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Hedgehog - like Proteins are Involved in a Novel Glia - Neuron Signalling Pathway in C. elegans
No full textComplex organisms rely on their nervous systems to sense their environment, process the gathered information, and execute appropriate responses. Glia play essential roles in this information transduction, and glia-neuron communication regulates sensation at every step. But despite this centrality of glia in nervous system function, many of the molecular mechanisms through which glial cells establish and maintain reciprocal communication with neurons are unknown. C. elegans sensory organs provide a good model to study glia-neuron crosstalk. The largest one in the animal is called the amphid and acts somewhat like a nose for the worm. Amphid neurons are integral to sensing odorants, salts, and other environmental cues that the animal needs to assess for successful feeding, reproduction, and escaping harmful conditions. In these roles, amphid neurons rely heavily on their two glial partners, a sheath and a socket glia of the amphid. These glia are involved in both the proper structural development and the functioning of amphid sensory organ throughout the animal\u27s life. In this thesis, I pursued various lines of inquiry that led me to a novel glia-neuron signalling pathway that regulates neuronal properties in very specific ways. Two key players in this mechanism are WRT-6, a Hedgehog-like protein secreted by the socket glia, and PTC-1, a Patched homolog found in the amphid neurons. It is believed that C. elegans, lacking bona fide homologs of Hedgehog and Smoothened, does not possess the canonical Hedgehog signalling pathway that has been evolutionarily conserved across a wide range of organisms. However, our results suggest that a non-canonical Hedgehog-like signalling might in fact be present in the worm\u27s nervous system, with a function in glia-neuron communication. Other players in this hypothesized pathway include SUP-17 and TSP-14 in the socket glia, FIG-1 and VAP-1 in the sheath glia, and Y9 in the amphid neurons. SUP-17 is a homolog of the ADAM10 metalloprotease, and TSP-14 is a homolog of TspanC8 tetraspanin family, which are known to modulate the maturation, localisation, and function of ADAM10. FIG-1 does not have a clear homolog in mammals, but its structure loosely resembles a mash-up of ADAM and ADAMTS proteins. VAP-1 belongs to the cysteine-rich secretory protein (CRISP)-related family, which have been implicated in cholesterol metabolism and cancer progression. Finally, Y9 is a novel 7-transmembrane protein I\u27ve described that is found on the cilia of a subset of the amphid neurons, which I speculate might function as a Smoothened-like protein. The findings from my thesis work favour a model in which these various glial and neuronal proteins affect each other\u27s cleavage, localisation, and/or function through direct and indirect mechanisms. Furthermore, I believe that a key molecular component of this complex regulation is cholesterol, or another sterol molecule, which is known to be central for canonical Hedgehog signalling. The sum of these interactions constitutes a Hedgehoglike signalling pathway between the glia and neurons of the amphid sensory organ. The neuronal processes controlled by this pathway are currently ill-understood, however one readout for them is whether the amphid neurons are able to incorporate lipophilic dye molecules to their plasma membranes. Uncovering more players and molecular mechanisms related to this signalling pathway has the potential not only to provide important insight into glia-neuron communication, but also to elucidate long-remaining mysteries regarding Hedgehog evolution in nematodes and potentially establish C. elegans as a promising model for the study of non-canonical Hh signalling in the nervous system
Molecular and Cellular Mechanisms of LIM Protein Mechanotransduction
No full textFor tissues to develop and maintain mechanical homeostasis, cells must be able to perceive and respond to mechanical cues in their local environments (mechanosense). While there has been significant progress in understanding the physiological significance of mechanosensation, the mechanisms by which proteins convert mechanical stimuli into biochemical signals (mechanotransduction) are poorly understood. The actin cytoskeleton serves as a nexus for cellular mechanotransduction, generating and transducing forces into intracellular signals that coordinate cell migration, cellular contractility, organelle dynamics, and gene expression. However, our understanding of the molecular mechanisms by which cytoskeletal mechanotransduction is achieved is limited. LIM domain proteins