Ciliary Plasma Membrane Research Articles

A Ciliary Branched Actin Network Drives Photoreceptor Disc Morphogenesis.

The light-detecting organelle of the photoreceptor cell is a modified primary cilium, called the outer segment. The outer segment houses hundreds of light-sensitive membrane, "discs," that are continuously renewed by the constant formation of new discs at the outer segment base and the phagocytosis of old ones from outer segment tips by the retinal pigment epithelium. In this chapter, we describe how an actin cytoskeleton network, residing precisely at the site of disc formation, provides the driving force that pushes out the ciliary plasma membrane to form each disc evagination that subsequently can mature into a bona fide disc. We highlight the functions of actin-binding proteins, particularly PCARE and Arp2/3, that are known to participate in disc formation. Finally, we describe a working model of disc formation built upon the many studies focusing on the role of actin during disc morphogenesis.

The route of the visual receptor rhodopsin along the cilium.

The photoreceptor outer segment is the most elaborate primary cilium, containing large amounts of rhodopsin (RHO) in disk membranes that grow from a connecting cilium. The movement of RHO along the connecting cilium precedes formation of the disk membranes. However, the route that RHO takes has not been clearly determined; some reports suggest that it follows an intracellular, vesicular route along the axoneme, possibly as an adaptation for the high load of delivery or the morphogenesis of the disk endomembranes. We addressed this question by studying RHO in cilia of IMCD3 cells and mouse rod photoreceptors. In IMCD3 cilia, fluorescence recovery after photobleaching (FRAP) experiments with fluorescently tagged RHO supported the idea of RHO motility in the ciliary plasma membrane and was inconsistent with the hypothesis of RHO motility within the lumen of the cilium. In rod photoreceptors, FRAP of RHO-EGFP was altered by externally applied lectin, supporting the idea of plasmalemmal RHO dynamics. Quantitative immunoelectron microscopy corroborated our live-cell conclusions, as RHO was found to be distributed along the plasma membrane of the connecting cilium, with negligible labeling within the axoneme. Taken together, the present findings demonstrate RHO trafficking entirely via the ciliary plasma membrane.This article has an associated First Person interview with the first author of the paper.

Peripherin diverts ciliary ectosome release to photoreceptor disc morphogenesis.

Formation of membrane discs in photoreceptor cells requires evagination of its ciliary plasma membrane by an unknown molecular mechanism. Salinas et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201608081) show that peripherin (also known as peripherin-2 or peripherin-2/rds) diverts membrane traffic to photoreceptor disc formation by inhibiting ectosome release from the cilium.

Rod disc renewal occurs by evagination of the ciliary plasma membrane that makes cadherin-based contacts with the inner segment

The outer segments of vertebrate rod photoreceptors are renewed every 10 d. Outer segment components are transported from the site of synthesis in the inner segment through the connecting cilium, followed by assembly of the highly ordered discs. Two models of assembly of discrete discs involving either successive fusion events between intracellular rhodopsin-bearing vesicles or the evagination of the plasma membrane followed by fusion of adjacent evaginations have been proposed. Here we use immuno-electron microscopy and electron tomography to show that rhodopsin is transported from the inner to the outer segment via the ciliary plasma membrane, subsequently forming successive evaginations that "zipper" up proximally, but at their leading edges are free to make junctions containing the protocadherin, PCDH21, with the inner segment plasma membrane. Given the physical dimensions of the evaginations, coupled with likely instability of the membrane cortex at the distal end of the connecting cilium, we propose that the evagination occurs via a process akin to blebbing and is not driven by actin polymerization. Disassembly of these junctions is accompanied by fusion of the leading edges of successive evaginations to form discrete discs. This fusion is topologically different to that mediated by the membrane fusion proteins, SNAREs, as initial fusion is between exoplasmic leaflets, and is accompanied by gain of the tetraspanin rim protein, peripherin.

