Brian Matsumoto

Rapid changes in the expression of glial cell proteins caused by experimental retinal detachment

We examined the expression of several proteins normally present in Müllers glia after the production of experimental retinal detachment in adult cats. Retinas were detached for one-half to seven days, after which the tissue was processed for correlative immunocytochemistry and biochemistry. Previous studies demonstrated that the intermediate filament proteins glial fibrillary acidic protein and vimentin, increase after long-term retinal detachment (30 to 60 days), whereas glutamine synthetase, carbonic anhydrase C, and cellular retinaldehyde-binding protein all decrease to barely detectable levels. Alterations in Müller cell protein expression are rapid and specific events that can be detected as early as two days after retinal detachment. By seven days, levels of protein expression are similar to those in the long-term retinal detachments. Within the first week after injury the Müller cell processes hypertrophy and begin forming glial scars, which indicates that early intervention may be required to halt or reverse the effects of detachment.

Evidence from normal and degenerating photoreceptors that two outer segment integral membrane proteins have separate transport pathways

Detachment of the neural retina from the retinal pigment epithelium induces photoreceptor degeneration. We studied the effects of this degeneration on the localization of two photoreceptor outer segment‐specific integral membrane proteins, opsin and peripherin/rds, in rod photoreceptors. Results from laser scanning confocal microscopic and electron microscopic immunolocalization demonstrate that these two proteins, normally targeted to the newly‐forming discs of the outer segments, accumulate in different sub‐cellular compartments during photoreceptor degeneration: opsin immunolabeling increases throughout the photoreceptor cells plasma membrane, while peripherin/rds immunolabeling occurs within cytoplasmic vesicles. The simplest hypothesis to explain our results is that these proteins are transported in different post‐Golgi transport vesicles and separately inserted into the plasma membrane. More complex mechanisms involve having the two co‐transported and then opsin finds its way into the plasma membrane but peripherin/rds does not, remaining behind in vesicles. Alternatively, both insert into the plasma membrane but peripherin/rds is recycled into cytoplasmic vesicles. We believe the data most strongly supports the first possibility. Although the transport pathways for these proteins have not been fully characterized, the presence of peripherin/rds‐positive vesicles adjacent to the striated rootlet suggests a transport role for this cytoskeletal element.

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 β‐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.

Resolution of subcellular detail in thick tissue sections: immunohistochemical preparation and fluorescence confocal microscopy.

Publisher Summary This chapter focuses on fluorescence confocal microscopy techniques for resolution of subcellular detail. Gentle procedures for specimen preparation and techniques for removing the out-of-focus fluorescence signal are equally important for maximizing resolution. The chapter illustrates the substitution of optical sectioning with the laser scanning confocal microscope (LSCM) for mechanical sectioning to generate the thin sections necessary for high contrast and resolution. LCSM allows the shallow depth of field of the light microscope to be used effectively. There are two other benefits of confocal microscopy: (1) resin or paraffin embedding is no longer necessary and consequently structural alteration and autofluorescence caused by these procedures is eliminated, and (2) three-dimensional information can be gathered with ease. The combination of immunofluorescence and LSCM makes the goal of viewing cytoskeletal arrays and other subcellular detail in three dimensions in intact tissues not only theoretically possible for the cell biologist, but also feasible.

Ectopic expression of alpha B-crystallin in Chinese hamster ovary cells suggests a nuclear role for this protein.

alpha B-crystallin (alpha B) is known to be a cytosolic, small heat shock-like multimeric protein that has anti-aggregation, chaperone-like properties. The expression of the alpha B-crystallin gene is developmentally regulated and is induced by a variety of stress stimuli. Importantly, alpha B-crystallin expression is enhanced during oncogenic transformation of cells, in a number of tumors, and most notably, in many neurodegenerative disorders, including Alzheimers disease and multiple sclerosis. Other than its perceived role as a structural protein in the ocular lens, the actual function of alpha B-crystallin in cellular physiology remains unknown. We have stably transfected CHO cells with an inducible alpha B-cDNA-MMTV-promoter construct that allows the synthesis of recombinant alpha B-crystallin only upon exposure of these cells to dexamethasone. Using immunostaining and conventional and confocal microscopy, we have examined the subcellular distribution of the ectopically expressed alpha B-crystallin. We find that in addition to being in the cytoplasm, the protein resides in the nuclear interior in the interphase nucleus. Double labeling with anti alpha B-crystallin and anti-tubulin, concanavallin, and wheat germ agglutinin, respectively, revealed that during cell division alpha B-crystallin is excluded from condensed chromatin and the nascent nuclei. However, the protein again appears in the newly formed nuclei after the completion of cytokinesis suggesting a conditional, regulatory role for alpha B-crystallin in the nucleus.

