GIPC3 couples to MYO6 and PDZ domain proteins, and shapes the hair cell apical region.

GIPC3 has been implicated in auditory function. Here, we establish that GIPC3 is initially localized to the cytoplasm of inner and outer hair cells of the cochlea and then is increasingly concentrated in cuticular plates and at cell junctions during postnatal development. Early postnatal Gipc3KO/KO mice had mostly normal mechanotransduction currents, but had no auditory brainstem response at 1 month of age. Cuticular plates of Gipc3KO/KO hair cells did not flatten during development as did those of controls; moreover, hair bundles were squeezed along the cochlear axis in mutant hair cells. Junctions between inner hair cells and adjacent inner phalangeal cells were also severely disrupted in Gipc3KO/KO cochleas. GIPC3 bound directly to MYO6, and the loss of MYO6 led to altered distribution of GIPC3. Immunoaffinity purification of GIPC3 from chicken inner ear extracts identified co-precipitating proteins associated with adherens junctions, intermediate filament networks and the cuticular plate. Several of immunoprecipitated proteins contained GIPC family consensus PDZ-binding motifs (PBMs), including MYO18A, which bound directly to the PDZ domain of GIPC3. We propose that GIPC3 and MYO6 couple to PBMs of cytoskeletal and cell junction proteins to shape the cuticular plate.

GIPC3 couples to MYO6 and PDZ domain proteins and shapes the hair cell apical region.

GIPC3 has been implicated in auditory function. Initially localized to the cytoplasm of inner and outer hair cells of the cochlea, GIPC3 increasingly concentrated in cuticular plates and at cell junctions during postnatal development. Early postnatal Gipc3 KO/KO mice had mostly normal mechanotransduction currents, but had no auditory brainstem response at one month of age. Cuticular plates of Gipc3 KO/KO hair cells did not flatten during development as did those of controls; moreover, hair bundles were squeezed along the cochlear axis in mutant hair cells. Junctions between inner hair cells and adjacent inner phalangeal cells were also severely disrupted in Gipc3 KO/KO cochleas. GIPC3 bound directly to MYO6, and the loss of MYO6 led to altered distribution of GIPC3. Immunoaffinity purification of GIPC3 from chicken inner ear extracts identified co-precipitating proteins associated with adherens junctions, intermediate filament networks, and the cuticular plate. Several of immunoprecipitated proteins contained GIPC-family consensus PDZ binding motifs (PBMs), including MYO18A, which binds directly to the PDZ domain of GIPC3. We propose that GIPC3 and MYO6 couple to PBMs of cytoskeletal and cell-junction proteins to shape the cuticular plate. Summary statement: The PDZ-domain protein GIPC3 couples the molecular motors MYO6 and MYO18A to actin cytoskeleton structures in hair cells. GIPC3 is necessary for shaping the hair cell’s cuticular plate and hence the arrangement of the stereocilia in the hair bundle.

TRPV6, TRPM6 and TRPM7 Do Not Contribute to Hair-Cell Mechanotransduction.

Hair cells of the inner ear transduce mechanical stimuli like sound or head movements into electrical signals, which are propagated to the central nervous system. The hair-cell mechanotransduction channel remains unidentified. We tested whether three transient receptor channel (TRP) family members, TRPV6, TRPM6 and TRPM7, were necessary for transduction. TRPV6 interacted with USH1C (harmonin), a scaffolding protein that participates in transduction. Using a cysteine-substitution knock-in mouse line and methanethiosulfonate (MTS) reagents selective for this allele, we found that inhibition of TRPV6 had no effect on transduction in mouse cochlear hair cells. TRPM6 and TRPM7 each interacted with the tip-link component PCDH15 in cultured eukaryotic cells, which suggested they might be part of the transduction complex. Cochlear hair cell transduction was not affected by manipulations of Mg2+, however, which normally perturbs TRPM6 and TRPM7. To definitively examine the role of these two channels in transduction, we showed that deletion of either or both of their genes selectively in hair cells had no effect on auditory function. We suggest that TRPV6, TRPM6 and TRPM7 are unlikely to be the pore-forming subunit of the hair-cell transduction channel.

