Actin filaments and photoreceptor membrane turnover

The shape and turnover of photoreceptor membranes appears to depend on associated actin filaments. In dipterans, the photoreceptor membrane is microvillar. It is turned over by the addition of new membrane at the bases of the microvilli and by subsequent shedding, mostly from the distal ends. Each microvillus contains actin filaments as a component of its cytoskeletal core. Two myosin I-like proteins co-localize with the actin filaments. It is suggested that one of the myosin I-like proteins might be linked to the microvillar membrane. By interacting with the actin filaments, this motor should move the membrane of a microvillus in a distal direction, thus providing a possible mechanism for the turnover of the membrane. A vertebrate photoreceptor cell contains a small cluster of actin filaments in its connecting cilium at the site where new transductive disk membranes are formed. Disruption of the actin filaments perturbs disk morphogenesis. The most likely explanation for this perturbation is that the process of initiating a new disk is inhibited. Conventional myosin (myosin II) is found in the connecting cilium with the same distribution as actin. A simple model is proposed to illustrate how the actin-myosin system of the connecting cilium might function to initiate the morphogenesis of a disk membrane.     

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 disc morphogenesis: The classical evagination model prevails

Vision begins in photoreceptor outer segments with light captured by opsins in continually synthesized disc membranes. The process by which rod photoreceptor discs are formed has been controversial. In this issue, Ding et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201508093) show conclusively that rod discs are formed by plasma membrane evagination.The vertebrate retina contains two types of photoreceptors, rod cells and cone cells, whose outer segments initiate phototransduction under night and daytime conditions, respectively. The outer segments of these cells lack ER, Golgi, and mitochondria and are filled with hundreds to a few thousand flattened membrane organelles, called photoreceptor discs, which are loaded with the molecular machinery of phototransduction. The structural organization of outer segments differs between rods and cones. Although cone outer segments contain “open” discs that are infoldings of the plasma membrane, rod outer segments possess “closed” discs that are completely separated from the plasma membrane.In 1967, in a paper that has been cited nearly 800 times, Richard Young reported the seminal finding that rod and cone outer segments are continually renewed (Young, 1967). Young’s classic experiment was elegantly simple: he injected [³H]methionine into a rat, mouse, and frog and performed autoradiograms of the excised retina on various days after the injection. He observed that the radiolabeled band moved along the outer segment as time after injection increased and ultimately disappeared at the apex of the cell (Fig. 1, republished from Young, 1967). (As Young was at the University of California, Los Angeles, this result was given the memorable moniker of “the UCLA marching band.”) Young’s seminal insight that outer segments are continually rebuilt posed a problem that has challenged photoreceptor cell biologists ever since: How are rod disc membranes initially formed? In this issue, Ding et al. present a compelling resolution to this question. Specifically, their work differentiates between currently competing models to determine whether rod discs are formed by evagination of plasma membrane at the base of the outer segment or by fusion of intracellular vesicles transported to the outer segment.Open in a separate windowFigure 1.Photoreceptor outer segments are continually renewed. Rats were injected with [³H]methionine, and radioautographs of photoreceptor cells were performed on various days after the injection. As time after injection increases (images 2–7), the radiolabel components are displaced from the inner segment along the outer segment toward the apex of the cell, revealing that the outer segment is continually renewed (figure republished from Young, 1967).The classic hypothesis of disc morphogenesis is that they are formed by evagination of basal outer segment plasma membrane (Steinberg et al., 1980). This hypothesis is based largely on evidence that one surface of the most basal discs of rods is open to the extracellular space, as shown by EM (Carter-Dawson and LaVail, 1979; Steinberg et al., 1980), with lipophilic dye fluorescence (Laties et al., 1976), and by analysis of membrane capacitance (Rüppel and Hagins, 1973). In addition, rods and cones might be expected to share a common machinery of disc formation. Because most cone discs are well established by EM, lipophilic dye imaging, and electrophysiology to be continuous with the plasma membrane, nascent rod discs would seem likely to also be part of the plasma membrane. Thus, according to the classic hypothesis, new discs in both photoreceptor