You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: Molecular Remodeling of Tip Links Underlies Mechanosensory Regeneration in Auditory Hair Cells}
Article:
Abstract:
Sound detection by inner ear hair cells requires tip links that interconnect mechanosensory stereocilia and convey force to yet unidentified transduction channels. Current models postulate a static composition of the tip link, with protocadherin 15( PCDH15) at the lower and cadherin 23( CDH23) at the upper end of the link. In terminally differentiated mammalian auditory hair cells, tip links are subjected to sound-induced forces throughout an organism's life. Although hair cells can regenerate disrupted tip links and restore hearing, the molecular details of this process are unknown. We developed a novel implementation of backscatter electron scanning microscopy to visualize simultaneously immuno-gold particles and stereocilia links, both of only a few nanometers in diameter. We show that functional, mechanotransduction-mediating tip links have at least two molecular compositions, containing either PCDH15/CDH23 or PCDH15/PCDH15. During regeneration, shorter tip links containing nearly equal amounts of PCDH15 at both ends appear first. Whole-cell patch-clamp recordings demonstrate that these transient PCDH15/PCDH15 links mediate mechanotransduction currents of normal amplitude but abnormal Ca2+-dependent decay( adaptation). The mature PCDH15/CDH23 tip link composition is re-established later, concomitant with complete recovery of adaptation. Thus, our findings provide a molecular mechanism for regeneration and maintenance of mechanosensory function in postmitotic auditory hair cells and could help identify elusive components of the mechanotransduction machinery.
Introduction:
Mechanosensory stereocilia bundles are arranged in rows of increasing height on the apical surface of the inner ear hair cells( Figure 1A). In mammals, auditory hair cells do not regenerate and therefore stereocilia bundles endure sound-induced deflections throughout an organism's life. When deflected by sound waves, stereocilia slide relative to each other[1] and tug on tiny extracellular tip link filaments[2]. Tip links are oriented obliquely, extending from the tops of shorter row stereocilia to the sides of stereocilia in the neighboring taller row( Figure 1A, inset). Current models of mechano-electrical transduction( MET) postulate that tip link tension controls the opening of the transduction channels that are located at or near the lower ends of the links[3]. Tip links are disrupted in vivo by intense acoustical stimulation[4] and can also be ablated in vitro by exposure to Ca2+-free extracellular medium, which results in the loss of hair cell mechanotransduction[5]. Although mammalian hair cells are terminally differentiated, broken tip links can regenerate and restore mechanosensitivity both in vitro[6] and in vivo[7]. However, the molecular mechanisms of this regenerative process are unknown, even though it is crucial for maintenance of hearing. Two protein components of the tip link have been identified, cadherin 23( CDH23)[8] and protocadherin 15( PCDH15)[9], which are both required for normal hearing and vision. Mutations of PCDH15 or CDH23 cause hearing loss or deaf-blindness[10]–[14]. It was proposed that the tip links are formed by a Ca2+-dependent heteromeric interaction between PCDH15 at the lower end of the link and CDH23 at the upper end[15]. This model is further supported by the evidence that tip link regeneration can be inhibited in the presence of soluble extracellular fragments of PCDH15 and CDH23[16]. However, disrupting the interaction of PCDH15 with CDH23 may have additional effects beyond tip link formation. A point mutation in PCDH15 that inhibits the PCDH15–CDH23 interaction in vitro results not only in the loss of tip links but also causes profound changes of the hair bundle structure[17], which were not observed in non-mechanosensitive hair cells of the mice lacking the TMC1/2 proteins, the latest candidates for MET channel subunits[18]. Furthermore, knocking out each of three major classes of PCDH15 alternative C-terminal splice isoforms( CD1, CD2, and CD3) demonstrated that none of them is uniquely required for tip link formation[19]. Therefore, it is still unclear whether or not a functional MET-mediating tip link always consists of a particular heteromeric PCDH15/CDH23 complex or tip links may have a different molecular composition. To explore the molecular mechanism of tip link regeneration, we developed backscatter electron scanning microscopy to simultaneously visualize nanometer-sized stereocilia links and immuno-gold particles localizing the tip link components PCDH15 and CDH23. We measured the distribution of PCDH15 and CDH23 on the surface of stereocilia and along the length of the tip links during their regeneration. To our surprise, nascent regenerating tip links had PC DH15 at both ends of the link. These newly formed shorter links mediated MET current with a normal amplitude but abnormal Ca2+-dependent decay( adaptation). Only later, the heteromeric PCDH15–CDH23 composition of tip links was re-established and the normal adaptation of MET current recovered. Thus, tip link regeneration involves a hitherto unknown remodeling mechanism, in which PCDH15 transiently substitutes for CDH23 to accelerate recovery of hair cell mechanosensitivity.
