Myosin-I Isozymes in Neonatal Rodent Auditory and Vestibular Epithelia

Abstract

Myosin isozymes are essential for hair cells, the sensory cells of the inner ear. Because a myosin-I subfamily member may mediate adaptation of mechanoelectrical transduction, we examined expression of all eight myosin-I isozymes in rodent auditory and vestibular epithelia. Using RT-PCR, we found prominent expression of three isozymes, Myo1b (also known as myosin-Ia or myr 1), Myo1c (myosin-Ib or myr 2), and Myo1e (myr 3). By contrast, Myo1a (brush-border myosin-I), Myo1d (myosin lc or myr 4), Myo1f, Myo1g, and Myo1h were less readily ampli®ed. Because sequence analysis demonstrated that the RT-PCR products encoded the appropriate isozymes, this represents the ®rst demonstration of expression of all eight mouse myosin-I genes. Using immunocytochemistry with isozyme-selective antibodies, we found that Myo1b was located at apical surfaces of supporting cells that surround hair cells in auditory epithelia of postnatal rats. In vestibular epithelia, Myo1b was present in a ring within the apical pole of the hair cell. In both cases, expression was prominent only immediately after birth. Myo1e was found in hair cells of the auditory and vestibular epithelia; this isozyme was enriched in the cuticular plate, the actin meshwork that anchors the stereocilia. Myo1c was found in hair-cell stereocilia, concentrated towards their tips; we con®rmed this localization by using adenovirus vectors to direct expression of a GFP-Myo1c tail fusion protein; this fusion protein localized to plasma membranes, often concentrating at stereociliary tips. Myo1c therefore remains the myosin isozyme best localized to carry out transducer adaptation.

Myosin 1c Participates in B Cell Cytoskeleton Rearrangements, Is Recruited to the Immunologic Synapse, and Contributes to Antigen Presentation

“…The expression of the eight class I myosins was analyzed by a one-step RT-PCR system (Invitrogen) according to manufacturer's instructions. The primers used were reported previously (19); these were designed to amplify regions that span introns (to avoid amplification of genomic DNA) and were validated with control tissues.…”

“…Beads were washed with cold TNE and boiled in sample buffer. SDS-PAGE and WB analysis were performed according to standard protocols, searching for Myo1c using monoclonal mT2 Ab (19 …”

“…The mice were produced at the Centro de Investigación y de Estudios Avanzados (Mexico City, Mexico) animal facility, and the animal care and use committee of Centro de Investigación y de Estudios Avanzados approved all experiments. Abs and reagents: R2652 (rabbit polyclonal IgG anti-Myo1c) and mT2 (mouse monoclonal IgG1 anti-Myo1c) (19), biotinylated NIM-R4 (monoclonal rat anti-MHC-II), biotinylated mouse anti-H-2K d (BD Pharmingen), biotinylated rat IgG or mouse IgG (Jackson ImmunoResearch), goat anti-lamin B, goat antiactin (both from Santa Cruz), mouse anti-b tubulin, Alexa488-labeled goat anti-rabbit IgG, HRP-anti-mouse IgG1 and HRP-anti-goat IgG, FITCphalloidin, Pacific Blue anti-B220, PE anti-CD3 (BD Pharmingen), DAPI, phalloidin-rhodamine (Molecular Probes), streptavidin-Cy3, streptavidin-HRP, streptavidin-Sepharose beads (Zymed), protein G-Sepharose beads (Invitrogen), and biotinylated cholera toxin B subunit (Sigma). Mouse cell lines: Ag8, BCL-1, WEHI-231, 70Z/3, BaF/3, 3T3, BW5147 and RMA-S, IC21, and P815.…”

SNP Genetic Diversity Within a Fragment of the Gene Myo15a Responsible for the Hearing Process in a Population of Farmed and Free-Living Animals of the Canidae Family/SNP Genetski Deverzitet U Okviru Fragmenta Gena Myo15a, Odgovornog Za Sluh U Populaciji Životinja Familije Canidae U Uslovima Farmskog I Slobodnog Uzgoja

“…Despite the store of genetic and expression data, the location of only seven myosins is known. Endogenous mouse myosin 1b was found to be present in helper cells, and myosin 1e was localized in sensory cells [13]. Endogenous myosin 5a was detected in afferent neurons in the guinea pig.…”

“…Analysis of expression of mouse tissue cDNA detected an mRNA transcript of myosins 1a, 1b, 1c, 1d, 1e, 1f, 1h, 3a, 3b, 5a, 6, 7a, 7b, 10, 15a, 16 and 18a [11][12][13][14]. Of these, at least 6 myosins are essential for hearing, as mutations in the genes MYO1A, MYO2A, MYO2B, MYO6, MYO7A and MYO15A cause deafness in humans and mice [14,15,16].…”