Three-dimensional organization of nascent rod outer segment disk membranes

The vertebrate photoreceptor cell contains an elaborate cilium that includes a stack of phototransductive membrane disks. The disk membranes are continually renewed, but how new disks are formed remains poorly understood. Here we used electron microscope tomography to obtain 3D visualization of the nascent disks of rod photoreceptors in three mammalian species, to gain insight into the process of disk morphogenesis. We observed that nascent disks are invariably continuous with the ciliary plasma membrane, although, owing to partial enclosure, they can appear to be internal in 2D profiles. Tomographic analyses of the basal-most region of the outer segment show changes in shape of the ciliary plasma membrane indicating an invagination, which is likely a first step in disk formation. The invagination flattens to create the proximal surface of an evaginating lamella, as well as membrane protrusions that extend between adjacent lamellae, thereby initiating a disk rim. Immediately distal to this initiation site, lamellae of increasing diameter are evident, indicating growth outward from the cilium. In agreement with a previous model, our data indicate that mature disks are formed once lamellae reach full diameter, and the growth of a rim encloses the space between adjacent surfaces of two lamellae. This study provides 3D data of nascent and mature rod photoreceptor disk membranes at unprecedented z-axis depth and resolution, and provides a basis for addressing fundamental questions, ranging from protein sorting in the photoreceptor cilium to photoreceptor electrophysiology.

Submembrane assembly and renewal of rod photoreceptor cGMP-gated channel: insight into the actin-dependent process of outer segment morphogenesis.

The photoreceptor outer segment (OS) is comprised of two compartments: plasma membrane (PM) and disk membranes. It is unknown how the PM renewal is coordinated with that of the disk membranes. Here we visualized the localization and trafficking process of rod cyclic nucleotide-gated channel α-subunit (CNGA1), a PM component essential for phototransduction. The localization was visualized by fusing CNGA1 to a fluorescent protein Dendra2 and expressing in Xenopus laevis rod photoreceptors. Dendra2 allowed us to label CNGA1 in a spatiotemporal manner and therefore discriminate between old and newly trafficked CNGA1-Dendra2 in the OS PM. Newly synthesized CNGA1 was preferentially trafficked to the basal region of the lateral OS PM where newly formed and matured disks are also added. Unique trafficking pattern and diffusion barrier excluded CNGA1 from the PM domains, which are the proposed site of disk membrane maturation. Such distinct compartmentalization allows the confinement of cyclic nucleotide-gated channel in the PM, while preventing the disk membrane incorporation. Cytochalasin D and latrunculin A treatments, which are known to disrupt F-actin-dependent disk membrane morphogenesis, prevented the entrance of newly synthesized CNGA1 to the OS PM, but did not prevent the entrance of rhodopsin and peripherin/rds to the membrane evaginations believed to be disk membrane precursors. Uptake of rhodopsin and peripherin/rds coincided with the overgrowth of the evaginations at the base of the OS. Thus F-actin is essential for the trafficking of CNGA1 to the ciliary PM, and coordinates the formations of disk membrane rim region and OS PM.

Live-Cell Imaging Evidence for the Ciliary Transport of Rod Photoreceptor Opsin by Heterotrimeric Kinesin-2

Primary cilia detect extracellular signals through membrane receptors and channels. The outer segment of a vertebrate photoreceptor cell represents the most elaborate of all primary cilia, containing extraordinarily large amounts of the visual receptor protein, opsin. Because of its high abundance, opsin represents a potential model system for the study of ciliary membrane receptors, including their transport. Here, we have analyzed the movement of ciliary opsin to test whether the highly conserved intraflagellar transport (IFT), as driven by heterotrimeric kinesin-2, is required. Results show that opsin can enter and move along the primary cilium of a nonphotoreceptor cell (an hTERT-RPE1 epithelial cell), suggesting that it can co-opt the basic anterograde motor system of cilia. Fluorescence recovery after photobleaching analysis of cilia of hTERT-RPE1 cells showed that the movement of ciliary opsin was comparable to that of the IFT protein, IFT88. Moreover, the movement of opsin in these cilia, as well as in cilia of mouse rod photoreceptor cells, was reduced significantly when KIF3A, the obligate motor subunit of heterotrimeric kinesin-2, was deficient. These studies therefore provide evidence from live-cell analysis that the conserved heterotrimeric kinesin-2 is required for the normal transport of opsin along the ciliary plasma membrane.

