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Item ANKRD24 organizes TRIOBP to reinforce stereocilia insertion points(JCB, 2022-02-17) Krey, Jocelyn F.; Liu, Chang; Belyantseva, Inna A.; Bateschell, Michael; Dumont, Rachel A.; Goldsmith, Jennifer; Chatterjee, Paroma; Morrill, Rachel S.; Fedorov, Lev M.; Foster, Sarah; Kim, Jinkyung; Nuttall, Alfred L.; Jones, Sherri M.; Choi, Dongseok; Friedman, Thomas B.; Ricci, Anthony J.; Zhao, Bo; Barr-Gillespie, Peter G.; Otolaryngology -- Head and Neck Surgery, School of MedicineThe stereocilia rootlet is a key structure in vertebrate hair cells, anchoring stereocilia firmly into the cell’s cuticular plate and protecting them from overstimulation. Using superresolution microscopy, we show that the ankyrin-repeat protein ANKRD24 concentrates at the stereocilia insertion point, forming a ring at the junction between the lower and upper rootlets. Annular ANKRD24 continues into the lower rootlet, where it surrounds and binds TRIOBP-5, which itself bundles rootlet F-actin. TRIOBP-5 is mislocalized in Ankrd24KO/KO hair cells, and ANKRD24 no longer localizes with rootlets in mice lacking TRIOBP-5; exogenous DsRed–TRIOBP-5 restores endogenous ANKRD24 to rootlets in these mice. Ankrd24KO/KO mice show progressive hearing loss and diminished recovery of auditory function after noise damage, as well as increased susceptibility to overstimulation of the hair bundle. We propose that ANKRD24 bridges the apical plasma membrane with the lower rootlet, maintaining a normal distribution of TRIOBP-5. Together with TRIOBP-5, ANKRD24 organizes rootlets to enable hearing with long-term resilience.Item Branch-Specific Microtubule Destabilization Mediates Axon Branch Loss during Neuromuscular Synapse Elimination(Elsevier, 2016-11-23) Brill, Monika S.; Kleele, Tatjana; Ruschkies, Laura; Wang, Mengzhe; Marahori, Natalia A.; Reuter, Miriam S.; Hausrat, Torben J.; Weigand, Emily; Fisher, Matthew; Ahles, Andrea; Engelhardt, Stefan; Bishop, Derron L.; Kneussel, Matthias; Misgeld, Thomas; Department of Cellular & Integrative Physiology, IU School of MedicineDevelopmental axon remodeling is characterized by the selective removal of branches from axon arbors. The mechanisms that underlie such branch loss are largely unknown. Additionally, how neuronal resources are specifically assigned to the branches of remodeling arbors is not understood. Here we show that axon branch loss at the developing mouse neuromuscular junction is mediated by branch-specific microtubule severing, which results in local disassembly of the microtubule cytoskeleton and loss of axonal transport in branches that will subsequently dismantle. Accordingly, pharmacological microtubule stabilization delays neuromuscular synapse elimination. This branch-specific disassembly of the cytoskeleton appears to be mediated by the microtubule-severing enzyme spastin, which is dysfunctional in some forms of upper motor neuron disease. Our results demonstrate a physiological role for a neurodegeneration-associated modulator of the cytoskeleton, reveal unexpected cell biology of branch-specific axon plasticity and underscore the mechanistic similarities of axon loss in development and disease.Item Collapsin Response Mediator Protein 1 (CRMP1) Is Required for High-Frequency Hearing(Elsevier, 2022) Li, Jinan; Liu, Chang; Zhao, Bo; Otolaryngology -- Head and Neck Surgery, School of MedicineCollapsin response mediator protein 1 (CRMP1), also known as dihydropyrimidinase-related protein 1, participates in cytoskeleton remodeling during axonal guidance and neuronal migration. In cochlear hair cells, the assembly and maintenance of the cytoskeleton is of great interest because it is crucial for the morphogenesis and maintenance of hair cells. Previous RNA sequencing analysis found that Crmp1 is highly expressed in cochlear hair cells. However, the expression profile and functions of CRMP1 in the inner ear remain unknown. In this study, the expression and localization of CRMP1 in hair cells was investigated using immunostaining, and was shown to be highly expressed in both outer and inner hair cells. Next, the stereocilia morphology of Crmp1-deficient mice was characterized. Abolishing CRMP1 did not affect the morphogenesis of hair cells. Interestingly, scanning electron microscopy detected hair cell loss at the