Epimorphic regeneration in the mammalian tympanic membrane.

Adult mammals are generally believed to have limited ability to regenerate complex tissues and instead, repair wounds by forming scars. In humans and across mammalian species, the tympanic membrane (TM) rapidly repairs perforations without intervention. Using mouse models, we demonstrate that the TM repairs itself through a process that bears many hallmarks of epimorphic regeneration rather than typical wound healing. Following injury, the TM forms a wound epidermis characterized by EGFR ligand expression and signaling. After the expansion of the wound epidermis that emerges from known stem cell regions of the TM, a multi-lineage blastema-like cellular mass is recruited. After two weeks, the tissue architecture of the TM is largely restored, but with disorganized collagen. In the months that follow, the organized and patterned collagen framework of the TM is restored resulting in scar-free repair. Finally, we demonstrate that deletion of Egfr in the epidermis results in failure to expand the wound epidermis, recruit the blastema-like cells, and regenerate normal TM structure. This work establishes the TM as a model of mammalian complex tissue regeneration.

Endoscopic Medial Reepithelization for Inflammatory Canal Stenosis.

OBJECTIVE: Inflammatory external auditory canal (EAC) Stenosis arises from infiltration of inflammatory cells, edema and eventual sclerosing of the medial EAC, leading to complete obstruction and conductive hearing loss. Current treatment includes surgical resection of the affected area with widening and reepithelization of the EAC via postauricular incision, but the condition is reported to recur with high frequency. Our aim was to assess the feasibility of endoscopic transcanal treatment as an alternative to postauricular canalplasty and understand its effect on recurrence rates. STUDY DESIGN: Retrospective case review. SETTING: Tertiary referral center. PATIENTS: Four patients were included who had bilateral conductive hearing loss and inflammatory canal stenosis, all with gross thickening of the tympanic membrane. INTERVENTIONS: Patients underwent endoscopic removal of obstructive tissue and reepithelization with split-thickness skin grafting. MAIN OUTCOME MEASURES: Postoperative air-bone gap (ABG), lack of recurrence, subjective reporting of hearing improvement, and lack of drainage. RESULTS: Eight of 8 ears (n = 4 patients) had significant improvement in hearing. No recurrence has been observed in any of the patients over a mean follow-up time of 90 months (range, 42-189 mo). Average reduction in ABG was 13.40 dB (SD = 9.0 dB) with a statistically significant difference between the pure tone average preoperative and postoperative ABG (p = 0.0008; n = 7). CONCLUSIONS: Endoscopic treatment of Inflammatory EAC stenosis obviates the need for postauricular incision and results in clinical improvement with a favorable recurrence rate.

A Hierarchy of Proliferative and Migratory Keratinocytes Maintains the Tympanic Membrane.

The tympanic membrane (TM) is critical for hearing and requires continuous clearing of cellular debris, but little is known about homeostatic mechanisms in the TM epidermis. Using single-cell RNA sequencing, lineage tracing, whole-organ explant, and live-cell imaging, we show that homeostatic TM epidermis is distinct from other epidermal sites and has discrete proliferative zones with a three-dimensional hierarchy of multiple keratinocyte populations. TM stem cells reside in a discrete location of the superior TM and generate long-lived clones and committed progenitors (CPs). CP clones exhibit lateral migration, and their proliferative capacity is supported by Pdgfra+ fibroblasts, generating migratory but non-proliferative progeny. Single-cell sequencing of the human TM revealed similar cell types and transcriptional programming. Thus, during homeostasis, TM keratinocytes transit through a proliferative CP state and exhibit directional lateral migration. This work forms a foundation for understanding TM disorders and modeling keratinocyte biology.

Dihydroxyacetone phosphate signals glucose availability to mTORC1.

The mechanistic target of rapamycin complex 1 (mTORC1) kinase regulates cell growth by setting the balance between anabolic and catabolic processes. To be active, mTORC1 requires the environmental presence of amino acids and glucose. While a mechanistic understanding of amino acid sensing by mTORC1 is emerging, how glucose activates mTORC1 remains mysterious. Here, we used metabolically engineered human cells lacking the canonical energy sensor AMP-activated protein kinase to identify glucose-derived metabolites required to activate mTORC1 independent of energetic stress. We show that mTORC1 senses a metabolite downstream of the aldolase and upstream of the GAPDH-catalysed steps of glycolysis and pinpoint dihydroxyacetone phosphate (DHAP) as the key molecule. In cells expressing a triose kinase, the synthesis of DHAP from DHA is sufficient to activate mTORC1 even in the absence of glucose. DHAP is a precursor for lipid synthesis, a process under the control of mTORC1, which provides a potential rationale for the sensing of DHAP by mTORC1.

Titrating gene expression using libraries of systematically attenuated CRISPR guide RNAs.

A lack of tools to precisely control gene expression has limited our ability to evaluate relationships between expression levels and phenotypes. Here, we describe an approach to titrate expression of human genes using CRISPR interference and series of single-guide RNAs (sgRNAs) with systematically modulated activities. We used large-scale measurements across multiple cell models to characterize activities of sgRNAs containing mismatches to their target sites and derived rules governing mismatched sgRNA activity using deep learning. These rules enabled us to synthesize a compact sgRNA library to titrate expression of ~2,400 genes essential for robust cell growth and to construct an in silico sgRNA library spanning the human genome. Staging cells along a continuum of gene expression levels combined with single-cell RNA-seq readout revealed sharp transitions in cellular behaviors at gene-specific expression thresholds. Our work provides a general tool to control gene expression, with applications ranging from tuning biochemical pathways to identifying suppressors for diseases of dysregulated gene expression.

