Filopodia are enriched in a cell cohesion molecule of Mr 80,000 and participate in cell-cell contact formation in Dictyostelium discoideum

During the early phase of Dictyostelium discoideum development, cells undergo chemotactic migration to form tight aggregates. A developmentally regulated surface glycoprotein of Mr 80,000 (gp80) has been implicated in mediating the EDTA-resistant type of cell cohesion at this stage. We have used a monoclonal antibody directed against gp80 to study the topographical distribution of gp80 on the cell surface. Indirect immunofluorescence studies showed that gp80 was primarily localized on the cell surface, with a higher concentration at contact areas. Immunoelectron microscopy was carried out by indirect labeling using protein A-gold, and a nonrandom distribution of gp80 was revealed. In addition to contact regions, gold particles were found preferentially localized on filopodia. Quantitative analysis using transmission electron microscopy (TEM) showed that approximately 60% more gold particles were localized in contact regions in comparison with the noncontact regions, and the filopodial surfaces had a twofold higher gold density. Both TEM and scanning electron microscopy showed that contact areas were enriched in filopodial structures. Filopodia often appeared to adhere to either smooth surfaces or similar filopodial structures of an adjacent cell. These observations suggest that the formation of stable cell-cell contacts involves at least four sequential steps in which filopodia and gp80 probably play an important role in the initial stages of recognition and cohesion among cells.     

Junctions between sinusoidal endothelial cells in fetal rat liver.

During a freeze-fracture study of tight junctions in fetal rat liver (Montesano et al., '75) unusual patterns of intramembranous particles were observed in regions of contact between sinusoidal endothelial cells. These patterns were mainly represented by arrays of particles, often associated with linear elevations or crests in the membrane A-face; they may represent abortive forms of tight junctions.     

Orthogonal arrays of particles in non-pigmented cells of rat ciliary epithelium: Relation to distribution of filipin- and digitonin-induced alterations of the basolateral membrane

Summary It has been suggested that orthogonal arrays of particles may increase the rigidity of plasma membrane, as does cholesterol. Therefore, using freeze-fractured non- pigmented ciliary epithelium, the distribution of such arrays was compared to the distribution of membrane deformations induced by the sterol-probes filipin and digitonin in different domains of the basolateral plasma membrane. The distribution of orthogonal arrays of particles was homogeneous between different regions of the basolateral membrane of the non-pigmented ciliary epithelium, while the number of filipin-induced alterations was nearly 4 times higher in the membrane domains not in contact with the basal lamina than in domains in contact with it. Contrary to the homogeneous distribution of arrays, digitonin-induced deformations also differed markedly in these two basolateral membrane domains. Considering that a marked positive response to sterol probes implies a high sterol content, we conclude that orthogonal arrays of particles can occur in plasma membrane regions well-provided with cholesterol and not in direct contact with the basal lamina. Other possible roles of these arrays are discussed.This paper was presented in part at the ARVO meeting, April– May 1986, Sarasota, USA     

Characterization and Distribution of O-Glycosylated Carbohydrates in the Cell Adhesion Molecule, Contact Site A, fromDictyostelium discoideum

This paper presents further investigation of the properties of carbohydrate II in the cell adhesion molecule, contact site A, fromDictyostelium discoideum.A purified contact site A was digested withAchromobacterprotease I to produce a 31-kDa fragment to which carbohydrate II was mainly bound and a 21-kDa fragment containing the NH₂terminus of contact site A, which was identified as Ala-Pro-Thr-Ile-Thr-Ala. The NH₂terminus of the 31-kDa fragment was Thr-Glu-Ala-Thr-Thr-Ser. It was estimated from the cDNA sequence data of contact site A that more than 20 Ser/Thr residues exist as target sites for the O-linked oligosaccharides in the 31-kDa fragment, but not for the N-linked oligosaccharides. These results suggest that carbohydrate II exists as clustered O-linked oligosaccharides in the COOH terminus of contact site A. The results of two-dimensional electrophoresis confirm that oligosaccharides of contact site A contain sialic acids. Immunoelectron microscopy was carried out to define the organelle in which O-glycosylation by carbohydrate II occurs and how carbohydrate II antigens are distributed on the cell surface. The results show that O-glycosylation can occur in the Golgi apparatus inD. discoideumas observed in other cells, although this O-glycosylation was inhibited by tunicamycin. Furthermore, gold particles were densely concentrated in cell–cell contact regions but sparsely distributed in noncontact regions.     