types are formed from outgrowths (evaginations) of the plasma membrane at the outer segment base. In both photoreceptor types, discs would begin life with one face exposed to the extracellular space, but at some point after formation, rod discs would pinch off from the outer segment plasma membrane to become self-contained and fully separated from the plasma membrane, whereas cones discs remain open. On the contrary, the vesicle fusion hypothesis postulates that nascent discs are born completely internalized in rods. Photoreceptor outer segments are now understood to be the plus end of a modified primary cilium (Bloodgood, 2009) and are joined to their inner segments by a narrow ciliary tube called the connecting cilium. This realization, combined with evidence of vesicles in the connecting cilium seen in electron micrographs, has been taken to support the model that vesicles are actively transported through the connecting cilium and generate nascent discs by membrane fusion at the base of the outer segment (Chuang et al., 2007, 2015).Ding et al. (2015) addressed these competing hypotheses with two distinct approaches. First, they treated sections of retinas of mice perfused with a membrane-staining mixture of tannic acid and uranyl acetate and performed EM. Because tannic acid penetrates intact membranes poorly, this treatment distinguishes between membranes exposed to the extracellular space and intracellular membrane structures. The researchers found that, like the plasma membrane, a small number of basal rod discs were intensely stained by tannic acid, whereas the staining of fully internalized discs was weak, confirming that newly formed rod discs are open to the extracellular space. Consistently and strikingly, EM analysis also revealed a single basal disc face (approximately five to seven discs north of the most basal disc) that is contiguous with the plasma membrane. Second, Ding et al. (2015) performed EM with an immunogold-tagged antibody raised against an intracellular epitope of peripherin, a protein that plays an essential role in disc stacking (Arikawa et al., 1992; Goldberg, 2006). Quantification of gold particle counts showed that the peripherin antibody closely associated intracellularly with the edges of fully internalized discs but was negligibly associated with the surface of nascent discs identified as facing the extracellular space, suggesting that peripherin redistributes along the rod disc edge upon its separation from the plasma membrane and enclosure into the outer segment. Finally, Ding et al. (2015) performed experiments using the fixation techniques reported by other investigators and demonstrated that artifacts of tissue fixation were responsible for the erroneous interpretation that basal discs are fully internalized and for the evidence supporting the vesicular fusion hypothesis.Other tools, such as superresolution microscopy of living rods stained with lipophilic dyes or fluorescent antibodies raised against epitopes on the extracellular face of the rod plasma membrane, could further test aspects of the evagination model of disc formation. Nonetheless, the work of Ding et al. (2015) unequivocally shows that basal rod discs are open to the extracellular space and provides a new system and conceptual framework for the investigation of the fundamental biological mechanism of plasma membrane evagination. As outer segment discs exhibit a specialized composition of lipids and phototransduction proteins, further work will also focus on how disc lipids and proteins are transported from the inner segment to the basal outer segment. The current hypotheses about such transport include (a) vesicular transport through the connecting cilium followed by fusion with the outer segment plasma membrane; (b) directed transport through the connecting cilium membrane after vesicle fusion at the base of the connecting cilium in the inner segment; and (c) exocytotic release from the inner segment followed by endocytotic capture in the outer segment. As the molecular details of disc formation and specialization become clearer, Richard Young’s “UCLA marching band” (Young, 1967) will continue to have a broad conceptual impact on the cell biology of photoreceptor development and cilia.     

Actin filaments in sensory hairs of inner ear receptor cells

Receptor cells in the ear are excited through the bending of sensory hairs which project in a bundle from their surface. The individual stereocilia of a bundle contain filaments about 5 nm in diameter. The identity of these filaments has been investigated in the crista ampullaris of the frog and guinea pig by a technique of decoration with subfragment-1 of myosin (S-1). After demembranation with Triton X-100 and incubation with S-1, "arrowhead" formation was observed along the filaments of the stereocilia and their rootlets and also along filaments in the cuticular plate inside the receptor cell. The distance between attached S-1 was 35 nm and arrowheads pointed in towards the cell soma. It is concluded that the filaments of stereocilia are composed of actin.     

