPIRe-1 (Allele-Specific, Proximity-Induced Reactivity-based inhibitor-1), a cell-permeable compound that selectively inhibits katanin with an engineered cysteine mutation. Only in cells expressing mutant katanin, ASPIRe-1 treatment increases the accumulation of CAMSAP2 at microtubule minus-ends, confirming specific on-target cellular activity. Importantly, ASPIRe-1 also selectively targets engineered cysteine mutants of VPS4B, FIGL1, and VCP. For the small molecule activator part of my thesis work, from a screen optimized to identify compounds that stimulate VCP ATPase activity as little as 20%, I discovered activators that represent five chemotypes. Minimal modification of the most potent hit, an isoindoline-based compound, resulted in VCP Activator 1 (VA1), a compound that dose-dependently stimulates VCP ATPase activity up to ~3-fold. Cryo-EM studies resulted instructures (~2.9-3.5 Å-resolution) of VCP in apo and ADP-bound states, and reveal VA1 binding an allosteric pocket near the C-terminus in both states. Finally, I engineered mutations in the VA1 binding site that confer resistance to VA1, and furthermore, modulate VCP ATPase activity to a similar level as VA1-mediated activation. Together, these findings suggest a chemical genetics approach to decipher AAA protein cellular functions and uncover a druggable allosteric site that can also be occupied by VCP\u27s C-terminal tail to control activity
Vulnerability to G - Quadruplexes in BRCA2 - Null Medulloblastoma: A Protective Role for the PIF1 Helicase
No full textThe reasons why cancer arises in children are not fully understood. Children with biallelic mutations in the essential DNA repair factor BRCA2 are highly predisposed to develop the most common pediatric brain cancer medulloblastoma (MB) before three years of age. Medulloblastoma is often the first malignancy in these children who have a high likelihood of developing several other cancers in their lifetime. MBs have a complex mutational landscape characterized by chromothripsis in p53-deficient tumors and a high level of somatic mutation in many other cases.We utilized a mouse model of spontaneous medulloblastoma development driven by BRCA2 loss in the central nervous system coupled with global inactivation of p53 to study the roles of BRCA2 in protecting from childhood cancer development. We endeavored to address how mutagenesis can occur in tumor-initiating cells in such a short period of time by identifying sources of DNA damage in the cell of origin of BRCA2-null MB–the cerebellar granule cell progenitors (GCPs). GCPs undergo a period of massive expansion in the postnatal cerebellum, stimulated by Sonic hedgehog (Shh) signaling as well as other mitogens and can be isolated from the cerebella of mice during this developmental window that peaks at P7. After multiple symmetric divisions, GCPs exit the cell cycle, migrate into the inner granule layer of the cerebellum, and differentiate into granule cells, the most numerous single neuron type in the brain. The critical developmental window of GCP expansion represents a state where highly proliferative GCPs are vulnerable to mutagenesis. G4-containing genomic regions are vulnerable to mutation and that the demands of tumor growth may cause positive selection of mutations in functionally relevant genes. From gene expression analysis ofBrca2-/-MBs, we identified that several G4-resolving helicases are upregulated in tumor. Of these helicases, the PIF1 helicase was present in tumor and absent from normal cerebellum, indicating its potential as a therapeutic target that could selectively affect tumor cells. To assess the role of PIF1 inBrca2-/-MB cells, we generated primary tumor cell lines and knocked out Pif1 with CRISPR/Cas9. PIF1 loss led to replication stress inBrca2-/-MB cells and downstream genomic instability. Treatment with PDS exacerbated the phenotypes of PIF1 deficiency, indicating that G4s that are unable to be resolved can lead to deleterious outcomes such as the development of micronuclei and aberrant mitoses. In Brca2-/-cells, G-quadruplexes represent a hurdle to normal replication that must be resolved by helicases like PIF1 to prevent mutagenesis. Down regulation of PIF1 could sensitize Brca2-/-cells to treatment with PDS and other G4-ligands and could represent a new vulnerability to exploit in Brca2-/-medulloblastoma
Transcriptomic Signatures of Projection Class Neurons for Vocal Control
