The surface-connected canalicular system in the sinus endothelial cells of rat spleen

The existence of a surface-connected canalicular system in the splenic sinus endothelial cells of the rat has been demonstrated by transmission electron microscopy with lanthanum nitrate acting as a tracer for the extracellular space. In addition, the three-dimensional arrangement of the canaliculi has been revealed by computer-aided reconstruction. The surface-connected canalicular system of the sinus endothelial cells consists of slender canaliculi that are branched, anastomosed, and that show continuity with the plasma membrane. They twist in and out among the organelles and are often found in close apposition to the spherical invaginations of the plasma membrane and run alongside them. Canaliculi which are not infiltrated by lanthanum nitrate take the form of electron-lucent tubules and are accompanied by numerous spherical invaginations of the plasma membrane. From a computer-aided reconstruction, the canaliculi, which invaginate from various sites of the plasma membrane, have been found to be continuous with each other and to penetrate to the surface of the sinus endothelial cell; they also branch and anastomose to form a complex network in the cytoplasm. Although the surface-connected canalicular system in blood platelets and thrombocytes is believed to function as the main route for the discharge of granules and the uptake of foreign materials and also to take part in the storage and transport of calcium, it is unclear at present whether the network of the surface-connected canalicular system in splenic sinus endothelial cells has any physiological significance.     

Dynamin-mediated Internalization of Caveolae

The dynamins comprise an expanding family of ubiquitously expressed 100-kD GTPases that have been implicated in severing clathrin-coated pits during receptor-mediated endocytosis. Currently, it is unclear whether the different dynamin isoforms perform redundant functions or participate in distinct endocytic processes. To define the function of dynamin II in mammalian epithelial cells, we have generated and characterized peptide-specific antibodies to domains that either are unique to this isoform or conserved within the dynamin family. When microinjected into cultured hepatocytes these affinity-purified antibodies inhibited clathrin-mediated endocytosis and induced the formation of long plasmalemmal invaginations with attached clathrin-coated pits. In addition, clusters of distinct, nonclathrin-coated, flask-shaped invaginations resembling caveolae accumulated at the plasma membrane of antibody-injected cells. In support of this, caveola-mediated endocytosis of labeled cholera toxin B was inhibited in antibody-injected hepatocytes. Using immunoisolation techniques an anti-dynamin antibody isolated caveolar membranes directly from a hepatocyte postnuclear membrane fraction. Finally, double label immunofluorescence microscopy revealed a striking colocalization between dynamin and the caveolar coat protein caveolin. Thus, functional in vivo studies as well as ultrastructural and biochemical analyses indicate that dynamin mediates both clathrin-dependent endocytosis and the internalization of caveolae in mammalian cells.     

Glucagon receptors in endothelial and Kupffer cells of mouse liver

To determine whether hepatic sinusoidal cells contain glucagon receptors and, if so, to study the significance of the receptors in the cells, binding of [125I]-glucagon to nonparenchymal cells (mainly endothelial cells and Kupffer cells) isolated from mouse liver was examined by quantitative autoradiography and biochemical methods. Furthermore, the pathway of intracellular transport of colloidal gold-labeled glucagon (AuG) was examined in vivo. Autoradiographic and biochemical results demonstrated many glucagon receptors in both endothelial cells and Kupffer cells, and more receptors being present in endothelial cells than in Kupffer cells. In vivo, endothelial cells internalized AuG particles into coated vesicles via coated pits and transported the particles to endosomes, lysosomes, and abluminal plasma membrane. Therefore, receptor-mediated transcytosis of AuG occurs in endothelial cells. The number of particles present on the abluminal plasma membrane was constant if the amount of injected AuG increased. Therefore, the magnitude of receptor-mediated transcytosis of AuG appears to be regulated by endothelial cells. Kupffer cells internalized the ligand into cytoplasmic tubular structures via plasma membrane invaginations and transported the ligand exclusively to endosomes and lysosomes, suggesting that the ligand is degraded by Kupffer cells.     

