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1.
PLoS Biol ; 21(4): e3001964, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-37011103

RESUMEN

Assembly of the hair bundle, the sensory organelle of the inner ear, depends on differential growth of actin-based stereocilia. Separate rows of stereocilia, labeled 1 through 3 from tallest to shortest, lengthen or shorten during discrete time intervals during development. We used lattice structured illumination microscopy and surface rendering to measure dimensions of stereocilia from mouse apical inner hair cells during early postnatal development; these measurements revealed a sharp transition at postnatal day 8 between stage III (row 1 and 2 widening; row 2 shortening) and stage IV (final row 1 lengthening and widening). Tip proteins that determine row 1 lengthening did not accumulate simultaneously during stages III and IV; while the actin-bundling protein EPS8 peaked at the end of stage III, GNAI3 peaked several days later-in early stage IV-and GPSM2 peaked near the end of stage IV. To establish the contributions of key macromolecular assemblies to bundle structure, we examined mouse mutants that eliminated tip links (Cdh23v2J or Pcdh15av3J), transduction channels (TmieKO), or the row 1 tip complex (Myo15ash2). Cdh23v2J/v2J and Pcdh15av3J/av3J bundles had adjacent stereocilia in the same row that were not matched in length, revealing that a major role of these cadherins is to synchronize lengths of side-by-side stereocilia. Use of the tip-link mutants also allowed us to distinguish the role of transduction from effects of transduction proteins themselves. While levels of GNAI3 and GPSM2, which stimulate stereocilia elongation, were greatly attenuated at the tips of TmieKO/KO row 1 stereocilia, they accumulated normally in Cdh23v2J/v2J and Pcdh15av3J/av3J stereocilia. These results reinforced the suggestion that the transduction proteins themselves facilitate localization of proteins in the row 1 complex. By contrast, EPS8 concentrates at tips of all TmieKO/KO, Cdh23v2J/v2J, and Pcdh15av3J/av3J stereocilia, correlating with the less polarized distribution of stereocilia lengths in these bundles. These latter results indicated that in wild-type hair cells, the transduction complex prevents accumulation of EPS8 at the tips of shorter stereocilia, causing them to shrink (rows 2 and 3) or disappear (row 4 and microvilli). Reduced rhodamine-actin labeling at row 2 stereocilia tips of tip-link and transduction mutants suggests that transduction's role is to destabilize actin filaments there. These results suggest that regulation of stereocilia length occurs through EPS8 and that CDH23 and PCDH15 regulate stereocilia lengthening beyond their role in gating mechanotransduction channels.


Asunto(s)
Mecanotransducción Celular , Estereocilios , Ratones , Animales , Estereocilios/metabolismo , Mecanotransducción Celular/fisiología , Actinas/metabolismo , Células Ciliadas Auditivas/metabolismo , Proteínas de Microfilamentos/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Cadherinas/genética , Cadherinas/metabolismo
2.
J Physiol ; 599(4): 1173-1198, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33151556

RESUMEN

KEY POINTS: Mechanoelectrical transduction at auditory hair cells requires highly specialized stereociliary bundles that project from their apical surface, forming a characteristic graded 'staircase' structure. The morphogenesis and maintenance of these stereociliary bundles is a tightly regulated process requiring the involvement of several actin-binding proteins, many of which are still unidentified. We identify a new stereociliary protein, the I-BAR protein BAIAP2L2, which localizes to the tips of the shorter transducing stereocilia in both inner and outer hair cells (IHCs and OHCs). We find that Baiap2l2 deficient mice lose their second and third rows of stereocilia, their mechanoelectrical transducer current, and develop progressive hearing loss, becoming deaf by 8 months of age. We demonstrate that BAIAP2L2 localization to stereocilia tips is dependent on the motor protein MYO15A and its cargo EPS8. We propose that BAIAP2L2 is a new key protein required for the maintenance of the transducing stereocilia in mature cochlear hair cells. ABSTRACT: The transduction of sound waves into electrical signals depends upon mechanosensitive stereociliary bundles that project from the apical surface of hair cells within the cochlea. The height and width of these actin-based stereocilia is tightly regulated throughout life to establish and maintain their characteristic staircase-like structure, which is essential for normal mechanoelectrical transduction. Here, we show that BAIAP2L2, a member of the I-BAR protein family, is a newly identified hair bundle protein that is localized to the tips of the shorter rows of transducing stereocilia in mouse cochlear hair cells. BAIAP2L2 was detected by immunohistochemistry from postnatal day 2.5 (P2.5) throughout adulthood. In Baiap2l2 deficient mice, outer hair cells (OHCs), but not inner hair cells (IHCs), began to lose their third row of stereocilia and showed a reduction in the size of the mechanoelectrical transducer current from just after P9. Over the following post-hearing weeks, the ordered staircase structure of the bundle progressively deteriorates, such that, by 8 months of age, both OHCs and IHCs of Baiap2l2 deficient mice have lost most of the second and third rows of stereocilia and become deaf. We also found that BAIAP2L2 interacts with other key stereociliary proteins involved in normal hair bundle morphogenesis, such as CDC42, RAC1, EPS8 and ESPNL. Furthermore, we show that BAIAP2L2 localization to the stereocilia tips depends on the motor protein MYO15A and its cargo EPS8. We propose that BAIAP2L2 is key to maintenance of the normal actin structure of the transducing stereocilia in mature mouse cochlear hair cells.