Polycystin-2 takes different routes to the somatic and ciliary plasma membrane

Polycystin-2 (also called TRPP2), an integral membrane protein mutated in patients with cystic kidney disease, is located in the primary cilium where it is thought to transmit mechanical stimuli into the cell interior. After studying a series of polycystin-2 deletion mutants we identified two amino acids in loop 4 that were essential for the trafficking of polycystin-2 to the somatic (nonciliary) plasma membrane. However, polycystin-2 mutant proteins in which these two residues were replaced by alanine were still sorted into the cilium, thus indicating that the trafficking routes to the somatic and ciliary plasma membrane compartments are distinct. We also observed that the introduction of dominant-negative Sar1 mutant proteins and treatment of cells with brefeldin A prevented the transport into the ciliary plasma membrane compartment, whereas metabolic labeling experiments, light microscopical imaging, and high-resolution electron microscopy revealed that full-length polycystin-2 did not traverse the Golgi apparatus on its way to the cilium. These data argue that the transport of polycystin-2 to the ciliary and to the somatic plasma membrane compartments originates in a COPII-dependent fashion at the endoplasmic reticulum, that polycystin-2 reaches the cis side of the Golgi apparatus in either case, but that the trafficking to the somatic plasma membrane goes through the Golgi apparatus whereas transport vesicles to the cilium leave the Golgi apparatus at the cis compartment. Such an interpretation is supported by the finding that mycophenolic acid treatment resulted in the colocalization of polycystin-2 with GM130, a marker of the cis-Golgi apparatus. Remarkably, we also observed that wild-type Smoothened, an integral membrane protein involved in hedgehog signaling that under resting conditions resides in the somatic plasma membrane, passed through the Golgi apparatus, but the M2 mutant of Smoothened, which is constitutively located in the ciliary but not in the somatic plasma membrane, does not. Finally, a dominant-negative form of Rab8a, a BBSome-associated monomeric GTPase, prevented the delivery of polycystin-2 to the primary cilium whereas a dominant-negative form of Rab23 showed no inhibitory effect, which is consistent with the view that the ciliary trafficking of polycystin-2 is regulated by the BBSome.

Myosin VIIa, the product of the Usher 1B syndrome gene, is concentrated in the connecting cilia of photoreceptor cells

Usher syndrome is the most common form of combined deafness and blindness. The gene that is defective in Usher syndrome 1B (USH1B) encodes for an unconventional myosin, myosin VIIa. To understand the cellular function of myosin VIIa and why defects in it lead to USH1B, it is essential to determine the precise cellular and subcellular localization of the protein. We investigated the distribution of myosin VIIa in human and rodent photoreceptor cells and retinal pigment epithelium (RPE), primarily by immunoelectron microscopy, using antibodies generated against two different domains of the protein. In both human and rodent retinae, myosin VIIa was detected in the apical processes of the RPE and in the cilium of rod and cone photoreceptor cells. Immunogold label was most concentrated in the connecting cilium. Here, myosin VIIa appeared to be distributed outside the ring of doublet microtubules near the ciliary plasma membrane. These observations indicate that a major role of myosin VIIa in the retina is in the photoreceptor cilium, perhaps in such a function as trafficking newly synthesized phototransductive membrane or maintaining the diffusion barrier between the inner and outer segments. Our results support the notion that defective ciliary function is the underlying cellular abnormality that leads to cellular degeneration in Usher syndrome. Cell Motil. Cytoskeleton 37:240–252, 1997. © 1997 Wiley-Liss Inc.

Myosin VIIa, the product of the Usher 1B syndrome gene, is concentrated in the connecting cilia of photoreceptor cells.

Usher syndrome is the most common form of combined deafness and blindness. The gene that is defective in Usher syndrome 1B (USH1B) encodes for an unconventional myosin, myosin VIIa. To understand the cellular function of myosin VIIa and why defects in it lead to USH1B, it is essential to determine the precise cellular and subcellular localization of the protein. We investigated the distribution of myosin VIIa in human and rodent photoreceptor cells and retinal pigment epithelium (RPE), primarily by immunoelectron microscopy, using antibodies generated against two different domains of the protein. In both human and rodent retinae, myosin VIIa was detected in the apical processes of the RPE and in the cilium of rod and cone photoreceptor cells. Immunogold label was most concentrated in the connecting cilium. Here, myosin VIIa appeared to be distributed outside the ring of doublet microtubules near the ciliary plasma membrane. These observations indicate that a major role of myosin VIIa in the retina is in the photoreceptor cilium, perhaps in such a function as trafficking newly synthesized phototransductive membrane or maintaining the diffusion barrier between the inner and outer segments. Our results support the notion that defective ciliary function is the underlying cellular abnormality that leads to cellular degeneration in Usher syndrome.

Molecular mechanisms of chemo-electrotransduction in the olfactory receptor cell.