Characean charasome-complex and plasmalemma vesicle development

Summary We report on an unusual phenomenon which occurs in some characean algae as a normal plasma membrane activity and also in association with charasome formation. The phenomenon of formation of coated invaginations of the plasma membrane was observed in twoChara and 6Nitella species. These invaginations are coated on their cytoplasmic surface, are 50–60 nm in diameter and rarely exceed 60 nm in length. They are abundant in the young cells ofChara andNitella and also occur in mature cells, but at a lower frequency.N. translucent is an exception in that coated invaginations were few in the young cells and absent in mature cells. Coated vesicles (50–60 nm diameter) were closely associated with these invaginations. Our observations suggest the vesicles may be derived from the invaginations by endocytosis.A close relationship was noted between the development of charasomes (plasmalemma modifications) and coated invaginations. Numerous coated invaginations are seen along the membranes of young charasomes; these invaginations appear to be associated with growth of the charasomes. Coated vesicles were not associated with the coated invaginations of the charasome membrane. The tubular network of cytoplasm and wall space seen in the mature charasome may be formed by fusion of coated invaginations of the developing charasomes, leaving cytoplasmic strands between the fused portions. Coated invaginations were not present along charasomes of the mature cells.     

Identification of Caveolae-like Structures on the Surface of Intact Cells Using Scanning Force Microscopy

Caveolae are small, functionally important membrane invaginations found on the surface of many different cell types. Using electron microscopy, caveolae can be unequivocally identified in cell membranes by virtue of their size and the presence of caveolin/VIP22 proteins in the caveolar coat. In this study we have applied for the first time scanning force microscopy (SFM), to visualize caveolae on the surface of living and fixed cells. By scanning the membranes of Chinese hamster ovary cells (CHO), using the tapping mode of the SFM in fluid, we could visualize small membrane pits on the cell membranes of living and fixed cells. Two populations of pits with mean diameters of around 100 nm and 200 nm were present. In addition, the location of many pits visualized with the SFM was coincident with membrane spots fluorescently labeled with a green fluorescent protein-caveolin-1 fusion protein. Scanning force microscopy on cells treated with methyl--cyclodextrin, an agent that sequesters cholesterol and disrupts caveolae, abolished pits with a measured diameter of 100 nm but left pits of around 200 nm diameter intact. Thus, the smallest membrane pits measured with the SFM in CHO cells were indeed very likely to be identical to caveolae. These experiments show for the first time that SFM can be used to visualize caveolae in intact cells.     

The shape of caveolae is omega-like after glutaraldehyde fixation and cup-like after cryofixation

Caveolae were defined as flask- or omega-shaped plasma membrane invaginations, abundant in adipocytes, fibroblasts, endothelial and smooth muscle cells. The major protein component of caveolar membranes is an integral membrane protein named caveolin. We compared the freeze-fracture behavior of caveolae in glutaraldehyde-fixed and cryofixed mouse fibroblast cells and found distinct differences. In glutaraldehyde-fixed cells almost all caveolae were cross-fractured through their pore and only very few caveolar membranes were membrane-fractured. We found the reverse situation in rapid frozen cells without any chemical fixation where most of the caveolae were membrane-fractured, showing different degrees of invagination from nearly flat to deeply invaginated. In ultrathin sections of glutaraldehyde-fixed heart endothelial cells, caveolae exhibit the well known omega-like shape. In high-pressure frozen, freeze-substituted and low temperature embedded heart endothelial cells, the caveolae frequently exhibit a cup-like shape without any constriction or pore. The cup-like caveolar shape could also be shown by tilt series analysis of freeze-fracture replicas obtained from cryofixed cells. Freeze-fracture immunolabeling of caveolin-1 revealed a lateral belt-like caveolin alignment. These findings point out that the constricted “neck” region of caveolae in most cases is an effect that is caused and intensified by the glutaraldehyde fixation. Our data indicate that caveolae in vivo show all degrees of invagination from nearly flat via cup-like depressed to in a few cases omega-like.     

