Immunocytochemical evidence that rod-connected horizontal cell axon terminals remodel in response to experimental retinal detachment in the cat.

PURPOSE: Cats have two types of horizontal cell (HC); one is axon-bearing (B-type), the other is axonless (A-type). We have previously described neurite sprouting from HCs in response to experimental retinal detachment. Here we sought to determine whether one or both types elaborate these outgrowths. METHODS: Sections as well as wholemounts of retinas detached for 3, 7 and 28 days together with control retinas were double or triple labeled with antibodies to the calcium binding proteins calretinin and calbindin, to the synaptic vesicle-associated membrane protein 2 (VAMP2), and to the 70 and 200 kDa subunits of the neurofilament protein. Digital immunofluorescence images were collected by both confocal and two-photon microscopy. RESULTS: In control retina, both HC types label with antibodies to calretinin and calbindin D, but only the A-type also intensely labels with the neurofilament protein antibody. After 3, 7 and 28 days of detachment, these staining patterns persist, but there is a moderate upregulation of neurofilament protein in the B-type cell. In the detached retina, HC processes sprout neurites that appear most commonly as a loose array of fine beaded processes rising from the outer plexiform layer (OPL) into the outer nuclear layer (ONL), or, especially at 28 days, as stout unbranching processes that often cross the ONL en route to the subretinal space where some expand and arborize. Both types are strongly calretinin-positive while being somewhat less positive for antibodies to calbindin D and neurofilament protein. Moreover, they all arise from similarly labeled processes in the distal-most domain of the OPL where the narrowly stratified field of axon terminal boutons of the B-type HC normally innervates rod spherules, two to three thousand per cell. Our data indicate that the HC sprouts apparently arise specifically from the axon terminal of the B-type cell since outgrowths were never seen arising from either type of HC perikaryon or from processes identifiable as A-type dendrites. CONCLUSIONS: The data described here point to the specific remodeling of the rod-connected axon terminals of the B-type cell through neurite outgrowth. Rods respond to detachment by withdrawing synaptic terminals from the OPL while cones do not. Those HC outgrowths that terminate within the ONL appear to retain their connection with the retracted terminals. Others apparently have lost their presynaptic targets and cross the ONL in association with hypertrophied Müller cell processes.

A role of histone H3 lysine 4 methyltransferase components in endosomal trafficking.

Histone lysine methyltransferase complexes are essential for chromatin organization and gene regulation. Whether any of this machinery functions in membrane traffic is unknown. In this study, we report that mammal Dpy-30 (mDpy-30), a subunit of several histone H3 lysine 4 (H3K4) methyltransferase (H3K4MT) complexes, resides in the nucleus and at the trans-Golgi network (TGN). The TGN targeting of mDpy-30 is mediated by BIG1, a TGN-localized guanine nucleotide exchange factor for adenosine diphosphate ribosylation factor GTPases. Altering mDpy-30 levels changes the distribution of cation-independent mannose 6-phosphate receptor (CIMPR) without affecting that of TGN46 or transferrin receptor. Our experiments also indicate that mDpy-30 functions in the endosome to TGN transport of CIMPR and that its knockdown results in the enrichment of internalized CIMPR and recycling endosomes near cell protrusions. Much like mDpy-30 depletion, the knockdown of Ash2L or RbBP5, two other H3K4MT subunits, leads to a similar redistribution of CIMPR. Collectively, these results suggest that mDpy-30 and probably H3K4MT play a role in the endosomal transport of specific cargo proteins.

Rho GTPases regulate rhabdom morphology in octopus photoreceptors.

In the cephalopod retina, light/dark adaptation is accompanied by a decrease/increase in rhabdom size and redistribution of rhodopsin and retinochrome. Rearrangements in the actin cytoskeleton probably govern changes in rhabdom size by regulating the degradation/formation of rhabdomere microvilli. Photopigment movements may be directed by microtubules present in the outer segment core cytoplasm. We believe that rhodopsin activation by light stimulates Rho and Rac signaling pathways, affecting these cytoskeletal systems and their possible functions in controlling rhabdom morphology and protein movements. In this study, we localized cytoskeletal and signaling proteins in octopus photoreceptors to determine their concurrence between the lighting conditions. We used toxin B from Clostridium difficile to inhibit the activity of Rho/Rac and observed its effect on the location of signaling proteins and actin and tubulin. In both lighting conditions, we found Rho in specific sets of juxtaposed rhabdomeres in embryonic and adult retinas. In the light, Rho and actin were localized along the length of the rhabdomere, but, in the dark, both proteins were absent from a space beneath the inner limiting membrane. Rac colocalized with tubulin in the outer segment core cytoplasm and, like Rho, the two proteins were also absent beneath the inner limiting membrane in the dark. The distribution of actin and Rho was affected by toxin B and, in dark-adapted retinas, actin and Rho distribution was similar to that observed in the light. Our results suggest that the Rho/Rac GTPases are candidates for the regulation of rhabdomere size and protein movements in light-dark-adapted octopus photoreceptors.

Cytoskeleton participation in subcellular trafficking of signal transduction proteins in rod photoreceptor cells.

Light sensitivity and adaptation, general characteristics of rod photoreceptor cell vision, allow rods to modulate their response depending on the lighting environment to which they are exposed. In dim light, rods are maximally sensitive, whereas, in bright light, rods are essentially inactive. In the retinas of dark-adapted mice, arrestin (an inhibitory protein) is located in the rod inner segment (RIS), and transducin (an activating protein) is located in the rod outer segment (ROS). In light-adapted retinas, the proteins have reciprocal localizations. In this study, our data demonstrate that the temporal and spatial changes in the subcellular localization of arrestin and beta-transducin are correlated with the amount of light to which the animals are exposed. By using the frog Xenopus laevis and immunofluorescence confocal microscopy, our results also show that in the dark-adapted retina some arrestin remains in the ROS. The data most dramatically demonstrate that this residual arrestin is highly concentrated in the connecting cilium, the axoneme, and the microtubules associated with the disc incisures. These data suggest a structure-function relationship between the light-dependent positional status of arrestin and the elements of the rod photoreceptor cytoskeleton. The massive, rapid, light-induced reciprocal changes in the subcellular concentrations of these proteins must directly affect phototransduction and appear to be a general phenomenon by which photoreceptor cells rapidly and transiently regulate the trafficking and subcellular concentration of a variety of signal transduction proteins within the RIS and ROS. Hereditary mutations in the components of the movement mechanism should lead to defects in vision and possibly blindness.

Heat shock protein 70 and heat shock protein 90 expression in light- and dark-adapted adult octopus retinas.

Light- and dark-adaptation leads to changes in rhabdom morphology and photopigment distribution in the octopus retina. Molecular chaperones, including heat shock proteins (Hsps), may be involved in specific signaling pathways that cause changes in photoreceptor actin- and tubulin-based cytoskeletons and movement of the photopigments, rhodopsin and retinochrome. In this study, we used immunoblotting, in situ RT-PCR, immunofluorescence and confocal microscopy to localize the inducible form of Hsp70 and the larger Hsp90 in light- and dark-adapted and dorsal and ventral halves of adult octopus retinas. The Hsps showed differences in distribution between the light and dark and in dorsal vs. ventral position in the retina. Double labeling confocal microscopy co-localized Hsp70 with actin and tubulin, and Hsp90 with the photopigment, retinochrome. Our results demonstrate the presence of Hsp70 and Hsp90 in otherwise non-stressed light- and dark-adapted octopus retinas. These Hsps may help stabilize the cytoskeleton, important for rhabdom structure, and are perhaps involved in the redistribution of retinochrome in conditions of light and dark.