Diurnal, localized exposure of phosphatidylserine by rod outer segment tips in wild-type but not Itgb5-/- or Mfge8-/- mouse retina.

In the mammalian retina, life-long renewal of light-sensitive photoreceptor outer segments (POS) involves circadian shedding of distal rod POS tips and their subsequent phagocytosis by the adjacent retinal pigment epithelium (RPE) every morning after light onset. Molecular mechanisms that promote or synchronize POS tip shedding have thus far remained unknown. Here we examined plasma membrane asymmetry of living POS by quantifying surface exposure of the membrane phospholipid phosphatidylserine (PS) using antibodies, annexin V, and pSIVA (polarity-sensitive indicator of viability and apoptosis), an annexin-based biosensor with switchable states of fluorescence. We found that isolated POS particles possess externalized PS, whose blockade or removal reduces their binding and engulfment by RPE in culture. Imaging of live photoreceptors in freshly dissected mouse retina detected PS externalization restricted to POS tips with discrete boundaries. In wild-type mice, frequency of rod tips exposing PS and length of tips with exposed PS peak shortly after light onset. In contrast, PS-marked POS tips do not vary in mice lacking the diurnal phagocytic rhythm of the RPE due to loss of either the phagocytosis receptor αvß5 integrin, expressed by the RPE but not by photoreceptors, or its extracellular ligand milk fat globule-EGF factor 8 (MFG-E8). These data identify a molecular distinction, localized PS exposure, that is specific to the surface of rod POS tips. Enhanced PS exposure preceding rod shedding and phagocytosis suggests that surface PS promotes these processes. Moreover, our results demonstrate that the diurnal rhythm of PS demarcation of POS tips is not intrinsic to rod photoreceptors but requires activities of the RPE as well.

Lack of effect of microfilament or microtubule cytoskeleton-disrupting agents on restriction of externalized phosphatidylserine to rod photoreceptor outer segment tips.

In the mammalian retina, life-long renewal of rod photoreceptor outer segments involves circadian shedding of distal outer segment tips and their prompt phagocytosis by the adjacent retinal pigment epithelium (RPE) every morning after light onset. Failure of this process causes retinal dystrophy in animal models and its decline likely contributes to retinal aging and some forms of degeneration of the human retina. We previously found that surface exposure of the membrane phospholipid phosphatidylserine (PS) is restricted to outer segment tips with discrete boundaries in mouse retina and that both frequency and length of tips exposing PS peak after light onset. Here, we sought to test mechanisms photoreceptors use to restrict PS specifically to their outer segment tips. To this end, we tested whether nocodazole or cytochalasin D, perturbing microtubule or F-actin microfilament cytoskeleton, respectively, affect localization of externalized PS at outer segment tips. Fluorescence imaging of PS exposed by rods in freshly dissected, live mouse retina showed normal PS demarcation of outer segment tips regardless of drug treatment. These results suggest that the mechanism that restricts externalized PS to rod tips is independent of F-actin and microtubule cytoskeletal systems.

Mice with early retinal degeneration show differences in neuropeptide expression in the suprachiasmatic nucleus.

BACKGROUND: In mammals, the brain clock responsible for generating circadian rhythms is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Light entrainment of the clock occurs through intrinsically photosensitive retinal ganglion cells (ipRGCs) whose axons project to the SCN via the retinohypothalamic tract. Although ipRGCs are sufficient for photoentrainment, rod and cone photoreceptors also contribute. Adult CBA/J mice, which exhibit loss of rod and cone photoreceptors during early postnatal development, have greater numbers of ipRGCs compared to CBA/N control mice. A greater number of photosensitive cells might argue for enhanced light responses, however, these mice exhibit attenuated phase shifting behaviors. To reconcile these findings, we looked for potential differences in SCN neurons of CBA/J mice that might underly the altered circadian behaviors. We hypothesized that CBA/J mice have differences in the expression of neuropeptides in the SCN, where ipRGCs synapse. The neuropeptides vasoactive intestinal peptide (VIP) and vasopressin (VP) are expressed by many SCN neurons and play an important role in the generation of circadian rhythms and photic entrainment. METHODS: Using immunohistochemistry, we looked for differences in the expression of VIP and VP in the SCN of CBA/J mice, and using a light-induced FOS assay, we also examined the degree of retinal innervation of the SCN by ipRGCs. RESULTS: Our data demonstrate greater numbers of VIP-and VP-positive cells in the SCN of CBA/J mice and a greater degree of light-induced FOS expression. CONCLUSIONS: These results implicate changes in neuropeptide expression in the SCN which may underlie the altered circadian responses to light in these animals.

The development of melanopsin-containing retinal ganglion cells in mice with early retinal degeneration.