Actin Turnover Is Required for Myosin-Dependent Mitochondrial Movements in Arabidopsis Root Hairs

Background

Previous studies have shown that plant mitochondrial movements are myosin-based along actin filaments, which undergo continuous turnover by the exchange of actin subunits from existing filaments. Although earlier studies revealed that actin filament dynamics are essential for many functions of the actin cytoskeleton, there are little data connecting actin dynamics and mitochondrial movements.

Methodology/Principal Findings

We addressed the role of actin filament dynamics in the control of mitochondrial movements by treating cells with various pharmaceuticals that affect actin filament assembly and disassembly. Confocal microscopy of Arabidopsis thaliana root hairs expressing GFP-FABD2 as an actin filament reporter showed that mitochondrial distribution was in agreement with the arrangement of actin filaments in root hairs at different developmental stages. Analyses of mitochondrial trajectories and instantaneous velocities immediately following pharmacological perturbation of the cytoskeleton using variable-angle evanescent wave microscopy and/or spinning disk confocal microscopy revealed that mitochondrial velocities were regulated by myosin activity and actin filament dynamics. Furthermore, simultaneous visualization of mitochondria and actin filaments suggested that mitochondrial positioning might involve depolymerization of actin filaments on the surface of mitochondria.

Conclusions/Significance

Base on these results we propose a mechanism for the regulation of mitochondrial speed of movements, positioning, and direction of movements that combines the coordinated activity of myosin and the rate of actin turnover, together with microtubule dynamics, which directs the positioning of actin polymerization events.     

Immunocytochemical localization of opsin in the cell membrane of developing rat retinal photoreceptors

Mature retinal rod photoreceptors sequester opsin in the disk and plasma membranes of the rod outer segment (ROS). Opsin is synthesized in the inner segment and is transferred to the outer segment along the connecting cilium that joins the two compartments. We have investigated early stages of retinal development during which the polarized distribution of opsin is established in the rod photoreceptor cell. Retinas were isolated from newborn rats, 3-21 d old, and incubated with affinity purified biotinyl-sheep anti-bovine opsin followed by avidin- ferritin. At early postnatal ages prior to the development of the ROS, opsin is labeled by antiopsin on the inner segment plasma membrane. At the fifth postnatal day, as ROS formation begins opsin was detected on the connecting cilium plasma membrane. However, the labeling density of the ciliary plasma membrane was not uniform: the proximal cilium was relatively unlabeled in comparison with the distal cilium and the ROS plasma membrane. In nearly mature rat retinas, opsin was no longer detected on the inner segment plasma membrane. A similar polarized distribution of opsin was also observed in adult human rod photoreceptor cells labeled with the same antibodies. These results suggest that some component(s) of the connecting cilium and its plasma membrane may participate in establishing and maintaining the polarized distribution of opsin.     

Membranes of retinal pigment epithelial cells in vitro are damaged in the phagocytotic process of the photoreceptor outer segment discs peroxidized by ferrous ions

The ferrous ions released from haemoglobin and storage-transferrin ions cause oxidative stress in the eyes. We observed the phagocytotic process of the photoreceptor outer segment discs peroxidized by ferrous ions in the retinal pigment epithelial (RPE) cells in vitro, and investigated how the ferrous ions influenced RPE in vitro and the photoreceptor outer segment discs. We obtained isolated photoreceptor outer segment discs using sucrose gradient of specific gravity after homogenizing porcine retinas. After bovine RPE cells were cultured with isolated photoreceptor outer segment discs containing FeCl2 for 5 and 24 h, we incubated the specimens with rhodamine phalloidin, antimouse alpha-tubulin antibody and antimouse Ig G (FITC and rhodamine labelled). We observed the specimens by a laser scanning microscopy, and made the ultrathin sections with or without 2% uranyl acetate and 2% lead acetate for examination by transmission electron microscopy. Actin filaments and microtubules of specialized cells such as RPE cells were actively involved in phagocytosis of the photoreceptor outer segment discs. Microtubules were damaged during the phagocytotic process of the photoreceptor outer segment discs peroxidized by ferrous ions. The peroxidation increased the granular and aggregated autofluorescence of the photoreceptor outer segment discs. The membranes of the disc and the phagosomes, and lysosomes in RPE cells were damaged by ferrous ions and had fine particles with high electron density staining without uranium acetate and lead citrate. The cytoskeletons such as actin filaments and microtubules, and the membranes of the phagosomes and the lysosomes in RPE cells in vitro were damaged during the phagocytotic process of the photoreceptor outer segment discs peroxidized by ferrous ions.     

