Uwe Wolfrum

Rhodopsin’s Carboxy-Terminal Cytoplasmic Tail Acts as a Membrane Receptor for Cytoplasmic Dynein by Binding to the Dynein Light Chain Tctex-1

The interaction of cytoplasmic dynein with its cargoes is thought to be indirectly mediated by dynactin, a complex that binds to the dynein intermediate chain. However, the roles of other dynein subunits in cargo binding have been unknown. Here we demonstrate that dynein translocates rhodopsin-bearing vesicles along microtubules. This interaction occurs directly between the C-terminal cytoplasmic tail of rhodopsin and Tctex-1, a dynein light chain. C-terminal rhodopsin mutations responsible for retinitis pigmentosa inhibit this interaction. Our results point to an alternative docking mechanism for cytoplasmic dynein, provide novel insights into the role of motor proteins in the polarized transport of post-Golgi vesicles, and shed light on the molecular basis of retinitis pigmentosa.

Protein quality control during aging involves recruitment of the macroautophagy pathway by BAG3

The Hsc/Hsp70 co‐chaperones of the BAG (Bcl‐2‐associated athanogene) protein family are modulators of protein quality control. We examined the specific roles of BAG1 and BAG3 in protein degradation during the aging process. We show that BAG1 and BAG3 regulate proteasomal and macroautophagic pathways, respectively, for the degradation of polyubiquitinated proteins. Moreover, using models of cellular aging, we find that a switch from BAG1 to BAG3 determines that aged cells use more intensively the macroautophagic system for turnover of polyubiquitinated proteins. This increased macroautophagic flux is regulated by BAG3 in concert with the ubiquitin‐binding protein p62/SQSTM1. The BAG3/BAG1 ratio is also elevated in neurons during aging of the rodent brain, where, consistent with a higher macroautophagy activity, we find increased levels of the autophagosomal marker LC3‐II as well as a higher cathepsin activity. We conclude that the BAG3‐mediated recruitment of the macroautophagy pathway is an important adaptation of the protein quality control system to maintain protein homeostasis in the presence of an enhanced pro‐oxidant and aggregation‐prone milieu characteristic of aging.

How Does the Eye Breathe? EVIDENCE FOR NEUROGLOBIN-MEDIATED OXYGEN SUPPLY IN THE MAMMALIAN RETINA

Visual performance of the vertebrate eye requires large amounts of oxygen, and thus the retina is one of the highest oxygen-consuming tissues of the body. Here we show that neuroglobin, a neuron-specific respiratory protein distantly related to hemoglobin and myoglobin, is present at high amounts in the mouse retina (∼100 μm). The estimated concentration of neuroglobin in the retina is thus about 100-fold higher than in the brain and is in the same range as that of myoglobin in the muscle. Neuroglobin is expressed in all neurons of the retina but not in the retinal pigment epithelium. Neuroglobin mRNA was detected in the perikarya of the nuclear and ganglion layers of the neuronal retina, whereas the protein was present mainly in the plexiform layers and in the ellipsoid region of photoreceptor inner segment. The distribution of neuroglobin correlates with the subcellular localization of mitochondria and with the relative oxygen demands, as the plexiform layers and the inner segment consume most of the retinal oxygen. These findings suggest that neuroglobin supplies oxygen to the retina, similar to myoglobin in the myocardium and the skeletal muscle.

Mutations in the gene encoding the basal body protein RPGRIP1L, a nephrocystin-4 interactor, cause Joubert syndrome.

Protein-protein interaction analyses have uncovered a ciliary and basal body protein network that, when disrupted, can result in nephronophthisis (NPHP), Leber congenital amaurosis, Senior-Løken syndrome (SLSN) or Joubert syndrome (JBTS). However, details of the molecular mechanisms underlying these disorders remain poorly understood. RPGRIP1-like protein (RPGRIP1L) is a homolog of RPGRIP1 (RPGR-interacting protein 1), a ciliary protein defective in Leber congenital amaurosis. We show that RPGRIP1L interacts with nephrocystin-4 and that mutations in the gene encoding nephrocystin-4 (NPHP4) that are known to cause SLSN disrupt this interaction. RPGRIP1L is ubiquitously expressed, and its protein product localizes to basal bodies. Therefore, we analyzed RPGRIP1L as a candidate gene for JBTS and identified loss-of-function mutations in three families with typical JBTS, including the characteristic mid-hindbrain malformation. This work identifies RPGRIP1L as a gene responsible for JBTS and establishes a central role for cilia and basal bodies in the pathophysiology of this disorder.

Vezatin, a novel transmembrane protein, bridges myosin VIIA to the cadherin–catenins complex

Defects in myosin VIIA are responsible for deafness in the human and mouse. The role of this unconventional myosin in the sensory hair cells of the inner ear is not yet understood. Here we show that the C‐terminal FERM domain of myosin VIIA binds to a novel transmembrane protein, vezatin, which we identified by a yeast two‐hybrid screen. Vezatin is a ubiquitous protein of adherens cell–cell junctions, where it interacts with both myosin VIIA and the cadherin–catenins complex. Its recruitment to adherens junctions implicates the C‐terminal region of α‐catenin. Taken together, these data suggest that myosin VIIA, anchored by vezatin to the cadherin–catenins complex, creates a tension force between adherens junctions and the actin cytoskeleton that is expected to strengthen cell–cell adhesion. In the inner ear sensory hair cells vezatin is, in addition, concentrated at another membrane–membrane interaction site, namely at the fibrillar links interconnecting the bases of adjacent stereocilia. In myosin VIIA‐defective mutants, inactivity of the vezatin–myosin VIIA complex at both sites could account for splaying out of the hair cell stereocilia.