Heterodimeric capping protein is required for stereocilia length and width regulation.

Control of the dimensions of actin-rich processes like filopodia, lamellipodia, microvilli, and stereocilia requires the coordinated activity of many proteins. Each of these actin structures relies on heterodimeric capping protein (CAPZ), which blocks actin polymerization at barbed ends. Because dimension control of the inner ear's stereocilia is particularly precise, we studied the CAPZB subunit in hair cells. CAPZB, present at ∼100 copies per stereocilium, concentrated at stereocilia tips as hair cell development progressed, similar to the CAPZB-interacting protein TWF2. We deleted Capzb specifically in hair cells using Atoh1-Cre, which eliminated auditory and vestibular function. Capzb-null stereocilia initially developed normally but later shortened and disappeared; surprisingly, stereocilia width decreased concomitantly with length. CAPZB2 expressed by in utero electroporation prevented normal elongation of vestibular stereocilia and irregularly widened them. Together, these results suggest that capping protein participates in stereocilia widening by preventing newly elongating actin filaments from depolymerizing.

Integration of Tmc1/2 into the mechanotransduction complex in zebrafish hair cells is regulated by Transmembrane O-methyltransferase (Tomt).

Transmembrane O-methyltransferase (TOMT/LRTOMT) is responsible for non-syndromic deafness DFNB63. However, the specific defects that lead to hearing loss have not been described. Using a zebrafish model of DFNB63, we show that the auditory and vestibular phenotypes are due to a lack of mechanotransduction (MET) in Tomt-deficient hair cells. GFP-tagged Tomt is enriched in the Golgi of hair cells, suggesting that Tomt might regulate the trafficking of other MET components to the hair bundle. We found that Tmc1/2 proteins are specifically excluded from the hair bundle in tomt mutants, whereas other MET complex proteins can still localize to the bundle. Furthermore, mouse TOMT and TMC1 can directly interact in HEK 293 cells, and this interaction is modulated by His183 in TOMT. Thus, we propose a model of MET complex assembly where Tomt and the Tmcs interact within the secretory pathway to traffic Tmc proteins to the hair bundle.

PDZD7-MYO7A complex identified in enriched stereocilia membranes.

While more than 70 genes have been linked to deafness, most of which are expressed in mechanosensory hair cells of the inner ear, a challenge has been to link these genes into molecular pathways. One example is Myo7a (myosin VIIA), in which deafness mutations affect the development and function of the mechanically sensitive stereocilia of hair cells. We describe here a procedure for the isolation of low-abundance protein complexes from stereocilia membrane fractions. Using this procedure, combined with identification and quantitation of proteins with mass spectrometry, we demonstrate that MYO7A forms a complex with PDZD7, a paralog of USH1C and DFNB31. MYO7A and PDZD7 interact in tissue-culture cells, and co-localize to the ankle-link region of stereocilia in wild-type but not Myo7a mutant mice. Our data thus describe a new paradigm for the interrogation of low-abundance protein complexes in hair cell stereocilia and establish an unanticipated link between MYO7A and PDZD7.

Tip-link protein protocadherin 15 interacts with transmembrane channel-like proteins TMC1 and TMC2.

The tip link protein protocadherin 15 (PCDH15) is a central component of the mechanotransduction complex in auditory and vestibular hair cells. PCDH15 is hypothesized to relay external forces to the mechanically gated channel located near its cytoplasmic C terminus. How PCDH15 is coupled to the transduction machinery is not clear. Using a membrane-based two-hybrid screen to identify proteins that bind to PCDH15, we detected an interaction between zebrafish Pcdh15a and an N-terminal fragment of transmembrane channel-like 2a (Tmc2a). Tmc2a is an ortholog of mammalian TMC2, which along with TMC1 has been implicated in mechanotransduction in mammalian hair cells. Using the above-mentioned two-hybrid assay, we found that zebrafish Tmc1 and Tmc2a can interact with the CD1 or CD3 cytoplasmic domain isoforms of Pcdh15a, and this interaction depends on the common region shared between the two Pcdh15 isoforms. Moreover, an interaction between mouse PCDH15-CD3 and TMC1 or TMC2 was observed in both yeast two-hybrid assays and coimmunoprecipitation experiments. To determine whether the Pcdh15-Tmc interaction is relevant to mechanotransduction in vivo, we overexpressed N-terminal fragments of Tmc2a in zebrafish hair cells. Overexpression of the Tmc2a N terminus results in mislocalization of Pcdh15a within hair bundles, together with a significant decrease in mechanosensitive responses, suggesting that a Pcdh15a-Tmc complex is critical for mechanotransduction. Together, these results identify an evolutionarily conserved association between the fish and mouse orthologs of PCDH15 and TMC1 and TMC2, supporting the notion that TMCs are key components of the transduction complex in hair cells.