Actin filaments in microridges of the oral mucosal epithelium in the carp Cyprinus carpio

Summary Actin filaments in the microridges on the surface of the fish oral mucosa taken from Cyprinus carpio were examined by electron microscopy after detergent extraction and decoration with myosin subfragment 1. After extraction with saponin, an irregular and densely packed meshwork of actin filaments was observed in the bases of the microridges, just lateral to the tight junctions with their fibrous undercoats. Actin filaments formed cores in the microridges and numerous linkages were seen between the filaments and the plasma membrane. Extraction with Triton X-100 and decoration with myosin subfragment 1 showed the ends of the actin filaments to be associated with the plasma membrane of the microridges, and in the bases of microridges the filament ends were anchored to intermediate filaments. Some actin filaments interconnected with the fibrous undercoats of the tight junctions. On the basis of these observations, the mechanism of the formation of microridges, including their pattern, is discussed.     

The connecting cilium inner scaffold provides a structural foundation that protects against retinal degeneration

Inherited retinal degeneration due to loss of photoreceptor cells is a leading cause of human blindness. These cells possess a photosensitive outer segment linked to the cell body through the connecting cilium (CC). While structural defects of the CC have been associated with retinal degeneration, its nanoscale molecular composition, assembly, and function are barely known. Here, using expansion microscopy and electron microscopy, we reveal the molecular architecture of the CC and demonstrate that microtubules are linked together by a CC inner scaffold containing POC5, CENTRIN, and FAM161A. Dissecting CC inner scaffold assembly during photoreceptor development in mouse revealed that it acts as a structural zipper, progressively bridging microtubule doublets and straightening the CC. Furthermore, we show that Fam161a disruption in mouse leads to specific CC inner scaffold loss and triggers microtubule doublet spreading, prior to outer segment collapse and photoreceptor degeneration, suggesting a molecular mechanism for a subtype of retinitis pigmentosa.

Inherited retinal degeneration due to loss of photoreceptor cells is a leading cause of human blindness. Ultrastructure expansion microscopy on mouse retina reveals the presence of a novel structure inside the photoreceptor connecting cilium, the inner scaffold, that protects the outer segment against degeneration.     

Identification of actin filaments in the rhabdomeral microvilli of Drosophila photoreceptors

The phototransductive microvilli of arthropod photoreceptors each contain an axial cytoskeleton. The present study shows that actin filaments are a component of this cytoskeleton in Drosophila. Firstly, actin was detected in the rhabdomeral microvilli and in the subrhabdomeral cytoplasm by immunogold labeling with antiactin. Secondly, the rhabdomeres were labeled with phalloidin, indicating the presence of filamentous actin. Finally, the actin filaments were decorated with myosin subfragment-1. The characteristic arrowhead complex formed by subfragment-1 decoration points towards the base of the microvilli, so that the fast growing end of each filament is at the distal end of the microvillus, where it is embedded in a detergent-resistant cap. Each microvillus contains more than one actin filament. Decorated filaments extend the entire length of each microvillus and project into the subrhabdomeral cytoplasm. This organization is comparable to that of the actin filaments in intestinal brush border microvilli. Similar observations were made with the photoreceptor microvilli of the crayfish, Procambarus. Our results provide an indication as to how any myosin that is associated with the rhabdomeres might function.     