No full textVocal learning is the ability to modify acoustic structure and syntax of vocalizations. This rare trait is thought to have independently evolved three times in birds and five times in mammals. Vocal learning species exhibit an underlying convergence in brain circuitry, including a direct, forebrain-to-brainstem projection critical for the motor production of learned vocalizations. The remarkable convergence in fore brain vocal circuitry is associated with convergent molecular specializations. In characterizing specializations for vocal motor control across species, we can expand our knowledge of vocal learning and the evolution of this trait. In the first section of this thesis, I generated a high-quality reference genome assembly for the common marmoset, anon-human primate that displays flexible vocal communication abilities in complex social settings as well as rudimentary neural circuits for vocal motor control. My findings demonstrate the power of using a trio-binning approach, in combination with long-read sequencing technologies, to produce a diploid genome with the two parental haplotypes assembled independently. This method captures the full range of heterozygous variations at high rates of accuracy between parental alleles in this species that exhibits naturally high levels of chimerism. The genome assembly was annotated and is now the National Center for Biotechnology Information (NCBI) reference assembly for the common marmoset. In the second section of this thesis, I generated and analyzed single nucleus RNA sequencing data sets from vocal motor and locomotor brain regions of marmoset and zebra finch (an avian model of vocal learning) to identify long-range projection classes of neurons, assess transcriptional markers, and make cross-species comparisons. In the marmoset, I identified layer 5 extratelencephalic-projecting (L5 ET) excitatory neurons of orofacial and hindlimb primary motor cortex. Compared to L5 ET hindlimb neurons, orofacial L5 ETs showed low fold, though significant, molecular specializations, including rudimentary expression patterns found in projection classes of neurons for vocal learning. In the finch, I identified excitatory neurons unique to the RA song nucleus, showing transcriptional patterns consistent with the direct projection neurons (RAnXIIts) associated with vocal production and vocal learning behavior. This class of neurons is marked by RBFOX1 expression and was annotated, with high confidence, as L5 ET neurons when mapped into a human M1 reference dataset. Cross-species integration of finch, marmoset, and human datasets showed one cluster including human L5 ET, marmoset L5 ET, and finch RAnXIIts RBFOX1+ neurons. Comparing these groups of neurons across species, I found distinct gene expression patterns of RAnXIIts neurons related to axonogenesis, calcium homeostasis, rapid firing, and ATP synthesis. This neuronal class also exhibits high expression of L5 ET markers that are primate-specific among mammals. Finally, cross-species genome alignments of RBFOX1promoter regions show high sequence similarities among vocal learning bird species. Together, the data I present here support a hypothesis that certain cell types of the avian vocal learning system evolved to incorporate molecular attributes making them functionally analogous to mammalian cortical layer neurons
Use of Genetically Encoded Tools to Interrogate Mechanisms of Glutathione Homeostasis from Mitochondria to Mice
Full text linkGlutathione is an evolutionarily ancient molecule that underlies many critical biological processes across all categories of life. It acts as a cofactor in some metabolic reactions, modifies proteins to modulate their activity, and participates in detoxification of xenobiotic compounds. Despite these wide-ranging functions, glutathione is best known as an antioxidant. Some consider it akin to the fountain of youth because it decreases throughout the course of aging while oxidative damage accumulates.In addition to aging, glutathione dysregulation occurs in a plethora of diseases, including neurodegenerative diseases like Alzheimer\u27s and Parkinson\u27s Disease, liver disease, and metabolic disease, among others. However, the specific roles of glutathione that contribute to disease progression remain poorly understood. In part, this is because the function of glutathione in cellular metabolism is still not fully characterized. Glutathione is thought to be critical for mitochondrial function, but it is not known how glutathione gets into mitochondria. Because of this, it has been very difficult to specifically deplete mitochondrial glutathione, resulting in circumstantial evidence that maintenance of glutathione abundance is critical for mitochondrial function. We set out to expand the tools available to characterize the function of compartmentalized glutathione pools. In the first half of this work, I engineered a bacterial enzyme to synthesize glutathione in mitochondria, effectively bypassing both endogenous glutathione synthesis and transport into mitochondria. Using this tool, I tried to identify the mitochondrial glutathione transporter by performing CRISPR-Cas9 screens that relied upon the assumption that the mitochondrial glutathione transporter(s) is/are essential for cell proliferation, and that enabling mitochondrial glutathione synthesis would enable identification of these no-longer essential genes. While this limited approach failed to identify the mitochondrial glutathione transporters, we identified new roles for several genes, including a mechanism of resistance