Discussion:
Our data revealed a hitherto unknown process of structural remodeling of broken tip links that is necessary for regeneration of MET in auditory hair cells( Figure 8). After stereocilia link disruption, CDH23 largely disappears while PCDH15 migrates from the side link area around the circumference of the stereocilium and becomes available for forming the upper end of a nascent tip link. At the same time, PCDH15 molecules at the lower end of a disrupted tip link also migrate circumferentially but remain at the stereocilia tip. Then, transient tip link filaments are assembled using PCDH15 molecules at both sides of the filament( Figure 8, red). These transient tip links mediate MET current with normal amplitude but an abnormal extent of adaptation. Finally, new or perhaps refolded/repaired CDH23 molecules( Figure 8, blue) replace PCDH15 at the upper end of the tip link, completing the regeneration of mature tip links and the concomitant recovery of MET adaptation. This final step seems to be essential for long-term maintenance of MET because CDH23 is apparently required for MET in mammalian hair cells[38]. The high affinity interaction between CDH23 and PCDH15[15] may favor the replacement of PCDH15 at the upper end of the tip link with newly synthesized or repaired CDH23, whose appearance in regenerating links is delayed relative to PCDH15. Because HL5614 antibodies recognize an extracellular epitope that seems to be common to numerous PCDH15 alternative splice isoforms[9] and because these isoforms may have redundant function[19], we cannot distinguish whether nascent regenerating tip links have at their ends identical or different PCDH15 isoforms. It is yet uncertain whether nonclassical cadherins like PCDH15 or CDH23 could form stable extracellular links through homomeric interaction. Evidence for CDH23–CDH23 homomeric interaction have been reported[8], but the same group later suggested that this interaction, along with homomeric PCDH15–PCDH15 interaction, is likely to have a low affinity[15], which is consistent with our data that PCDH15–PCDH15 tip links are only temporary intermediates in the tip link regeneration process. A recent report also provided no direct evidence for homomeric interaction between the first EC domains of either CDH23 or PCDH15[39]. However, heteromeric interaction of different PCDH15 isoforms is still possible. Alternative splicing of the Pcdh15 gene can generate several protein products that differ at the N-terminus( Figure S7) and provides a dazzling variety of potential heteromeric interactions between different PCDH15 isoforms, which are known to be targeted specifically to different stereocilia compartments[9]. In addition, a secreted isoform of PCDH15 lacking the transmembrane domain[9],[40] may also be recruited to help span the distance between adjacent stereocilia. It is yet unclear exactly how molecular remodeling of tip links affects the extent of adaptation of MET responses. It is possible that the decreased length of PCDH15–PCDH15 tip links changes the angle of inclination of tip link and therefore affects the extent of adaptation( Figure S8A–B). However, such changes are predicted to affect the slope of the MET current–displacement curve, which was not observed in our experiments( Figure 4D). The extent of adaptation may also decrease if the elastic harmonin-based complex connecting the upper end of the tip link to the cytoskeleton is replaced to a complex with nonlinear saturating stiffness, for example with PCDH15 that is bound to the cytoskeleton with molecules that “slip down” along the actin core when tip link tension reaches a certain level( Figure S8C). Indeed, recent studies identified harmonin and THMS as interacting partners of CDH23 and PCDH15, correspondingly[41],[42]. These very different interacting partners may contribute to different mechanical properties of macromolecular complexes connecting CDH23 and PCDH15 to the actin cores of stereocilia. Another possibility is that CDH23 at the upper end of the tip link may affect homomeric interaction of parallel PCDH15 filaments at the lower end of the link, thereby affecting the adaptation machinery, or recruit additional adaptation-related components of tip link, which can be seen as a third “foot” at the lower end of the link[43]. In any case, our data open new perspectives to study the molecular basis of MET machinery by characterizing redistribution of the proteins normally associated with PCDH15 and CDH23 during tip link regeneration. Delayed acquisition of fully developed adaptation is a common finding of studies