are a superfamily of cytoskeleton-associated proteins involved in cytoskeletal remodeling and transcriptional regulation, but how mechanical forces modulate their functions is unclear. Here, I employed an interdisciplinary toolkit of structural, biophysical, and cell biology methods to investigate mechanisms of cytoskeletal mechanotransduction through LIM proteins. In a collaborative project, we characterized the LIM domain superfamily and tested the hypothesis that LIM proteins are mechanosensitive actin binding proteins. Using an imaging-based screen of mechanically-stretched fibroblasts, I identified the zyxin, FHL, and paxillin families of tandem LIM domain proteins to be mechanoresponsive cytoskeletal proteins. A single conserved phenylalanine in each LIM domain was identified to be necessary for mechanosensitivity. Through development of an in vitro force reconstitution system, we discovered that LIM proteins are the first class of actin binding proteins to only bind actin filaments when they are under force. We show that strained actin binding acts as a cytoplasmic sink to retain FHL2 from shuttling into the nucleus, potentially inhibiting its activity as a transcriptional co-regulator. These studies unearthed a novel force-activated actin-binding mechanism, shedding light on how LIM proteins can associate with the cytoskeleton despite lacking canonical actin-binding domains, and established a pathway that could mediate cytoskeleton-to-nucleus mechanotransduction by FHL2. These studies set the stage for investigating how force-activated binding by LIM proteins could coordinate downstream mechanotransduction. Thus, I next focused on investigating how these force-activated binding events could mediate stress fiber repair: a paradigmatic example of cytoskeletal mechanotransduction mediated by the LIM protein zyxin. While cellular studies have uncovered the molecular components involved in stress fiber repair, the precise molecular mechanism by which zyxin coordinates the recognition of tensed actin filaments with downstream effector functions of repair proteins is unknown. Through reconstitution of the stress fiber repair reaction with purified proteins, I found that zyxin and other mechanosensitive LIM proteins form force-activated assemblies that bridge actin fragments, acting as a molecular bandage and sustaining the stress across broken filaments. We hypothesize that these force-activated assemblies are polymers of LIM proteins, representing a new class of force-evoked biomolecular assembly. I found that these force-activated zyxin assemblies also serve as hubs for the binding and recruitment of stress fiber repair factors. Through a minimal reconstitution of a contractile stress fiber-like network, I found that force-activated zyxin assemblies coordinate actin nucleation and crosslinking by repair factors VASP and α-actinin, respectively, to orchestrate stress fiber repair at the filament network scale. Recent studies have shown that zyxin localization is a strong molecular marker of high traction stresses in cells. Based on our findings that zyxin only binds actin filaments in the presence of myosin-evoked forces in vitro, we hypothesized that zyxin-enriched regions of the cytoskeleton would feature myosin-evoked actin filament (F-actin) conformations. Notably, studies applying our in vitro force reconstitution assays on EM grids revealed striking force-evoked superhelical actin filament segments. Thus, I investigated whether these F-actin structures are present at zyxin enriched cytoskeletal structures, such as focal adhesions and stress fibers, using cryo-electron tomography. In a collaborative project, we found oscillatory F-actin structures enriched at adhesions and stress fibers high in zyxin, reminiscent of myosin-evoked superhelical F-actin. These results suggest that myosin-evoked forces can generate superhelical F-actin structures in cells that may be sensed by mechanosensitive actin binding proteins. Most recently, I investigated the cytoskeleton-to-nucleus mechanotransduction pathway of FHL2, a mechanosensitive LIM protein reported to be a transcriptional co-regulator. Using multi-omics, cell biology, and biochemical assays, I characterized the cytoskeletal and nuclear functions of FHL2. While the nuclear functions of FHL2 remain elusive, I found that FHL2 is negative regulator of cytoskeletal mechanics, modulating focal adhesion morphology and cellular contractility. Collectively, the research presented in this thesis sheds new insights to the molecular mechanisms of LIM protein mechanotransduction through strained actin binding. Moreover, these studies reveal how mechanical homeostasis is coordinated by the LIM protein zyxin while providing new tools for studying the mechanisms of cytoskeletal mechanotransduction and the assembly of contractile networks. Lastly, my cellular studies of FHL2 provide a molecular atlas of its potential functions as a signal relay between the cytoskeleton and the nucleus
