Identification with a recombinant antibody of an inner-ear cytokeratin, a marker for hair-cell differentiation

Abstract

Extensive biochemical characterization of cells in the inner ear has been hampered by a lack of tools with which to identify inner-ear proteins. By using a single-chain antibody fragment isolated from a bacteriophage-displayed library, we have identified a cytokeratin that is abundant in nonsensory cells of the frog inner ear. Although the progenitors of hair cells exhibit strong immunoreactivity to this cytokeratin, the signal declines in immature hair cells and vanishes as the cells mature. The correlation between diminished immunoreactivity and hair-cell differentiation indicates that the cytokeratin is down-regulated during the transition from a nonsensory to a sensory cell and suggests that the marker is an early index of hair-cell differentiation. O ur awareness of faint sounds and weak accelerations rests on the sensitivity of hair cells, which respond to subnanometer deflections of their hair bundles (1). The exquisite sensitivity of hair cells unfortunately renders them acutely susceptible to trauma from acoustical overstimulation. In addition, aminoglycoside antibiotics and some chemotherapeutic agents can damage or kill these sensory cells. Because they are postmitotic, hair cells can be replaced only by the differentiation of precursors. Although hair cells do not regenerate in the adult mammalian cochlea, replacement does occur in mammalian vestibular organs and more extensively in nonmammalian receptor organs (2, 3).Little is known about the proteins involved in hair-cell differentiation and function. Although the identification and characterization of inner-ear proteins are impeded by the paucity of cells, the isolation of specific reagents directed against inner-ear proteins makes biochemical studies feasible. To generate such reagents, we produced a bacteriophage-displayed antibodyfragment library directed against proteins of the bullfrog inner ear (4). From this library, we isolated a single-chain Fv fragment (scFv) that specifically labels a protein expressed almost exclusively in the inner ear. We describe here the further characterization and identification of this protein as a cytokeratin whose expression declines during hair-cell differentiation. Materials and MethodsThe production of a library of bacteriophage-displayed scFvs and the selection of a clone producing an scFv with a high specificity for an inner-ear antigen have been described (4). For the present study, soluble scFvs from clone scFv-278 were isolated by the procedures in that reference.Immunocytochemistry on Saccular Whole Mounts. Bullfrog sacculi were isolated and the otolithic membranes were mechanically removed. Sacculi were fixed for 1 h at 4°C in 4% (wt͞vol) formaldehyde in PBS (68 mM NaCl͞58 mM Na 2 HPO 4 ͞17 mM NaH 2 PO 4 , pH 7.4), rinsed in PBS, permeabilized for 1 h in PBS containing 1% (vol͞vol) Triton X-100, and washed in PBS. Samples were blocked for 2 h in PBS containing 2.5% (vol͞vol) Liquid Block (Amersham Pharmacia) and 3% (vol͞vol) goat serum, then incubated overnight at 4°C in a mixture of 1.8 mg͞liter aff...

Closure of supporting cell scar formations requires dynamic actin mechanisms

“…In amphibian, avian and mammalian vestibular systems, cytokeratins are preferentially localized in supporting cells (Cyr et al, 2000;Meiteles and Raphael, 1994;Stone et al, 1996). However, unlike previous studies, we also observed weak puncta of cytokeratin labeling in hair cells, possibly In sections from explants fixed 18 h after a 6-h incubation with gentamicin, cellular extrusions ( * ) are frequently seen above the sensory epithelium.…”

“…Cytokeratin expression is down regulated during hair cell differentiation (Cyr et al, 2000;Raphael et al, 1987). The upregulation of cytokeratins in hair cell extrusions indicates the epithelial origin of these sensory cells, and associated apoptotic cell death mechanisms (Caulin et al, 1997;Leers et al, 1999).…”

Hair cell recovery in mitotically blocked cultures of the bullfrog saccule

“…After fixation, these cultures were labeled with phalloidin, a marker of filamentous actin, and immunolabeled with antisera against myosin VI, a known HC marker (84), and a single-chain antibody fragment against a novel cytokeratin expressed in bullfrog SCs (85). The morphology, ultrastructure, and immunolabeling of cells in these cultures were then examined with confocal and transmission electron microscopy.…”

Structure and functions of keratin proteins in simple, stratified, keratinized and cornified epithelia

“…Merkel cells express K8, K18 and K19 (Moll et al 1984;Narisawa et al 1994;Larouche et al 2008), and smooth muscle cells in the human placenta cross-react with an anti-K8 antibody, thus indicating that they contain K8 (Miettinen & Fetsch, 2000) or at least a molecule with an epitope that binds to this antibody. Similarly, in the transition from a non-sensory to a specialized sensory cell, the synthesis of keratins is downregulated (Cyr et al 2000).…”