basal cochlear region, an area responsible for high-frequency auditory perception, in Crmp1-deficient mice. Correspondingly, an auditory brainstem response test showed that mice lacking CRMP1 had progressive hearing loss at high frequencies. In summary, these data suggest that CRMP1 is required for high-frequency auditory perception.Item Conditional Myh9 and Myh10 inactivation in adult mouse renal epithelium results in progressive kidney disease(American Society for Clinical Investigation, 2020-11-05) Otterpohl, Karla L.; Busselman, Brook W.; Ratnayake, Ishara; Hart, Ryan G.; Hart, Kimberly R.; Evans, Claire M.; Phillips, Carrie L.; Beach, Jordan R.; Ahrenkiel, Phil; Molitoris, Bruce A.; Surendran, Kameswaran; Chandrasekar, Indra; Pathology and Laboratory Medicine, School of MedicineActin-associated nonmuscle myosin II (NM2) motor proteins play critical roles in a myriad of cellular functions, including endocytosis and organelle transport pathways. Cell type–specific expression and unique subcellular localization of the NM2 proteins, encoded by the Myh9 and Myh10 genes, in the mouse kidney tubules led us to hypothesize that these proteins have specialized functional roles within the renal epithelium. Inducible conditional knockout (cKO) of Myh9 and Myh10 in the renal tubules of adult mice resulted in progressive kidney disease. Prior to overt renal tubular injury, we observed intracellular accumulation of the glycosylphosphatidylinositol-anchored protein uromodulin (UMOD) and gradual loss of Na+ K+ 2Cl– cotransporter from the apical membrane of the thick ascending limb epithelia. The UMOD accumulation coincided with expansion of endoplasmic reticulum (ER) tubules and activation of ER stress and unfolded protein response pathways in Myh9&10-cKO kidneys. We conclude that NM2 proteins are required for localization and transport of UMOD and loss of function results in accumulation of UMOD and ER stress–mediated progressive renal tubulointerstitial disease. These observations establish cell type–specific role(s) for NM2 proteins in regulation of specialized renal epithelial transport pathways and reveal the possibility that human kidney disease associated with MYH9 mutations could be of renal epithelial origin.Item Cytoskeletal organization in the osteoclast(2000) Babb, Sherry G.Item Disruption of both ROCK1 and ROCK2 genes in cardiomyocytes promotes autophagy and reduces cardiac fibrosis during aging(Wiley, 2019-06) Shi, Jianjian; Surma, Michelle; Yang, Yang; Wei, Lei; Pediatrics, School of MedicineIn this study, we investigated the pathophysiological impact of Rho-associated coiled-coil–containing protein kinase (ROCK)1 and ROCK2 double deletion vs. single deletion on cardiac remodeling. Utilizing a cardiomyocyte-specific and tamoxifen-inducible MerCreMer recombinase (MCM), 3 mouse lines (MCM/ROCK1fl/fl/ROCK2fl/fl, MCM/ROCK1fl/fl, and MCM/ROCK2fl/fl) were generated. As early as 5 d after inducible deletion, the double ROCK knockout hearts exhibited reduced phosphorylation of myosin light chain (MLC) and focal adhesion kinase (FAK), supporting a role for ROCK activity in regulating the nonsarcomeric cytoskeleton. Moreover, the autophagy marker microtubule-associated proteins 1A-1B light chain 3B was increased in the double ROCK knockout, and these early molecular features persisted throughout aging. Mechanistically, the double ROCK knockout promoted age-associated or starvation-induced autophagy concomitant with reduced protein kinase B (AKT), mammalian target of rapamycin (mTOR), Unc-51–like kinase signaling, and cardiac fibrosis. In contrast, ROCK2 knockout hearts showed increased phosphorylated (p)-MLC and p-FAK levels, which were mostly attributable to a compensatory ROCK1 overactivation. Autophagy was inhibited at the baseline accompanying increased mTOR activity, leading to increased cardiac fibrosis in the ROCK2 knockout hearts. Finally, the loss of ROCK1 had no significant effect on p-MLC and p-FAK levels, mTOR signaling, or autophagy at baseline. In summary, deletions of ROCK isoforms in cardiomyocytes have different, even opposite, effects on endogenous ROCK activity and the MLC/FAK/AKT/mTOR signaling pathway, which is involved in autophagy and fibrosis of the heart.Item Elastase alters contractility and promotes an inflammatory synthetic phenotype in airway smooth