Serine Metabolism Leverages the Integrated Stress Response to Direct Stem Cell Fate During Tissue Regeneration
No full textEpidermal stem cells constantly rejuvenate the skin\u27s barrier, which excludes harmful microbes and prevents dehydration. While hair follicle stem cells (HFSCs) normally regenerate hair, they must also reconstruct and thereafter maintain the overlying epidermis upon a barrier breach, such as a wound. How these fate choices are balanced to restore physiologic function to damaged tissue remains poorly understood. Here, I uncover the non-essential amino acid serine as a surprising regulator in this process. Utilizing dietary intervention and HFSC-specific loss-of-function studies, I demonstrate that under serine-depleted conditions HFSCs delay hair growth. Upon injury, serine-restricted HFSCs skew their fate towards epidermal re-epithelialization while concomitantly limiting hair growth. Combining temporal single-cell RNA sequencing, loss-of-function genetics, and pharmacological intervention, I show that serine deficiency augments an injury-activated integrated stress response (ISR). Moreover, this response accelerates re-epithelization and rapidly restores the skin\u27s barrier at the wound edge. Altogether, my findings demonstrate that stem cells use serine metabolism and the ISR as regulators of tissue regeneration, offering potential for dietary and pharmacological intervention to accelerate wound healing
Replicative Senescence is ATM-Driven, Reversible, and Delayed by Reduced ATM Activity at Low Oxygen
No full textSomatic human cells have a limited proliferative capacity, dividing a certain number of times before entering a predictable proliferative arrest. Telomeres become shorter with every division until a few become \u27critically-short\u27 and activate the DNA Damage Response (DDR), thereby inducing this replicative senescence. However, the nature of the activated DDR is unclear. ATM kinase activation at critically short telomeres has been implicated in the induction of senescence, suggesting that the shortest telomeres lack su]icient TRF2 to repress ATM signaling. However, ATM-deficient cells also undergo replicative senescence, raising the question of POT1 deficiency and subsequent ATR signaling. Moreover, low oxygen culture conditions impart an extension of fibroblast proliferative lifespan, indicating the nature of the critically-short telomere, or the response to that telomere, may be context-dependent. The permanence of senescence is also unclear. Traditionally, senescence has been considered an irreversible state, but evidence suggests interventions specific to the senescent-inducing signal may allow some arrested cells to re-enter the cell cycle. Finding the main driver of replicative senescence – and interrupting that signal – would allow the purported inflexibility of proliferative arrest to be tested. Using specific inhibitors of ATM, ATR, Chk2, and Chk1, we show that replicative senescence is solely due to ATM signaling, and that deficiency in TRF2 is the primary reason for ATM activation at critically short telomeres. In further support of this, we find that ataxia-telangiectasia cells (deficient in functional ATM expression) tend to bypass a traditional replicative senescence, instead entering telomeric crisis. Using FUCCI live-cell imaging, we establish that ATM inhibition allows a subset of wildtype senescent cells to re- enter the cell cycle and progress through several cell divisions. RNA-seq and p38 inhibitor culturing show ATM-inhibited post-senescent fibroblasts eventually enter a second replicative arrest due to p38 α/β-p53-p21 pathway induction. We also show that cells maintained at low (3%) oxygen have a greater tolerance for critically-short telomeres, despite unaltered shelterin expression. Instead, this greater tolerance of short telomeres is due to a diminished ability of ATM to respond to DSBs, despite normal expression of ATM pathway components. Our data is consistent with hypoxia-induced sequestration of ATM in inactive dimers, explaining why cells are more tolerant to critically short telomeres and have an extended proliferative lifespan when maintained at physiological oxygen levels
GPCR to Channel Communication in a Membrane Signaling Pathway
