Therefore, we identified levels of tyrosine phosphorylation of FAK and MerTK that usually imply activation of these kinases. that adopted circadian photoreceptor dropping in wild-type RPE. Rhythmic activation of focal adhesion and Mer tyrosine kinases that mediate wild-type retinal phagocytosis was also completely absent in retina. These results demonstrate an essential part for v5 integrin receptors and their downstream signaling pathways in synchronizing retinal phagocytosis. Furthermore, they determine the integrin mouse strain as a new animal model of age-related retinal dysfunction. mice accompanied by build up of RPE lipofuscin, a cardinal feature of RPE ageing and disease. Quantification of POS clearance demonstrates that lack of v5 integrin primarily affects the phagocytic function of the RPE: RPE in vitro fails to phagocytose POS and RPE in vivo completely lacks the characteristic burst of phagocytosis in response to early morning rod dropping. Furthermore, our experiments identify a stringent temporal rules of focal adhesion kinase (FAK) and MerTK activities in the retina that correlates with circadian photoreceptor dropping. Strikingly, these synchronized signaling events are completely abolished in v5-deficient retina. Collectively, these data provide the 1st direct evidence that v5 integrinCdependent signaling is essential for retinal function by controlling RPE phagocytosis. Materials and Methods Animals. Earlier characterization of mice showed that PRIMA-1 these mice are viable and fertile, and they display no obvious morphologic abnormalities (24C26). PRIMA-1 and wild-type mice of the same genetic background (129T2/SvEmsJ; The Jackson Laboratory) were housed under cyclic 12-h light/12-h dark light conditions (light onset at 6.00 h) and fed ad libitum. All methods including animals were authorized by the Weill Medical College Institutional Animal Care and Use Committee. For experiments, mice were killed by CO2 asphyxiation. Lens and cornea were removed from enucleated eyeballs. Refreshing eyecups were processed for microscopy or Western blotting as explained below. Electroretinography. Electroretinograms (ERGs) of five and seven wild-type mice that were precisely age matched were recorded monthly between the age groups of 4 and 12 mo. Mice were dark adapted over night before anesthesia by i.p. injection of 100 mg/kg ketamine and 10 mg/kg xylazine. After topical attention anesthesia (0.5% proparacaine hydrochloride) and pupil dilation (10% phenylephrine hydrochloride and 1% tropicamide), full-field scotopic ERGs were recorded using a custom-made gold wire corneal contact lens electrode scaled to the mouse eye (provided by T. Mittag, Mount Sinai School of Medicine, PRIMA-1 New York, NY) and subdermal research (forehead) and floor (back) electrodes as explained previously (27). A photostimulator mounted inside a reflective dome (Ganzfeld) was used to deliver 10-s white flashes with full intensity adobe flash stimuli of 1 1.5 cd-s/m2 (UTAS-2000; LKC Systems). Neutral denseness filters, ranging from ?2.4 to 0 log neutral denseness filter (log ND) in 0.4 log unit methods were used to decrease light stimuli. Stimuli were presented in order of increasing intensity. At least three independent reactions were generated and offered as means SD for each of the seven intensities tested. A-wave amplitudes were measured from your baseline to the trough of the a-wave. B-wave amplitudes were measured from your trough of the a-wave to the peak of the b-wave. Immunofluorescence and Light Microscopy. 10-m-thick frozen sections from paraformaldehyde-fixed eyecups were prepared and stained with antibodies precisely according to founded methods (18, 28). For methyl green staining, 8-m sections were slice from eyecups fixed in formaldehyde/ethanol/acetic acid and inlayed in paraffin. RGB light microscopy images were acquired using IP lab on a microscope (Axiovert 35; Carl Zeiss MicroImaging, Inc.) having a CCD video camera (SenSys) and recompiled in Photoshop 7.0 (Adobe). Cultured PRIMA-1 cells were fixed in ice-cold methanol and processed as explained previously (29). Antibodies used were 5 integrin monoclonal antibody, polyclonal antibodies to 5 integrin (provided by L.F. Reichardt, University or college of California, San Francisco, San Francisco, CA) and to ZO-1 (Zymed). Secondary antibodies Rabbit Polyclonal to MIPT3 were purchased from Molecular Probes. Wide-field fluorescence images were acquired using MetaMorph (Universal Imaging) on an epifluorescence microscope (model C600; Nikon) with a cooled CCD camera (Princeton) or on a confocal microscopy system (model TSP2; Leica) and recompiled in Photoshop 7.0. Electron Microscopy. Eyecups were fixed in 2.5% glutaraldehyde and 0.2% picric acid in 0.1 M cacodylate buffer, pH 7.3. PRIMA-1 Samples were post-fixed in 1% osmium tetroxide for 1 h, dehydrated in acetone, and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate. Specimens were examined at 80 kV with an electron microscope (model 100 CXII; JEOL). Phagosomes were counted in electron micrographs of 0.5-m retinal cross sections. For each time point and strain, three continuous stretches of RPE/photoreceptors of at least 1 mm in length, each prepared from different eyes, were examined. Structures were counted as phagosomes only if they (a) resided in the RPE cytoplasm and (b) contained lamellar membranes of the same appearance as those of intact photoreceptor.
Therefore, we identified levels of tyrosine phosphorylation of FAK and MerTK that usually imply activation of these kinases
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