Spreading of wheat germ agglutinin-induced erythrocyte contact by formation of spatially discrete contacts.

The time dependence of agglutination and cell-cell contact spreading in human erythrocytes exposed to wheat germ agglutinin (WGA) was characterized by light and electron microscopy. Cells (3 x 10(7)/mL) had a threshold lectin concentration in the range of 0.6-2.0 micrograms/mL for initial cell contact. Spreading was essentially completed within 60 and 2 min in undisturbed and gently agitated suspensions, respectively. The cells in large WGA agglutinates retained features of their initial disk form in contrast to the convex outlines of polycation or polyethylene glycol-induced agglutinates. Spreading of contact area was accompanied by development of a pattern of discrete contact regions separated by a distance of the order of 1 micron. Freeze fracture electron microscopy and studies with ferritin-labeled WGA showed no significant aggregation of intramembrane particles or specific lectin receptors under conditions when contact spreading occurred. It is argued that flow stress effects on cells in suspended agglutinates give rise to a situation where opposite membranes, at the leading edge of cell contact, are separated by a thin aqueous layer. When this intercellular water layer exceeds a critical length, it becomes unstable. The layer breaks up by surface wave development to form an array of intracellular water spaces. Formation of the aqueous spaces causes opposite membrane regions to move synchronously toward each other. Lectin molecules crosslink the wave crests to give spatially periodic contact points.     

STRUCTURAL AND FUNCTIONAL HETEROGENEITY OF THE SURFACE OF RAT LEUKEMIA CELLS

Rat leukemia cells IRC 741 in suspension culture form single cytoplasmic protrusions by which the cells preferentially adhere to one another. The induction and/or maintenance of these protrusions is sensitive to changes in intercellular contact, pH, temperature, and nutritional conditions. The protrusions are stable, rigid structures which take part in intercellular adhesion but not in adhesion to substrata. Movement on substrata occurs by means of ruffling membranes formed on the main cell body. This asymmetry in cellular form and function is associated with specialized cell surface regions. Ultrastructurally, the cell surface over the protrusions lacks microvilli, and is covered with a 3,000–4,000-Å thick cell coat consisting of 200–500-Å electron-dense particles in an amorphous matrix. In contrast, the surface over the main cell body has microvilli and a 200-Å wide cell coat which lacks particles. The extracellular particles overlying the protrusions have electron-lucent cores, are protease- and pepsin-resistant, and do not stain with colloidal iron, while the matrix in which they are embedded is sensitive to proteolytic enzymes and contains acidic moieties. The negative surface charge density over the protrusions is higher than that over the main cell body, as shown by the orientation of the cells in an electric field. The unexpected observation that a region of higher charge density is one of increased intercellular adhesiveness might be explained, in part, by the rigidity of the protrusions and by the very small radius of curvature of the overlying extracellular particles. The protrusions permit the observation of discrete regions, differing in charge density, on the surface of living leukemia cells.     