SAMTOR is an S-adenosylmethionine sensor for the mTORC1 pathway.

mTOR complex 1 (mTORC1) regulates cell growth and metabolism in response to multiple environmental cues. Nutrients signal via the Rag guanosine triphosphatases (GTPases) to promote the localization of mTORC1 to the lysosomal surface, its site of activation. We identified SAMTOR, a previously uncharacterized protein, which inhibits mTORC1 signaling by interacting with GATOR1, the GTPase activating protein (GAP) for RagA/B. We found that the methyl donor S-adenosylmethionine (SAM) disrupts the SAMTOR-GATOR1 complex by binding directly to SAMTOR with a dissociation constant of approximately 7 µM. In cells, methionine starvation reduces SAM levels below this dissociation constant and promotes the association of SAMTOR with GATOR1, thereby inhibiting mTORC1 signaling in a SAMTOR-dependent fashion. Methionine-induced activation of mTORC1 requires the SAM binding capacity of SAMTOR. Thus, SAMTOR is a SAM sensor that links methionine and one-carbon metabolism to mTORC1 signaling.

KICSTOR recruits GATOR1 to the lysosome and is necessary for nutrients to regulate mTORC1.

The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth that responds to diverse environmental signals and is deregulated in many human diseases, including cancer and epilepsy. Amino acids are a key input to this system, and act through the Rag GTPases to promote the translocation of mTORC1 to the lysosomal surface, its site of activation. Multiple protein complexes regulate the Rag GTPases in response to amino acids, including GATOR1, a GTPase activating protein for RAGA, and GATOR2, a positive regulator of unknown molecular function. Here we identify a protein complex (KICSTOR) that is composed of four proteins, KPTN, ITFG2, C12orf66 and SZT2, and that is required for amino acid or glucose deprivation to inhibit mTORC1 in cultured human cells. In mice that lack SZT2, mTORC1 signalling is increased in several tissues, including in neurons in the brain. KICSTOR localizes to lysosomes; binds and recruits GATOR1, but not GATOR2, to the lysosomal surface; and is necessary for the interaction of GATOR1 with its substrates, the Rag GTPases, and with GATOR2. Notably, several KICSTOR components are mutated in neurological diseases associated with mutations that lead to hyperactive mTORC1 signalling. Thus, KICSTOR is a lysosome-associated negative regulator of mTORC1 signalling, which, like GATOR1, is mutated in human disease.

The CASTOR Proteins Are Arginine Sensors for the mTORC1 Pathway.

Amino acids signal to the mTOR complex I (mTORC1) growth pathway through the Rag GTPases. Multiple distinct complexes regulate the Rags, including GATOR1, a GTPase activating protein (GAP), and GATOR2, a positive regulator of unknown molecular function. Arginine stimulation of cells activates mTORC1, but how it is sensed is not well understood. Recently, SLC38A9 was identified as a putative lysosomal arginine sensor required for arginine to activate mTORC1 but how arginine deprivation represses mTORC1 is unknown. Here, we show that CASTOR1, a previously uncharacterized protein, interacts with GATOR2 and is required for arginine deprivation to inhibit mTORC1. CASTOR1 homodimerizes and can also heterodimerize with the related protein, CASTOR2. Arginine disrupts the CASTOR1-GATOR2 complex by binding to CASTOR1 with a dissociation constant of ~30 µM, and its arginine-binding capacity is required for arginine to activate mTORC1 in cells. Collectively, these results establish CASTOR1 as an arginine sensor for the mTORC1 pathway.

Sestrin2 is a leucine sensor for the mTORC1 pathway.

Leucine is a proteogenic amino acid that also regulates many aspects of mammalian physiology, in large part by activating the mTOR complex 1 (mTORC1) protein kinase, a master growth controller. Amino acids signal to mTORC1 through the Rag guanosine triphosphatases (GTPases). Several factors regulate the Rags, including GATOR1, aGTPase-activating protein; GATOR2, a positive regulator of unknown function; and Sestrin2, a GATOR2-interacting protein that inhibits mTORC1 signaling. We find that leucine, but not arginine, disrupts the Sestrin2-GATOR2 interaction by binding to Sestrin2 with a dissociation constant of 20 micromolar, which is the leucine concentration that half-maximally activates mTORC1. The leucine-binding capacity of Sestrin2 is required for leucine to activate mTORC1 in cells. These results indicate that Sestrin2 is a leucine sensor for the mTORC1 pathway.

The Sestrins interact with GATOR2 to negatively regulate the amino-acid-sensing pathway upstream of mTORC1.

The mechanistic target of rapamycin complex 1 (mTORC1) kinase is a major regulator of cell growth that responds to numerous environmental cues. A key input is amino acids, which act through the heterodimeric Rag GTPases (RagA or RagB bound to RagC or RagD) in order to promote the translocation of mTORC1 to the lysosomal surface, its site of activation. GATOR2 is a complex of unknown function that positively regulates mTORC1 signaling by acting upstream of or in parallel to GATOR1, which is a GTPase-activating protein (GAP) for RagA or RagB and an inhibitor of the amino-acid-sensing pathway. Here, we find that the Sestrins, a family of poorly understood growth regulators (Sestrin1-Sestrin3), interact with GATOR2 in an amino-acid-sensitive fashion. Sestrin2-mediated inhibition of mTORC1 signaling requires GATOR1 and the Rag GTPases, and the Sestrins regulate the localization of mTORC1 in response to amino acids. Thus, we identify the Sestrins as GATOR2-interacting proteins that regulate the amino-acid-sensing branch of the mTORC1 pathway.