Estrogen receptor α regulates non-canonical autophagy that provides stress resistance to neuroblastoma and breast cancer cells and involves BAG3 function.

Breast cancer is a heterogeneous disease and approximately 70% of newly diagnosed breast cancers are estrogen receptor (ER) positive. Out of the two ER types, α and ß, ERα is the only ER that is detectable by immunohistochemistry in breast cancer biopsies and is the predominant subtype expressed in breast tumor tissue. ER-positive tumors are currently treated with anti-hormone therapy to inhibit ER signaling. It is well known that breast cancer cells can develop endocrine resistance and resistance to anti-hormone therapy and this can be facilitated via the autophagy pathway, but so far the description of a detailed autophagy expression profile of ER-positive cancer cells is missing. In the present study, we characterized tumor cell lines ectopically expressing ERα or ERß as well as the breast cancer-derived MCF-7 cell line endogenously expressing ERα but being ERß negative. We could show that ERα-expressing cells have a higher autophagic activity than cells expressing ERß and cells lacking ER expression. Additionally, for autophagy-related gene expression we describe an ERα-specific 'autophagy-footprint' that is fundamentally different to tumor cells expressing ERß or lacking ER expression. This newly described ERα-mediated and estrogen response element (ERE)-independent non-canonical autophagy pathway, which involves the function of the co-chaperone Bcl2-associated athanogene 3 (BAG3), is independent of classical mammalian target of rapamycin (mTOR) and phosphatidylinositol 3 kinase (PI3K) signaling networks and provides stress resistance in our model systems. Altogether, our study uncovers a novel non-canonical autophagy pathway that might be an interesting target for personalized medicine and treatment of ERα-positive breast cancer cells that do not respond to anti-hormone therapy and classical autophagy inhibitors.

PTC124-mediated translational readthrough of a nonsense mutation causing Usher syndrome type 1C.

We investigated the therapeutic potential of the premature termination codon (PTC) readthrough-inducing drug PTC124 in treating the retinal phenotype of Usher syndrome, caused by a nonsense mutation in the USH1C gene. Applications in cell culture, organotypic retina cultures, and mice in vivo revealed significant readthrough and the recovery of protein function. In comparison with other readthrough drugs, namely the clinically approved readthrough-inducing aminoglycoside gentamicin, PTC124 exhibits significant better retinal biocompatibility. Its high readthrough efficiency in combination with excellent biocompatibility makes PTC124 a promising therapeutic agent for PTCs in USH1C, as well as other ocular and nonocular genetic diseases.

RPGR ORF15 isoform co-localizes with RPGRIP1 at centrioles and basal bodies and interacts with nucleophosmin.

The ORF15 isoform of RPGR (RPGR(ORF15)) and RPGR interacting protein 1 (RPGRIP1) are mutated in a variety of retinal dystrophies but their functions are poorly understood. Here, we show that in cultured mammalian cells both RPGR(ORF15) and RPGRIP1 localize to centrioles. These localizations are resistant to the microtubule destabilizing drug nocodazole and persist throughout the cell cycle. RPGR and RPGRIP1 also co-localize at basal bodies in cells with primary cilia. The C-terminal (C2) domain of RPGR(ORF15) (ORF15(C2)) is highly conserved across 13 mammalian species, suggesting that it is a functionally important domain. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we show that this domain interacts with a 40 kDa shuttling protein nucleophosmin (NPM). The RPGR(ORF15)-NPM interaction was confirmed by (i) yeast two-hybrid analyses; (ii) binding of both recombinant and native HeLa cell NPM to RPGR(ORF15) fusion proteins in vitro; (iii) co-immunoprecipitation of native NPM, RPGR(ORF15) and RPGRIP1 from bovine retinal extracts and of native HeLa cell NPM and transfected RPGR(ORF15) from cultured cells and (iv) co-localization of NPM and RPGR(ORF15) at metaphase centrosomes in cultured cells. NPM is a multifunctional protein chaperone that shuttles between the nucleoli and the cytoplasm and has been associated with licensing of centrosomal division. RPGR and RPGRIP1 join a growing number of centrosomal proteins involved in human disease.

Identification of novel molecular components of the photoreceptor connecting cilium by immunoscreens.