No full textG-protein-coupled receptors (GPCRs) regulate a variety of downstream effector proteins, making them essential for many physiological processes. For example, in the heart, the activation of beta-adrenergic receptors (βARs) and the stimulatory G protein (Gαs) leads to the opening of downstream hyperpolarization-activated cyclic nucleotide–gated (HCN) channels, resulting in an accelerated heart rate. In contrast, activation of M2 muscarinic receptors (M2Rs) and the inhibitory G protein (Gαi) opens G protein-gated inward rectifier K⁺ (GIRK) channels, slowing the heart rate. In addition to the Gαs and Gαi pathways, there is also the Gαq signaling pathway. In this pathway, activation of Gq-coupled GPCRs can lead to the activation of phospholipase Cβ (PLCβ). This enzyme can then hydrolyze phosphatidylinositol 4,5- bisphosphate (PI(4,5)P2) and thus decrease inward rectifier K+(Kir) channel activity. While the major components of these signaling pathways have been extensively studied, many unknowns remain regarding how GPCRs regulate channels. How do different signaling lipids regulate the activity of ion channels? To address this question, the first part of the thesis presents an analysis of how phosphatidylinositol lipids regulate the gating of Kir2.2 channels. In this study, I employed the planar lipid bilayer system to study how PI(4,5)P2 concentration affects the single-channel kinetics of Kir2.2. I show that Kir2.2 displays bursting behavior in the presence of PI(4,5)P2. Increasing PI(4,5)P2 concentration shortens Kir2.2 interburst duration and lengthens burst duration, without affecting the kinetics within the burst. To study the contribution of each phosphate on PI(4,5)P2 in activating the channel, I tested the response of Kir2.2 to different phosphoinositides (PIPs). I show that 5\u27 phosphate is essential to Kir2.2 gating. Other PIPs without 5\u27 phosphate can compete with PI(4,5)P2 but cannot activate Kir2.2. A cell typically has multiple signaling pathways operating simultaneously, often even sharing the same components. All these proteins constantly move within the cell membrane. How does each protein recognize its downstream targets and ensure that the signaling process functions specifically? In the second part of my thesis, using electron microscopy I show that five membrane proteins – three GPCRs, an ion channel, and an enzyme – form self-clusters under natural expression levels in a cardiac-derived cell line. The cluster size distributions imply that these proteins self-oligomerize through weak interactions. I then investigated the biological function of protein clustering. In this part, I examined the role of protein distributions in the M2R-GIRK channel signaling. I found that a positive bias exists for GIRK clusters to be near M2R clusters. I conclude that M2R clusters increase Gβγ concentration locally. The correlation between the electron microscopy findings and electrophysiology suggests that only a small fraction of GIRK channels—those close to M2R clusters—can be regulated by M2R. By invoking weak interactions that permit transient binding of M2R to M2R and GIRK to GIRK and M2R to GIRK the distribution patterns and electrophysiological properties of the HL-1 cell membrane are replicated in a reaction-diffusion simulation. Based on the simulation, I propose that both protein clustering and the positive bias are crucial to the M2R-GIRK channel signaling pathway
Neuromast Optogenetics Reveals Rules of Spatial Encoding in the Zebrafish Lateral Line
No full textFor animals to respond effectively to their environment, their sensory circuits must learn to distinguish between similar patterns of sensory information. A lingering question in neuroscience is how neuronal circuits achieve this performance. In this study, I use the posterior lateral line (pLL) of larval zebrafish to address this question. In the wild, the pLL must differentiate among a variety of hydrodynamic stimuli. However, understanding how fish distinguish between different water-flow stimuli has been challenging due to difficulties in stimulating individual neuromasts. To tackle this, I introduce a novel method in this thesis that combines single-neuromast optogenetics with whole-brain calcium imaging in zebrafish larvae. By optogenetically stimulating individual neuromasts, I observe that second-order circuits in the medial octavolateralis nucleus (MON) exhibit diverse selectivity properties to neuromast input, despite an expected lack of spatiotopy. I further demonstrate that complex combinations of neuromast stimulation are represented by sparse ensembles of neurons within the MON and show that neuromast input integrates in the zebrafish brain through non-linear means. Based on my results, I discuss the implications and limitations of this experimental system, suggest new strategies for enhancement, and detail future directions for using single-neuromast optogenetics to better understand the integrative encoding of directional flow and the developmental capacity of central circuits associated with the pLL. My approach offers an innovative method for spatiotemporally interrogating the zebrafish lateral line system and presents a valuable model for studying whole-brain sensory encoding
