Ablation of Whirlin Long Isoform Disrupts the USH2 Protein Complex and Causes Vision and Hearing Loss

Mutations in whirlin cause either Usher syndrome type II (USH2), a deafness-blindness disorder, or nonsyndromic deafness. The molecular basis for the variable disease expression is unknown. We show here that only the whirlin long isoform, distinct from a short isoform by virtue of having two N-terminal PDZ domains, is expressed in the retina. Both long and short isoforms are expressed in the inner ear. The N-terminal PDZ domains of the long whirlin isoform mediates the formation of a multi-protein complex that includes usherin and VLGR1, both of which are also implicated in USH2. We localized this USH2 protein complex to the periciliary membrane complex (PMC) in mouse photoreceptors that appears analogous to the frog periciliary ridge complex. The latter is proposed to play a role in photoreceptor protein trafficking through the connecting cilium. Mice carrying a targeted disruption near the N-terminus of whirlin manifest retinal and inner ear defects, reproducing the clinical features of human USH2 disease. This is in contrast to mice with mutations affecting the C-terminal portion of whirlin in which the phenotype is restricted to the inner ear. In mice lacking any one of the USH2 proteins, the normal localization of all USH2 proteins is disrupted, and there is evidence of protein destabilization. Taken together, our findings provide new insights into the pathogenic mechanism of Usher syndrome. First, the three USH2 proteins exist as an obligatory functional complex in vivo, and loss of one USH2 protein is functionally close to loss of all three. Second, defects in the three USH2 proteins share a common pathogenic process, i.e., disruption of the PMC. Third, whirlin mutations that ablate the N-terminal PDZ domains lead to Usher syndrome, but non-syndromic hearing loss will result if they are spared

Rootletin Interacts with C-Nap1 and May Function as a Physical Linker between the Pair of Centrioles/Basal Bodies in Cells

Rootletin, a major structural component of the ciliary rootlet, is located at the basal bodies and centrosomes in ciliated and nonciliated cells, respectively. Here we investigated its potential role in the linkage of basal bodies/centrioles and the mechanism involved in such linkages. We show that rootletin interacts with C-Nap1, a protein restricted at the ends of centrioles and functioning in centrosome cohesion in interphase cells. Their interaction in vivo is supported by their colocalization at the basal bodies/centrioles and coordinated association with the centrioles during the cell cycle. Ultrastructural examinations demonstrate that rootletin fibers connect the basal bodies in ciliated cells and are present both at the ends of and in between the pair of centrioles in nonciliated cells. The latter finding stands in contrast with C-Nap1, which is present only at the ends of the centrioles. Transient expression of C-Nap1 fragments dissociated rootletin fibers from the centrioles, resulting in centrosome separation in interphase. Overexpression of rootletin in cells caused multinucleation, micronucleation, and irregularity of nuclear shape and size, indicative of defects in chromosome separation. These data suggest that rootletin may function as a physical linker between the pair of basal bodies/centrioles by binding to C-Nap1

A Modified Micro-Macro Constitutive Model for Porous Rocks with Pressure-Sensitive Matrix by considering a New Hardening Law

This paper aims mainly at providing an incremental elastoplastic constitutive model for heterogeneous porous rock-like materials in the frame of micromechanics. The studied material is considered to be made up of randomly distributed spherical pores embedded in a pressure-sensitive solid matrix obeying Drucker–Prager yield function. The effective elastic properties of porous rocks are obtained by the use of Mori and Tanaka homogenization scheme, which are on function of the bulk and shear moduli of the solid matrix and of the value of porosity. For the macroscopic nonlinear phase, a limit analysis-based macroscopic criterion is adopted to derive the basic constitutive rule by considering an associated plastic flow rule. In order to capture the typical hardening effects of rocks, an originally proposed hardening function of the solid matrix is also taken into consideration, which is related on the accumulated equivalent plastic strain. In order to verify its accuracy, the proposed micro-macro constitutive model is implemented by a numerical procedure including elastic predictions and plastic corrections and compared to experimental results of triaxial compression tests of sandstone with different confining pressures. It is observed that the numerical simulation is in accord with the experimental data, indicating that the obtained model is able to predict the main mechanical behaviours of rock-like materials

The Ciliary Rootlet Maintains Long-Term Stability of Sensory Cilia

The striated ciliary rootlet is a prominent cytoskeleton originating from basal bodies of ciliated cells. Although a familiar structure in cell biology, its function has remained unresolved. In this study, we carried out targeted disruption in mice of the gene for rootletin, a component of the rootlet. In the mutant, ciliated cells are devoid of rootlets. Phototransduction and ciliary beating in sensory and motile cilia initially exhibit no apparent functional deficits. However, photoreceptors degenerate over time, and mutant lungs appear prone to pathological changes consistent with insufficient mucociliary clearance. Further analyses revealed a striking fragility at the ciliary base in photoreceptors lacking rootlets. In vitro assays suggest that the rootlet is among the least dynamic of all cytoskeletons and interacts with actin filaments. Thus, a primary function of the rootlet is to provide structural support for the cilium. Inasmuch as photoreceptors elaborate an exceptionally enlarged sensory cilium, they are especially dependent on the rootlet for structural integrity and long-term survival

Whirlin knockout mice have morphological defects around the PMC in photoreceptors revealed by electron microscopy.

<p>(A) A representative image showing the normal ultrastructure around the PMC in the wild-type photoreceptor (whirlin^+/+). (B–F) In whirlin knockout mice (whirlin^−/−), abnormal distance (E) and membrane fusion (empty arrows, B–E) between the apical inner segment and the connecting cilium were found. In addition, a large amount of vacuoles (filled arrows, D–F) were accumulated around the PMC. (D,D′) show the same cell at different sectioning levels. OS, outer segments; CC, connecting cilia; IS, inner segments. Scale bars, 200 nm. (G) All the above abnormalities exist in a small fraction of photoreceptors randomly distributed in the retina of whirlin knockout mice. The number of mice analyzed in each group is indicated in the bottom of each bar. Mean ± SEM; **, p<0.01.</p