Preparation of proteins
The following coding sequences were amplified and cloned into a pGST//1 vector (derived from pGEX-4T-1; Amersham) for expression in Escherichia coli
prokaryotic cells or in pFast Bac (GE Healthcare) for expression in SF9 insect cells (baculovirus system): full-length harmonin-a (accession no. AF228924) and its Nter (residues 1–79), Nter-PDZ1 (1–203), Nter-PDZ1-PDZ2 (1–299), PDZ3 (434–545) truncated fragments; the cytoplasmic region of cadherin-23 either including the sequence encoded by exon68 (CDH23 + exon68, residues 847–1114 and 847–1110; accession no. AY563163.1) or lacking this sequence (CDH23Δexon68, residues 847–1079 and 847–1075; accession no. AY563164.1). Proteins were produced in BL21(DE3)codonPlus-RP E. coli
cells. The glutathione S
-transferase (GST)-tagged proteins were purified using a glutathione Sepharose 4B column, followed by size-exclusion chromatography. For the different harmonin-a constructs, the GST tag was removed using recombinant tobacco etch virus NIa proteinase. The cadherin-23 fragments were eluted from a glutathione Sepharose 4B column with reduced glutathione. The integrity of the different cadherin-23 fragments was verified by mass spectrometry (QSTAR XL QqTOF mass spectrometer; AB-MDS-Sciex, Thornill, Canada). For some SPR experiments, the CDH23 + exon68 fragment was produced in SF9 cells using the baculovirus expression system. The His6
-tagged CDH23 + exon68 fragment was purified using Ni-NTA agarose beads, followed by size-exclusion chromatography. The His6
-tagged tail of myosin VIIa was purified as previously described (15
The following primary antibodies were used: rabbit polyclonal antibodies against myosin VIIa (15
), harmonin-b (15
) or cadherin-23 (14
), mouse monoclonal IgG against Myc (mAb 9E10 from Santa Cruz Biotechnology) and IgM against PI(4,5)P2
(mAb 2C11 from Molecular Probes, Eugene). For harmonin distribution studies in hair cells, a rabbit antiserum (harmonin-H1; 1:500) was produced against the Nter fragment. Primary antibodies were detected using Alexa488- or Cy3-conjugated secondary antibodies (Molecular Probes). F-actin was visualized with TRITC-conjugated phalloidin (Sigma).
In vitro binding experiments
To explore the role of Ca2+ in harmonin/cadherin-23 complex formation, CDH23 + exon68 was incubated with glutathione beads, and then incubated with untagged harmonin-a in 50 mm HEPES (pH 7.2), 150 mm KCl, 1 mm Tris(2-carboxyethyl)phosphine (TCEP) at 4°C for 2 h. The concentration of free Ca2+ was estimated by Igor Pro software (Wavemetrics, Portland, OR, USA).
Cell culture and immunocytofluorescence analysis
The cDNA constructs encoding full-length harmonin-a and CDH23 + exon68 were cloned into the pEGFP vector, and the construct encoding the PH domain (residues 16–132) of phospholipase C (BC015249) was cloned into the pCMV-Myc vector. For transient transfection experiments, HeLa cells were transfected with the DNA of interest using lipofectamine™ 2000 (Invitrogen), and cells were grown for 24 h before fixation. For PI(4,5)P2 enrichment at the plasma membrane, cells were incubated with 100 µm H2O2 for 20 min before fixation. For immunocytofluorescence experiments, cell samples were fixed using 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), stained with a primary antibody for 1 h, washed in PBS three times for 5 min, stained with a secondary antibody for 1 h, washed as above and mounted using Fluorosave (Calbiochem, USA).
Cochlear dissection and immunostaining
Whole-mount preparations of the mouse organ of Corti were obtained as follows. Animals were killed by exposure to CO2 followed by decapitation, and the inner ears were removed and placed in PBS. The organ of Corti was exposed by removing the stria vascularis, and the tissues were fixed by immersion in 4% PFA in PBS for 1 h. The samples were washed three times in PBS, incubated at room temperature for 1 h in PBS containing 20% goat serum and stained overnight with the primary antibodies diluted in PBS containing 1% BSA. The samples were washed three times in PBS and incubated with either Alexa 488-conjugated or Cy3-conjugated goat anti-rabbit Fab'2 antibodies diluted in PBS containing 20% goat serum, at room temperature for 1 h. After three washes in PBS, the tectorial membrane was carefully removed, and the pieces were mounted using Fluorosave. Samples were analyzed by means of a Zeiss LSM510 Meta confocal microscope.
Lipids and preparation of vesicles
PI, phosphatidylinositol 4-phosphate (PI4P), phosphatidylinositol 5-phosphate (PI5P), PI(4,5)P2, phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylcholine (PC) were purchased from Avanti Polar Lipids (AL, USA). Phospholipids were mixed in the following molar ratios: 35% PC, 13% PS, 35% PE and 17% either PI or PIP. Large unilamellar vesicles (LUVs) were prepared by the rehydration of argon-dried lipids in buffer A (20 mm HEPES pH 7.2, 150 mm KCl, 1 mm TCEP), followed by sonication, filtration on a 0.4 µm pore size filter (Millipore) and extrusion through a 100 nm pore size filter in a mini-extruder (Avanti).
SPR experiments were performed on a Biacore 2000 system (GE Healthcare), equilibrated at 25°C with buffer A. For cadherin-23/harmonin-a interactions, 300–600 resonance units (RU ≈ pg/mm2) of GST-fused CDH23 constructs were captured on an anti-GST antibody-coupled CM5 sensorchip surface (GE Healthcare), over which the full-length harmonin-a and its fragments were then flowed at 20 µl/min for 90 s. For harmonin-a/myosin VIIa and cadherin-23/myosin VIIa interactions, harmonin-a (1500 RU) and CDH23 (1700 RU) were covalently immobilized on a CM5 sensorchip, over which the myosin VIIa tail was flowed at 30 µl/min for 120 s. Protein–lipid interactions were monitored using a lipophilic L1 sensorchip (GE Healthcare) on which 7000–8500 RU of the different LUVs were captured, and proteins were then flowed over the LUV at 10 µl/min for 300 s.