to the most prevalently used glutathione synthesis inhibitor. To identify alternative molecules that may inhibit glutathione synthesis, I designed and performed a high-throughput chemical screen. I was able to identify several candidate molecules, although they are not direct inhibitors of glutathione synthesis. Further identification of the targets of these inhibitors may provide additional insight into the function of glutathione in cellular metabolism. Ultimately, in collaboration with several of my colleagues, we used alternative approaches to identify two putative mitochondrial transporters required for mitochondrial glutathione import. We found that, as expected, mitochondrial glutathione is required for cell viability. However, we found that this was due to glutathione\u27s role as a critical cofactor in iron sulfur biosynthesis, not due to its role as an antioxidant, emphasizing the importance of considering unbiased approaches to determine the function of glutathione in disease progression. In the second half of this work, I developed two mouse models with unregulated glutathione availability. I was surprised to find that that constitutively high glutathione is in compatible with embryonic development, demonstrating that homeostatic mechanisms of maintaining glutathione levels are critical for viability. However, unregulated glutathione synthesis was compatible with life in adult animals and resulted in an increase of GSH up to 5-fold in some tissues. Interestingly, this increase in GSH was compatible with normal tissue metabolism, underscoring that regulation of glutathione synthesis is required for some essential embryonic processes. Future studies with these mouse models with high glutathione will enable interrogation of glutathione sufficiency in disease progression
Influence of Mouse Motor Cortex on Vocal Musculature
Full text linkLearned vocal communication and spoken language are complex behaviors we have long sought to understand. Studying the neurobiology of speech and language will be advanced by investigating its component traits in model organisms. Most vertebrates share common brainstem circuits for vocalization, and they produce innate vocal repertoires over which the animals have little to no control. Vocal learners can imitate heard sounds, and thus have a high degree of control over their vocalizations. Vocal learning species have a direct projection from the pallial forebrain to the brainstem vocal motor neurons, which facilitate their vocal dexterity. Songbirds have been the standard model of vocal learning, but there are technical limits to our ability to test various hypotheses about the development and evolution of vocal learning circuits. Mice, which are more closely related to humans, have been found to exhibit some rudimentary features that are like those seen in vocal learning species. These include: 1) a direct, but sparse, projection from the primary motor cortex(M1) to the laryngeal motor neurons in the brainstem, termed the laryngeal motor cortex (LMC); 2) cortical lesions that damage this direct projecting population alter the frequency distribution of vocalizations; and 3) change their vocal syntax based on social context. Although mouse ultrasonic vocalizations (USVs) have received a great deal of attention and interest, the role these cortical circuits and the mechanisms by which they might affect USVs have been little explored. Here, we investigate the role of the motor cortex of mice in controlling vocal musculature, and we develop new methods that will allow us to gain more insights into the control mice have over their USVs. We performed intracortical microstimulation (ICMS) with paired electromyographic (EMG) recordings to test whether the direct projection previously identified in mice can generate laryngeal muscle contractions. Simultaneously, we recorded EMG signals from the anterior digastric, a jaw opening muscle, as a control. We found that the LMC population of neurons can generate laryngeal muscle contractions. We also identified the orofacial motor cortex (OMC) as another region of M1 that can generate laryngeal muscle contractions, although weaker than from LMC. The muscles responded with different latencies from the LMC and OMC stimulations suggestive of both indirect and direct brainstem motor neuron projections, respectively. Using a retrograde transsynaptic virus, we show that the region of M1 containing the LMC has neurons that represent the larynx as well as jaw and forelimb muscles; in a small proportion of neurons, two muscles were represented by single neurons. Using an anterograde transsynaptic tracer from the OMC, we found that there are direct projections to the motor neurons of the jaw. Lastly, chemical lesions of OMC led to a modest change in the number of USVs per sequence. To test the degree of control mice have over their USVs, we developed a suite of tools to train mice in an operant vocal task. To detect and classify USVs in real-time, we developed a software