Regulation of Chromatin Multi-Scale Organization and Function by Linker Histone H1
No full textThe three-dimensional organization of the genome is critical for regulating gene expression and maintaining cellular identity. The organizing principles underlying this 3D structure are a subject of much study, and it has proven to be a complex system to unravel. There are multiple scales of genome organization, ranging from single basepair modifications to chromatin compartments spanning many megabasepairs, and innumerable factors influence and regulate genome structure and function. One factor that appears to play a key role is linker histone H1, which is the most prevalent chromatin-binding protein in the cell. It has long been thought to be responsible for compacting chromatin and repressing transcription, though many historical studies of H1 were performed in vitro, and the details of its role in cells remain unclear. H1 is essential in mammals, and stable depletion in cells has previously resulted in disruption of higher-order chromatin structure, epigenetic landscape, and, to varying degrees, gene regulation. Here, we show that acute H1 depletion results in decompaction of the chromatin fiber, accompanied by transcriptional derepression and increased chromatin accessibility. In contrast to stable H1 knockdown, long-range chromatin structure and the epigenetic landscape are only modestly affected in our acute depletion system, indicating that these are downstream effects of H1 depletion. These findings show that the primary function of H1 in cells is influencing short-range chromatin structure and transcription, and further unravel the relationship between genome structure and function by showing that major gene regulation can occur without strong shifts in the epigenetic landscape or long-range chromatin organization
Recovering from Dormancy: Fixation of CSM6-Inducing Spacers in the Bacterial Population
No full textOrganisms from every kingdom of life constantly need to contend with their parasites to survive and reproduce. Bacteriophages (phages) are the viruses that infect bacteria and often outnumber them in most ecological niches. As a result, prokaryotes have evolved a myriad of defense systems to restrict the propagation of phages as well as other parasitic mobile genetic elements such as plasmids. Genetic loci known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated cas genes serve as one such defense system in both bacteria and archaea. A hallmark of CRISPR immunity is its adaptive nature in which memories of prior infections are acquired and stored. CRISPR loci contain clusters of characteristic repeat sequences separated by short spacer sequences which are derived from prior invading genetic material and form the basis of these acquired memories. During an immune response, spacer sequences are transcribed into guide RNAs and will recognize complementary sequences on invading genomes. Upon target recognition and binding, guide RNAs direct Cas effector proteins and complexes to the sites of infection where restriction of the phage can take place. The staphylococcal type III-A CRISPR-Cas immune system uses guide RNAs to locate target sequences on nascent viral transcripts instead of on DNA. Due to the transcription dependent nature of target sequence recognition, this response can display two distinct mechanisms of immunity depending on how soon the target is transcribed after infection. When the CRISPR-Cas system is triggered by a target sequence on an early-expressed phage transcript, direct degradation of the associated viral DNA by the DNase domain of Cas10 occurs. This rapid activation of immunity leads to a robust response which cures the host from infection before the phage can replicate. In contrast, when the guide RNA targets a late-expressed transcript, successful defense requires the additional activity of Csm6, a non-specific RNase which cleaves both invader and host RNA in the cell. Csm6 activity results in mass RNA degradation which triggers dormancy in the infected cell. How dormancy protects from infection and whether it can be relieved is not known. Abortive infection mechanisms of immunity are common in prokaryotes and confer defense at the population level – infected cells will sacrifice themselves to inhibit the propagation of the invader, saving uninfected cells in the population from infection. It has long been assumed that Csm6 confers immunity through an