investigating formation of the mechanotransduction apparatus either after tip link disruption[6],[16] or during development[30],[36],[37]. However, there are different interpretations of these data. According to one model, the fully functional mechanotransduction machinery is assembled somewhere around the base of the hair bundle and transported by myosin-based motors to the tips of stereocilia, where it undergoes final “refinement”[37]. Another model postulates that the mechanotransduction apparatus is assembled at its final location at the tips of the stereocilia from components that arrive there in a stepwise progression[30]. We did not find evidence for upward movement of stereocilia links or PCDH15 molecules along the stereocilium( Figure 1), which is consistent with direct assembly of the transduction apparatus at the tips of stereocilia. On the other hand, replacement of PCDH15 with CDH23 at the final step of molecular remodeling of the tip links may be considered as a “refinement” of the transduction apparatus. We should note, however, one caveat of the direct comparison of our data with the previously published reports. In this study, we investigated regeneration of mechanotransduction in young postnatal mouse IHCs, capitalizing on the unique sensitivity of all stereocilia links in these cells to BAPTA treatment. Previous reports described regeneration or development of mechanotransduction in different hair cell types—e. g., hair cells of chick basilar papilla[6],[36], mouse vestibular hair cells[25], and cochlear outer hair cells of rat[30] and mouse[16],[37]. The diversity of preparations suggests caution in comparing results. For example, an unusually fast recovery of MET current–displacement relationship( Figure 4D) may result from specific mechanical properties of the IHC bundles or from better control of vertical positioning of the stimulating probe in our experiments( Figure 3). Our data reconcile two previous molecular models of tip link formation. One model proposed that the tip link has a PCDH15–CD3 isoform at the lower end and a PCHD15–CD1 isoform at the upper end of the link[9]. A more recent model suggests that the tip link is built with PCDH15 and CDH23 at the lower and upper ends of the link, correspondingly[15]. We show that the molecular composition of the tip links is not static. During tip link regeneration, the hair cell temporarily utilizes PCDH15 molecules at both ends of the link to recover MET responses until mature PCDH15–CDH23 links are established. It is generally believed that regeneration of tip links and mechanotransduction in vitro recapitulates the assembly of the transduction apparatus in vivo( also see Figures 6 and 7)[16],[25],[36],[37]. Therefore, the two-step molecular remodeling process that we have discovered may underlie formation of the tip links in the developing hair bundle, their turnover in the mature bundle, and their regeneration after intense acoustical stimulation in order to safeguard hearing.
The inner ear detects sound when stereocilia , the mechanosensory projections on the apical surface of the hair cells , are deflected and tug on tiny extracellular tip links . These links interconnect stereocilia and convey forces to the mechanosensitive transduction channels . Current models postulate a static composition of the tip link with protocadherin 15 ( PCDH15 ) at the link's bottom end and cadherin 23 ( CDH23 ) at the upper end . Tip links are subjected to substantial sound-induced forces . Although hair cells can renew ( regenerate ) disrupted tip links and restore hearing , the molecular details of this process are unknown . Our study provides mechanistic insight into tip link regeneration . We used backscatter scanning electron microscopy to monitor the distribution of immuno-gold labeled molecular components of the tip links during their re-formation and a conventional whole-cell patch-clamp technique to follow the concomitant recovery of mechano-electrical transduction . According to our data , the mechanotransduction machinery is initially re-established by the formation of functional ( mechanotransduction-mediating ) links of a previously unknown composition , PCDH15–PCDH15 . Transition to the PCDH15–CDH23 composition underlies final maturation of mechanotransduction . This two-step mechanism of tip link regeneration was unexpected . As tip links are continuously stressed by loud sounds and regenerated throughout an organism's life , we provide a plausible molecular mechanism for the life-long maintenance of mechanosensory function in nonregenerating cochlear hair cells .