The perception of odors begins with depolarization of the olfactory receptor cells. This transduction process is mediated by G-protein-linked activation of adenylate cyclase, which produces a membrane current by direct gating of cationic channels (cAMP-gated channels) located in ciliary plasma membrane. Opening of these cAMP-gated channels allows monovalent as well as divalent cations to enter the ciliary cytoplasm, and this Ca2+ influx serves two important functions. First, the Ca2+ influx activates a Cl- current, which stabilizes the transduction current during fluctuations in extracellular cation concentration and increases the total current activated by cAMP. Second, the Ca2+ influx mediates adaptation by providing negative feedback at several stages in the transduction mechanism.

Gating properties of the cAMP-gated channel in toad olfactory receptor cells.

1. Inside-out membrane patches were excised from different parts of isolated olfactory receptor cells of the toad, and the gating properties of cAMP-gated channels were investigated under low concentrations of divalent cations. 2. At +50 mV, an outward current was observed when 1 mM cAMP was applied to the cytoplasmic side of membrane patches excised from cilia. cGMP had a similar effect. The dose-response relation for the cAMP-induced current could be fitted with the Hill equation with a coefficient of 1.5 for both cAMP and cGMP, and half-maximal concentration (K1/2) values of 19 microM (cAMP) and 16 microM (cGMP). 3. cAMP at low concentration (1 microM) induced step-like currents representing the opening of individual channels with a unitary conductance of about 30 pS. The I-V relation for the unitary events showed a weak outward rectification. 4. The relation between variance and mean current amplitude was well described with a parabola. Even at a saturating ligand concentration (1 mM cAMP) the current fluctuations did not disappear, indicating that fully liganded channels still switched between open and closed states. The maximum open probability was about 80% (+40 to +60 mV). 5. The current fluctuations at 1 mM cAMP were analysed with power spectral analysis. At +50 mV, frequency components lower than 10 Hz were well described with a Lorentzian function with a corner frequency of 1.7 +/- 0.2 Hz (+/- S.D.). In addition, higher flat frequency components were observed. At -50 mV the corner frequency became 0.6 +/- 0.1 Hz. 6. Membrane patches having a single cAMP-gated channel were obtained from the dendro-somatic membrane. These channels were very similar to the ciliary channels in electrophysiological characteristics. 7. The single cAMP-gated channel current showed flickering bursts that were interrupted with gaps. In saturating cAMP condition (1 mM), both burst- and gap-time histograms were fitted with single-exponential functions with time constants of 148 and 141 ms, respectively. 8. The channels were present at a high density of around 1750 microns-2 on the ciliary plasma membrane, as compared to 6 microns-2 on dendro-somatic membrane.

Evidence for the transport of opsin in the connecting cilium and basal rod outer segment in rat retina: rapid-freeze, deep-etch and horseradish peroxidase labelling studies.

In order to clarify the pathway of opsin transport in the connecting cilium and basal rod outer segment, we examined rat rod cells by a rapid-freeze and deep-etch technique and also examined the uptake of horseradish peroxidase into isolated retina. The distribution of intramembrane particles on the P-face of the cilium indicated that the ciliary plasma membrane has similar opsin content to the basal rod outer segment plasma membrane. Dilated cisternae were detected below the stack of disk membranes at the basal rod outer segment in fresh retina. The fine structure of the P-face and true surface of these cisternae was identical to that of the disk membrane. Uptake of horseradish peroxidase was detected in the cisternae or in both cisternae and most basal disk, indicating that the cisternae are formed prior to the disk membrane. In the distal part of connecting cilium, we found axially oriented infoldings on the P-face of the plasma membrane, and subplasmalemmal tubules or cisternae adjacent and parallel to them. Such subplasmalemmal membranes were labeled by exogenous horseradish peroxidase, suggesting that the infoldings are invaginating plasma membrane. These results may indicate that opsin molecules are conveyed on the ciliary plasma membrane, and that this opsin-rich plasma membrane is internalized in the distal connecting cilium to form dilated cisternae, which subsequently change to the disk membranes.

Localization of peripherin/rds in the disk membranes of cone and rod photoreceptors: relationship to disk membrane morphogenesis and retinal degeneration.

