ILDR1 null mice, a model of human deafness DFNB42, show structural aberrations of tricellular tight junctions and degeneration of auditory hair cells.

In the mammalian inner ear, bicellular and tricellular tight junctions (tTJs) seal the paracellular space between epithelial cells. Tricellulin and immunoglobulin-like (Ig-like) domain containing receptor 1 (ILDR1, also referred to as angulin-2) localize to tTJs of the sensory and non-sensory epithelia in the organ of Corti and vestibular end organs. Recessive mutations of TRIC (DFNB49) encoding tricellulin and ILDR1 (DFNB42) cause human nonsyndromic deafness. However, the pathophysiology of DFNB42 deafness remains unknown. ILDR1 was recently reported to be a lipoprotein receptor mediating the secretion of the fat-stimulated cholecystokinin (CCK) hormone in the small intestine, while ILDR1 in EpH4 mouse mammary epithelial cells in vitro was shown to recruit tricellulin to tTJs. Here we show that two different mouse Ildr1 mutant alleles have early-onset severe deafness associated with a rapid degeneration of cochlear hair cells (HCs) but have a normal endocochlear potential. ILDR1 is not required for recruitment of tricellulin to tTJs in the cochlea in vivo; however, tricellulin becomes mislocalized in the inner ear sensory epithelia of ILDR1 null mice after the first postnatal week. As revealed by freeze-fracture electron microscopy, ILDR1 contributes to the ultrastructure of inner ear tTJs. Taken together, our data provide insight into the pathophysiology of human DFNB42 deafness and demonstrate that ILDR1 is crucial for normal hearing by maintaining the structural and functional integrity of tTJs, which are critical for the survival of auditory neurosensory HCs.

The ultrastructure of Placus striatus and a revision of the family Placidae (Ciliophora).

The first ultrastructural description of the genus Placus is presented. Each somatic kinetosome has a cone-shaped axosomal plate, nonoverlapping postciliary microtubules, an anteriorly directed kinetodesmal fiber, and a radial ribbon of transverse microtubules, which extend laterally under the ciliary furrow and insert in the cortical ridge. A closed ring of paired kinetosomes encircles the cytostome. A brosse begins adjacent to the oral pairs and extends posteriorly for one-fourth to one-half the cell's length. Autapomorphies for Placus include bowling pin-shaped toxicysts extruded onto a distinct area of the cell surface immediately posterior to the brosse, and a brosse kinety consisting of a single row of paired cilia. Placus and its sister taxon Spathidiopsis both have spiraling kineties composed of single cilia inserted into the side of the ciliary furrow. Spathidiopsis can be distinguished from Placus because it has a brosse consisting of two kineties (i.e. one composed of paired cilia, the other of single cilia), a row of rod-shaped toxicyst-bearing palps extending around one side of the oral area and along the length of the brosse, and a mid-cell cortical inpocketing containing toxicysts and a segment of the brosse. A revised listing of species assigned to the family Placidae is given.

A molecular and ultrastructural description of Spathidiopsis buddenbrocki and the phylogenetic position of the family Placidae (Ciliophora).

Spathidiopsis and Placus are the only two genera within the family Placidae. The family has been placed in the class Prostomatea and order Prorodontida because its members have somatic monokinetids with a radial transverse ribbon, a straight non-overlapping postciliary ribbon, and anteriorly directed non-overlapping kinetodesmal fibril, an apical cytostome lacking specialized oral cilia, a brosse, and toxicysts. To confirm the stability of this placement, ultrastructural morphology and small subunit rRNA gene sequences of Spathidiopsis socialis, Spathidiopsis buddenbrocki, and Placus striatus were determined. These data were combined with information from other ciliates, and phylogenetic trees were generated using maximum-likelihood and maximum-parsimony methods. The analyses confirmed the family Placidae to be a monophyletic group in the Prostomatea with the Placidae a sister group to a Cryptocaryon + Coleps + Prorodon clade.

An actin molecular treadmill and myosins maintain stereocilia functional architecture and self-renewal.

We have previously shown that the seemingly static paracrystalline actin core of hair cell stereocilia undergoes continuous turnover. Here, we used the same approach of transfecting hair cells with actin-green fluorescent protein (GFP) and espin-GFP to characterize the turnover process. Actin and espin are incorporated at the paracrystal tip and flow rearwards at the same rate. The flux rates (approximately 0.002-0.04 actin subunits s(-1)) were proportional to the stereocilia length so that the entire staircase stereocilia bundle was turned over synchronously. Cytochalasin D caused stereocilia to shorten at rates matching paracrystal turnover. Myosins VI and VIIa were localized alongside the actin paracrystal, whereas myosin XVa was observed at the tips at levels proportional to stereocilia lengths. Electron microscopy analysis of the abnormally short stereocilia in the shaker 2 mice did not show the characteristic tip density. We argue that actin renewal in the paracrystal follows a treadmill mechanism, which, together with the myosins, dynamically shapes the functional architecture of the stereocilia bundle.

Phylogeny and systematic position of Zosterodasys (Ciliophora, Synhymeniida): a combined analysis of ciliate relationships using morphological and molecular data.

The Synhymeniida is characterized both by a band of somatic dikinetids, the synhymenium, extending across the surface of the cell and by a ventral cell mouth lacking specialized feeding cilia but subtended by a well-developed cyrtos. The synhymeniids have been hypothesized to be members of the class Nassophorea but our previous ultrastructural study of the synhymeniid genus Zosterodasys did not show any clear synapomorphies that would permit definitive placement in the Nassophorea or as a sister taxon to any of the other ciliate groups possessing a cyrtos. In the present study, simultaneous analysis of morphological and small subunit rDNA molecular data indicates that the Synhymeniida are sister to the class Phyllopharyngea and that this clade is, in turn, sister to the remaining Nassophorea, although this result is sensitive to dataset inclusion and alignment parameters. While this suggests that taxa with a ventral cyrtos might be united into a named taxon (e.g. resurrecting the Hypostomata), additional data are needed to reach a definitive conclusion.

