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First published online 16 September 2008
doi: 10.1242/jcs.028134

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II-βV spectrin bridges the plasma membrane and cortical lattice in the lateral wall of the auditory outer hair cells

¹ Institut Pasteur, Unité de Génétique et Physiologie de l'Audition, 25 rue du Dr Roux, 75015 Paris, France
² INSERM UMRS587, F75015 Paris, France
³ UPMC Paris 06, F75015 Paris, France

View larger version (73K): [in this window] [in a new window]   Fig. 1. Spectrin subunits in the auditory organ. (A) Spectrins exist as heterotetramers composed of - and β-spectrin subunits [(, β)2]. The and β subunits present the same structural modular organization, mainly made up of multiple triple-helical repeat units: 21 for subunits, 17 for conventional β subunits and 30 for βV. Other domains, such as the Src-homology domain (SH3) and the EF-hand calmodulin-binding domain in II spectrin, and the N-terminal calponin homology (CH) domains and C-terminal pleckstrin-homology (PH) domain in β spectrins, are indicated. (B) RT-PCR analysis on mouse organ-of-Corti tissue samples at P10 shows the presence of transcripts encoding the II-spectrin and all known β-spectrin subunits, i.e. βI, βII, βIII, βIV and βV. (C-F) Whole mounts of organs of Corti (OC) from P8-P10 mice. Actin filaments (F-actin, red) are labeled with rhodamin-conjugated phalloidin. The II- and βII-spectrin subunits (green; C,D) are detected both in the sensory hair cells and their supporting cells. (E) By contrast, the βV spectrin subunit is detected in the sensory hair cells only. A strong βV spectrin labeling is observed in OHCs (see inset). (F) Serial confocal planes (0.6-µm thick) at different levels of the OHC (see middle panel) show that βV spectrin is restricted to the cortical lattice, along the cell lateral wall. Scale bars: 50 µm (C,E); 5 µm (D,F).

View larger version (65K): [in this window] [in a new window]   Fig. 2. Spectrin subunits are present in different subcellular regions in outer and inner hair cells. (A-D) Cross-sections through organs of Corti from P8-P10 mice. The mammalian cochlea contains two classes of hair cells: inner and outer hair cells (IHCs and OHCs, respectively; labeled with an anti-myosin-VIIa antibody, B). Whereas the II- and βII-spectrin subunits are detected in both types of hair cells and flanking supporting cells (A,C), βV spectrin is restricted to the hair cells (D). Asterisks indicate positions of the sensory hair cells. (E-I) Isolated hair cells. (E) In IHCs, βV spectrin displays a cytoplasmic staining, with a strong submembrane labeling at the neck region of the cell. (F) βII spectrin is detected in the cuticular plate. (G) In OHCs, the βV spectrin labeling extends all along the lateral wall, similar to that of prestin. (H,I) In these cells, the βII-spectrin subunit predominates in the cuticular plate (H), whereas the II subunit is detected in both the cuticular plate and along the lateral wall (I). Scale bars: 5 µm.

View larger version (84K): [in this window] [in a new window]   Fig. 3. Ultrastructural distribution of βV spectrin in the lateral wall of OHCs. (A-D) βV spectrin immunogold labeling in the lateral wall of a P15 OHC (A-C). Gold particles are not observed at the apical junctional complex (AJC) or in the ribbon (R) synapse area (A,D). M, mitochondria. Scale bars: 100 nm.

View larger version (45K): [in this window] [in a new window]   Fig. 4. βV spectrin binds to F-actin and forms a complex with II spectrin and band 4.1 in vivo. (A) GST and GST-tagged βV spectrin fragments used in the binding experiments (Coomassie-blue staining following SDS-PAGE). (B,C) βV spectrin–band-4.1 interaction. The endogenous II spectrin binds to GST-βVR29 (B) and is immunoprecipitated by the anti-βV spectrin antibody, but not by the preimmune serum or by protein G alone (C). (D) F-actin co-sedimentation assay. The soluble (S) and pellet (P) fractions obtained upon ultracentrifugation are indicated. GST-tagged βVCH is not recovered in the pellet fraction (lane 3). When the same amount of GST-tagged βVCH is incubated with F-actin at 37°C for 30 minutes (lanes 5 and 6), almost all GST-tagged βVCH is recovered with F-actin in the pellet fraction after ultracentrifugation (lane 6). GST alone was used as a negative control (lanes 7-10). (E,F) βV spectrin–band-4.1 interaction. (E) In vitro binding assay. GST-tagged βVCH directly interacts with the SAB domain of band 4.1. (F) Co-immunoprecipitation assay. The anti-βV spectrin antibody immunoprecipitates band 4.1 from lysates of P8 mouse organs of Corti.

