The sensitivity and frequency selectivity of the mammalian cochlea involves a mechanical amplification process called electromotility, which requires prestin-dependent length changes of the outer hair cell (OHC) lateral wall in response to changes in membrane electric potential. The cortical lattice, the highly organized cytoskeleton underlying the OHC lateral plasma membrane, is made up of F-actin and spectrin. Here, we show that αII and two of the five β-spectrin subunits, βII and βV, are present in OHCs. βII spectrin is restricted to the cuticular plate, a dense apical network of actin filaments, whereas βV spectrin is concentrated at the cortical lattice. Moreover, we show that αII-βV spectrin directly interacts with F-actin and band 4.1, two components of the OHC cortical lattice. βV spectrin is progressively recruited into the cortical lattice between postnatal day 2 (P2) and P10 in the mouse, in parallel with prestin membrane insertion, which itself parallels the maturation of cell electromotility. Although βV spectrin does not directly interact with prestin, we found that addition of lysates derived from mature auditory organs, but not from the brain or liver, enables βV spectrin-prestin interaction. Using this assay, βV spectrin, via its PH domain, indirectly interacts with the C-terminal cytodomain of prestin. We conclude that the cortical network involved in the sound-induced electromotility of OHCs contains αII-βV spectrin, and not the conventional αII-βII spectrin.Supplementary material available online at http://jcs.biologists.org/cgi/content/full/121/20/3347/DC1 Key words: βV spectrin, Prestin, Outer hair cell, Cortical lattice, Electromotility Summary αII-βV spectrin bridges the plasma membrane and cortical lattice in the lateral wall of the auditory outer hair cells

Mutations in the myosin VIIa gene cause Usher syndrome type IB (USH1B), characterized by deaf-blindness. A delay of opsin trafficking has been observed in the retinal photoreceptor cells of myosin VIIa-deficient mice. We identified spectrin βV, the mammalian β-heavy spectrin, as a myosin VIIa- and rhodopsin-interacting partner in photoreceptor cells. Spectrin βV displays a polarized distribution from the Golgi apparatus to the base of the outer segment, which, unlike that of other β spectrins, matches the trafficking route of opsin and other phototransduction proteins. Formation of spectrin βV-rhodopsin complex could be detected in the differentiating photoreceptors as soon as their outer segment emerges. A failure of the spectrin βV-mediated coupling between myosin VIIa and opsin molecules thus probably accounts for the opsin transport delay in myosin VIIa-deficient mice. We showed that spectrin βV also associates with two USH1 proteins, sans (USH1G) and harmonin (USH1C). Spectrins are supposed to function as heteromers of α and β subunits, but fluorescence resonance energy transfer and in vitro binding experiments indicated that spectrin βV can also form homodimers, which likely supports its αII-independent βV functions. Finally, consistent with its distribution along the connecting cilia axonemes, spectrin βV binds to several subunits of the microtubule-based motor proteins, kinesin II and the dynein complex. We therefore suggest that spectrin βV homomers couple some USH1 proteins, opsin and other phototransduction proteins to both actin- and microtubule-based motors, thereby contributing to their transport towards the photoreceptor outer disks.