TRIOBP-5 sculpts stereocilia rootlets and stiffens supporting cells enabling hearing.

TRIOBP remodels the cytoskeleton by forming unusually dense F-actin bundles and is implicated in human cancer, schizophrenia, and deafness. Mutations ablating human and mouse TRIOBP-4 and TRIOBP-5 isoforms are associated with profound deafness, as inner ear mechanosensory hair cells degenerate after stereocilia rootlets fail to develop. However, the mechanisms regulating formation of stereocilia rootlets by each TRIOBP isoform remain unknown. Using 3 new Triobp mouse models, we report that TRIOBP-5 is essential for thickening bundles of F-actin in rootlets, establishing their mature dimensions and for stiffening supporting cells of the auditory sensory epithelium. The coiled-coil domains of this isoform are required for reinforcement and maintenance of stereocilia rootlets. A loss of TRIOBP-5 in mouse results in dysmorphic rootlets that are abnormally thin in the cuticular plate but have increased widths and lengths within stereocilia cores, and causes progressive deafness recapitulating the human phenotype. Our study extends the current understanding of TRIOBP isoform-specific functions necessary for life-long hearing, with implications for insight into other TRIOBPopathies.

The 133-kDa N-terminal domain enables myosin 15 to maintain mechanotransducing stereocilia and is essential for hearing.

The precise assembly of inner ear hair cell stereocilia into rows of increasing height is critical for mechanotransduction and the sense of hearing. Yet, how the lengths of actin-based stereocilia are regulated remains poorly understood. Mutations of the molecular motor myosin 15 stunt stereocilia growth and cause deafness. We found that hair cells express two isoforms of myosin 15 that differ by inclusion of an 133-kDa N-terminal domain, and that these isoforms can selectively traffic to different stereocilia rows. Using an isoform-specific knockout mouse, we show that hair cells expressing only the small isoform remarkably develop normal stereocilia bundles. However, a critical subset of stereocilia with active mechanotransducer channels subsequently retracts. The larger isoform with the 133-kDa N-terminal domain traffics to these specialized stereocilia and prevents disassembly of their actin core. Our results show that myosin 15 isoforms can navigate between functionally distinct classes of stereocilia, and are independently required to assemble and then maintain the intricate hair bundle architecture.

Changes in plasma membrane structure and electromotile properties in prestin deficient outer hair cells.

Cochlear outer hair cells (OHCs) rapidly change their length and stiffness when their membrane potential is altered. Prestin, the motor protein for this electromotility, is present along the OHC lateral plasma membrane where there is a high density of intra-membrane protein particles (IMPs). However, it is not known to what extent prestin contributes to this unusual dense population of proteins and overall organization of the membrane to generate the unique electromechanical response of OHCs. We investigated the relationship of prestin with the IMPs, the underlying cortical cytoskeletal lattice, and electromotility in prestin-deficient mice. Using freeze-fracture, we observed a reduction in density and size of the IMPs that correlates with the reduction and absence of prestin in the heterozygous and homozygous mice, respectively. We also observed a reduction or absence of electromotility-related charge density, axial stiffness, and piezoelectric properties of the OHC. A comparison of the charge density with the number of IMPs suggests that prestin forms tetramers in the wild type but is likely to form lower number oligomers in the prestin-deficient OHCs from the heterozygous mice. Interestingly, the characteristic actin-based cortical cytoskeletal lattice that underlies the membrane is absent in the prestin-null OHCs, suggesting that prestin is also required for recruiting or maintaining the cortical cytoskeletal lattice. These results suggest that the majority of the IMPs are indeed prestin and that electrically evoked length and stiffness changes are interrelated and dependent on both prestin and on the cortical actin cytoskeletal lattice of the OHC lateral membrane.

Growth hormone promotes glomerular lipid accumulation in bGH mice.

BACKGROUND: Bovine growth hormone (bGH) transgenic mice develop progressive glomerulosclerosis and exhibit abnormalities in hepatic lipid metabolism. We have previously shown that growth hormone up-regulates the low-density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) in mouse mesangial cells. However, a role of lipid abnormalities in bGH kidney disease has not yet been demonstrated. METHODS: Groups of bGH mice (5 and 11 months old) presenting with, respectively, moderate and severe degrees of glomerulosclerosis were compared to age-matched controls. Neutral lipid content in kidney cortex was determined by oil red-O staining, serum cholesterol, and triglycerides by enzymatic assays, relative mRNA expression of LDL receptors, HMGR, and scavenger receptor by real-time reverse transcription-polymerase chain reaction (RT-PCR), and HMGR protein expression by immunoblotting. Two younger (5 and 12 weeks old) groups of mice were used to study scavenger receptor expression at earlier time points. RESULTS: Serum cholesterol was significantly increased in bGH mice at 5 months, but triglycerides were lower than control levels at both 5 and 11 months. Renal cortex HMGR expression was elevated at the mRNA but not at the protein level in the 11-month-old bGH group compared to controls. However, glomerular neutral lipid staining and scavenger receptor mRNA expression were markedly increased in all bGH mice, including those at 5 weeks of age compared to respective controls. CONCLUSION: The bGH mouse exhibits an increased mesangial lipid content and elevated scavenger receptor mRNA expression as early as at 5 weeks of age, suggesting that an increased kidney uptake of oxidized LDL could play a role in the development of glomerulosclerosis in this mouse model.