Biophysical characteristics of cells cultured on cholesteryl ester liquid crystals.

This study aimed at examining the biophysical characteristics of human derived keratinocytes (HaCaT) cultured on cholesteryl ester liquid crystals (CELC). CELC was previously shown to improve sensitivity in sensing cell contractions. Characteristics of the cell integrin expressions and presence of extracellular matrix (ECM) proteins on the liquid crystals were interrogated using various immunocytochemical techniques. The investigation was followed by characterization of the chemical properties of the liquid crystals (LC) after immersion in cell culture media using Fourier transform infrared spectroscopy (FTIR). The surface morphology of cells adhered to the LC was studied using atomic force microscopy (AFM). Consistent with the expressions of the integrins α2, α3 and ß1, extracellular matrix proteins (laminin, collagen type IV and fibronectin) were found secreted by the HaCaT onto CELC and these proteins were also secreted by cells cultured on the glass substrates. FTIR analysis of the LC revealed the existence of spectrum assigned to cholesterol and ester moieties that are essential compounds for the metabolizing activities of keratinocytes. The immunostainings indicated that cell adhesion on the LC is mediated by self-secreted ECM proteins. As revealed by the AFM imaging, the constraint in cell membrane spread on the LC leads to the increase in cell surface roughness and thickness of cell membrane. The biophysical expressions of cells on biocompatible CELC suggested that CELC could be a new class of biological relevant material.

Mechanotransduction: the elusive hair cell transduction channel revealed?

A family of transmembrane proteins has been shown to modulate both the calcium permeability and single-channel conductance of the vertebrate hair-cell mechanosensor, implicating them directly in inner ear mechanosensation.

Interfacial study of cell adhesion to liquid crystals using widefield surface plasmon resonance microscopy.

Widefield surface plasmon resonance (WSPR) microscopy provides high resolution imaging of interfacial interactions. We report the application of the WSPR imaging system in the study of the interaction between keratinocytes and liquid crystals (LC). Imaging of fixed keratinocytes cultured on gold coated surface plasmon substrates functionalized with a thin film of liquid crystals was performed in air using a 1.45NA objective based system. Focal adhesion of the cells adhered to glass and LC were further studied using immunofluorescence staining of the vinculin. The imaging system was also simulated with 2×2 scattering matrix to investigate the optical reflection of the resonant plasmonic wave via the glass/gold/cell and glass/gold/LC/cell layers. WSPR imaging indicated that keratinocytes are less spread and formed distinct topography of cell-liquid crystal couplings when cultured on liquid crystal coated substrates. The simulation indicates that glass/LC shifted the surface plasmon excitation angle to 75.39° as compared to glass/air interface at 44°. The WSPR microcopy reveals that the cells remodelled their topography of adhesion at different interfaces.

Regulation of PI3K signalling by the phosphatidylinositol transfer protein PITPalpha during axonal extension in hippocampal neurons.