Induction of caveolae in the apical plasma membrane of Madin-Darby canine kidney cells

In this paper, we have analyzed the behavior of antibody cross-linked raft-associated proteins on the surface of MDCK cells. We observed that cross-linking of membrane proteins gave different results depending on whether cross-linking occurred on the apical or basolateral plasma membrane. Whereas antibody cross-linking induced the formation of large clusters on the basolateral membrane, resembling those observed on the surface of fibroblasts (Harder, T., P. Scheiffele, P. Verkade, and K. Simons. 1998. J. Cell Biol. 929-942), only small ( approximately 100 nm) clusters formed on the apical plasma membrane. Cross-linked apical raft proteins e.g., GPI-anchored placental alkaline phosphatase (PLAP), influenza hemagglutinin, and gp114 coclustered and were internalized slowly ( approximately 10% after 60 min). Endocytosis occurred through surface invaginations that corresponded in size to caveolae and were labeled with caveolin-1 antibodies. Upon cholesterol depletion the internalization of PLAP was completely inhibited. In contrast, when a non-raft protein, the mutant LDL receptor LDLR-CT22, was cross-linked, it was excluded from the clusters of raft proteins and was rapidly internalized via clathrin-coated pits.Since caveolae are normally present on the basolateral membrane but lacking from the apical side, our data demonstrate that antibody cross-linking induced the formation of caveolae, which slowly internalized cross-linked clusters of raft-associated proteins.     

Localization of ryanodine receptor 3 in the sinus endothelial cells of the rat spleen

The ultrastructural localization of ryanodine receptors (RyR) in sinus endothelial cells of the rat spleen was examined by confocal laser scanning and electron microscopy by using isoform-specific antibodies to each of the RyR isoforms. Immunofluorescence microscopy of tissue cryosections revealed RyR3 to be localized, with a strand-like form, in the superficial layer and within the cytoplasm of endothelial cells. Antibodies to RyR1 and RyR2 did not react indicating RyR3 was the predominant isoform. RyR3 was observed over the cortical layer of actin filaments in the apical part and beneath stress fibers in the basal part of the endothelial cells. The distribution of Ca²⁺-storing tubulovesicular-structures within endothelial cells was established by tissue sections treated with osmium ferricyanide selectively to stain the sarcoplasmic reticulum and transverse tubules in muscle cells; electron microscopy revealed densely stained tubulovesicular structures located throughout the sinus endothelial cells and interconnected at various sites. These structures closely apposed the plasma membrane at the apical, lateral, and basal surfaces of the cells and occasionally ran closely parallel to the plasma membrane and near to the mitochondria. Immunogold electron microscopy revealed RyR in the membranes of the nucleus, tubulovesicular structures, and subplasmalemmal cisternae. In the subplasmalemmal cisternae at the apical, lateral, and basal surfaces, RyR was detected on the membranes near to the plasma membrane. Labeling was also present on the membranes of tubulovesicular structures near to caveolae and on the cristae of the mitochondria. Thus, RyR probably participates in Ca²⁺ signal transduction and/or mechanosignal transduction in sinus endothelial cells.This work was supported by Grant-in-Aid for Scientific Research (C), Japan.     