muscle tissues(American Physiological Society, 2018-04-01) Lockett, Angelia D.; Wu, Yidi; Gunst, Susan J.; Cellular and Integrative Physiology, School of MedicineNeutrophil elastase is secreted by inflammatory cells during airway inflammation and can elicit airway hyperreactivity in vivo. Elastase can degrade multiple components of the extracellular matrix. We hypothesized that elastase might disrupt the connections between airway smooth muscle (ASM) cells and the extracellular matrix and that this might have direct effects on ASM tissue responsiveness and inflammation. The effect of elastase treatment on ASM contractility was assessed in vitro in isolated strips of canine tracheal smooth muscle by stimulation of tissues with cumulatively increasing concentrations of acetylcholine (ACh) and measurement of contractile force. Elastase treatment potentiated contractile responses to ACh at low concentrations but suppressed the maximal contractile force generated by the tissues without affecting the phosphorylation of myosin regulatory light chain (RLC). Elastase also promoted the secretion of eotaxin and the activation of Akt in ASM tissues and decreased expression of smooth muscle myosin heavy chain, consistent with promotion of a synthetic inflammatory phenotype. As the degradation of matrix proteins can alter integrin engagement, we evaluated the effect of elastase on the assembly and activation of integrin-associated adhesion junction complexes in ASM tissues. Elastase led to talin cleavage, reduced talin binding to vinculin, and suppressed activation of the adhesome proteins paxillin, focal adhesion kinase, and vinculin, indicating that elastase causes the disassembly of adhesion junction complexes and the inactivation of adhesome signaling proteins. We conclude that elastase promotes an inflammatory phenotype and increased sensitivity to ACh in ASM tissues by disrupting signaling pathways mediated by integrin-associated adhesion complexes.Item Functional variants in the LRRK2 gene confer shared effects on risk for Crohn's disease and Parkinson's disease(American Association for the Advancement of Science, 2018-01-10) Hui, Ken Y.; Fernandez-Hernandez, Heriberto; Hu, Jianzhong; Schaffner, Adam; Pankratz, Nathan; Hsu, Nai-Yun; Chuang, Ling-Shiang; Carmi, Shai; Villaverde, Nicole; Li, Xianting; Rivas, Manual; Levine, Adam P.; Bao, Xiuliang; Labrias, Philippe R.; Haritunians, Talin; Ruane, Darren; Gettler, Kyle; Chen, Ernie; Li, Dalin; Schiff, Elena R.; Pontikos, Nikolas; Barzilai, Nir; Brant, Steven R.; Bressman, Susan; Cheifetz, Adam S.; Clark, Lorraine N.; Daly, Mark J.; Desnick, Robert J.; Duerr, Richard H.; Katz, Seymour; Lencz, Todd; Myers, Richard H.; Ostrer, Harry; Ozelius, Laurie; Payami, Haydeh; Peter, Yakov; Rioux, John D.; Segal, Anthony W.; Scott, William K.; Silverberg, Mark S.; Vance, Jeffery M.; Ubarretxena-Belandia, Iban; Foroud, Tatiana; Atzmon, Gil; Pe’er, Itsik; Ioannou, Yiannis; McGovern, Dermot P.B.; Yue, Zhenyu; Schadt, Eric E.; Cho, Judy H.; Peter, Inga; Medical and Molecular Genetics, School of MedicineCrohn's disease (CD), a form of inflammatory bowel disease, has a higher prevalence in Ashkenazi Jewish than in non-Jewish European populations. To define the role of nonsynonymous mutations, we performed exome sequencing of Ashkenazi Jewish patients with CD, followed by array-based genotyping and association analysis in 2066 CD cases and 3633 healthy controls. We detected association signals in the LRRK2 gene that conferred risk for CD (N2081D variant, P = 9.5 × 10-10) or protection from CD (N551K variant, tagging R1398H-associated haplotype, P = 3.3 × 10-8). These variants affected CD age of onset, disease location, LRRK2 activity, and autophagy. Bayesian network analysis of CD patient intestinal tissue further implicated LRRK2 in CD pathogenesis. Analysis of the extended LRRK2 locus in 24,570 CD cases, patients with Parkinson's disease (PD), and healthy controls revealed extensive pleiotropy, with shared genetic effects between CD and PD in both Ashkenazi Jewish and non-Jewish cohorts. The LRRK2 N2081D CD risk allele is located in the same kinase domain as G2019S, a mutation that is the major genetic cause of familial and sporadic PD. Like the G2019S mutation, the N2081D variant was associated with increased kinase activity, whereas neither N551K nor