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.     

Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells

The association of actin filaments with membranes is now recognized as an important parameter in the motility of nonmuscle cells. We have investigated the organization of one of the most extensive and highly ordered actin filament-membrane complexes in nature, the brush border of intestinal epithelial cells. Through the analysis of isolated, demembranated brush borders decorated with the myosin subfragment, S1, we have determined that all the microvillar actin filaments have the same polarity. The S1 arrowhead complexes point away from the site of attachment of actin filaments at the apical tip of the microvillar membrane. In addition to the end-on attachment of actin filaments at the tip of the microvillus, these filaments are also connected to the plasma membrane all along their lengths by periodic (33 nm) cross bridges. These bridges were best observed in isolated brush borders incubated in high concentrations of Mg++. Their visibility is attributed to the induction of actin paracrystals in the filament bundles of the microvilli. Finally, we present evidence for the presence of myosinlike filaments in the terminal web region of the brush border. A model for the functional organization of actin and myosin in the brush border is presented.     

Morphogenesis of the photoreceptor outer segment during postnatal development in the mouse (BALB/c) retina

Summary Disc formation of rod photoreceptor cells in developing BALB/c mice retinas was studied by rapid freeze, freeze-substitution, freeze-etching, immunocytochemistry, and myosin S-1 decoration methods. Freeze-substituted photoreceptor cells contained variously shaped vesicles in the apical swelling of the connecting cilium or the base of the outer segment during postnatal development. Rapid freezing successfully arrested pinocytosis; the fusion of small vesicles to give large ones, and the compression of certain vesicles (0.3–0.6 m) appears to lead gradually to the formation of the so-called discs. We therefore propose that membranous discs are formed by the fusion of small pinocytotic vesicles and their subsequent compression. Discs formed in this way were partially stacked, but were ordered at random during the early developmental stages. During development, a partial stack of discs was progressively rearranged to a regular form as seen in mature outer segments. Cytoskeletal actin was expected to be involved in the disc formation; it was demonstrated in the distal axoneme of the connecting cilium during development and showed no change in its distribution. However, the polarity of the actin filaments, as revealed by myosin S-1 decoration in early developmental stages, was much more variable than in the adult. Barbed ends of actin filaments were associated with the plasma membrane or the membrane of vesicles. We also found actin filaments coiled up helically on ciliary microtubules.     

Ultrastructural changes of the microvillar cytoskeleton in the photoreceptor of the crayfish Orconectes limosus related to different adaptation conditions

In the retina of crayfish microvilli of seven of the eight photoreceptor cells build highly organized structures, the rhabdoms. Cytoskeletal elements inside the microvilli were investigated in conventional and slightly extracted electron microscopical preparations. In conventional preparations the ultrastructure of these cytoskeletal elements depended on the adaptational state of the animal. They appeared as central filament-like structures inside each microvillus when dark-adapted retinae were prepared and fixed at night in the absence of calcium. Changes of these conditions (light, daytime, or calcium concentration) impaired the detectability of these central filaments; in light-adapted eyes prepared at midday they were rarely seen. Nevertheless, single microvillar filaments were present in light-adapted retinae after mild cell permeabilization with the saponin beta-escin. They appeared as a regular structure in each microvillus, often attached to the membrane. Their fine structure was consistent with the ultrastructure of single actin filaments as indicated by fast-Fourier-analysis and further supported by the presence of anti-actin immunoreactivity in electron microscopical and immunocytochemical preparations. These results indicate that microvillar filaments are not necessarily destroyed by light as previously described; we suggest that their appearance inside the microvillus might be altered by the properties of associated, maybe sidearm-like proteins.     