Dependence of myoblast fusion on a cortical actin wall and nonmuscle myosin IIA

Cell-cell fusion is a fundamental cellular process that is essential for development as well as fertilization. Myoblast fusion to form multinucleated skeletal muscle myotubes is a well studied, yet incompletely understood example of cell-cell fusion that is essential for formation of contractile skeletal muscle tissue. Studies in this report identify several novel cytoskeletal events essential to an early phase of myoblast fusion among cultured murine myoblasts. During myoblast pairing and alignment, cortical actin filaments organize into a dense actin wall structure that parallels and extends the length of the plasma membrane of the bipolar, aligned cells. As fusion progresses, gaps appear within the actin wall at sites of vesicle accumulation, the vesicles pair across the aligned myoblasts, cell-cell contacts and fusion pores form. Inhibition of nonmuscle myosin IIA (NM-MHC-IIA) motor activity prevents formation of this cortical actin wall, as well as the appearance of vesicles at a membrane proximal location, and myoblast fusion. These results suggest that early formation of a subplasmalemmal actin wall during myoblast alignment is a critical event for myoblast fusion that supports bipolar membrane alignment and temporally regulates trafficking of vesicles to the nascent fusion sites during skeletal muscle myoblast differentiation.     

Correlative immuno-electron-microscopic and immunofluorescent localization of actin in sensory and supporting cells of the inner ear by use of a low-temperature embedding resin

Summary The cochleas from chinchilla inner ears were processed in the cold through Lowicryl K4M, and cured by UV light. Thick (2 m) sections were reacted with primary antibodies raised against actin, and anti-actin antibodies localized by FITC epifluorescence. On thin sections from the same blocks anti-actin antibodies were localized ultrastructurally with secondary antibodies coupled to colloidal gold.In the hair cells, actin was present in the stereocilia and cuticular plate, regions where thin filaments were observed by electron microscopy. Colloidal gold was uniformly distributed over these regions and over the stereocilia rootlets demonstrating that actin was present in this region although previously in permeabilized cells, the rootlet was not decorated with myosin subfragment S-1. Actin was present in the pillar and Deiters supporting cells at the reticular lamina and at the basilar membrane, where a meshwork of thin filaments was seen by electron microscopy. Colloidal gold particles were also localized over the thin processes of the pillar and Deiters cells, and over the region of the Deiters cell which envelops the base of the outer hair cell. In these regions actin co-localized with microtubules along the entire length of the supporting cells.     

The Fine Structure of the Retina Studied with the Electron Microscope : IV. Morphogenesis of Outer Segments of Retinal Rods

The morphogenesis of the outer segments of retinal rods was studied mainly in the kitten before the opening of the eye, and the probable sequence of the morphogenetic stages is deduced. Since the development of retinal rods is not synchronous, the deductions were based on observations of many single and serial sections. One centriole extends ciliary tubules of about 0.5 µ long, in the growing primitive cilium. Beyond this length, each ciliary tubule becomes a row of small vesicles (called "ciliary vesicles" in this paper), which penetrate into the distal region of the cilium. Where the ciliary vesicles establish contact with the plasma membrane of the distal region of the cilium, more or less deep infoldings of the plasma membrane are observed. In the distal region can be seen rows of tubular or vesicular structures. A few of these membranous structures are continuous with the bottoms of the infoldings. At the following stage, the infoldings disappear and the ciliary vesicles lose contact with the distal plasma membrane. Nonetheless, the formation of the tubular structures continues in the distal region of the primitive outer segment. The tubular structures appear to be transformed into the primitive rod sacs by sidewise enlargement. At a subsequent time, presumably, these primitive rod sacs flatten and are rearranged into a position perpendicular to the long axis of the outer segment. The detailed structure of the basal body of the connecting cilium was also studied by means of serial sections.     

Submembrane cytoskeleton of pigmented glial cells,primary pigment cells and crystalline cone cells in the honeybee compound eye

Summary The organization of the submembrane cytoskeleton of non-photoreceptive, accessory cells in the honeybee compound eye was examined using light-microscopic (phallotoxin labeling, immunohistochemistry) and electron-microscopic (decoration with myosin fragments) techniques. The crystalline cone cells contain numerous peripheral actin filaments oriented longitudinally with antiparallel polarity. Bundles of microtubules lie under the plasma membrane of primary pigment cells, in close apposition to the crystalline cone; they are interspersed with only a few actin filaments. Pigmented glial cells (secondary pigment cells) contain a two-dimensional filament/particle web lining their entire plasma membranes. Both filamentous actin and -spectrin are localized within the cortex of these cells, indicating that they are web components. The results demonstrate that the three cell types contain different cortical cytoskeletons, implying different functional properties.     