R1398H variants on the protective haplotype altered kinase activity. We also confirmed that R1398H, but not N551K, increased guanosine triphosphate binding and hydrolyzing enzyme (GTPase) activity, thereby deactivating LRRK2. The presence of shared LRRK2 alleles in CD and PD provides refined insight into disease mechanisms and may have major implications for the treatment of these two seemingly unrelated diseases.Item IMPAIRED FUNCTION OF FANCONI ANEMIA TYPE C DEFICIENT MACROPHAGES(2012-03-16) Liu, Ying; Haneline, Laura S.; Dent, Alexander L.; He, Johnny J.; Srour, Edward F.; Yoder, Mervin C.Fanconi anemia (FA) is a genetic disorder characterized by bone marrow (BM) failure. Previous studies suggest that FA patients exhibit alterations in immunologic function. However, it is unclear whether the immune defects are immune cell autonomous or secondary to leucopenia from evolving BM failure. The aim of the current study was to determine whether FA type C deficient (Fancc-/-) macrophages exhibit impaired function and contribute to an altered inflammatory response. In this study, primary peritoneal macrophage function and the inflammatory response of Fancc-/- immune cells after in vivo intraperitoneal (IP) administration of lipopolysaccharide (LPS) were assessed. Fancc-/- peritoneum exhibit normal macrophage distribution at baseline. However, Fancc-/- macrophages exhibit reduced adhesion both on fibronectin and endothelial cells, impaired migration toward monocyte chemotactic protein-1 (MCP-1) and macrophages-colony stimulating factor (M-CSF), and altered phagocytosis of E.coli and ImmunoglobulinG (IgG)-labeled latex beads compared to WT. An altered F-actin reorganization and impaired activation of RhoA were observed in Fancc-/- macrophages. After single LPS injection IP, Fancc-/- mice exhibited decreased macrophage recruitment, reduced peripheral inflammatory monocytes and impaired myeloid colony formation in presence of M-CSF. Upon M-CSF stimulation, Fancc-/- BM derived macrophages (BMDM) showed a decreased phosphorylation of AKT and ERK compared to WT, leading to reduced proliferation. Collectively, these data suggest that Fancc-/- macrophages and subsequent defects in adhesion, migration, phagocytosis, and recruitment in vivo. These data also support a Fancc-/- macrophage cells autonomous defect predisposing to an altered inflammatory response.Item Lysine Acetylation Is Widespread on Proteins of Diverse Function and Localization in the Protozoan Parasite Toxoplasma gondii(American Society for Microbiology, 2012) Jeffers, Victoria; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of MedicineWhile histone proteins are the founding members of lysine acetylation substrates, it is now clear that hundreds of other proteins can be acetylated in multiple compartments of the cell. Our knowledge of the scope of this modification throughout the kingdom of life is beginning to emerge, as proteome-wide lysine acetylation has been documented in prokaryotes, Arabidopsis thaliana, Drosophila melanogaster, and human cells. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify parasite peptides enriched by immunopurification with acetyl-lysine antibody, we produced the first proteome-wide analysis of acetylation for a protozoan organism, the opportunistic apicomplexan parasite Toxoplasma gondii. The results show that lysine acetylation is abundant in the actively proliferating tachyzoite form of the parasite, which causes acute toxoplasmosis. Our approach successfully identified known acetylation marks on Toxoplasma histones and α-tubulin and detected over 400 novel acetylation sites on a wide variety of additional proteins, including those with roles in transcription, translation, metabolism, and stress responses. Importantly, an extensive set of parasite-specific proteins, including those found in organelles unique to Apicomplexa, is acetylated in the parasite. Our data provide a wealth of new information that improves our understanding of the evolution of this vital regulatory modification while potentially revealing novel therapeutic avenues. We conclude from this study that lysine acetylation was prevalent in the early stages of eukaryotic cell evolution and occurs on proteins involved in a remarkably diverse array of cellular functions, including those that are specific to parasites.