The Actin Network in the Ciliary Stalk of Photoreceptors Functions in the Generation of New Outer Segment Discs

Cytochalasin D (CD) interferes with the morphogenesis of outer segment disc membrane in photoreceptors. Disruption of either the actin network in the ciliary stalk, where membrane evagination is initiated, or the actin core of the calycal processes, whose position could define the disc perimeter, could be responsible. We have attempted to determine which of these local F‐actin populations is involved in membrane morphogenesis and what step in the process is actin‐dependent. Biocytin accumulation in nascent discs, detected by fluorescent avidin and laser scanning confocal microscopy (LSCM), provided a means of labeling abnormal discs and a measure of disc membrane addition. F‐actin content and distribution were assessed using fluorescent phalloidin and LSCM. First, we examined the effects of a range of CD dosages (0.1, 1.0, or 10.0 μM) on rod photoreceptors in Xenopus laevis eyecup cultures. Ectopic outgrowth of discs, evaluated by LSCM and transmission electron microscopy (TEM), occurred at each concentration. Phalloidin labeling intensified in the ciliary stalk with increasing CD concentration, indicating F‐actin aggregation. In contrast, it diminished in the calycal processes, indicating dispersal; TEM showed that calycal process collapse ensued. Disruption was evident at a lower concentration in the ciliary stalk (0.1 μM) than in the calycal processes (1.0 μM). TEM confirmed that the calycal processes remained intact at 0.1 μM. Thus, CDs action on the ciliary stalk network is sufficient to disrupt disc morphogenesis. Second, we examined the effect of CD on temperature‐induced acceleration of the rate of disc formation. In the absence of CD, a 10°C temperature shift increased the disc formation rate nearly three‐fold. CD (5 μM) caused a 94% inhibition (P < 0.025) of this response; yet, the rate of membrane addition to ectopically growing discs exhibited the expected three‐fold increase. Thus, CDs action interferes with the generation of new discs.

Preparation of Retinas for Studying Photoreceptors with Confocal Microscopy

Publisher Summary Laser scanning confocal microscopy (LSCM) is an alternate method available for obtaining the optical equivalent of thin fluorescent sections. The optical principle of confocal microscopy is simple, but a large investment in hardware and software is required. LSCM optically isolates fluorescence emission to the image plane, providing a means of obtaining high-resolution images of photoreceptor cells in thick sections. This chapter describes the procedures and techniques for preparing retinas for confocal microscopy. The chapter also provides a description of the protocol and a detailed paradigm for each procedure. A variety of proteins has been successfully stained with these techniques, including cytoskeletal proteins, integral membrane proteins, and soluble cytoplasmic proteins. In addition, the basic procedure has been applied with lectins as well as fluorescent phalloidins and phallotoxins. The ability to stain a diverse population of retinal proteins with a variety of fluorescent probes demonstrates the general applicability of this technique for localizing any protein within the retina.

Reconstitution of the photoreceptor-pigment epithelium interface: L-glutamate stimulation of adhesive interactions and rod disc shedding after recombination of dissociated Xenopus laevis eyecups.

In order to investigate adhesive interactions between photoreceptor and pigment epithelial cells, we have mechanically separated neural retinas from Xenopus laevis eyecups and then recombined the tissues in vitro. When tissue pairs are incubated in a defined medium, cell-cell contact is achieved within 3 hr. However, the average proportion of reassembled eyecups in which photoreceptor outer segments interdigitate with epithelial microvilli is limited. Furthermore, rod disc shedding does not take place in these cultures, even following a dark to light transition. When recombined tissues are placed in medium supplemented with 12 mM L-glutamate, retinal reattachment is enhanced and there is a four-fold increase in epithelial phagosome content. The positive effect of excitatory amino acid exposure on shedding, however, is restricted to regions where visual and epithelial cells interdigitate. These results indicate that re-establishment of cell contact may be necessary for shedding of apical disc membranes prior to their engulfment by the epithelium. While reattachment is not affected by pre-incubation of separated tissues in normal medium, rod photoreceptors fail to undergo membrane turnover in response to L-glutamate if a delay of 1 hr or more is interposed between isolation of the retina and its recombination with the pigment epithelium. This is probably due to a decline in retinal function in culture, since a similar preincubation of the pigment epithelium prior to reassembly with a freshly isolated retina does not inhibit the shedding response.(ABSTRACT TRUNCATED AT 250 WORDS)

Light-/dark-induced changes in rhabdom structure in the retina of Octopus bimaculoides

Abstract. We examined rhabdom structure and the distribution of filamentous actin in the photoreceptor outer segments of the retina of Octopus bimaculoides. Animals were dark- or light-adapted, fixed, embedded and sectioned for light and electron microscopy. Statistical analyses were used to compare relative cross-sectional areas of rhabdom microvilli and core cytoplasm within and between the two lighting conditions. Dark-/light-adapted rhabdoms were also prepared for confocal laser scanning microscopy and labeled with fluorescence-tagged phalloidin. Results show differences in the morphology of dark- and light-adapted octopus rhabdoms with the cross-sectional areas of the rhabdoms increasing in dark-adapted retinas and diminishing in the light. Comparisons between the lighting conditions show that an avillar portion of the photoreceptor outer segment membrane, prominent in the light-adapted retina, is recruited to form new rhabdomere microvilli in dark-adapted eyes. Filamentous actin was associated with the avillar membrane in light-adapted retinas, which may indicate that actin and other microvillus core proteins remain linked to the avillar membrane to support rapid microvillus formation in the dark. Photopigment redistributions also occur in light- and dark-adapted retinas, and this study suggests that these changes must be coordinated with the simultaneous breakdown and reformation of the rhabdomere microvilli.

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.