Phosphatidylinositol transfer proteins (PITPs) mediate the transfer of phosphatidylinositol (PtdIns) or phosphatidylcholine (PtdCho) between two membrane compartments, thereby regulating the interface between signalling, phosphoinositide (PI) metabolism and membrane traffic. Here, we show that PITPalpha is enriched in specific areas of the postnatal and adult brain, including the hippocampus and cerebellum. Overexpression of PITPalpha, but not PITPbeta or a PITPalpha mutant deficient in binding PtdIns, enhances laminin-dependent extension of axonal processes in hippocampal neurons, whereas knockdown of PITPalpha protein by siRNA suppresses laminin and BDNF-induced axonal growth. PITPalpha-mediated axonal outgrowth is sensitive to phosphoinositide 3-kinase (PI3K) inhibition and shows dependency on the Akt/GSK-3/CRMP-2 pathway. We conclude that PITPalpha controls the polarized extension of axonal processes through the provision of PtdIns for localized PI3K-dependent signalling.

Differential expression of a C-terminal splice variant of phosphatidylinositol transfer protein beta lacking the constitutive-phosphorylated Ser262 that localizes to the Golgi compartment.

Mammalian PITPbeta (phosphatidylinositol transfer protein beta) is a 272-amino-acid polypeptide capable of transferring PtdIns, PtdCho and SM (sphingomyelin) between membrane bilayers. It has been reported that Ser262 present in the C-terminus of PITPbeta is constitutively phosphorylated and determines Golgi localization. We provide evidence for the expression of an sp (splice) variant of PITPbeta (PITPbeta-sp2) where the C-terminal 15 amino acids of PITPbeta-sp1 are replaced by an alternative C-terminus of 16 amino acids. PITPbeta-sp1 is the product of the first 11 exons, whereas PITPbeta-sp2 is a product of the first 10 exons followed by the twelfth exon--exon 11 being 'skipped'. Both splice variants are capable of PtdIns and PtdCho transfer, with PITPbeta-sp2 being unable to transport SM. PITPbeta is ubiquitously expressed, with the highest amounts of PITPbeta found in HL60 cells and in rat liver; HL60 cells express only PITPbeta-sp1, whereas rat liver expresses both sp variants in similar amounts. In both cell types, PITPbeta-sp1 is constitutively phosphorylated and both the PtdIns and PtdCho forms of PITPbeta-sp1 are present. In contrast, PITPbeta-sp2 lacks the constitutively phosphorylated Ser262 (replaced with glutamine). Nonetheless, both PITPbeta variants localize to the Golgi and, moreover, dephosphorylation of Ser262 of PITPbeta-sp1 does not affect its Golgi localization. The presence of PITPbeta sp variants adds an extra level of proteome complexity and, in rat liver, the single gene for PITPbeta gives rise to seven distinct protein species that can be resolved on the basis of their charge differences.

Phosphorylation of a distinct structural form of phosphatidylinositol transfer protein alpha at Ser166 by protein kinase C disrupts receptor-mediated phospholipase C signaling by inhibiting delivery of phosphatidylinositol to membranes.

Phosphatidylinositol transfer protein alpha (PITPalpha) participates in the supply of phosphatidylinositol (PI) required for many cellular events including phospholipase C (PLC) beta and gamma signaling by G-protein-coupled receptors and receptor-tyrosine kinases, respectively. Protein kinase C has been known to modulate PLC signaling by G-protein-coupled receptors and receptor-tyrosine kinases, although the molecular target has not been identified in most instances. In each case phorbol myristate acetate pretreatment of HL60, HeLa, and COS-7 cells abrogated PLC stimulation by the agonists formyl-Met-Leu-Phe, ATP, and epidermal growth factor, respectively. Here we show that phosphorylation of PITPalpha at Ser166 resulted in inhibition of receptor-stimulated PLC activity. Ser166 is localized in a small pocket between the 165-172 loop and the rest of the protein and was not solvent-accessible in either the PI- or phosphatidylcholine-loaded structures of PITPalpha. To allow phosphorylation at Ser166, a distinct structural form is postulated, and mutation of Thr59 to alanine shifted the equilibrium to this form, which could be resolved on native PAGE. The elution profile observed by size exclusion chromatography of phosphorylated PITPalpha from rat brain or in vitro phosphorylated PITPalpha demonstrated that phosphorylated PITPalpha is structurally distinct from the non-phosphorylated form. Phosphorylated PITPalpha was unable to deliver its PI cargo, although it could deliver phosphatidylcholine. We conclude that the PITPalpha structure has to relax to allow access to the Ser166 site, and this may occur at the membrane surface where PI delivery is required for receptor-mediated PLC signaling.