The outer segments of vertebrate rod photoreceptor cells consist of an ordered stack of membrane disks, which, except for a few nascent disks at the base of the outer segment, is surrounded by a separate plasma membrane. Previous studies indicate that the protein, peripherin or peripherin/rds, is localized along the rim of mature disks of rod outer segments. A mutation in the gene for this protein has been reported to be responsible for retinal degeneration in the rds mouse. In the present study, we have shown by immunogold labeling of rat and ground squirrel retinas that peripherin/rds is present in the disk rims of cone outer segments as well as rod outer segments. Additionally, in the basal regions of rod and cone outer segments, where disk morphogenesis occurs, we have found that the distribution of peripherin/rds is restricted to a region that is adjacent to the cilium. Extension of its distribution from the cilium coincides with the formation of the disk rim. These results support the model of disk membrane morphogenesis that predicts rim formation to be a second stage of growth, after the first stage in which the ciliary plasma membrane evaginates to form open nascent disks. The results also indicate how the proteins of the outer segment plasma membrane and the disk membranes are sorted into their separate domains: different sets of proteins may be incorporated into membrane outgrowths during different growth stages of disk morphogenesis. Finally, the presence of peripherin/rds protein in both cone and rod outer segment disks, together with the phenotype of the rds mouse, which is characterized by the failure of both rod and cone outer segment formation, suggest that the same rds gene is expressed in both types of photoreceptor cells.

Actin filaments in the photoreceptor cilium of the rds mutant mouse

An actin filament meshwork was recently demonstrated within the ciliary axoneme at the base of the photoreceptor outer segment (OS) in rat retina. Individual filaments of a uniform polarity extended from the cilium and entered into the bottom of the OS disc stack where they associated with the plasma membrane in the region of new disc assembly. This and other studies have indicated that an actin-mediated mechanism may regulate OS disc morphogenesis. The homozygous rds mouse exhibits an absence of OS formation, although cilia do develop and opsin is contained within the ciliary plasma membrane. The rds abnormality is believed to result from a defect in OS disc assembly. Immunogold labeling has shown that actin is situated within the distal end of rds photoreceptor cilia, as well as in the distal cilium of normal mice prior to the onset of OS differentiation. However, anti-actin antibodies do not distinguish between monomer and filamentous actin. In the current study, neural retinas from rds and control mice were permeabilized with saponin, incubated with myosin subfragment-1 (S-1), and prepared for electron microscopy. Following this treatment, a meshwork of myosin S-1 decorated actin filaments could be observed within the axoneme in the distal end of each rds photoreceptor cilium. As in normal visual cells, actin filaments exited the axoneme by passing between pairs of microtubule doublets. These filaments had the correct polarity, with all arrowheads pointing toward the axoneme, and they associated with the ciliary plasma membrane in the region where OS disc morphogenesis would normally occur. Additionally, filament-containing membranous structures were observed in close proximity to the distal cilium. Some of these may correspond to the opsin-rich vesicles previously identified in the subretinal space of the rds mouse.

Light microscopic and ultrastructural study of the development of the saccus vasculosus in the rainbow trout, Oncorhynchus mykiss.

This study using light and electron microscopy indicates that the saccus vasculosus is distinguishable in 9-mm embryos and grows continuously throughout embryonic development to the adult stage. In the saccus vasculosus, epithelial mitoses are observed in all stages studied. Phases of centriologenesis, ciliogenesis, and globule formation have been characterized in developing coronet cells. During the phase of centriologenesis, new centrioles appear in association with pre-existing centrioles and not on deuterosomes. After ciliogenesis, each cilium differentiates to a globule almost at the same time as the other cilia of the coronet cell. The inner membrane system of the globules seems to derive from the ciliary plasma membrane. This membrane system often produces membrane whorls during the development. The different phases of coronet cell development have been found in the same individual and in all the stages studied except the 9-mm embryo. Cerebrospinal fluid-contacting neurons are observed in the saccus epithelium from the 12-mm embryos on and are distinguishable from coronet cells in their early formative stages. The three cell types of the saccus vasculosus increase continuously in number during development. Nerve processes are found in the saccus vasculosus of embryos, whereas differentiated synapses appear later in the fry. The significance of continued coronet cell formation is discussed in relation to a putative coronet cell and/or a globule renewal cycle in the adult.

Effect of acoustic overstimulation on the glycocalyx and the ciliary interconnections in the organ of Corti: high resolution scanning electron microscopic investigation.