LUMENAL PLASMA MEMBRANE OF THE URINARY BLADDER : I. Three-Dimensional Reconstruction from Freeze-Etch Images

To determine the three-dimensional structure of the lumenal membrane of transitional epithelium, a study was made of sectioned, negatively stained, and freeze-etched specimens from intact epithelium and membrane fractions from rabbit urinary bladder. Particulate membrane components are confined to plaque regions within which the unit membrane is asymmetric, having a thicker outer leaflet. Transversely fractured freeze-etched plaques display a thick (~80 A), particulate lumenal leaflet and a thin (~40 A) cytoplasmic one. Four different faces of the two leaflets can be distinguished: two complementary, split, inner membrane faces exposed by freeze-cleaving the bilayer and two external (lumenal and cytoplasmic) membrane surfaces revealed by deep-etching. On the split, inner face of the lumenal leaflet appear polygonal plaques of hexagonally arranged particles. These fit into holes observed on the complementary, split, innerface of the cytoplasmic leaflet. The particles, which have a center-to-center spacing of ~160 A, also seem to protrude from the external surface of the lumenal leaflet, where their subunits (~50 A in diameter) are revealed by freeze-etching and negative staining. The plaques are separated from each other by smooth-surfaced regions, which cleave like simple lipid bilayers. Since the array of plaque particles covers only ~73% of the membrane surface area, whereas 27% is taken up by particle-free interplaque regions, the presence of particles cannot in itself entirely account for the permeability barrier of the lumenal membrane. Although no particles are observed protruding from the cytoplasmic surface of the membrane, cytoplasmic filaments are attached to it by short, cross-bridge-like filaments that seem to contact the particles within the membrane. These long cytoplasmic filaments cross-link adjacent plaques. Therefore, we suggest that at least one function of the particles is to serve as anchoring sites for cytoplasmic filaments, which limit the expansion of the lumenal membrane during distention of the bladder, thereby preventing it from rupturing. The particle-free interplaque regions probably function as hinge areas between the stiff plaques, allowing the membrane to fold up when the bladder is contracted.     

ELECTRON MICROSCOPY OF SYNAPTIC STRUCTURE OF OCTOPUS BRAIN

The well known type of synapse between a presynaptic process containing vesicles and a "clear" postsynaptic process can be commonly observed in the various lobes of the brain of Octopus. The presynaptic vesicles are aggregated near regions of the synaptic membranes which show specialisation and asymmetric "thickening" indicating functional polarisation, and here chemical transmission is presumed to take place. In addition, in the vertical lobe a very interesting serial arrangement of synaptic contacts occurs. Presynaptic bags, formed from varicosities of fibres from the superior frontal lobe, contact the trunks of amacrine cells in the manner just described. The trunks, however, although apparently postsynaptic are themselves packed with synaptic vesicles. The trunks, in turn, make "presynaptic" contacts with clear spinous processes of other neurons of yet undetermined origin. Typical polarised membrane specialisations occur at the contact regions. The trunk vesicles aggregated closest to the contact regions have a shell of particles round their walls. At present, there is no way of telling whether the membrane conductance to the various ions is differently affected at either of the transmission sites, and, if an inhibitory mechanism is involved, whether it is of the presynaptic or postsynaptic variety.     

Significance of exine shedding in Cupressaceae-type pollen

In conifers, which have non-saccate Cupressaceae-type pollen, the pollen must land on a pollination drop or be picked up by the pollination drop from the surface of the cone near the ovule before it can be taken into the ovule. After contact with the drop, the pollen intine absorbs moisture from the drop, expands and the exine is shed. In this study the significance of the shedding of the exine is interpreted from experiments in which simulated pollination drops and micropyles were used to determine the movement of pollen and other particles in suspension. The non-expanded pollen, which can be observed upon contact with the pollination drop, sheds the exine, which then functions as a non-elastic particle, while the pollen from which the exine was shed swells and functions as an elastic particle because it is enclosed by the flexible intine. Non-elastic particles are not easily transferred through narrow passages (the micropyle and micropylar canal) and tend to plug these passages. However, elastic particles, such as the swollen pollen, are easily transferred along narrow passages even when non-elastic particles are present. The simulated experiments demonstrate that exine shedding is an important feature in getting pollen through the narrow micropyle and micropylar canal to the nucellus of the ovule.     