Identification of phospholipase B from Dictyostelium discoideum reveals a new lipase family present in mammals, flies and nematodes, but not yeast.

The social amoeba Dictyostelium discoideum exhibits high activities of phospholipase and lysophospholipase [Ferber, Munder, Fischer and Gerisch (1970) Eur. J. Biochem. 14, 253-257]. We assayed Dictyostelium lysates to demonstrate the presence of a highly active phospholipase B (PLB) enzyme that removed both fatty-acid chains from phosphatidylcholine and produced the water-soluble glycerophosphorylcholine. We purified the PLB activity from Dictyostelium cytosol using standard agarose media (size exclusion and ion exchange), and combined this with an affinity purification step using myristoylated ARF1 (ADP-ribosylation factor 1), a protein which has a single fatty acid at its N-terminus. Two proteins co-purified (48 kDa and 65 kDa), and the 48 kDa protein was digested with trypsin, peptide fragments were separated by reverse-phase chromatography, and the resultant peptides were sequenced by Edman degradation. From the peptide sequences obtained, database searches revealed a gene which encodes a protein of 65 kDa with unknown function. The 48 kDa protein therefore appears to be a fragment of the full-length 65 kDa product. Expression of the gene in Escherichia coli confirmed that it encodes a PLB. Characterization of its substrate specificity indicated that, in addition to phosphatidylcholine deacylation, the enzyme also hydrolysed phosphatidylinositol and phosphatidylethanolamine. The PLB identified in the present study is not related to existing PLBs found in bacteria, fungi or mammals. There are, however, genes similar to Dictyostelium PLB in mammals, flies, worms and Giardia, but not in yeast. We therefore have identified a novel family of intracellular PLBs.

Structure-function analysis of human [corrected] phosphatidylinositol transfer protein alpha bound to phosphatidylinositol.

Phosphatidylinositol transfer protein alpha (PITPalpha) selectively transports and promotes exchange of phosphatidylinositol (PI) and phosphatidylcholine (PC) between lipid bilayers. In higher eukaryotes PITPalpha is required for cellular functions such as phospholipase C-mediated signaling, regulated exocytosis, and secretory vesicle formation. We have determined the crystal structure of human PITPalpha bound to its physiological ligand, PI, at 2.95 A resolution. The structure identifies the critical side chains within the lipid-headgroup binding pocket that define the exquisite specificity for PI. Mutational analysis of the PI binding pocket is in good agreement with the structural data and allows manipulation of functional properties of PITPalpha. Surprisingly, there are no major conformational differences between PI- and PC-loaded PITPalpha, despite previous predictions. In the crystal, PITPalpha-PI is dimeric, with two identical dimers in the asymmetric unit. The dimer interface masks precisely the sequence we identify as contributing to PITPalpha membrane interaction. Our structure represents a soluble, transport-competent form of PI-loaded PITPalpha.

EGF regulation of PITP dynamics is blocked by inhibitors of phospholipase C and of the Ras-MAP kinase pathway.

Phosphatidylinositol transfer proteins (PITP) function in signal transduction and in membrane traffic. Studies aimed at elucidating the mechanism of action of PITP have yielded a singular theme; the activity of PITP stems from its ability to transfer phosphatidylinositol (PI) from its site of synthesis to sites of cellular activity and to stimulate the local synthesis of phosphorylated forms of PI. The participation of various phosphoinositides in EGF signal transduction and in the trafficking of the EGF receptors is well documented. Using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET) between EGFP-PITP proteins and fluorescently labeled phospholipids, we report that PITPalpha and PITPbeta can dynamically interact with PI or PC at the plasma membrane when stimulated with EGF. Additionally, PITPbeta is localized at the Golgi, and EGF stimulation resulted in enhanced FRET. Inhibitors of the PLC and the Ras/MAP kinase pathway were both able to inhibit the EGF-stimulated interaction of PITPalpha with PI at the plasma membrane. The mobility of PITP proteins was determined by using fluorescence recovery after photobleaching (FRAP), and EGF stimulation reduced the mobility at the plasma membrane. We conclude that the dynamic behavior of PITPalpha and PITPbeta in vivo is a regulated process involving multiple mechanisms.