In mammals, the neuronal pathways by which rod and cone photoreceptors mediate vision have been well documented. The roles that classical photoreceptors play in photoentrainment, however, have been less clear. In mammals, intrinsically photosensitive retinal ganglion cells (ipRGCs) that express the photopigment melanopsin project directly to the suprachiasmatic nucleus of the hypothalamus, the site of the circadian clock, and thereby contribute to non-image-forming responses to light. Classical photoreceptors are not necessary for photoentrainment as loss of rods and cones does not eliminate light entrainment. Conflicting evidence arose, however, when attenuated phase-shifting responses were observed in the retinal-degenerate CBA/J mouse. In this study, we examined the time course of retinal degeneration in CBA/J mice and used these animals to determine if maturation of the outer retina regulates the morphology, number and distribution of ipRGCs. We also examined whether degeneration during the early development of the outer retina can alter the function of the adult circadian system. We report that dendritic stratification and distribution of ipRGCs was unaltered in mice with early retinal degeneration, suggesting that normal development of the outer retina was not necessary for these processes. We found, however, that adult CBA/J mice have greater numbers of ipRGCs than controls, implicating a role for the outer retinal photoreceptors in regulating developmental cell death of ipRGCs.

Long continuous actin bundles in Drosophila bristles are constructed by overlapping short filaments.

The actin bundles essential for Drosophila bristle elongation are hundreds of microns long and composed of cross-linked unipolar filaments. These long bundles are built from much shorter modules that graft together. Using both confocal and electron microscopy, we demonstrate that newly synthesized modules are short (1-2 microm in length); modules elongate to approximately 3 microm by growing over the surface of longitudinally adjacent modules to form a graft; the grafted regions are initially secured by the forked protein cross-bridge and later by the fascin cross-bridge; actin bundles are smoothed by filament addition and appear continuous and without swellings; and in the absence of grafting, dramatic alterations in cell shape occur that substitutes cell width expansion for elongation. Thus, bundle morphogenesis has several components: module formation, elongation, grafting, and bundle smoothing. These actin bundles are much like a rope or cable, made by overlapping elements that run a small fraction of the overall length, and stiffened by cross-linking.

Actin filament bundles in Drosophila wing hairs: hairs and bristles use different strategies for assembly.

Actin filament bundles can shape cellular extensions into dramatically different forms. We examined cytoskeleton formation during wing hair morphogenesis using both confocal and electron microscopy. Hairs elongate with linear kinetics (approximately 1 microm/h) over the course of approximately 18 h. The resulting structure is vividly asymmetric and shaped like a rose thorn--elongated in the distal direction, curved in two dimensions with an oval base and a round tip. High-resolution analysis shows that the cytoskeleton forms from microvilli-like pimples that project actin filaments into the cytoplasm. These filaments become cross-linked into bundles by the sequential use of three cross-bridges: villin, forked and fascin. Genetic loss of each cross-bridge affects cell shape. Filament bundles associate together, with no lateral membrane attachments, into a cone of overlapping bundles that matures into an oval base by the asymmetric addition of bundles on the distal side. In contrast, the long bristle cell extension is supported by equally long (up to 400 microm) filament bundles assembled together by end-to-end grafting of shorter modules. Thus, bristle and hair cells use microvilli and cross-bridges to generate the common raw material of actin filament bundles but employ different strategies to assemble these into vastly different shapes.

The role actin filaments play in providing the characteristic curved form of Drosophila bristles.

Drosophila bristles display a precise orientation and curvature. An asymmetric extension of the socket cell overlies the newly emerging bristle rudiment to provide direction for bristle elongation, a process thought to be orchestrated by the nerve dendrite lying between these cells. Scanning electron microscopic analysis of individual bristles showed that curvature is planar and far greater near the bristle base. Correlated with this, as development proceeds the pupa gradually recedes from the inner pupal case (an extracellular layer that encloses the pupa) leading to less bristle curvature along the shaft. We propose that the inner pupal case induces elongating bristles to bend when they contact this barrier. During elongation the actin cytoskeleton locks in this curvature by grafting together the overlapping modules that comprise the long filament bundles. Because the bristle is curved, the actin bundles on the superior side must be longer than those on the inferior side. This is accomplished during grafting by greater elongation of superior side modules. Poor actin cross-bridging in mutant bristles results in altered curvature. Thus, the pattern of bristle curvature is a product of both extrinsic factors-the socket cell and the inner pupal case--and intrinsic factors--actin cytoskeleton assembly.

Actin filament turnover regulated by cross-linking accounts for the size, shape, location, and number of actin bundles in Drosophila bristles.

Drosophila bristle cells are shaped during growth by longitudinal bundles of cross-linked actin filaments attached to the plasma membrane. We used confocal and electron microscopy to examine actin bundle structure and found that during bristle elongation, snarls of uncross-linked actin filaments and small internal bundles also form in the shaft cytoplasm only to disappear within 4 min. Thus, formation and later removal of actin filaments are prominent features of growing bristles. These transient snarls and internal bundles can be stabilized by culturing elongating bristles with jasplakinolide, a membrane-permeant inhibitor of actin filament depolymerization, resulting in enormous numbers of internal bundles and uncross-linked filaments. Examination of bundle disassembly in mutant bristles shows that plasma membrane association and cross-bridging adjacent actin filaments together inhibits depolymerization. Thus, highly cross-bridged and membrane-bound actin filaments turn over slowly and persist, whereas poorly cross-linked filaments turnover more rapidly. We argue that the selection of stable bundles relative to poorly cross-bridged filaments can account for the size, shape, number, and location of the longitudinal actin bundles in bristles. As a result, filament turnover plays an important role in regulating cytoskeleton assembly and consequently cell shape.