Hirofumi Sakaguchi

Cadherin 23 and protocadherin 15 interact to form tip-link filaments in sensory hair cells.

Hair cells of the inner ear are mechanosensors that transduce mechanical forces arising from sound waves and head movement into electrochemical signals to provide our sense of hearing and balance. Each hair cell contains at the apical surface a bundle of stereocilia. Mechanoelectrical transduction takes place close to the tips of stereocilia in proximity to extracellular tip-link filaments that connect the stereocilia and are thought to gate the mechanoelectrical transduction channel. Recent reports on the composition, properties and function of tip links are conflicting. Here we demonstrate that two cadherins that are linked to inherited forms of deafness in humans interact to form tip links. Immunohistochemical studies using rodent hair cells show that cadherin 23 (CDH23) and protocadherin 15 (PCDH15) localize to the upper and lower part of tip links, respectively. The amino termini of the two cadherins co-localize on tip-link filaments. Biochemical experiments show that CDH23 homodimers interact in trans with PCDH15 homodimers to form a filament with structural similarity to tip links. Ions that affect tip-link integrity and a mutation in PCDH15 that causes a recessive form of deafness disrupt interactions between CDH23 and PCDH15. Our studies define the molecular composition of tip links and provide a conceptual base for exploring the mechanisms of sensory impairment associated with mutations in CDH23 and PCDH15.

NMII Forms a Contractile Transcellular Sarcomeric Network to Regulate Apical Cell Junctions and Tissue Geometry

Nonmuscle myosin II (NMII) is thought to be the master integrator of force within epithelial apical junctions, mediating epithelial tissue morphogenesis and tensional homeostasis. Mutations in NMII are associated with a number of diseases due to failures in cell-cell adhesion. However, the organization and the precise mechanism by which NMII generates and responds to tension along the intercellular junctional line are still not known. We discovered that periodic assemblies of bipolar NMII filaments interlace with perijunctional actin and α-actinin to form a continuous belt of muscle-like sarcomeric units (∼400-600 nm) around each epithelial cell. Remarkably, the sarcomeres of adjacent cells are precisely paired across the junctional line, forming an integrated, transcellular contractile network. The contraction/relaxation of paired sarcomeres concomitantly impacts changes in apical cell shape and tissue geometry. We show differential distribution of NMII isoforms across heterotypic junctions and evidence for compensation between isoforms. Our results provide a model for how NMII force generation is effected along the junctional perimeter of each cell and communicated across neighboring cells in the epithelial organization. The sarcomeric network also provides a well-defined target to investigate the multiple roles of NMII in junctional homeostasis as well as in development and disease.

Dynamic compartmentalization of protein tyrosine phosphatase receptor Q at the proximal end of stereocilia: Implication of myosin VI-based transport

Hair cell stereocilia are apical membrane protrusions filled with uniformly polarized actin filament bundles. Protein tyrosine phosphatase receptor Q (PTPRQ), a membrane protein with extracellular fibronectin repeats has been shown to localize at the stereocilia base and the apical hair cell surface, and to be essential for stereocilia integrity. We analyzed the distribution of PTPRQ and a possible mechanism for its compartmentalization. Using immunofluorescence we demonstrate that PTPRQ is compartmentalized at the stereocilia base with a decaying gradient from base to apex. This distribution can be explained by a model of transport directed toward the stereocilia base, which counteracts diffusion of the molecules. By mathematical analysis, we show that this counter transport is consistent with the minus end-directed movement of myosin VI along the stereocilia actin filaments. Myosin VI is localized at the stereocilia base, and exogenously expressed myosin VI and PTPRQ colocalize in the perinuclear endosomes in COS-7 cells. In myosin VI-deficient mice, PTPRQ is distributed along the entire stereocilia. PTPRQ-deficient mice show a pattern of stereocilia disruption that is similar to that reported in myosin VI-deficient mice, where the predominant features are loss of tapered base, and fusion of adjacent stereocilia. Thin section and freeze-etching electron microscopy showed that localization of PTPRQ coincides with the presence of a dense cell surface coat. Our results suggest that PTPRQ and myosin VI form a complex that dynamically maintains the organization of the cell surface coat at the stereocilia base and helps maintain the structure of the overall stereocilia bundle.

Tip links in hair cells: molecular composition and role in hearing loss.