Characterization of intestinal microvillar membrane disks: detergent- resistant membrane sheets enriched in associated brush border myosin I (110K-calmodulin)

The actin bundle within each microvillus of the intestinal brush border (BB) is tethered laterally to the membrane by bridges composed of BB myosin I. Avian BB myosin I, formerly termed 110K-calmodulin, consists of a heavy chain with an apparent Mr of 110 kD and three to four molecules of calmodulin "light chains." Recent studies have shown that this complex shares many properties with myosin including mechanochemical activity. In this report, the isolation and characterization of a membrane fraction enriched in bound BB myosin I is described. This membrane fraction, termed microvillar membrane disks, was purified from ATP extracts of nonionic detergent-treated microvilli prepared from avian intestinal BBs. Ultrastructural analysis revealed that these membranes are flat, disk-shaped sheets with protrusions which are identical in morphology to purified BB myosin I. The disks exhibit actin-activated Mg-ATPase activity and bind and cross-link actin filaments in an ATP-dependent fashion. The mechanochemical activity of the membrane disks was assessed using the Nitella bead movement assay (Sheetz, M. P., and J. A. Spudich. 1983. Nature [Lond.]. 303:31-35). These preparations were shown to be free of significant contamination by conventional BB myosin. Latex beads coated with microvillar membrane disks move in a myosin-like fashion along Nitella actin cables at rates of 12-60 nm/s (average rate of 33 nm/s); unlike purified BB myosin I, the movement of membrane disk-coated beads was most reproducibly observed in buffers containing low Ca2+.     

Ezrin regulates microvillus morphogenesis by promoting distinct activities of Eps8 proteins

The mechanisms that regulate actin filament polymerization resulting in the morphogenesis of the brush border microvilli in epithelial cells remain unknown. Eps8, the prototype of a family of proteins capable of capping and bundling actin filaments, has been shown to bundle the microvillar actin filaments. We report that Eps8L1a, a member of the Eps8 family and a novel ezrin-interacting partner, controls microvillus length through its capping activity. Depletion of Eps8L1a leads to the formation of long microvilli, whereas its overexpression has the opposite effect. We demonstrate that ezrin differentially modulates the actin-capping and -bundling activities of Eps8 and Eps8L1a during microvillus assembly. Coexpression of ezrin with Eps8 promotes the formation of membrane ruffles and tufts of microvilli, whereas expression of ezrin and Eps8L1a induces the clustering of actin-containing structures at the cell surface. These distinct morphological changes are neither observed when a mutant of ezrin defective in its binding to Eps8/Eps8L1a is coexpressed with Eps8 or Eps8L1a nor observed when ezrin is expressed with mutants of Eps8 or Eps8L1a defective in the actin-bundling or -capping activities, respectively. Our data show a synergistic effect of ezrin and Eps8 proteins in the assembly and organization of actin microvillar filaments.     

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.     

WASp is required for the correct temporal morphogenesis of rhabdomere microvilli

Microvilli are actin-based fingerlike membrane projections that form the basis of the brush border of enterocytes and the Drosophila melanogaster photoreceptor rhabdomere. Although many microvillar cytoskeletal components have been identified, the molecular basis of microvillus formation is largely undefined. Here, we report that the Wiskott-Aldrich syndrome protein (WASp) is necessary for rhabdomere microvillus morphogenesis. We show that WASp accumulates on the photoreceptor apical surface before microvillus formation, and at the time of microvillus initiation WASp colocalizes with amphiphysin and moesin. The loss of WASp delays the enrichment of F-actin on the apical photoreceptor surface, delays the appearance of the primordial microvillar projections, and subsequently leads to malformed rhabdomeres.     

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.     

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.     

Ultrastructural study of sensory cells of the proboscidial glandular epithelium of Riseriellus occultus (Nemertea,Heteronemertea)

Only one sensory cell type has been observed within the glandular epithelium of the proboscis in the heteronemertine Riseriellus occultus. These bipolar cells are abundant and scattered singly throughout the proboscis length. The apical surface of each dendrite bears a single cilium enclosed by a ring of six to eight prominent microvilli. The cilium has the typical 9×2 + 2 axoneme arrangement and is equipped with a cross-striated vertical rootlet extending from the basal body. No accessory centriole or horizontal rootlet was observed. Large, modified microvilli (stereovilli) surrounding the cilium are joined together by a system of fine filaments derived from the glycocalyx. Each microvillus contains a bundle of actin-like filaments which anchor on the indented inner surface of a dense, apical ring situated beneath the level of the ciliary basal body. The tip of the cilium is expanded and modified to form a bulb-like structure which lies above the level where the surrounding microvilli terminate. In the region where the cilium emerges from the microvillar cone, the membrane of the microvillar apices makes contact with a corresponding portion of the ciliary membrane. At this level microvilli and cilium are apparently firmly linked by junctional systems resembling adherens junctions. The results suggest that these sensory cells may be mechanoreceptors. © 1996 Wiley-Liss, Inc.     