Cytoplasmic actin in neuronal processes as a possible mediator of synaptic plasticity

We have demonstrated that, after permeation with saponin and decoration with S-1 myosin subfragment, the cytoplasmic actin is organized in filaments in dendritic spines, dendrites, and axon terminals of the dentate molecular layer. The filaments are associated with the plasma membrane and the postsynaptic density with their barbed ends and also in parallel with periodical cross bridges. In the spine stalks and dendrites, the actin filaments are organized in long strands. Given the contractile properties of actin, these results suggest that the cytoplasmic actin may be involved in various forms of experimentally induced synaptic plasticity by changing the shape or volume of the pre- and postsynaptic side and by retracting and sprouting synapses.     

Electron microscope observations on the submicroscopic organization of the retinal rods

The submicroscopic organization of the retinal rods of the rabbit has been studied with high resolution electron microscopy in thin longitudinal and cross-sections. The outer rod segment consists of a stack of flattened sacs or cisternae each of them limited by a thin homogeneous membrane of about 30 A. The membrane of the rod sacs is attached to the surface membrane and is also in continuity with short tubular stalks of about 100 to 150 A which apparently end in relation with the connecting cilium. The bundle of filaments that constitute the connection between the outer and the inner segments is described under the name of connecting cilium. This fibrous component has a structure that is very similar to that of the cilium. It shows 9 pairs of peripheral filaments of about 160 A in diameter, a matrix material, and a surface membrane. Very infrequently two central single filaments are observed. The connecting cilium has a typical basal body in the inner segment; its distal end penetrates the outer segment, where it establishes some structural relation to the rod sacs. The relationships and submicroscopic organization of the connecting cilium were studied in longitudinal and in cross-sections passing at different levels of the rod segments. The inner rod segment shows two distinct regions: a distal and a proximal one. The distal region, corresponding to the ellipsoid of classical histology is mainly composed of longitudinally packed mitochondria. It also contains the basal body of the cilium, vacuoles of the endoplasmic reticulum, dense particles, and intervening matrix with very fine filaments. In the proximal region of the inner segment the mitochondria are lacking and within the matrix it is possible to recognize elements of the Golgi complex, vacuoles of the endoplasmic reticulum, dense particles and numerous neuroprotofibrils of 160 to 200 A in diameter which collect and form a definite bundle at the exit of the rod fiber. The interpretation of the connecting fibers as a portion of a cilium and of the outer segment as a differentiation of the distal part of a primitive cilium are discussed. The importance of the continuity of the surface membranes of the outer segment, connecting cilium, and inner segment is emphasized and its possible physiological role is discussed.     

Interactions between actin filaments and between actin filaments and membranes in quick-frozen and deeply etched hair cells of the chick ear

Replicas of the apical surface of hair cells of the inner ear (vestibular organ) were examined after quick freezing and rotary shadowing. With this technique we illustrate two previously undescribed ways in which the actin filaments in the stereocilia and in the cuticular plate are attached to the plasma membrane. First, in each stereocilium there are threadlike connectors running from the actin filament bundle to the limiting membrane. Second, many of the actin filaments in the cuticular plate are connected to the apical cell membrane by tiny branched connecting units like a "crow's foot." Where these "feet" contact the membrane there is a small swelling. These branched "feet" extend mainly from the ends of the actin filaments but some connect the lateral surfaces of the actin filaments as well. Actin filaments in the cuticular plate are also connected to each other by finer filaments, 3 nm in thickness and 74 +/- 14 nm in length. Interestingly, these 3-nm filaments (which measure 4 nm in replicas) connect actin filaments not only of the same polarity but of opposite polarities as documented by examining replicas of the cuticular plate which had been decorated with subfragment 1 (S1) of myosin. At the apicolateral margins of the cell we find two populations of actin filaments, one just beneath the tight junction as a network, the other at the level of the zonula adherens as a ring. The latter which is quite substantial is composed of actin filaments that run parallel to each other; adjacent filaments often show opposite polarities, as evidenced by S1 decoration. The filaments making up this ring are connected together by the 3-nm connectors. Because of the polarity of the filaments this ring may be a "contractile" ring; the implications of this is discussed.     