Calcium pump of the plasma membrane is localized in caveolae

The Ca2+ pump in the plasma membrane plays a key role in the fine control of the cytoplasmic free Ca2+ concentration. In the present study, its subcellular localization was examined with immunocytochemical techniques using a specific antibody generated against the erythrocyte membrane Ca2+ pump ATPase. By immunofluorescence microscopy of cultured cells, the labeling with the antibody was seen as numerous small dots, often distributed in linear arrays or along cell edges. Immunogold EM of cryosections revealed that the dots correspond to caveolae, or smooth invaginations of the plasma membrane. The same technique applied to mouse tissues in vivo showed that the Ca2+ pump is similarly localized in caveolae of endothelial cells, smooth muscle cells, cardiac muscle cells, epidermal keratinocytes and mesothelial cells. By quantitative analysis of the immunogold labeling, the Ca2+ pump in capillary endothelial cells and visceral smooth muscle cells was found to be concentrated 18-25-fold in the caveolar membrane compared with the noncaveolar portion of the plasma membrane. In renal tubular and small intestinal epithelial cells, which have been known to contain the Ca2+ pump but do not have many caveolae, most of the labeling was randomly distributed in the basolateral plasma membrane, although caveolae were also positively labeled. The results demonstrate that the caveolae in various cells has the plasmalemmal Ca2+ pump as a common constituent. In conjunction with our recent finding that an inositol 1,4,5-trisphosphate receptor-like protein exists in the caveolae (Fujimoto, T., S. Nakade, A. Miyawaki, K. Mikoshiba, and K. Ogawa. 1992. J. Cell Biol. 119:1507-1513), it is inferred that the smooth plasmalemmal invagination is an apparatus specialized for Ca2+ intake and extrusion from the cytoplasm.     

Localization of caveolin-3 in the sinus endothelial cells of the rat spleen

The localization of caveolins in the sinus endothelial cells of the rat spleen has been demonstrated by confocal laser scanning and electron microscopy. Caveolin-3, a muscle-specific caveolin, was detected by Western blot analysis and immunofluorescence microscopy of isolated sinus endothelial cells and tissue cryosections of the spleen. During the immunofluorescence microscopy of isolated endothelial cells, both caveolin-3 and caveolin-1 were found. In tissue cryosections of the spleen, caveolin-3, as well as caveolin-1 and -2, was present in the contours and cytoplasm of the cells. Immunogold electron microscopy of tissue cryosections revealed caveolin-3, -1, and -2 to be present in caveolae in the apical, lateral, and basal plasma membranes and some vesicular profiles in the cytoplasm of sinus endothelial cells. Furthermore, caveolin-3 was colocalized with caveolin-1 in the same caveolae in the apical, lateral, and basal plasma membranes. Stress fibers and tubulovesicular structures were situated in the vicinity of caveolae labeled with anti-caveolin-3, anti-caveolin-1, and anti-caveolin-2 antibodies. It is speculated that caveolae in sinus endothelial cells play an important role in the constriction of stress fibers.     

Plasma membrane coat and a coated vesicle-associated reticulum of membranes: their structure and possible interrelationship in Chara corallina

Primary fixation with buffered glutaraldehyde plus 2.0 mM CaCl2 and 0.1% tannic acid results in the preservation of certain portions of the plasma membrane coat of Chara when seen with the electron microscope. Such a coat is not observable after fixation with glutaraldehyde alone. The coat appears to be present on all the above ground, vegetative cells of the male plant. Within complex invaginations of the plasma membrane, which are known as charasomes, the coat has two structural components, a central core that is either tubular or solid and a fibrous or granular peripheral region that surrounds the core. The coat material appears to be at least partially derived, via exocytosis, from the contents of single membrane-bound organelles known as glycosomes. Glycosomes seem to originate from within an assemblage of membranes and coated vesicles that can be described, in purely structural terms, as a partially coated reticulum. Such a reticulum is distinguishable from Golgi stacks because the reticulum (a) is not composed of stacked membranes, (b) is extensively involved with large, clearly detailed coated vesicles and coated invaginations, (c) is closely associated with glycosomes, and (d) is only slightly stained by the zinc-iodide- osmium tetraoxide reagent.     