The local deletion of a microvillar cytoskeleton from photoreceptors of tipulid flies during membrane turnover

The distal regions of the photoreceptor microvilli of tipulid flies are shed to extracellular space during membrane turnover. Before abscission, the microvillar tips undergo a transformation: they become deformed, and after conventional fixation for electron microscopy are relatively electron-lucent compared to the stable, basal microvillar segments. We now show that the electron-lucent segment is an empty bag of membrane whose P-face after freeze-etch preparation appears as densely particulate as the remainder of the microvillus. Transformation is achieved by the local deletion of a microvillar cytoskeleton which consists of a single, axial filament linked to the plasma membrane by side-arms. The filament may be partially preserved by the chelation of Ca2+; the provision of a divalent cation (Mg2+ or Ba2+) stabilizes the side-arms during subsequent fixation, as has been shown previously for the rhabdomeral cytoskeleton of blowflies. Incubation of the isolated retina in the presence of 0.25 mM Ca2+ at room temperature for 10-20 min causes proteolysis of the cytoskeleton which is blocked by as little as 0.5 mM of the thiol protease inhibitors Ep-475 and Ep-459. Loss of the cytoskeleton is accompanied by deformation of all regions of the microvilli. Local deletion of the cytoskeleton from the transformed zone of the normal rhabdom is sufficient to explain deformation of the microvillar tips, but not their subsequent abscission. The intimate association between a Ca2+-activated thiol protease and the cytoskeleton implied by the great rapidity of proteolysis calls for a reassessment of published studies of membrane turnover by radioautography, and of the nature of light-induced damage to arthropod photoreceptor membranes.     

The Eyes of Mesostoma ehrenbergi (Focke, 1836) (Platyhelminthes,Rhabdocoela). Fine Structure and Photoreceptor Membrane Turnover

Abstract Each pigment-cup eye of Mesostoma ehrenbergi consists of two photoreceptor cells, the anterior cell being bilobate. the posterior almost linear, and of a multicellular pigment cup. The nuclei of the photoreceptor cells are located inside the medial region of the brain. Thin cytoplasmic photoreceptor projections provided with neurosecretory-like granules are interposed between the inner surface of the eye cup and the distal extremity of the microvilli. The breakdown and renewal of microvillar membranes was analysed. Membrane turnover is a continuous process. At dusk and during the night abscission of photoreceptive membranes occurs. At dawn the membrane fragments are degraded to granular material, which is then endocytosed into the submicrovillar cytoplasm as coated vesicles. These vesicles form multivesicular bodies. The degradation of multivesicular body content occurs during the following light hours. The dark period is correlated with membrane synthesis for elongation of reticular membranes, which are converted into ellipsoid bodies. The formation of new microvillar membranes occurs at the base of the microvillar border, and involves the fusion with the old microvillar membranes of small vesicles detached from the tubular endoplasmic membranes and from the flattened concentric cisternae of ellipsoid bodies. The correlations with daily cycles of other invertebrates are discussed.     

The brush border cytoskeleton is not static: in vivo turnover of proteins

The shape and stability of intestinal epithelial cell microvilli are maintained by a cytoskeletal core composed of a bundle of actin filaments with several associated proteins. The core filaments are intimately associated with the overlying plasma membrane, in which there occur rapid turnover of proteins and constant incorporation of new membrane. Previous work has shown that starvation or inhibition of protein synthesis results in modulation of microvillar length, which indicates that there may be cytoskeletal protein turnover. We demonstrate herein, by means of in vivo pulse labeling with radioactive amino acids, that turnover of brush border cytoskeletal proteins occurs in mature absorptive cells. Turnover of cytoskeletal proteins appears to be quite slow relative to membrane protein turnover, which suggests that the turnover of these two microvillar compartments is not coupled. We thus conclude that cytoskeletal protein turnover may be a factor used to maintain normal length and stability of microvilli and that the cytoskeleton cannot be considered a static structure.     