A Class VI Unconventional Myosin Is Associated with a Homologue of a Microtubule-binding Protein,Cytoplasmic Linker Protein–170, in Neurons and at the Posterior Pole of Drosophila Embryos

Abstract. Coordination of cellular organization requires the interaction of the cytoskeletal filament systems. Recently, several lines of investigation have suggested that transport of cellular components along both microtubules and actin filaments is important for cellular organization and function. We report here on molecules that may mediate coordination between the actin and microtubule cytoskeletons. We have identified a 195-kD protein that coimmunoprecipitates with a class VI myosin, Drosophila 95F unconventional myosin. Cloning and sequencing of the gene encoding the 195-kD protein reveals that it is the first homologue identified of cytoplasmic linker protein (CLIP)–170, a protein that links endocytic vesicles to microtubules. We have named this protein D-CLIP-190 (the predicted molecular mass is 189 kD) based on its similarity to CLIP-170 and its ability to cosediment with microtubules. The similarity between D-CLIP-190 and CLIP-170 extends throughout the length of the proteins, and they have a number of predicted sequence and structural features in common. 95F myosin and D-CLIP-190 are coexpressed in a number of tissues during embryogenesis in Drosophila. In the axonal processes of neurons, they are colocalized in the same particulate structures, which resemble vesicles. They are also colocalized at the posterior pole of the early embryo, and this localization is dependent on the actin cytoskeleton. The association of a myosin and a homologue of a microtubule-binding protein in the nervous system and at the posterior pole, where both microtubule and actin-dependent processes are known to be important, leads us to speculate that these two proteins may functionally link the actin and microtubule cytoskeletons.Global organization of the cell and the coordination of its physiology requires interaction between different cytoskeletal systems. During interphase, a typical eukaryotic cell has microtubules emanating from the centrosome located near the nucleus, which extend to the periphery of the cell, presumably interacting with the cortical actin filament meshwork. Microtubules during interphase are thought to be mainly required for the organization of the membrane systems (e.g., vesicular traffic and organelle movement). The actin-rich cortex is important for maintaining cell shape and for cellular movement.There is increasing evidence of coordination between the actin and the microtubule cytoskeletons (Langford, 1995; Koonce, 1996). Data from a number of systems suggests that many cell types use a combination of microtubule and actin filament–based transport in vesicle and organelle trafficking. It is well established that microtubules are required for long distance transport of cellular components. In contrast, the actin cytoskeleton is thought to be required for more local traffic. The best evidence for transport along both cytoskeletal systems is in neurons. Vesicles appear to be transported along actin filaments in mammalian growth cones (Evans and Bridgman, 1995). Furthermore, gelsolin, which promotes depolymerization of actin filaments, has been shown to inhibit fast axonal transport in this system (Brady et al., 1984). In extruded squid axoplasm, Kuznetsov et al. (1992) observed what appeared to be the same vesicle moving along microtubules and then, subsequently, along microfilaments. Inhibitor studies provide evidence that mitochondria can move along both actin filaments and microtubules in neurons in vivo (Morris and Hollenbeck, 1995). These data support the idea that actin filament and microtubule-based transport cooperate to achieve proper organization of cellular components.The same phenomenon may be occurring in other cell types. In yeast, the mutant phenotype of the MYO2 gene, which encodes an unconventional myosin, is suppressed by overexpression of a kinesin-related protein. These two proteins are colocalized in regions of active growth where a polarized arrangement of actin plays an important role (Lillie and Brown, 1992, 1994). Microtubules are not normally required for this growth. Thus, the basis for suppression is not completely understood. However, the phenotypic suppression suggests that perhaps microtubule-based transport can substitute for actin filament–based transport, under some conditions. In polarized epithelial cells, Fath et al. (1994) have isolated a population of vesicles containing both myosin and microtubule motors. They speculate that proper transport of vesicles relies on both microtubule and actin filament–based transport.Previously, it has been shown that a class VI unconventional myosin, the Drosophila 95F unconventional myosin, transports particles along actin filaments during the syncytial blastoderm stage of Drosophila embryonic development (Mermall et al., 1994). 95F myosin activity is required for normal embryonic development (Mermall and Miller, 1995). 95F myosin is also associated with particulate structures in other cells of the embryo later in development where it may also be involved in actin-based transport. To investigate further the transport catalyzed by 95F myosin, we have begun studies to identify proteins associated with 95F myosin that might be cargoes or regulators. In this work, we have identified a protein that coimmunoprecipitates with 95F myosin. Sequence analysis reveals that this protein is the Drosophila homologue of cytoplasmic linker protein (CLIP)¹–170. CLIP-170 is believed to function as a linker between endocytic vesicles and microtubules (Pierre et al., 1992). We have named this associated protein D-CLIP-190. Colocalization of 95F myosin and D-CLIP-190 at the subcellular level at several times in development and in cultured embryonic cells provides support for the in vivo association of these two proteins. The association of a myosin and a homologue of a microtubule-binding protein suggests that these two proteins may act to coordinate the interaction between actin filaments and microtubules.     