Spiral Coating of the Endothelial Caveolar Membranes as Revealed by Electron Tomography and Template Matching

Caveolae are invaginations of the plasma membrane involved in multiple cellular processes, including transcytosis. In this paper we present an extensive 3-D electron tomographic study of the endothelial caveolar system in situ . Analysis of large cellular volumes of (high-pressure frozen, freeze-substituted and epon-embedded) human umbilical vein endothelial cells (HUVECs) provided a notable view on the architecture of the caveolar system that comprises – as confirmed by 3-D immunolabeling for caveolin of 'intact' cells – bona fide caveolae, free plasmalemmal vesicles, racemose invaginations and free multi-caveolar bodies. Application of template matching to tomograms allowed the 3-D localization of caveolar membrane coatings in a robust manner. In this way we observed that bona fide endothelial caveolae, cryofixed and embedded in their cellular context, show a spiral organization of the coating as shown in the past for chemically fixed and freeze-etched caveolae from fibroblasts. Meticulous 3-D analysis further revealed that the coatings are distributed in triads of spirals over the caveolar bulb and neck. Remarkably, this coating distribution is consistently present over the membranes of the other members of the caveolar system in HUVECs. The novel observations that we present clarify the ultrastructural complexity of the 'intact' caveolar system, setting a detailed morphological basis for its functional diversity.     

The antibody-induced clustering and endocytosis of HLA antigens on cultured human fibroblasts

It has previously been shown by immunofluorescence experiments that the cross-linking of HLA antigens into patches (by antibody reagents directed to human β₂-microglobulin) on the surfaces of cultured human fibroblasts leads to the lining up of the patches over the actomyosin-containing stress fibers lying immediately under the surface membrane. These experiments have now been extended to the resolution of the electron microscope by the use of ferritin-conjugated antibody. The results show that a substantial part of the HLA surface clusters that form by 5 min after the addition of the antibody reagents is found in small uncoated surface invaginations which are subsequently endocytosed and ultimately fuse with lysosomal bodies. At no stage in this process is there any indication that coated pits or coated vesicles participate. These and other results suggest, therefore, that there are at least two distinct mechanisms for the ligand-induced endocytosis and lysosomal processing of membrane components, one involving coated pits and the other the noncoated invaginations described in this paper. Transmembrane associations of clusters with intracellular actomyosin-containing structures may have a role in the endocytosis of these noncoated invaginations.     

Visualizing caveolin-1 and HDL in cholesterol-loaded aortic endothelial cells

Caveolae are vesicular invaginations of the plasma membranes that regulate signal transduction and transcytosis, as well as cellular cholesterol homeostasis. Our previous studies indicated that the removal of cholesterol from aortic endothelial cells and smooth muscle cells in the presence of HDL is associated with plasmalemmal invaginations and plasmalemmal vesicles. The goal of the present study was to investigate the location and distribution of caveolin-1, the main structural protein component of caveolae, in cholesterol-loaded aortic endothelial cells after HDL incubation. Confocal microscopic analysis demonstrated that the caveolin-1 appeared to colocalize with HDL-fluorescein 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) conjugates on the cell surface. No free HDL-DiI conjugates were revealed in the cytoplasm. Immunoelectron microscopy further demonstrated that caveolin-1 gold (15 nm) conjugates colocalized with HDL gold (10 nm) conjugates in the plasmalemmal invaginations. These morphological results indicated that caveolae are the major membrane domains facilitating the transport of excess cholesterol to HDL on the cell surface of aortic endothelial cells.     

A Highlights from MBoC Selection: Asymmetric formation of coated pits on dorsal and ventral surfaces at the leading edges of motile cells and on protrusions of immobile cells

Clathrin/AP2-coated vesicles are the principal endocytic carriers originating at the plasma membrane. In the experiments reported here, we used spinning-disk confocal and lattice light-sheet microscopy to study the assembly dynamics of coated pits on the dorsal and ventral membranes of migrating U373 glioblastoma cells stably expressing AP2 tagged with enhanced green fluorescence (AP2-EGFP) and on lateral protrusions from immobile SUM159 breast carcinoma cells, gene-edited to express AP2-EGFP. On U373 cells, coated pits initiated on the dorsal membrane at the front of the lamellipodium and at the approximate boundary between the lamellipodium and lamella and continued to grow as they were swept back toward the cell body; coated pits were absent from the corresponding ventral membrane. We observed a similar dorsal/ventral asymmetry on membrane protrusions from SUM159 cells. Stationary coated pits formed and budded on the remainder of the dorsal and ventral surfaces of both types of cells. These observations support a previously proposed model that invokes net membrane deposition at the leading edge due to an imbalance between the endocytic and exocytic membrane flow at the front of a migrating cell.     