Fluid pressure induces osteoclast differentiation comparably to titanium particles but through a molecular pathway only partly involving TNFα

In contrast to the well-understood inflammatory pathway driven by TNFα, by which implant-derived particles induce bone resorption, little is known about the process in which loosening is generated as a result of force-induced mechanical stimulus at the bone-implant interface. Specifically, there is no knowledge as to what cells or signaling pathways couple mechanical stimuli to bone resorption in context of loosening. We hypothesized that different stimuli, i.e., fluid flow versus wear particles, act through different cytokine networks for activation and localization of osteoclasts. By using an animal model in which osteoclasts and bone resorption were induced by fluid pressure or particles, we were able to detect distinct differences in osteoclast localization and inflammatory gene expression between fluid pressure and titanium particles. Fluid pressure recruits and activates osteoclasts with bone marrow contact away from the fluid pressure exposure zone, whereas titanium particles recruit and activate osteoclasts in areas in direct contact to particles. Fluid pressure induced weaker expression of the selected inflammatory related genes, although the eventual degree of osteoclast induction was similar in both models. Using TNFαRa (4 mg/kg) (Enbrel) and dexamethasone (2 mg/kg) as specific and more general suppressors of inflammation we showed that the TNFαRa failed to generate statistically impaired osteoclast generation while dexamethasone was much more potent. These results demonstrate that fluid pressure induces osteoclasts at a different localization than titanium particles by a molecular pathway less associated with TNFα and the innate system, which open up for other pathways controlling pressure induced osteoclastogenesis.     

Effect on the adhesion and locomotion of mouse fibroblasts by their interacting with differently charged substrates: A quantitative study by ultrastructural method

Attachment of fibroblastic cells to differently charged substrates was observed by electron microscopy. Cells attached with large contact regions to less negatively charged substrates. When the negativity of the substrate charge increased, the total area of the contact regions decreased. The degree of adhesion was estimated by the ‘contact index’, which is the percentage of the contact regions in comparison with the circumference of the cells in vertical sections. The results in this paper show that the contact index has a fine correlation with both the substrate charge and cell locomotion. This indicates that electrostatic interaction at the cell surface is an important factor in controlling cell locomotion and cell adhesion.     

Fouling of nanostructured insect cuticle: adhesion of natural and artificial contaminants

The adhesional properties of contaminating particles of scales of various lengths were investigated for a wide range of micro- and nanostructured insect wing cuticles. The contaminating particles consisted of artificial hydrophilic (silica) and spherical hydrophobic (C(18)) particles, and natural pollen grains. Insect wing cuticle architectures with an open micro-/nanostructure framework demonstrated topographies for minimising solid-solid and solid-liquid contact areas. Such structuring of the wing membranes allows for a variety of removal mechanisms to contend with particle contact, such as wind and self-cleaning droplet interactions. Cuticles exhibiting high contact angles showed considerably lower particle adhesional forces than more hydrophilic insect surfaces. Values as low as 3 nN were recorded in air for silica of ~28 nm in diameter and <25 nN for silica particles 30 μm in diameter. A similar adhesional trend was also observed for contact with pollen particles.     

Convergent setal morphology in sand-covering spiders suggests a design principle for particle capture

Sicarius and Homalonychus are unrelated, desert-dwelling spiders that independently evolved the ability to cover themselves in fine sand particles, making them cryptic against their background. Observations that particles associate with these spiders' setae inspired us to investigate the role of setal microstructure in particle capture and retention. Here we report that Sicarius and Homalonychus convergently evolved numerous high aspect ratio, flexible fibres that we call 'hairlettes' protruding from the setal shaft. We demonstrate that particles attach more densely to regions of Homalonychus with hairlettes than to other regions of the same animal where hairlettes are absent, and document close contact of hairlettes to sand particles that persists after applying force. Mathematical models further suggest that adhesion of hairlettes to sand particles is a sufficient mechanism of particle capture and retention. Together, these data provide the first evidence that hairlettes facilitate sand retention through intermolecular adhesion to particles. Their independent evolutionary origins in Sicarius and Homalonychus suggest that the unique setal structure is adaptive and represents a general biomechanical mechanism for sand capture to cuticle. This discovery has implications for the design of inventions inspired by this system, from camouflage to the management of granular systems.     