Purpose of reviewTip links are thought to be an essential element of the mechanoelectrical transduction (MET) apparatus in sensory hair cells of the inner ear. The molecules that form tip links have recently been identified, and the analysis of their properties has not only changed our view of MET but also suggests that tip-link defects can cause hearing loss. Recent findingsStructural, histological and biochemical studies show that the extracellular domains of two deafness-associated cadherins, cadherin 23 (CDH23) and protocadherin 15 (PCDH15), interact in trans to form the upper and lower part of each tip link, respectively. High-speed Ca2+ imaging suggests that MET channels are localized exclusively at the lower end of each tip link. Biochemical and genetic studies provide evidence that defects in tip links cause hearing impairment in humans. SummaryThe identification of the proteins that form tip links have shed new light on the molecular basis of MET and the mechanisms causing hereditary deafness, noise-induced hearing loss and presbycusis.

Cooperation of p40phox with p47phox for Nox2-based NADPH Oxidase Activation during Fcγ Receptor (FcγR)-mediated Phagocytosis MECHANISM FOR ACQUISITION OF p40phox PHOSPHATIDYLINOSITOL 3-PHOSPHATE (PI(3)P) BINDING

Background: p40phox acquires PI(3)P-binding capabilities through arachidonic acid-induced and H2O2-induced conformational changes in phagocytes. Results: In addition to conformational changes induced by H2O2 in the cytoplasm, p40phox can acquire PI(3)P binding following membrane targeting. Conclusion: p40phox has novel mechanisms inducing its conformation changes, apart from p47phox. Significance: This study demonstrates both p40phox and p47phox synchronously function as “carriers” and “adaptors” of Nox2-based NADPH oxidase assembly through their conformation changes. During activation of the phagocyte (Nox2-based) NADPH oxidase, the cytoplasmic Phox complex (p47phox-p67phox-p40phox) translocates and associates with the membrane-spanning flavocytochrome b558. It is unclear where (in cytoplasm or on membranes), when (before or after assembly), and how p40phox acquires its PI(3)P-binding capabilities. We demonstrated that in addition to conformational changes induced by H2O2 in the cytoplasm, p40phox acquires PI(3)P-binding through direct or indirect membrane targeting. We also found that p40phox is essential when p47phox is partially phosphorylated during FcγR-mediated oxidase activation; however, p40phox is less critical when p47phox is adequately phosphorylated, using phosphorylation-mimicking mutants in HEK293Nox2/FcγRIIa and RAW264.7p40/p47KD cells. Moreover, PI binding to p47phox is less important when the autoinhibitory PX-PB1 domain interaction in p40phox is disrupted or when p40phox is targeted to membranes. Furthermore, we suggest that high affinity PI(3)P binding of the p40phox PX domain is critical during its accumulation on phagosomes, even when masked by the PB1 domain in the resting state. Thus, in addition to mechanisms for directly acquiring PI(3)P binding in the cytoplasm by H2O2, p40phox can acquire PI(3)P binding on targeted membranes in a p47phox-dependent manner and functions both as a “carrier” of the cytoplasmic Phox complex to phagosomes and an “adaptor” of oxidase assembly on phagosomes in cooperation with p47phox, using positive feedback mechanisms.

Protein localization by actin treadmilling and molecular motors regulates stereocilia shape and treadmilling rate.

We present a physical model that describes the active localization of actin-regulating proteins inside stereocilia during steady-state conditions. The mechanism of localization is through the interplay of free diffusion and directed motion, which is driven by coupling to the treadmilling actin filaments and to myosin motors that move along the actin filaments. The resulting localization of both the molecular motors and their cargo is calculated, and is found to have an exponential (or steeper) profile. This localization can be at the base (driven by actin retrograde flow and minus-end myosin motors), or at the stereocilia tip (driven by plus-end myosin motors). The localization of proteins that influence the actin depolymerization and polymerization rates allow us to describe the narrow shape of the stereocilia base, and the observed increase of the actin polymerization rate with the stereocilia height.

Maintenance of stereocilia and apical junctional complexes by Cdc42 in cochlear hair cells.