Changes in ciliary interconnections in the organ of Corti are described after acoustic overstimulation using a special high resolution scanning electron microscope and tannic acid-osmium staining technique, giving an almost three dimensional view. Guinea pigs were exposed to a 3.85 kHz pure tone at an intensity of 120 dB for 22.5 minutes. The first detectable change was a disarrangement of the cilia with a loosening of the interconnections. The ciliary plasma membrane presented with an abnormally smooth appearance. The tip links connecting the tips of the stereocilia to their taller neighbours were also affected showing elongation or even disappearance. The fine granules which cover the tips of the tallest stereocilia of the outer hair cells were decreased. These findings suggest that acoustic overstimulation may affect the carbohydrate metabolism exceeding to degeneration of ciliary interconnections resulting in a disarrangement and detachment of cilia. The tip links, which may participate in sensory cell transduction, seem also to be affected by acoustic overstimulation.

Organization of actin filaments and immunocolocalization of alpha-actinin in the connecting cilium of rat photoreceptors.

A small discrete concentration of actin filaments in the connecting cilium of vertebrate photoreceptors appears to have a role in the morphogenesis of the phototransductive disk membranes (Williams et al., '88). We have visualized these actin filaments in rat rod photoreceptors by decorating them with myosin subfragment-1. At the site of disk morphogenesis, we observed a cluster of short filaments, with various orientations and their faster growing (barbed) ends at the ciliary plasma membrane. Their association with the liplike structure of an early nascent disk is consistent with their apparent involvement in the initiation of disk morphogenesis. A few longer decorated filaments extended along the core the connecting cilium, away from the site of disk morphogenesis, implying that they might have some function other than the shaping of a new disk. Most of the antiactin label was found in the region of the short filaments. The alpha-actinin immunolabel coincided with that of actin, suggesting that the filaments may be crosslinked by alpha-actinin.

Immunogold localization of actin in developing photoreceptor cilia of normal and rds mutant mice

Recent immunocytochemical studies have localized actin to the distal cilium of mature vertebrate photoreceptors. Since this is the site where the ciliary plasma membrane evaginates to form new outer segment discs, the results suggest that an actin-mediated contractile mechanism or cytoskeletal network might regulate some aspect of outer segment disc morphogenesis. In the present study, immunoelectron microscopy was used to localize actin to the developing cilia of normal and rds mutant mouse photoreceptors. In normal mice, actin could not be localized to newly projecting cilia, but an actin-rich domain was demonstrated within the distal, bulbous ending of elongated cilia just prior to outer segment development. These results suggest that actin is not important for ciliary growth, but that it may be necessary for the subsequent differentiation of an outer segment. In the rds mutant mouse, there is an absence of outer segment formation, although cilia appear to develop normally. Rhodamine phalloidin staining of cryostat sections demonstrated a normal F-actin distribution within the rds retina. Utilizing immunogold labeling of developing rds photoreceptors, actin was localized to the distal, bulbous ending of elongated cilia. This result indicates that actin is situated within its normal domain in rds cilia.

Disruption of microfilament organization and deregulation of disk membrane morphogenesis by cytochalasin D in rod and cone photoreceptors.

Morphogenesis of photoreceptor outer segment disks appears to occur by an evagination of the ciliary plasma membrane (Steinberg et al., J Comp Neurol 190:501-519, '80). We tested if polymerized actin (F-actin) was necessary for the regulation of this postulated process by incubating Xenopus eyecups with 5 or 25 microM cytochalasin D for 6-28 hours. During the second hour, the incubation medium contained 3H-leucine. Both concentrations of cytochalasin resulted in: 1) dissolution of the rhodamine-phalloidin labeling pattern of photoreceptors, and 2) collapse of the calycal processes (which are normally filled with actin filaments) and disappearance of the inner segment microfilaments. In addition, the few most basal rod and cone outer segment disks appeared several times their normal diameter. These oversized disks had incorporated 3H-leucine and extended along the margin of the outer or inner segment. The nature of the overgrown disks is consistent only with a morphogenetic process involving evaginations of the ciliary plasma membrane. Deregulation by cytochalasin D was manifest by excessive growth of a few nascent disks rather than normal growth of many. Therefore, the normal network of actin filaments is apparently not necessary for continued evagination of the membrane, but it does seem to be an essential part of the mechanism that initiates the evagination of the ciliary plasma membrane and/or the mechanism that controls how far nascent disks grow.