Meckelin 3 Is Necessary for Photoreceptor Outer Segment Development in Rat Meckel Syndrome

Ciliopathies lead to multiorgan pathologies that include renal cysts, deafness, obesity and retinal degeneration. Retinal photoreceptors have connecting cilia joining the inner and outer segment that are responsible for transport of molecules to develop and maintain the outer segment process. The present study evaluated meckelin (MKS3) expression during outer segment genesis and determined the consequences of mutant meckelin on photoreceptor development and survival in Wistar polycystic kidney disease Wpk/Wpk rat using immunohistochemistry, analysis of cell death and electron microscopy. MKS3 was ubiquitously expressed throughout the retina at postnatal day 10 (P10) and P21. However, in the mature retina, MKS3 expression was restricted to photoreceptors and the retinal ganglion cell layer. At P10, both the wild type and homozygous Wpk mutant retina had all retinal cell types. In contrast, by P21, cells expressing rod- and cone-specific markers were fewer in number and expression of opsins appeared to be abnormally localized to the cell body. Cell death analyses were consistent with the disappearance of photoreceptor-specific markers and showed that the cells were undergoing caspase-dependent cell death. By electron microscopy, P10 photoreceptors showed rudimentary outer segments with an axoneme, but did not develop outer segment discs that were clearly present in the wild type counterpart. At p21 the mutant outer segments appeared much the same as the P10 mutant outer segments with only a short axoneme, while the wild-type controls had developed outer segments with many well-organized discs. We conclude that MKS3 is not important for formation of connecting cilium and rudimentary outer segments, but is critical for the maturation of outer segment processes.     

Biosynthesis and vectorial transport of opsin on vesicles in retinal rod photoreceptors

Retinal rod photoreceptor cells absorb light at one end and establish synaptic contacts on the other. Light sensitivity is conferred by a set of membrane and cytosol proteins that are gathered at one end of the cell to form a specialized organelle, the rod outer segment (ROS). The ROS is composed of rhodopsin-laden, flattened disk-shaped membranes enveloped by the cell's plasma membrane. Rhodopsin is synthesized on elements of the rough endoplasmic reticulum and Golgi apparatus near the nucleus in the inner segment. From this synthetic site, the membrane-bound apoprotein, opsin, is released from the Golgi in the membranes of small vesicles. These vesicles are transported through the cytoplasm of the inner segment until they reach its apical plasma membrane. At that site, opsin-laden vesicles appear to fuse near the base of the connecting cilium that joins the inner and outer segments. This fusion inserts opsin into the plasma membrane of the photoreceptor. Opsin becomes incorporated into the disk membrane by a process of membrane expansion and fusion to form the flattened disks of the outer segment. Within the disks, opsin is highly mobile, and rapidly rotates and traverses the disk surface. Despite its mobility in the outer segment, quantitative electron microscopic, immunocytochemical, and autoradiographic studies of opsin distribution demonstrate that little opsin is detectable in the inner segment plasma membrane, although its bilayer is in continuity with the plasma membrane of the outer segment. The photoreceptor successfully establishes the polarized distribution of its membrane proteins by restricting the redistribution of opsin after vectorially transporting it to one end of the cell on post-Golgi vesicles.