Mapping and quantitative analysis of gephyrin cytoplasmic trafficking pathways in motoneurons, using an optimized Transmission Electron Microscopy Color Imaging (TEMCI) procedure

In the present study, an optimized Transmission Electron Microscopy Color Imaging (TEMCI) procedure was used to map and quantify the pathways involved in the trafficking and subcellular targeting of gephyrin in identified abducens motoneurons. Gephyrin is a scaffolding protein, which plays a crucial role in the clustering of the GABA_A and glycine receptors to the cytoskeleton. TEMCI associated several accurate tools: (i) nanogold immunodetection of gephyrin in motoneurons identified on the basis of their immunoreactivity to Choline Acetyl Transferase, (ii) low magnification color scale coding of gephyrin densities on series of ultrathin sections of motoneurons, which gave a map of the cytoplasmic distribution of the protein, (iii) statistical analysis of the subcellular distribution of the immunolabeling. The color map of gephyrin densities in the cell bodies reflected the distribution of inhibitory synapses over the membrane. The TEMCI analysis of motoneurons with various patterns of synaptic covering made it possible to visualize for the first time the cytoplasmic transport pathway of gephyrin towards its target at synaptic contact. A high magnification quantitative analysis, including the study of 109 inhibitory synapses, showed that most gephyrin-associated immunogold particles (67%) were located in the subsynaptic regions facing the active zones, and the second most densely occupied regions were the perisynaptic regions (19.5% of immunogold particles). A consistent proportion of the gephyrin (11.5%), significantly higher than densities present in the rest of the cytoplasm (2%), was detected in the extrasynaptic submembrane region.     

Identification of the dimer interface of the lactose transport protein from Streptococcus thermophilus

The lactose transporter from Streptococcus thermophilus catalyses the symport of galactosides and protons. The carrier domain of the protein harbours the contact sites for dimerization, and the individual subunits in the dimer interact functionally during the transport reaction. As a first step towards the elucidation of the mechanism behind the cooperation between the subunits, regions involved in the dimer interface were determined by oxidative and chemical cross-linking of 12 cysteine substitution mutants. Four positions in the protein were found to be susceptible to intermolecular cross-linking. To ensure that the observed cross-links were not the result of randomly colliding particles, the cross-linking was studied in samples in which either the concentration of LacS in the membrane was varied or the oligomeric state was manipulated. These experiments showed that the cross-links were formed specifically within the dimer. The four regions of the protein located at the dimer interface are close to the extracellular ends of transmembrane segments V and VIII and the intracellular ends of transmembrane segments VI and VII.     

Fouling of nanostructured insect cuticle: adhesion of natural and artificial contaminants

The adhesional properties of contaminating particles of scales of various lengths were investigated for a wide range of micro- and nanostructured insect wing cuticles. The contaminating particles consisted of artificial hydrophilic (silica) and spherical hydrophobic (C₁₈) particles, and natural pollen grains. Insect wing cuticle architectures with an open micro-/nanostructure framework demonstrated topographies for minimising solid–solid and solid–liquid contact areas. Such structuring of the wing membranes allows for a variety of removal mechanisms to contend with particle contact, such as wind and self-cleaning droplet interactions. Cuticles exhibiting high contact angles showed considerably lower particle adhesional forces than more hydrophilic insect surfaces. Values as low as 3 nN were recorded in air for silica of ～28 nm in diameter and <25 nN for silica particles 30 μm in diameter. A similar adhesional trend was also observed for contact with pollen particles.     