BAG3 mediates chaperone‐based aggresome‐targeting and selective autophagy of misfolded proteins

Increasing evidence indicates the existence of selective autophagy pathways, but the manner in which substrates are recognized and targeted to the autophagy system is poorly understood. One strategy is transport of a particular substrate to the aggresome, a perinuclear compartment with high autophagic activity. In this paper, we identify a new cellular pathway that uses the specificity of heat‐shock protein 70 (Hsp70) to misfolded proteins as the basis for aggresome‐targeting and autophagic degradation. This pathway is regulated by the stress‐induced co‐chaperone Bcl‐2‐associated athanogene 3 (BAG3), which interacts with the microtubule‐motor dynein and selectively directs Hsp70 substrates to the motor and thereby to the aggresome. Notably, aggresome‐targeting by BAG3 is distinct from previously described mechanisms, as it does not depend on substrate ubiquitination.

Myosin VIIa, the product of the Usher 1B syndrome gene, is concentrated in the connecting cilia of photoreceptor cells

Usher syndrome is the most common form of combined deafness and blindness. The gene that is defective in Usher syndrome 1B (USH1B) encodes for an unconventional myosin, myosin VIIa. To understand the cellular function of myosin VIIa and why defects in it lead to USH1B, it is essential to determine the precise cellular and subcellular localization of the protein. We investigated the distribution of myosin VIIa in human and rodent photoreceptor cells and retinal pigment epithelium (RPE), primarily by immunoelectron microscopy, using antibodies generated against two different domains of the protein. In both human and rodent retinae, myosin VIIa was detected in the apical processes of the RPE and in the cilium of rod and cone photoreceptor cells. Immunogold label was most concentrated in the connecting cilium. Here, myosin VIIa appeared to be distributed outside the ring of doublet microtubules near the ciliary plasma membrane. These observations indicate that a major role of myosin VIIa in the retina is in the photoreceptor cilium, perhaps in such a function as trafficking newly synthesized phototransductive membrane or maintaining the diffusion barrier between the inner and outer segments. Our results support the notion that defective ciliary function is the underlying cellular abnormality that leads to cellular degeneration in Usher syndrome.

MyRIP, a novel Rab effector, enables myosin VIIa recruitment to retinal melanosomes

Defects of the myosin VIIa motor protein cause deafness and retinal anomalies in humans and mice. We report on the identification of a novel myosin‐VIIa‐interacting protein that we have named MyRIP (myosin‐VIIa‐ and Rab‐interacting protein), since it also binds to Rab27A in a GTP‐dependent manner. In the retinal pigment epithelium cells, MyRIP, myosin VIIa and Rab27A are associated with melanosomes. In transfected PC12 cells, overexpression of MyRIP was shown to interfere with the myosin VIIa tail localization. We propose that a molecular complex composed of Rab27A, MyRIP and myosin VIIa bridges retinal melanosomes to the actin cytoskeleton and thereby mediates the local trafficking of these organelles. The defect of this molecular complex is likely to account for the perinuclear mislocalization of the melanosomes observed in the retinal pigment epithelium cells of myosinVIIa‐defective mice.

Mutations in LCA5, encoding the ciliary protein lebercilin, cause Leber congenital amaurosis.

Leber congenital amaurosis (LCA) causes blindness or severe visual impairment at or within a few months of birth. Here we show, using homozygosity mapping, that the LCA5 gene on chromosome 6q14, which encodes the previously unknown ciliary protein lebercilin, is associated with this disease. We detected homozygous nonsense and frameshift mutations in LCA5 in five families affected with LCA. In a sixth family, the LCA5 transcript was completely absent. LCA5 is expressed widely throughout development, although the phenotype in affected individuals is limited to the eye. Lebercilin localizes to the connecting cilia of photoreceptors and to the microtubules, centrioles and primary cilia of cultured mammalian cells. Using tandem affinity purification, we identified 24 proteins that link lebercilin to centrosomal and ciliary functions. Members of this interactome represent candidate genes for LCA and other ciliopathies. Our findings emphasize the emerging role of disrupted ciliary processes in the molecular pathogenesis of LCA.

A core cochlear phenotype in USH1 mouse mutants implicates fibrous links of the hair bundle in its cohesion, orientation and differential growth

The planar polarity and staircase-like pattern of the hair bundle are essential to the mechanoelectrical transduction function of inner ear sensory cells. Mutations in genes encoding myosin VIIa, harmonin, cadherin 23, protocadherin 15 or sans cause Usher syndrome type I (USH1, characterized by congenital deafness, vestibular dysfunction and retinitis pigmentosa leading to blindness) in humans and hair bundle disorganization in mice. Whether the USH1 proteins are involved in common hair bundle morphogenetic processes is unknown. Here, we show that mouse models for the five USH1 genetic forms share hair bundle morphological defects. Hair bundle fragmentation and misorientation (25-52° mean kinociliary deviation, depending on the mutant) were detected as early as embryonic day 17. Abnormal differential elongation of stereocilia rows occurred in the first postnatal days. In the emerging hair bundles, myosin VIIa, the actin-binding submembrane protein harmonin-b, and the interstereocilia-kinocilium lateral link components cadherin 23 and protocadherin 15, all concentrated at stereocilia tips, in accordance with their known in vitro interactions. Soon after birth, harmonin-b switched from the tip of the stereocilia to the upper end of the tip link, which also comprises cadherin 23 and protocadherin 15. This positional change did not occur in mice deficient for cadherin 23 or protocadherin 15. We suggest that tension forces applied to the early lateral links and to the tip link, both of which can be anchored to actin filaments via harmonin-b, play a key role in hair bundle cohesion and proper orientation for the former, and in stereociliary elongation for the latter.