Spermine increases phosphatidylinositol 4,5-bisphosphate content in permeabilized and nonpermeabilized HL60 cells.

The polyamine spermine (N,N'bis[3-aminopropyl]-1,4-butanediamine) activates phosphatidylinositol-4-phosphate 5-kinase (PtdIns(4)P5K) and phosphatidylinositol 4-kinase (PtdIns4K) in vitro. Spermine concentration increases that occur in proliferating cells were approximated in streptolysin O-permeabilized HL60 cells. When phospholipase C was activated by GTPgammaS in the presence of PITPalpha, 0.1-1.2 mM spermine evoked increases in PtdIns(4,5)P(2) contents in a dose-dependent manner to 110-170% of control and concomitantly decreased inositol phosphate formation by 10-50%. Spermine-induced increases in PtdIns(4,5)P(2) content in permeabilized cells also occurred during GTPgammaS stimulation in the absence of PITPalpha, were augmented in the presence of PITPalpha, occurred in unstimulated cells and were additive to PtdIns(4,5)P(2) formation evoked by ARF1, another activator of phosphoinositide kinases. Slowly developing spermine-evoked increases in PtdIns(4,5)P(2) contents occurred in nonpermeabilized cells that were abolished in the presence of a spermine transport inhibitor. Data are consistent with spermine at physiological concentrations evoking a PITPalpha-dependent shift in formation of PtdIns(4,5)P(2) from compartments that contained an active phospholipase C to compartments that were separated from an active PLC and from PtdIns(4,5)P(2) formed by ARF1.

Mechanism of ADP ribosylation factor-stimulated phosphatidylinositol 4,5-bisphosphate synthesis in HL60 cells.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is required both as a substrate for the generation of lipid-derived second messengers as well as an intact lipid for many aspects of cell signaling, endo- and exocytosis, and reorganization of the cytoskeleton. ADP ribosylation factor (ARF) proteins regulate PI(4,5)P(2) synthesis, and here we have examined whether this is due to direct activation of Type I phosphatidylinositol 4-phosphate (PIP) 5-kinase or indirectly by phosphatidate (PA) derived from phospholipase D (PLD) in HL60 cells. ARF1 and ARF6 are both expressed in HL60 cells and can be depleted from the cells by permeabilization. Both ARFs increased the levels of PIP(2) (PI(4,5)P(2), PI(3,5)P(2), or PI(3,4)P(2) isomers) at the expense of PIP when added back to permeabilized cells. The PIP(2) could be hydrolyzed by phospholipase C, identifying it as PI(4,5)P(2). However, the ARF1-stimulated pool of PI(4,5)P(2) was accessible to the phospholipase C more efficiently in the presence of phosphatidylinositol transfer protein-alpha. To examine the role of PLD in the regulation of PI(4,5)P(2) synthesis, we used butanol to diminish the PLD-derived PA. PI(4,5)P(2) synthesis stimulated by ARF1 was not blocked by 0.5% butanol but could be blocked by 1.5% butanol. Although 0.5% butanol was optimal for maximal transphosphatidylation, PA production was still detectable. In contrast, 1.5% butanol was found to inhibit the activation of PLD by ARF1 and also decrease PIP levels by 50%. Thus the toxicity of 1.5% butanol prevented us from concluding whether PA was an important factor in raising PI(4,5)P(2) levels. To circumvent the use of alcohols, an ARF1 point mutant was identified (N52R-ARF1) that could selectively activate PIP 5-kinase alpha activity but not PLD activity. N52R-ARF1 was still able to increase PI(4,5)P(2) levels but at reduced efficiency. We therefore conclude that both PA derived from the PLD pathway and ARF proteins, by directly activating PIP 5-kinase, contribute to the regulation of PI(4,5)P(2) synthesis at the plasma membrane in HL60 cells.