The connecting cilium of photoreceptor cells is the only intracellular link between the morphologically, functionally and biochemically different compartments of the inner and outer segments. The non-motile modified cilium plays an important role in the organization and the function of photoreceptor cells, namely in delivery and turnover of enzymes and substrates of the visual transduction cascade, and the photosensitive membranes of the outer segment. The protein components of the cilium participate in the intracellular transport through the cilium, in the outer segment disk morphogenesis and in the maintenance of discrete membrane domains. In order to identify yet unknown cytoskeletal components of the connecting cilium, a combined biochemical and molecular biological strategy was applied. For this purpose, antibodies were raised against proteins of photoreceptor cell axonemes. Using this AX-4-antiserum, a rat retina cDNA expression library was immunoscreened and clones encoding partial sequences of (i) already known photoreceptor specific proteins; (ii) ubiquitously expressed proteins; (iii) clones with homologies to retinal ESTs; and (iv) clones coding for cytoskeletal proteins were isolated. Further analysis revealed that these candidate clones have homologies to Drosophila flightless I, mouse APC-binding protein EB2, human microtubule associated protein 4 (MAP4), human centrin 3, human cytoplasmic dynein intermediate chain 2C, and human dynamitin.The immunoscreening approach used here was successfully applied to isolate genes encoding yet unknown cytoskeletal proteins of photoreceptor cell axonemes. The obtained information will provide further insight into the role of the connecting cilium in photoreceptor cell function.

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 alpha-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.

Rhodopsin transport in the membrane of the connecting cilium of mammalian photoreceptor cells.

The transport of the photopigment rhodopsin from the inner segment to the photosensitive outer segment of vertebrate photoreceptor cells has been one of the main remaining mysteries in photoreceptor cell biology. Because of the lack of any direct evidence for the pathway through the photoreceptor cilium, alternative extracellular pathways have been proposed. Our primary aim in the present study was to resolve rhodopsin trafficking from the inner to the outer segment. We demonstrate, predominantly by high-sensitive immunoelectron microscopy, that rhodopsin is also densely packed in the membrane of the photoreceptor connecting cilium. Present prominent labeling of rhodopsin in the ciliary membrane provides the first striking evidence that rhodopsin is translocated from the inner segment to the outer segment of wild type photoreceptors via the ciliary membrane. At the ciliary membrane rhodopsin co-localizes with the unconventional myosin VIIa, the product of human Usher syndrome 1B gene. Furthermore, axonemal actin was identified in the photoreceptor cilium, which is spatially co-localized with myosin VIIa and opsin. This actin cytoskeleton of the cilium may provide the structural bases for myosin VIIa-linked ciliary trafficking of membrane components, including rhodopsin.

Photoreceptor autophagy: effects of light history on number and opsin content of degradative vacuoles.

PURPOSE: To investigate whether regulation of rhodopsin levels as a response to changed lighting environment is performed by autophagic degradation of opsin in rod inner segments (RISs). METHODS: Groups of albino rats were kept in 3 lux or 200 lux. At 10 weeks of age, one group was transferred from 3 lux to 200 lux, another group was switched from 200 lux to 3 lux, and two groups remained in their native lighting (baselines). Rats were killed at days 1, 2, and 3 after switching. Another group was switched from 3 lux to 200 lux, and rats were killed at short intervals after the switch. Numbers of autophagic vacuoles (AVs) in RISs were counted, and immunogold labeling was performed for opsin and ubiquitin in electron microscopic sections. RESULTS: The number of AVs increased significantly after switching from 3 lux to 200 lux at days 1 and 2 and declined at day 3, whereas the reverse intensity change did not cause any increase. Early time points after change from 3 lux to 200 lux showed a significant increase of AVs 2 and 3 hours after switching. Distinct opsin label was observed in AVs of rats switched to 200 lux. Ubiquitin label was present in all investigated specimens and was also seen in AVs especially in 200-lux immigrants. CONCLUSIONS: Earlier studies had shown that an adjustment to new lighting environment is performed by changes in rhodopsin levels in ROSs. Autophagic degradation of opsin or rhodopsin may subserve, at least in part, the adaptation to abruptly increased habitat illuminance by removing surplus visual pigment.

Interaction of glutamic-acid-rich proteins with the cGMP signalling pathway in rod photoreceptors.

The assembly of signalling molecules into macromolecular complexes (transducisomes) provides specificity, sensitivity and speed in intracellular signalling pathways. Rod photoreceptors in the eye contain an unusual set of glutamic-acid-rich proteins (GARPs) of unknown function. GARPs exist as two soluble forms, GARP1 and GARP2, and as a large cytoplasmic domain (GARP' part) of the beta-subunit of the cyclic GMP-gated channel. Here we identify GARPs as multivalent proteins that interact with the key players of cGMP signalling, phosphodiesterase and guanylate cyclase, and with a retina-specific ATP-binding cassette transporter (ABCR), through four, short, repetitive sequences. In electron micrographs, GARPs are restricted to the rim region and incisures of discs in close proximity to the guanylate cyclase and ABCR, whereas the phosphodiesterase is randomly distributed. GARP2, the most abundant splice form, associates more strongly with light-activated than with inactive phosphodiesterase, and GARP2 potently inhibits phosphodiesterase activity. Thus, the GARPs organize a dynamic protein complex near the disc rim that may control cGMP turnover and possibly other light-dependent processes. Because there are no similar GARPs in cones, we propose that GARPs may prevent unnecessary cGMP turnover during daylight, when rods are held in saturation by the relatively high light levels.