View larger version (59K): [in this window] [in a new window]   Fig. 5. Concomitant time-course distribution of βV spectrin and prestin in OHCs. (A) A model illustrating the role of the II-βV spectrin network in the OHC lateral wall. The II-βV spectrin heterotetramers crosslink adjacent circumferential actin filaments, while extending out of the plane (25 nm) to reach the plasma membrane, which is densely packed with the prestin motor protein. (B) An F-actin-labeled cochlea illustrating the different positions (base, middle and apex) along the cochlea longitudinal axis. (C) Whole-mount immunofluorescence analysis of βV spectrin and prestin distributions in the basal (0-20%), middle (40-60%) and apical (80-100%) turns of the cochlea. Consistent with the progressive maturation of the cochlea, the intensities of βV spectrin and prestin stainings in OHCs increase from P2 to P10, and from the basal to the apical zone of the cochlea. Scale bars: 5 µm.

View larger version (52K): [in this window] [in a new window]   Fig. 6. (A) Intensity of OHC immunolabeling with anti-βV spectrin (squares) and anti-prestin (circles) antibodies plotted against age (days after birth). The values obtained for the apical turns (80-100%, see Fig. 5B) were averaged, and then normalized to the corresponding average value obtained in P20 mice. (B-D) Parallel distribution of βV spectrin and prestin within differentiating OHCs. (B) At P3, the bulk of βV spectrin staining is diffuse within the cytoplasm. At this stage, II spectrin can be detected in the cuticular plate and along the cell lateral wall. In some OHCs from the base of the cochlea, prestin can be detected, essentially at the apico-lateral cell membrane. (C) At P5, βV spectrin and prestin are both detected in the forming cortical lattice. (D) From P8 onwards, the labeling extends along the entire length of the OHC lateral wall. Scale bars: 5 µm.

View larger version (57K): [in this window] [in a new window]   Fig. 7. βV spectrin indirectly interacts with prestin. (A,B,D) In vitro binding assays. (A) Neither GST-tagged βVCH nor GST-tagged βVR29 directly interact with full-length prestin. (B) By contrast, the interaction between GST-tagged βVR29 and prestin can be detected when protein extracts derived from P90 organs of Corti (OC) are added to the binding solution. (C-E) Mapping of the βV spectrin-binding region on prestin and of the prestin-binding region on βV spectrin. (C) Schematic diagram of the transmembrane topology of prestin and its deletion constructs used in the pull-down assays. Prestin is a 10- to 12-transmembrane-domain protein, with relatively large cytoplasmic N and C termini. The numbers refer to the corresponding positions of amino acid residues in the rat prestin. (D) In the presence of P35 OC lysates, both the full-length prestin (prestin-FL) and the prestin C-terminal cytoplasmic domain (prestin-C) bind to either GST-tagged βVR29 or GST-tagged βVPH. (E) Western blot. In the reciprocal experiment, GST-tagged prestin-C529 (aa 529-744) binds to the myc-tagged βVR26 (aa 3012-3674) only in the presence of OC lysates.

View larger version (55K): [in this window] [in a new window]   Fig. 8. Specificity, time and spatial dependency of the βV spectrin–prestin association. (A) In the presence of OC lysates, only SLC26A5-C (prestin-C), but not SLC26A4-C (pendrin-C), SLC26A6-C or SLC26A7-C, binds to GST-tagged βVR29. (B) This interaction cannot be detected when prestin-FL lacks either the STAS domain (Del632) or the last 35 amino acid residues (Del709). (C) In the presence of protein extracts derived from P0 organs of Corti (a stage at which OHC electromotility is not yet present), prestin does not associate with GST-tagged βVR29. The interaction can be seen only when lysates from P4, P8 or P35 organs of Corti are used. (D) βV spectrin–prestin interaction in the presence of different mouse tissue lysates. An interaction cannot be detected when using lysates derived from the brain, liver or HEK293 cells. Vest, vestibule (balance organ). The same amounts of proteins were used at each developmental stage, as shown by western blot analysis of the lysates with an anti-actin antibody (C,D).

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