Ultrastructural analysis of the organization and distribution of insulin receptors on the surface of 3T3-L1 adipocytes: rapid microaggregation and migration of occupied receptors

Monomeric ferritin-insulin and high-resolution electron microscopic analysis were used to study the organization, distribution, and movement of insulin receptors on differentiated 3T3-L1 adipocytes. Analysis of the binding to prefixed cells showed that insulin initially occupied single and paired receptors preferentially located on microvilli. The majority of receptors (60%) were found as single molecules and 30% were pairs. In 1 min at 37% C, 50% of the receptors on nonfixed cells were found on the intervillous plasma membrane and more than 70% of the total receptors had microaggregated. By 30 min only 7% of the receptors were single or paired molecules on microvilli. The majority were on the intervillous membrane, with 95% of those receptors in groups. The receptor groups on the intervillous plasma membrane could be found in both noncoated invaginations and coated pits. The concentration of occupied receptors in the noncoated invaginations and the coated pits was similar; however, ten times more noncoated invaginations than coated pits contained occupied insulin receptors. The observations in this study contrast with those reported on rat adipocytes using identical techniques (Jarett and Smith, 1977). Insulin receptors on adipocytes were initially grouped and randomly distributed over the entire cell surface and did not microaggregate into larger groups. Insulin receptors on rat adipocytes were found in noncoated invaginations but were excluded from the coated pits. The differences in the organization and behavior of the insulin receptor between rat and 3T3-L1 adipocytes suggest that the mechanisms regulating the initial organization of insulin receptors and the aggregation of occupied receptors may be controlled by tissue-specific processes. Since both of these cell types are equally insulin sensitive, the differences in the initial organization and distribution of the insulin receptors on the cell surface may not be related to the sensitivity or biological responsiveness of these cells to insulin but may affect other processes such as receptor regulation and internalization. On the other hand, the microaggregates of occupied receptors on both cell types may relate to biological responsiveness.     

An internalization-competent influenza hemagglutinin mutant causes the redistribution of AP-2 to existing coated pits and is colocalized with AP-2 in clathrin free clusters

Image correlation spectroscopy and cross correlation spectroscopy were used to demonstrate that approximately 25% of the internalization-competent influenza virus hemagglutinin mutant, HA+8, is colocalized with clathrin and AP-2 at the plasma membrane of intact cells, while wild-type HA (which is excluded from coated pits) does not colocalize with either protein. Clathrin and AP-2 clusters were saturated when HA+8 was overexpressed, and this was accompanied by a redistribution of AP-2 into existing coated pits. However, de novo coated pit formation was not observed. In nontreated cells, the number of clusters of clathrin or AP-2 colocalized with HA+8 was always comparable. Hypertonic treatment which disperses the clathrin lattices resulted in more clusters containing AP-2 and HA+8 than clathrin and HA+8. Less colocalization of HA+8 with clathrin was also observed after cytosol acidification, which causes the formation of deeply invaginated pits, where the HA+8 may be inaccessible to extracellular labeling by antibodies, and blocks coated vesicle budding. However, cytosol acidification elevated the number of clusters containing both HA+8 and AP-2, suggesting an increase in their level of association outside of the deep invaginations. Our results imply that AP-2 and HA+8 can colocalize in clusters devoid of clathrin, at least in cells treated to alter the clathrin lattice structure. Although we cannot ascertain whether this also occurs in untreated cells, we propose that AP-2 binding to membrane proteins carrying internalization signals can occur prior to the binding of AP-2 to clathrin. While such complexes can in principle serve to recruit clathrin for the formation of new coated pits, the higher affinity of the internalization signals for clathrin-associated AP-2 [Rapoport, I., et al. (1997) EMBO J. 16, 2240-2250] makes it more likely that once the AP-2-membrane protein complexes form, they are quickly recruited into existing coated pits.     