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.

Expression of centrin isoforms in the mammalian retina.

Centrin is a calcium-binding phosphoprotein of centrosomes, mitotic spindle poles, and flagellar basal apparatus. Indirect immunofluorescence studies in human and rat retinas reveal centrin localization in two distinct cellular structures: at centrosomes of nonciliated neuronal cells as well as in basal bodies, and in larger amounts in the highly modified cilium--the connecting cilium--of photoreceptor cells. Western blot analyses of mammalian retinal proteins show two closely migrating centrin bands at about 20 kDa, the previously described molecular weight of centrins. Using isoform specific primers in PCR, the expression of two related but distinct forms of centrin (centrin 1 and centrin 2), can be identified in the retina of human and rat as well as in the mammalian testis, tissues where cilia are present. However, only one isoform (centrin 2) is expressed in nondifferentiated, nonciliated retinal cells (retinoblastoma cells), as well as in rat liver, skeletal muscle, and cardiac muscle. These observations suggest centrin 2 message may be universally expressed while centrin 1 message may be restricted to retina and testis which contain cells that have differentiated cilia or flagella, or their modifications.

Myosin VIIa as a common component of cilia and microvilli.

The distribution of myosin VIIa, which is defective or absent in Usher syndrome 1B, was studied in a variety of tissues by immunomicroscopy. The primary aim was to determine whether this putative actin-based mechanoenzyme is a common component of cilia. Previously, it has been proposed that defective ciliary function might be the basis of some forms of Usher syndrome. Myosin VIIa was detected in cilia from cochlear hair cells, olfactory neurons, kidney distal tubules, and lung bronchi. It was also found to cofractionate with the axonemal fraction of retinal photoreceptor cells. Immunolabeling appeared most concentrated in the periphery of the transition zone of the cilia. This general presence of a myosin in cilia is surprising, given that cilia are dominated by microtubules, and not actin filaments. In addition to cilia, myosin VIIa was also found in actin-rich microvilli of different types of cell. We conclude that myosin VIIa is a common component of cilia and microvilli.

Molecular cloning of Drosophila Rh6 rhodopsin: the visual pigment of a subset of R8 photoreceptor cells.

By screening retinal cDNA libraries for photoreceptor-specifically expressed genes we have isolated and sequenced a cDNA clone encoding the rhodopsin (Rh6) of a subset of R8 photoreceptor cells of the Drosophila compound eye. Compared to the other visual pigments of Drosophila, this rhodopsin is equally homologous to Rh1 and Rh2 (51% amino acid identity) but shows only 32% and 33% amino acid identity with Rh3 and Rh4, respectively. The open reading frame codes for a protein of 369 amino acids (MW = 41691). The primary structure of Rh6 displays sites typical for rhodopsin molecules in general, for example, a chromophore binding site in transmembrane domain VII, sequence motifs in the intracellular loops 2 and 3 required for the binding of a heterotrimeric G-protein, and a glycosylation site near the N-terminus which seems to be important for protein transport and maturation. Since R8 cells are founder cells in the developing compound eye, the isolation of a rhodopsin gene expressed in these cells may aid the understanding of terminal differentiation of photoreceptor cells.

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.

Isolation of genes encoding photoreceptor-specific proteins by immunoscreening with antibodies directed against purified blowfly rhabdoms.

The proteins which perform and regulate key steps in phototransduction are assumed to be localized in the rhabdomeric membrane of invertebrate photoreceptor cells. We have employed antibodies raised against rhabdoms purified from blowfly eyes in order to isolate copy deoxyribonucleic acid (cDNA) clones encoding proteins that are required in the phototransduction machinery. By immunoscreening a Calliphora retinal cDNA library, we obtained clones of genes coding for five different proteins. As revealed by partial cDNA sequence analysis, three of these genes represent the Calliphora homologs of Drosophila trp, inaC and InaD, while the other two displayed no homology to known genes. Northern blot analysis confirmed that trp, inaC and InaD transcripts were present in RNA isolated from the retina, but not in RNA isolated from brain or thorax. Specific antibodies directed against trp, inaC and InaD protein were raised using recombinantly expressed proteins or synthetic peptides. Western blot analyses revealed that trp, inaC and InaD protein are specifically associated with the rhabdomeral photoreceptor membrane. Extraction of membranes with buffers of different ionic strengths suggested that the trp gene product is an integral membrane protein, whilst the inaC and InaD gene products are peripherally bound membrane proteins. This demonstrates that the immunoscreening approach used here can be successfully applied to isolate genes that code for either integral or peripheral photoreceptor membrane proteins.