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.     

A new role for the architecture of microvillar actin bundles in apical retention of membrane proteins

Actin-bundling proteins are identified as key players in the morphogenesis of thin membrane protrusions. Until now, functional redundancy among the actin-bundling proteins villin, espin, and plastin-1 has prevented definitive conclusions regarding their role in intestinal microvilli. We report that triple knockout mice lacking these microvillar actin-bundling proteins suffer from growth delay but surprisingly still develop microvilli. However, the microvillar actin filaments are sparse and lack the characteristic organization of bundles. This correlates with a highly inefficient apical retention of enzymes and transporters that accumulate in subapical endocytic compartments. Myosin-1a, a motor involved in the anchorage of membrane proteins in microvilli, is also mislocalized. These findings illustrate, in vivo, a precise role for local actin filament architecture in the stabilization of apical cargoes into microvilli. Hence, the function of actin-bundling proteins is not to enable microvillar protrusion, as has been assumed, but to confer the appropriate actin organization for the apical retention of proteins essential for normal intestinal physiology.     

Brush border motility. Microvillar contraction in triton-treated brush borders isolated from intestinal epithelium

The brush border of intestinal epithelial cells consists of an array of tightly packed microvilli. Within each microvillus is a bundle of 20-30 actin filaments. The basal ends of the filament bundles are embedded in and interconected by a filamentous meshwork, the terminal web, which lies directly beneath the microvilli. When calcium and ATP are added to isolated brush borders that have been treated with the detergent, Triton X-100, the microvillar filament bundles rapidly retract into and through the terminal web region. Biochemical studies of brush border contractile proteins suggest that the observed microvillar contraction is actomyosin mediated. We have shown previously that the major protein of the brush border's actin (Tilney, L. G., and M. S. Mooseker. 1971. Proc. Natl. Acad. Sci. U. S. A. 68:2611-2615). The brush border also contains a protein with the same molecular weight as the heavy chain subunit of myosin (200, 000 daltons). In addition, preparations of demembranated brush borders exhibit potassium-EDTA ATPase activity of 0.02 mumol phosphate/mg-min (22 degrees C); this assay is diagnostic for myosin-like ATPase isolated from vertebrate sources. Other proteins of the brush border include a 30,000 dalton protein with properties similar to those of tropomyosin, and a protein with the same molecular weight as the Z band protein, alpha-actinin (95,000 daltons). How these observations bear on the basis for microvillar movements in vivo is discussed within the framework of our recent model for the organization of actin and myosin in the brush border (Mooseker, M. S., and L. G. Tilney. 1975. J. Cell Biol. 67:725-743).     

Direct involvement of ezrin/radixin/moesin (ERM)-binding membrane proteins in the organization of microvilli in collaboration with activated ERM proteins.

Ezrin/radixin/moesin (ERM) proteins have been thought to play a central role in the organization of cortical actin-based cytoskeletons including microvillar formation through cross-linking actin filaments and integral membrane proteins such as CD43, CD44, and ICAM-2. To examine the functions of these ERM-binding membrane proteins (ERMBMPs) in cortical morphogenesis, we overexpressed ERMBMPs (the extracellular domain of E-cadherin fused with the transmembrane/cytoplasmic domain of CD43, CD44, or ICAM-2) in various cultured cells. In cultured fibroblasts such as L and CV-1 cells, their overexpression significantly induced microvillar elongation, recruiting ERM proteins and actin filaments. When the ERM-binding domains were truncated from these molecules, their ability to induce microvillar elongation became undetectable. In contrast, in cultured epithelial cells such as MTD-1A and A431 cells, the overexpression of ERMBMPs did not elongate microvilli. However, in the presence of EGF, overexpression of ERMBMPs induced remarkable microvillar elongation in A431 cells. These results indicated that ERMBMPs function as organizing centers for cortical morphogenesis by organizing microvilli in collaboration with activated ERM proteins. Furthermore, immunodetection with a phosphorylated ERM-specific antibody and site-directed mutagenesis suggested that ERM proteins phosphorylated at their COOH-terminal threonine residue represent activated ERM proteins.     