abortive infection mechanism. Here my thesis work shows that Csm6 triggers a growth arrest in the host that hinders viral propagation, initially providing immunity at the population level by allowing the continued replication of uninfected cells. My work also demonstrates that Csm6-induced dormancy has selective advantages for cells as it leads to broad immunity against untargeted phages. This selective advantage offers an explanation as to why cells may opt to acquire spacers that rely on Csm6 activity. Finally, my work shows that Csm6-induced dormancy is reversible in a subset of cells through the eventual degradation of the phage DNA, explaining how such spacers are maintained within the population. Collectively, my thesis work addresses a long-standing conundrum in the field and reveals that Csm6 does not operate solely through abortive infection. Instead, the type III-A CRISPR-Cas system has a built-in mechanism that allows for exit from Csm6-induced dormancy which enable the subsistence of spacers that provide broad-spectrum immunity
Sequential Transcriptional Gates in The Thalamo-Cortical Circuit Coordinate Memory Consolidation
No full textHow are memories maintained over weeks, months or even years? The molecular mechanisms that enable memories to persist over long time-scales remain poorly understood. As a point of entry, recent work in our lab revealed that beyond the hippocampus, where memories are initially formed over hours/days, the thalamo-cortical circuit is important for the gradual stabilization of memories over weeks/months. In this thesis, I aimed to reveal the molecular mechanisms operating in the thalamo-cortical circuit that may be responsible for extending memory time-scales. I began by developing a virtual reality-based behavioral paradigm where, by varying the frequency of learned associations, mice formed multiple memories but only consolidated some, while forgetting others, over the span of weeks. I then profiled the molecular programs that diverge between consolidated and forgotten memories across the anterior thalamus (ANT) and anterior cingulate cortex (ACC). Transcriptomic analyses identified distinct waves of transcription (cellular macrostates), unique to consolidated memories, that defined memory persistence. Notably, a select set of transcriptional regulators—Camta1 and Tcf4 in the ANT, and Ash1l in the ACC—orchestrated region specific molecular programs that enabled entry into these macrostates. Targeted CRISPR-knockout studies revealed that while these transcriptional regulators had no effects on memory formation, they had prominent time-dependent roles in memory stabilization. In particular, Camta1 was required for initial memory maintenance over days, while Tcf4 and the histone methyl-transferase, Ash1l, were required for sustaining memory at later stages, extending into weeks. How might these transcription factors function to extend memory time-scales? Further mechanistic studies through ChIP-sequencing revealed that Camta1 primarily targets genes involved in synaptic plasticity, while Tcf4 regulates genes associated with longer-lived adhesion and structural elements. Ash1l epigenetically regulates both synaptic and structural gene programs, thus functioning not necessarily to create new targets but rather to prime and prolong pre-existing plasticity and structural gene programs required for synaptic persistence. This study highlights the ANT–ACC circuit as a crucial circuit for memory maintenance and puts forth a model where transcriptional programs acting on progressively longer time-scales across the ANT-ACC support continuous memory stabilization ultimately shaping stable cortical memory ensembles
Inquiries into the Mycobacterial Caseinolytic Protease System
No full textDespite progress over the last century, Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), remains the leading cause of death from an infectious disease worldwide. The high global burden of Mtb, alongside its protracted treatment regimen and insidious rise of multi-drug resistance, underscores the need for non-canonical drug targets and pathways to improve antitubercular chemotherapy. Mtb\u27s success as a pathogen is linked to its ability to infect and evade clearance by the human immune system despite a wide range of noxious host stressors. Along with transcriptional responses, the maintenance of protein homeostasis is critical to this adaptability and pathogenicity. Previous transposon insertion sequencing (TnSeq) screens, in conjunction with our laboratory\u27s work on mycobacterial genetic vulnerability, identified the caseinolytic protease (Clp) system, a central component of the proteostasis network, as essential and highly vulnerable in Mtb. As an