ABSTRACT Cdc42 is a key regulator of dynamic actin organization. However, little is known about how Cdc42-dependent actin regulation influences steady-state actin structures in differentiated epithelia. We employed inner ear hair-cell-specific conditional knockout to analyze the role of Cdc42 in hair cells possessing highly elaborate stable actin protrusions (stereocilia). Hair cells of Atoh1–Cre;Cdc42flox/flox mice developed normally but progressively degenerated after maturation, resulting in progressive hearing loss particularly at high frequencies. Cochlear hair cell degeneration was more robust in inner hair cells than in outer hair cells, and began as stereocilia fusion and depletion, accompanied by a thinning and waving circumferential actin belt at apical junctional complexes (AJCs). Adenovirus-encoded GFP–Cdc42 expression in hair cells and fluorescence resonance energy transfer (FRET) imaging of hair cells from transgenic mice expressing a Cdc42-FRET biosensor indicated Cdc42 presence and activation at stereociliary membranes and AJCs in cochlear hair cells. Cdc42-knockdown in MDCK cells produced phenotypes similar to those of Cdc42-deleted hair cells, including abnormal microvilli and disrupted AJCs, and downregulated actin turnover represented by enhanced levels of phosphorylated cofilin. Thus, Cdc42 influenced the maintenance of stable actin structures through elaborate tuning of actin turnover, and maintained function and viability of cochlear hair cells.

Deletion of Tricellulin Causes Progressive Hearing Loss Associated with Degeneration of Cochlear Hair Cells

Tricellulin (also known as MARVELD2) is considered as a central component of tricellular tight junctions and is distributed among various epithelial tissues. Although mutations in the gene encoding tricellulin are known to cause deafness in humans (DFNB49) and mice, the influence of its systemic deletion in vivo remains unknown. When we generated tricellulin-knockout mice (Tric−/−), we found an early-onset rapidly progressive hearing loss associated with the degeneration of hair cells (HCs); however, their body size and overall appearance were normal. Tric−/− mice did not show any morphological change pertaining to other organs such as the gastrointestinal tract, liver, kidney, thyroid gland and heart. The endocochlear potential (EP) was normal in Tric−/− mice, suggesting that the tight junction barrier is maintained in the stria vascularis, where EP is generated. The degeneration of HCs, which occurred after the maturation of EP, was prevented in the culture medium with an ion concentration similar to that of the perilymph. These data demonstrate the specific requirement of tricellulin for maintaining ion homeostasis around cochlear HCs to ensure their survival. The Tric−/− mouse provides a new model for understanding the distinct roles of tricellulin in different epithelial systems as well as in the pathogenesis of DFNB49.

A Newly Established Neuronal ρ-0 Cell Line Highly Susceptible to Oxidative Stress Accumulates Iron and Other Metals RELEVANCE TO THE ORIGIN OF METAL ION DEPOSITS IN BRAINS WITH NEURODEGENERATIVE DISORDERS

From human neuroblastoma-derived SILA cells we have established a ρ-0 cell line that is deficient in both respiration and mitochondrial DNA. Lactate dehydrogenase activity, lactate production, and growth in the medium without glucose indicate that these cells shift from aerobic to anaerobic metabolism. Electron microscopic observations revealed abnormal mitochondria with unique cristae structures. Staining with MitoTracker dye showed that the mitochondrial transmembrane potential was reduced by 30–40% from the parent cell levels. These cells were markedly susceptible to H2O2 and died apparently by a necrotic mechanism, a process blocked by deferoxamine in the parent cells but not ρ-0 cells. Analysis by inductively coupled plasma-mass spectrometry revealed an approximately 3-fold accumulation of iron in the ρ-0 cells at confluence (n = 4–6, three clones, *p < 0.05). Iron and four other metals were all elevated in the cells of one of the ρ-0 clones and were similar to control levels in the control cybrid cells, which were replenished with normal mitochondrial DNA. Their sensitivity to H2O2 was also similar to that of the parent cells. These results indicate that a newly established neuronal related ρ-0 cell line is highly susceptible to active oxygen species and that these toxicity effects appear to be related to an accumulation of transition metals, which probably occurs through the respiratory impairment.

Spatiotemporal patterns of Musashi1 expression during inner ear development.

Musashi1 (Msi 1) is an RNA binding protein associated with asymmetric cell divisions in neural progenitor cells. To investigate the involvement of Msi1 in the inner ear development, we studied the expression of Msi1 in mouse inner ears with RT-PCR and immunohistochemistry. Immunohistochemistry revealed that Msi1 was expressed in all otocyst cells at embryonic day (E) 10 and 12. Msi1 immunoreactivity became lost in hair cells after E14 in vestibule and after E16 in cochlea, whereas it persisted in supporting cells until adulthood. The subcellular localization of Msi1 changed from “cytoplasmic predominance” to “nuclear predominance” during the first 2 weeks after birth. The present data suggested that Msi may play a role in inner ear development.