Characterization of the 110-kdalton actin-calmodulin-, and membrane-binding protein from microvilli of intestinal epithelial cells

One of the major proteins of the chicken intestinal microvillus is a calmodulin-binding protein of 105-110 kdaltons which has been tentatively identified as the bridge linking the microvillar filament bundle laterally to the membrane. We have treated isolated, membrane- intact brush borders with ATP and obtained solubilization of the 110- kdalton protein, calmodulin (CM), myosin, and lesser amounts of several other cytoskeletal proteins. Electron micrographs of ATP-extracted brush borders showed loss of the linkers between the actin filament bundle and the microvillar membrane, with "ballooning" of the membrane away from the filament bundle, particularly at the tip end. In brush borders treated with calcium and trifluoperazine to solubilize CM, precise arrangement and morphology of lateral bridges was unperturbed, but ATP treatment would no longer solubilize the 110-kdalton protein. This result suggests that associated CM is necessary for the ATP- induced solubilization of the 110-kdalton protein. A 110-kdalton protein-CM complex, with 110-kdalton protein: CM ratios of 1:1-2, was partially purified from ATP-extracts of brush borders by a combination of gel filtration and hydroxylapatite chromatography. The 110-kdalton protein-CM complex is an irregular, elongated molecule that ranged in size from 5 X 8 nm to 8 X 14 nm, with a Stokes' radius of 6.1 nm. This 110-kdalton protein-CM complex exhibited no Mg++-ATPase activity and no detectable myosin light chain kinase activity. In co-sedimentation assays, the 110-kdalton protein-CM bound to F-actin in the absence but not the presence of ATP. Both the interaction of the complex with actin and the binding of CM to the 110-kdalton protein were calcium- independent. Negative stains of F-actin and 110-kdalton protein-CM in the absence of ATP showed loosely organized aggregates of actin with the 110-kdalton protein-CM complex coating the surface of the filaments. On the basis of our data, and in agreement with previous calculations (Matsudaira, P.T., and D.R. Burgess, 1979, J. Cell Biol. 83:667-673), we suggest that the lateral bridge of the microvillus is composed of a dimer of the 110-kdalton protein with four associated calmodulins.     

ERM (Ezrin/Radixin/Moesin)-based Molecular Mechanism of Microvillar Breakdown at an Early Stage of Apoptosis

Breakdown of microvilli is a common early event in various types of apoptosis, but its molecular mechanism and implications remain unclear. ERM (ezrin/radixin/moesin) proteins are ubiquitously expressed microvillar proteins that are activated in the cytoplasm, translocate to the plasma membrane, and function as general actin filament/plasma membrane cross-linkers to form microvilli. Immunofluorescence microscopic and biochemical analyses revealed that, at the early phase of Fas ligand (FasL)–induced apoptosis in L cells expressing Fas (LHF), ERM proteins translocate from the plasma membranes of microvilli to the cytoplasm concomitant with dephosphorylation. When the FasL-induced dephosphorylation of ERM proteins was suppressed by calyculin A, a serine/threonine protein phosphatase inhibitor, the cytoplasmic translocation of ERM proteins was blocked. The interleukin-1β–converting enzyme (ICE) protease inhibitors suppressed the dephosphorylation as well as the cytoplasmic translocation of ERM proteins. These findings indicate that during FasL-induced apoptosis, the ICE protease cascade was first activated, and then ERM proteins were dephosphorylated followed by their cytoplasmic translocation, i.e., microvillar breakdown. Next, to examine the subsequent events in microvillar breakdown, we prepared DiO-labeled single-layered plasma membranes with the cytoplasmic surface freely exposed from FasL-treated or nontreated LHF cells. On single-layered plasma membranes from nontreated cells, ERM proteins and actin filaments were densely detected, whereas those from FasL-treated cells were free from ERM proteins or actin filaments. We thus concluded that the cytoplasmic translocation of ERM proteins is responsible for the microvillar breakdown at an early phase of apoptosis and that the depletion of ERM proteins from plasma membranes results in the gross dissociation of actin-based cytoskeleton from plasma membranes. The physiological relevance of this ERM protein–based microvillar breakdown in apoptosis will be discussed.