attractive, emergent anti-tubercular target, a deeper mechanistic understanding of the Clp complex in vivo is instrumental in refining ongoing drug discovery efforts and improving our understanding of Clp\u27s role in Mtb pathogenesis. The Clp system in Mtb is comprised of a two-tiered proteolytic barrel of homoheptameric ClpP1 and ClpP2 rings that degrades protein substrates delivered by homohexameric AAA+ unfoldases, ClpX and ClpC1. In contrast to other organisms, all components of the Clp system in Mtb are highly vulnerable and essential for growth in axenic culture and mouse models of infection. Due to the technical challenges associated with studying essential genes in mycobacteria, few bona-fide substrates of the Clp system have been identified. Furthermore, because of these challenges, correlations between seminal biochemical and structural studies with in vivo function are scarce. With the development of an inducible and tunable CRISPR interference (CRISPRi) platform in our laboratory, we were uniquely positioned to systematically investigate key questions in mycobacterial Clp biology, including which processes are regulated by the Clp system and contribute to its essentiality. By complementing knockdown of endogenous clp components with CRISPRi-resistant alleles encoding biochemically functional mutations, we unexpectedly found that only the proteolytic activity of ClpP1, but not ClpP2, is essential for Clp substrate degradation, Mtb growth, and murine infection. Our observations not only support a revised model of the Mtb Clp system, where ClpP2 essentiality stems from scaffolding chaperone binding while ClpP1 provides the essential proteolytic activity of the complex, but also hold important implications for the current development of inhibitors toward this therapeutic target. Through a combination of methods including co-immunoprecipitation coupled with mass spectrometry (co-IP/MS) and in vivo substrate accumulation experiments, several new ClpC1 substrates and interactors were identified in the non-pathogenic, Mtb model organism M. smegmatis (Msmeg). RbpA, an essential transcription factor, was highly enriched and found to be regulated post-translationally by ClpC1 through recognition of an N-terminal degron sequence. Such hits contribute to the short list of documented mycobacterial Clp substrates and directly implicate the ClpC1 chaperone in transcriptional regulation. Fluorescent degradation assays successfully identified novel C-terminal ClpC1 regulated degrons in a higher throughput manner, with certain degron sequences degraded more strongly than others. We found that ClpC1 interactors span broad biological roles emphasizing the central role of this complex in mycobacterial proteostasis. To better understand the processes underlying clpC1 essentiality, a genetic interaction screen was undertaken in a hypomorphic clpC1 knockdown background. Genes were identified and validated through growth assays that were either sensitized to knockdown, termed collateral vulnerabilities, or experienced enhanced fitness, positive interactors. Among the major collateral vulnerabilities were genes involved in cell envelope biosynthetic processes, consistent with morphological defects observed by microscopy upon clp disruption. Positive interactors were diverse in function and likely have complex interactions with clpC1. A highly vulnerable gene and strong positive interactor, gatD, involved in lipid II and peptidoglycan biosynthesis, was found to be a putative novel ClpC1 substrate through recognition of a C-terminal degron. A plate suppressor screen found that suppression of the succinate dehydrogenase, sdh, or succinyl-CoA synthase, suc, operons partially rescues an otherwise lethal strong clpC1 knockdown strain, suggesting ClpC1 may be involved in cellular respiration. That repression of no single gene fully rescues the strong growth defect imparted by strong clpC1 knockdown suggests clpC1 essentiality likely stems from pleiotropic functions in the bacterium, in contrast to findings of clp essentiality in the model bacterium Caulobacter crescentus. Taken together, the results presented in this thesis deepen our understanding of the mycobacterial Clp system in vivo. By fine tuning the expression of endogenous clp genes, the functional essentiality of its composite members was studied, novel substrates and degrons were characterized, and pathways directly or indirectly regulated by the protease complex were identified. By better defining the broad roles of the Clp system in mycobacterial physiology, we hope these studies contribute to a better understanding of its function in the proteostasis network and aid in the development of improved treatments against this recalcitrant pathogen