Centrin in the photoreceptor cells of mammalian retinae.

Photoreceptor cells of vertebrate retinae are highly specialized ciliary cells. Their non-motile ciliated structure is restricted to the so-called connecting cilium at the joint between the light sensitive outer segment and the metabolically active inner segment. Extensive bidirectional intracellular transport between both segments is forced to occur through this tight connecting cilium. In the present study it is shown that the CA2+-binding, phospho-protein centrin is present in mammalian retinae. Western blot and immunoprecipitation reveal that anti-centrin antibodies react with purified photoreceptor cell fractions of retinae in bands at a molecular weight of 20 kDa, the molecular weight of centrins found in other cells. Indirect immunofluorescence analysis of cryosections through retinae of different mammalian species show that centrin is present only in centrosomes and basal bodies but also more extensively at the linkage between the inner and the outer segment of the photoreceptor cells. Immunocytological studies on isolated rod cells and immunoelectron microscopy clearly demonstrate a unique presence of centrin in the connecting cilium of photoreceptor cells. High molecular identity between centrins in lower eukaryotes and mammals indicates that centrin may play a role in cellular motility and/or in microtubule severing in the mammalian retina.

Opsin maturation and targeting to rhabdomeral photoreceptor membranes requires the retinal chromophore.

The function of the retinal chromophore in the transcription, translation and targeting of opsin was investigated in fly photoreceptor cells R1-6. Carotenoid deprivation and light-dependent depletion of photoreceptor cells in 11-cis (3-OH) retinal reduced not only the rhodopsin content but also the opsin density of the rhabdomeral membrane by as much as 96%. Electron microscopy revealed that rhabdomeral membranes which lack opsin are morphologically intact, but the cells show a modest proliferation of endoplasmic reticulum and zipper-like differentiations of the plasma membrane adjacent to the microvilli. Opsin mRNA levels, quantified by Northern blot analysis with opsin sense cRNA as an external standard, remain relatively constant (7 x 10(-19) moles opsin mRNA per cell), irrespective of the chromophore-dependent, large changes in the opsin content. Labeling of newly synthesized opsin after injection of eyes with [35S]methionine reveals that opsin mRNA is translated in rhodopsin-depleted cells to the same extent as in rhodopsin-rich cells. Molecular weight changes of metabolically radiolabeled opsin, indicative for the posttranslational processing of the nascent opsin to the mature opsin form, suggest that opsin processing is delayed in chromophore-deprived photoreceptor cells. Time courses of opsin labeling reveal that newly synthesized opsin is degraded faster in chromophore-deprived cells than in cells with a high chromophore supply. These results strongly suggest that the chromophore does not regulate opsin gene transcription, but is required for the processing of opsin and its targeting to the rhabdomeral photoreceptor membranes.

Cytoskeletal elements in arthropod sensilla and mammalian photoreceptors.

Ciliary receptor cells, typified by cilia or modified cilia, are very common in the animal kingdom. In addition to the cytoskeleton of their ciliary processes these receptors possess other specific prominent cytoskeletal elements. Two representative systems are presented: i) scolopidia, mechanosensitive sensilla of various arthropod species; and ii) photoreceptor cells of the retina of the bovine eye. Two cytoskeletal structures are characteristic for arthropod scolopidia: a scolopale typifies the innermost auxiliary cell, and long ciliary rootlets are extending well into the sensory cells. The latter element is also characteristic for the inner segment of the photoreceptor cells in bovine. The scolopale of scolopidia is mainly composed of actin filaments. In the absence of myosin, the uniform polarity of the actin filaments and their association with tropomyosin all indicate a stabilizing role of the filament bundles within the scolopale. This function and a certain elasticity of actin filament bundles may be important during stimulation of the sensilla. The ciliary rootlets of both systems originate at the basal bodies at the ciliary base of the sensory cells and project proximally. These rootlets are composed of longitudinally oriented, fine filaments forming a characteristic regular cross-striation. An alpha-actinin immunoreactivity was detected within the ciliary rootlets of scolopidia. In addition, antibodies to centrin react with the rootlets of both types of receptors. Since centrin is largely responsible for the contraction of the flagellar rootlets in green algae, contraction may also occur in the ciliary rootlets of insect sensilla and vertebrate photoreceptors. In both systems, contraction or relaxation of the ciliary rootlets could serve in sensory transduction or adaptation.