called Analysis of Mouse Vocal Communication (AMVOC). We combined AMVOC with other open-source hardware and software to design an operant training paradigm to that lets us test the volitional control of mouse vocal behavior. We provide a proof-of-principle application of this system to train mice to increase vocalizations for food reward. Mice had been assumed to lack a functional cortical representation of the larynx, and similar assumptions have been made about other vocal non-learners. In contrast, the results of my thesis provide evidence that M1 in a vocal non-learner can influence vocal musculature, consistent with the continuum hypothesis of vocal learning. We also demonstrate that the representations of muscles for different behaviors across mouse M1 are highly intermixed, sharing both cortical space and single neurons. These results offer new insights into the origin and evolution of laryngeal control by cortical circuits, suggesting that these circuits are both more commonly distributed across mammalian species and that they may have arisen earlier than was previously hypothesized. Further, the results presented here will allow us to better understand the shared features of vocal production that are integral to better understanding human speech
Breaking Down Microtubule Formation: Characterizing the Biochemical and Cellular Functions of the γ-Tubulin Ring Complex
Full text linkThe γ-Tubulin ring complex (γ-TuRC) is an essential regulator of the microtubule cytoskeleton. It is composed of\u3e30 individual proteins that include the major component, γ-tubulin, as well as γ-tubulin complex proteins 2-6 (GCP2-6), mitotic-spindle organizing proteins associated with a ring of γ-tubulin proteins 1 and 2 (MOZART, or MZT1 and MZT2), and an actin molecule. This ~2.2MDa assembly regulates microtubule dynamics by facilitating the nucleation of new microtubules, modulating microtubule minus-end dynamics by acting as a minus-end cap, and anchoring microtubules to specify their cellular localization. These three major activities of the γ-TuRC, nucleation, capping, and anchoring, contribute to the dynamic nature of individual microtubules and of larger microtubule networks that are critical for cellular activities, including cell motility, in trace have spurred biochemical and cellular characterization of its nucleation activity. However, it remains unclear how the remaining major γ-TuRC activities contribute to microtubule dynamics. In the first part of this thesis, I characterize the capping activity of the γ-TuRC. Using biochemical assays, I examine the association of recombinantly expressed and purified γ-TuRC at microtubule minus-ends either following a nucleation event, or on pre-formed microtubules. Using total internal reflection fluorescence (TIRF) microscopy, the dynamics of this association can be quantified in terms of the number of microtubules that are capped, and the length of time the cap persists at the minus-end under these two conditions. Additionally, I purified a recombinant γ-TuRC composed of GTP-binding deficient γ-tubulin in order to examine the GTP-binding dependency of the γ-TuRC\u27s capping activity. As opposed to the γ-TuRC\u27s nucleation activity, which is GTP-binding dependent, I found that the γ-TuRC\u27s capping activity is GTP-binding independent. By expressing GTP-binding deficient γ-tubulin in HeLa cells depleted of endogenous γ-tubulin protein, I characterized the role of the γ-TuRC\u27s capping activity in dividing cells. While cells expressing the GTP-binding γ-tubulin mutant could not form bipolar mitotic spindles and became arrested in mitosis, fixed- and live-cell imaging experiments showed that expression of this mutant rescued non-centrosomal microtubule formation, which was lost under γ-tubulin knockdown conditions. Together, these data suggest that the γ-TuRC\u27s capping activity is GTP-binding independent and plays a role in non-centrosomal microtubule formation during mitosis. In the second part of my thesis, I perform studies towards further characterizing the γ-TuRC. First, I use affinity-purification followed by mass spectrometry to characterize the composition of γ-TuRCs purified from a HeLa cell line overexpressing GFP-tagged γ-tubulin. Further mass spectrometry analysis identified interacting proteins that co-purified with γ-tubulin, some of which have a known function related to the microtubule cytoskeleton or cell division. Second, I performed cell biology experiments to examine how the composition of the γ-TuRC may affect its activities.Recent work has suggested that the N-terminal domains of GCP6, named the N-helical domains (NHD) and the belt domain, are needed to maintain the structural integrity of the γ-TuRC, and that without these domains, specific components of the complex are lost. Cells expressing these N-terminally truncated GCP6 constructs displayed partial γ-tubulin-containing complexes relative to cells expressing full-length GCP6. Furthermore, cells expressing GCP6 truncated of the NHDs and the belt domain showed loss of centrosomal GCP6 and γ-tubulin specifically during mitosis, while their centrosomal localization persisted during interphase. Together, these data suggest that different components of the γ-TuRC may mediate the localization and anchoring of the γ-TuRC at specific cellular sites, such as the centrosome