Endocytic vesicles and surface invaginations in cultured vascular endothelium: a morphometric comparison

Ruthenium red staining of plasma membrane glycoproteins of confluent cultured arterial endothelial cells revealed that the limiting membrane of many apparently discrete cytoplasmic vesicles was continuous with the plasmalemma. Surface invaginations accessible to ruthenium red appeared as vesicles when sectioned out of the plane of attachment to the cell surface, Morphometric analysis of ruthenium red-positive (RR+) and ruthenium red-negative vesicles (RR-) indicated that 47.2% of the total apparent vesicle population was RR+ and that those infoldings accounted for 19.6 +/- 1.4% of the cell surface in transverse sections. Whereas 14.9% of the true vesicles (ruthenium red-negative) were coated vesicles, only 1.1% of RR+ "vesicles" were coated pits. These studies show that although many deep infoldings of the cell surface may be misinterpreted as vesicles, almost all are uncoated. The existence of discrete coated vesicles (independent of coated pits) in vascular endothelium in vitro is readily apparent.     

Caveolin and its cellular and subcellular immunolocalisation in lung alveolar epithelium: implications for alveolar epithelial type I cell function

Caveolae are flask-shaped invaginations of the plasmalemma which pinch off to form discrete vesicles within the cell cytoplasm. Biochemically, caveolae may be distinguished by the presence of a protein, caveolin, that is the principal component of filaments constituting their striated cytoplasmic coat. Squamous alveolar epithelial type I (ATI) cells, comprising approximately 95% of the surface area of lung alveolar epithelium, possess numerous plasmalemmal invaginations and cytoplasmic vesicles ultrastructurally indicative of caveolae. However, an ultrastructural appearance does not universally imply the biochemical presence of caveolin. This immunocytochemical study has utilised a novel application of confocal laser scanning and electron microscopy unequivocally to localise caveolin-1 to ATI cells. Further, cytoplasmic vesicles and flask-shaped membrane invaginations in the ATI cell were morphologically identified whose membranes were decorated with anti-caveolin-1 immunogold label. Coexistent with this, however, in both ATI and capillary endothelial cells could be seen membrane invaginations morphologically characteristic of caveolae, but which lacked associated caveolin immunogold label. This could reflect a true biochemical heterogeneity in populations of morphologically similar plasmalemmal invaginations or an antigen threshold requirement for labelling. The cuboidal alveolar epithelial type II cell (ATII) also displayed specific label for caveolin-1 but with no ultrastructural evidence for the formation of caveolae. The biochemical association of caveolin with ATI cell vesicles has broad implications for the assignment and further study of ATI cell function.     

Host cell caveolae act as an entry-port for group A streptococci

This study identified caveolae as an entry port for group A streptococci into epithelial and endothelial cells. Scanning electron microscopy as well as ultrathin sections of infected cells demonstrated accumulation of small omega-shaped cavities in the host cell membrane close to adherent streptococci. During invasion, invaginations were formed that subsequently revealed intracellular compartments surrounding streptococci. Caveolin-1 was shown to be present in the membrane of invaginations and the compartment membranes. These compartments were devoid of any classic endosomal/lysosomal marker proteins and can thus be described as caveosomes. Disruption of caveolae with methyl-beta-cyclodextrin and filipin abolished host cell invasion. Importantly, streptococci inside caveosomes avoid fusion with lysosomes. Expressing of SfbI protein on the surface of the non-invasive S. gordonii resulted in identical morphological alterations on the host cell as for S. pyogenes. Incubation of HUVEC cells with purified recombinant sole SfbI protein also triggered accumulation of cavity-like structures and formation of membrane invaginations. Tagged to colloidal gold-particles, SfbI protein was shown to cluster following membrane contact. Thus, our results demonstrate that host cell caveolae initiate the invasion process of group A streptococci and that the streptococcal invasin SfbI is the triggering factor that activates the caveolae-mediated endocytic pathway.