Gap junctions between microvilli of an oocyte and follicle cells in the teleost (Plecoglossus altivelis)

In the teleost, Plecoglossus altivelis, intercellular junctions between microvilli of an oocyte and follicle cells were studied by electron microscopy. Microvilli, which were radiated from an oocyte and arrived at the surface of follicle cells, established contact with follicle cells. These contact areas appeared to be a seven-layered membrane with an overall thickness of about 18 microns by standard fixation. In freeze-fracture replicas, many small aggregates of intramembraneous particles were revealed on the cleavage faces of cytoplasmic membranes of follicle cells. These morphological evidences suggest that in the teleost gap junctions exist between the oocyte and follicle cells, especially on the surface of follicle cells.     

Membrane specializations in the paired spermatozoa of dytiscid water beetles

The paired spermatozoa of the dytiscid beetles Dytiscus marginalis and Hydaticus seminiger were studied by electron microscopy with the aim of examining whether the regions of the cell membrane in the zones of sperm conjugation might differ from other regions and to explore whether these cells had any other specialized domains of the cell membrane that could be recognized by the freeze-fracturing technique. The spermatozoa are conjugated along one side of the sperm head and proximal tail portion, called the ventral side. The cell membrane was seen to contain tightly packed intramembranous particles (IMPs) that were predominantly located in the external membrane face (the E-face). In thin sections the cell membrane had a ladder-like appearance at these regions and a specialized type of glycocalyx seen as a fluffy material containing granules. Other specialized membrane domains could also be recorded: a ribbon of particles in the protoplasmic face (P-face) of the dorsal side of the spermatozoon at the proximal tail portion and regularly arranged particle rows in the P-face of the distal tail portion. These domains corresponded to regions where the glycocalyx is prominent. Both the E-face and the P-face of the cell membrane were seen to contain numerous intramembranous particles, which suggests an active function for both membrane leaflets; this is in contrast to the situation in most cells where the particles are mainly in the P-face. The functions of the intramembranous particles in the specialized domains of the cell membrane remains unknown. Some particles may represent receptors or ion gates, others proteins with a mechanical function.     

Bioaerosole – was wir alles einatmen

Bioaerosols Bioaerosols represent essential constituents of the ambient atmosphere and include high numbers of components derived from plants, animals, and microorganisms. The size of these components commonly ranges from several nanometers to hundreds of micrometers. When particles of a bioaerosol establish contact with human subjects, they may be taken up via the respiratory system. Whilst larger particles are chiefly separated from the inhaled air stream in the nose and mouth and, thus, have a negligible chance to penetrate to the lung, small particles are partly characterized by high deposition probabilities in the bronchi and alveoli. High amounts of biogenic material deposited in these regions are again removed by the clearance mechanisms of the lung. Bioaerosol particles are regarded to induce infectious diseases, allergic reactions and coupled insufficiencies as well as toxic reactions.     

Actin and RNA are components of the chromatoid bodies in spermatids of the rat

Chromatoid bodies present in spermatocytes and spermatids of the rat show directed movements around spermatid nuclei during differentiation. This transient organelle contains RNA and establishes contact to intranuclear material and to the acrosomal complex. In order to determine possible components of motility and to verify the presence of RNA, we used a recently developed low-temperature embedding resin combined with protein A-gold and enzyme-gold techniques for studies at the ultrastructural level. All chromatoid bodies analyzed display high concentrations of gold particles over the electron-dense regions when labeled with antiactin. In contrast, RNase-gold particles were localized mainly in the electron-translucent areas. Corresponding controls were always negative. The results suggest a relationship between the impressive motility of the chromatoid body and actin present in the organelle. In addition, specific localization of RNA supports earlier findings that consider the chromatoid body an essential element for differentiation during spermiogenesis.