Asunto(s)
Sordera , Proteínas de la Membrana , Estereocilios , Animales , Sordera/genética , Células Ciliadas Auditivas Internas , Células Ciliadas Auditivas Externas , Proteínas de la Membrana/genética , Ratones , Proteínas de Microfilamentos
3.
Neuron ; 111(20): 3195-3210.e7, 2023 10 18.
Artículo en Inglés | MEDLINE | ID: mdl-37543036

RESUMEN

OSCA/TMEM63s form mechanically activated (MA) ion channels in plants and animals, respectively. OSCAs and related TMEM16s and transmembrane channel-like (TMC) proteins form homodimers with two pores. Here, we uncover an unanticipated monomeric configuration of TMEM63 proteins. Structures of TMEM63A and TMEM63B (referred to as TMEM63s) revealed a single highly restricted pore. Functional analyses demonstrated that TMEM63s are bona fide mechanosensitive ion channels, characterized by small conductance and high thresholds. TMEM63s possess evolutionary variations in the intracellular linker IL2, which mediates dimerization in OSCAs. Replacement of OSCA1.2 IL2 with TMEM63A IL2 or mutations to key variable residues resulted in monomeric OSCA1.2 and MA currents with significantly higher thresholds. Structural analyses revealed substantial conformational differences in the mechano-sensing domain IL2 and gating helix TM6 between TMEM63s and OSCA1.2. Our studies reveal that mechanosensitivity in OSCA/TMEM63 channels is affected by oligomerization and suggest gating mechanisms that may be shared by OSCA/TMEM63, TMEM16, and TMC channels.


Asunto(s)
Interleucina-2 , Canales Iónicos , Animales , Interleucina-2/genética , Interleucina-2/metabolismo , Canales Iónicos/metabolismo , Mutación/genética
4.
Mol Biol Cell ; 33(4): br6, 2022 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-35044843

RESUMEN

Brain-specific angiogenesis inhibitor 1-associated protein 2-like protein 2 (BAIAP2L2), a membrane-binding protein required for the maintenance of mechanotransduction in hair cells, is selectively retained at the tips of transducing stereocilia. BAIAP2L2 trafficked to stereocilia tips in the absence of EPS8, but EPS8 increased the efficiency of localization. A tripartite complex of BAIAP2L2, EPS8, and MYO15A formed efficiently in vitro, and these three proteins robustly targeted to filopodia tips when coexpressed in cultured cells. Mice lacking functional transduction channels no longer concentrated BAIAP2L2 at row 2 stereocilia tips, a result that was phenocopied by blocking channels with tubocurarine in cochlear explants. Transduction channels permit Ca2+ entry into stereocilia, and we found that membrane localization of BAIAP2L2 was enhanced in the presence of Ca2+. Finally, reduction of intracellular Ca2+ in hair cells using BAPTA-AM led to a loss of BAIAP2L2 at stereocilia tips. Taken together, our results show that a MYO15A-EPS8 complex transports BAIAP2L2 to stereocilia tips, and Ca2+ entry through open channels at row 2 tips retains BAIAP2L2 there.


Asunto(s)
Calcio , Mecanotransducción Celular , Proteínas de la Membrana , Estereocilios , Animales , Calcio/metabolismo , Células Ciliadas Auditivas , Mecanotransducción Celular/fisiología , Proteínas de la Membrana/metabolismo , Ratones
5.
J Cereb Blood Flow Metab ; 37(10): 3278-3299, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-28816095

RESUMEN

Traumatic brain injury (TBI) is an expanding public health epidemic with pathophysiology that is difficult to diagnose and thus treat. TBI biomarkers should assess patients across severities and reveal pathophysiology, but currently, their kinetics and specificity are unclear. No single ideal TBI biomarker exists. We identified new candidates from a TBI CSF proteome by selecting trauma-released, astrocyte-enriched proteins including aldolase C (ALDOC), its 38kD breakdown product (BDP), brain lipid binding protein (BLBP), astrocytic phosphoprotein (PEA15), glutamine synthetase (GS) and new 18-25kD-GFAP-BDPs. Their levels increased over four orders of magnitude in severe TBI CSF. First post-injury week, ALDOC levels were markedly high and stable. Short-lived BLBP and PEA15 related to injury progression. ALDOC, BLBP and PEA15 appeared hyper-acutely and were similarly robust in severe and mild TBI blood; 25kD-GFAP-BDP appeared overnight after TBI and was rarely present after mild TBI. Using a human culture trauma model, we investigated biomarker kinetics. Wounded (mechanoporated) astrocytes released ALDOC, BLBP and PEA15 acutely. Delayed cell death corresponded with GFAP release and proteolysis into small GFAP-BDPs. Associating biomarkers with cellular injury stages produced astroglial injury-defined (AID) biomarkers that facilitate TBI assessment, as neurological deficits are rooted not only in death of CNS cells, but also in their functional compromise.


Asunto(s)
Astrocitos/patología , Biomarcadores/análisis , Lesiones Traumáticas del Encéfalo/líquido cefalorraquídeo , Proteínas Reguladoras de la Apoptosis , Astrocitos/química , Conmoción Encefálica , Lesiones Traumáticas del Encéfalo/diagnóstico , Células Cultivadas , Proteína de Unión a los Ácidos Grasos 7/sangre , Fructosa-Bifosfato Aldolasa/sangre , Humanos , Péptidos y Proteínas de Señalización Intracelular/sangre , Cinética , Fosfoproteínas/sangre , Proteoma/análisis , Proteínas Supresoras de Tumor/sangre
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