The Nanomechanics of Protocadherin 15, A Protein Essential for Human Hearing
Full text linkMechanical force controls the opening and closing of mechanotransductive ion channels atop the hair bundles in the inner ear. A mechanical element called the gating spring modulates the mechanotransduction channel\u27s open probability by changing the force transmitted to the channels. The molecular identity of the gating spring is yet unconfirmed, but a leading candidate is the filamentous tip link connecting the mechanotransduction channel to the tallest neighboring stereocilium. The tip link is essential to mechanotransduction: when it is broken, mechanotransduction is abolished, and when it is allowed to regenerate, mechanotransduction returns. Each tip link comprises four protein molecules: a dimer of protocadherin 15 and a dimer of cadherin 23, both of which are stabilized by Ca2+ binding. Further underscoring the role of the tip link in hearing, there are numerous mutations of its constituent proteins that result in deafness. My thesis work has focused on protocadherin 15, the lower portion of the tip link that connects at its C-terminus to the mechanotransduction channel. I was interested in answering several questions: does protocadherin 15 have the appropriate properties to be a component of the gating spring? What factors control its mechanical response? What is its stiffness? How does it soften under force? And how do these answers change in the case of a deafness-causing mutation in protocadherin 15? In order to answer these questions, I used an optical-trap system with sub-nanometer spatial resolution and microsecond temporal resolution to investigate the mechanics of protocadherin 15 at a single-molecule level. To augment this approach I also undertook electron microscopic studies to investigate the structure of wildtype and mutated protocadherin 15. I found that both the mechanics and structure of protocadherin 15 are dependent on Ca2+ and that protocadherin 15 undergoes limited unfolding at a physiological level of Ca2+. My experimentally determined stiffness for protocadherin 15 accords with published values of the gating spring\u27s stiffness, which implies that protocadherin 15 is able to modulate its stiffness without undergoing large unfolding events in physiological Ca2+ conditions. In the case of a point mutation that causes non-syndromic hearing loss, the structure of protocadherin 15 is more conformationally heterogenous, and the protein undergoes frequent unfolding events at all levels of Ca2+. The frequent unfolding events suggest that the mutated protocadherin 15 has lost the ability to maintain appropriate tension under physiological forces, which could prevent the proper opening of the mechanotransduction channel and result in deafness fort those with this mutation. This work shows that the maintenance of appropriate tension in the gating spring is critical to the appropriate conveyance of force to the mechanotransduction channel
Pioneer Factors Compete for Epigenetic Factors in Switching Stem Cell Fates
Full text linkDuring development, progenitor cells can activate one cell fate while simultaneously silencing another, a process that is tightly regulated in adult tissues. However, this process is often derailed in diseases such as cancers, leading to uncontrolled growth and malignancy. At the crossroads of fate-switching, pioneer factors are a class of transcription factors equipped to bind cognate motifs in closed chromatin. Once they access the closed chromatin, pioneer factors can either act as transcriptional activators or repressors of cell fates by recruiting co-activators or co-repressors. Nevertheless, whether and how a single factor can simultaneously silence the old fate while activating the new cell fate remains largely unknown. Here, I tackled this question with SOX9, a master regulator that diverts embryonic epidermal stem cells (EpdSCs) into becoming hair follicle stem cells (HFSCs). I triggered a temporal fate-switching by engineering mice to re-activate SOX9 in adult EpdSCs. Combining epigenetic, proteomic, and functional analyses, I interrogated the ensuing transcriptional and epigenetic dynamics, slowed temporally by the mature EpdSC niche microenvironment. My findings demonstrate that SOX9 plays a dual role in the fate-switching process. As it binds and opens key HFSC enhancers, it also redistributes co-factors away from EpdSC enhancers, leading to their silencing. Furthermore, in the absence of its normal regulation, prolonged expression of SOX9 in EpdSCs leads to the activation of downstream transcriptional regulators associated with basal cell carcinoma