Membrane Contact Area Research Articles

Model of cell electrofusion. Membrane electroporation, pore coalescence and percolation.

High electric field impulses (1-20 kV/cm, 1-20 microseconds) may trigger fusion between adhering cells or lipid vesicles (electrofusion). In this paper a qualitative model of electrofusion is proposed consistent with both electron and light microscopic data. Electrofusion is considered as a multistep process comprising tight membrane-contact formation, membrane electroporation as well as an alternating series of subsequent fast collective and slow diffusive fusion stages. The following sequence of steps is suggested: The electric field pulse enforces (via polarization) a tight contact between the membranes of the cells or vesicles to be fused. During tight-contact formation between the opposing membrane surfaces the membrane-adherent water layers are partially squeezed out from the intermembraneous space. Pores are formed in the double membrane contact area (electroporation) involving lateral diffusion and rotation of the lipid molecules in both adhering membrane parts. With increasing pore density, pore-pore interactions lead to short-range coalescence of double membrane pores resulting in ramified cracks; especially small tongues and loops are formed. At supercritical pore density long-range coalescence of the pores occurs (percolation) producing one large double membrane loop (or tongue) and subsequently one large hole in the contact area. After switching off the electric field, the smaller pores, tongues and loops reseal and water flows back into the intermembraneous space of the double membrane in the contact area. As a consequence of the increasing membrane-membrane separation due to water backflow, cooperative rounding of the edges of remaining larger tongues and holes occurs. This results in the formation of an intercellular cytoplasm bridge (channel) concomitant with the disappearance of the contact line between the fusing cells. The membrane parts surrounded by continuous loop-like cracks may separate from the system and may finally form vesicles. Our electrofusion model comprises a strong linkage between the membrane pore formation by high electric fields (electroporation) and the process of electrofusion. Additionally, both pore-pore interactions as well as protein-protein interactions in the contact area of the fusing cells are explicitly introduced. The model provides a qualitative molecular description of basic experimental observations such as the production of membrane fragments, of smaller inside-out vesicles and the formation of larger intercellular cytoplasm bridges.

Increased uterine vascular permeability at the time of embryonic attachment in the pig.

The temporal relationship between embryonic attachment and endometrial vascular permeability was investigated in the gilt. Light and electron microscopy failed to reveal structural differences between Day 10 cycling and pregnant maternal epithelia, including evidence of blastocyst contact. Chorionic adhesion was preserved at mesometrial regions in 3 of 5 Day 13 pregnant animals and appeared to be related to localized differentiation of the underlying maternal epithelium. In order to study uterine vascular permeability, 44 gilts between Days 11 and 19 of the cycle and pregnancy were injected i.v. with a 0.5% solution of Evans Blue (2.5 ml/kg body weight). Examination of excised uteri under ultraviolet light revealed a well-defined zone of endometrial fluorescence corresponding to extravascular content of the dye. Exclusive to pregnant gilts, this response appeared in conjunction with blastocyst elongation at Day 12, and was consistently confined to areas of embryonic membrane contact thereafter. The changes in endometrial morphology and vascular permeability suggest involvement of some embryonic factor(s) acting in a localized manner. Increased histotrophe production is probably facilitated by the flux of plasma constituents to maternal epithelial cells. Coincidence of increased uterine vascular permeability at the site of attachment with elevated blood flow would enhance transport of nutrients toward the conceptus and allow access of blastocyst-induced products to the maternal circulation.

Nonspecific cytotoxic cells in fish ( [formula omitted][formula omitted]) IV. Target cell binding and recycling capacity

The morphology of nonspecific cytotoxic cells (NCC) was identified. NCC were purified by target cell conjugate formation and density gradient separation. NCC are monocyte-like. They have reniform nuclei and a low nucleus/cytoplasm ratio. Cytoplasmic granules were not seen after giemsa staining. Scanning electron microscopy demonstrated moderate surface villi and target cell attachment occurred via long membraneous filament-like surface projections extending to the target cell membranes. Transmission electron microscopy of effector: target cell conjugates revealed membrane contact areas without fusion or fragmentation. The nucleus of the NCC had accentuated peripheral chromation and a prominent Golgi apparatus; the cytoplasm contained osmiophilic granules. Michaelis-Menten and Lineweaver-Burk transformation of target cell binding revealed a Vmax of 11–15,000 and a Km of 40,000. The percentage of NCC bound to target cells was 16–18%. Results of these studies were combined with the conjugate experiment to obtain an estimated percentage of active NCC (5–7%). A maximum recycling capacity of .16–.30 indicated that once attachment by NCC to the target cell occurred (and a lethal signal delivered by an effector cell), either the NCC did not recycle or a long lag period was required to restore its cytotoxic capability.

Free energy potential for aggregation of mixed phosphatidylcholine/phosphatidylserine lipid vesicles in glucose polymer (dextran) solutions

The energetics of lipid vesicle-vesicle aggregation in dextran (36,000 mol wt) solutions have been studied with the use of micromechanical experiments. The affinities (free energy reduction per unit area of contact) for vesicle-vesicle aggregation were determined from measurements of the tension induced in an initially flaccid vesicle membrane as it adhered to another vesicle. The experiments involved controlled aggregation of single vesicles by the following procedure: two giant (approximately 20 micron diam) vesicles were selected from a chamber on the microscope stage that contained the vesicle suspension and transferred to a second chamber that contained a dextran (36,000 mol wt) salt solution (120 mM); the vesicles were then maneuvered into position for contact. One vesicle was aspirated with sufficient suction pressure to create a rigid sphere outside the pipette; the other vesicle was allowed to spread over the rigid vesicle surface. The aggregation potential (affinity) was derived from the membrane tension vs. contact area. Vesicles were formed from mixture of egg lecithin (PC) and phosphatidylserine (PS). For vesicles with a PC/PS ratio of 10:1, the affinity showed a linear increase with concentration of dextran; the values were on the order of 10(-1) ergs/cm2 at 10% by weight in grams. Similarly, pure PC vesicle aggregation was characterized by an affinity value of 1.5 X 10(-1) ergs/cm2 in 10% dextran by weight in grams. In 10% by weight in grams solutions of dextran, the free energy potential for vesicle aggregation decreased as the surface charge (PS) was increased; the affinity extrapolated to zero at a PC/PS ratio of 2:1. When adherent vesicle pairs were transferred into a dextran-free buffer, the vesicles did not spontaneously separate. They maintained adhesive contact until forceably separated, after which they would not read here. Thus, it appears that dextran forms a "cross-bridge" between the vesicle surfaces.

Inhibition of limb chondrogenesis in vitro by vitamin A: Alterations in cell surface characteristics

Mesenchyme cells derived from embryonic mouse limb buds were cultured at high cell density. During the first 24 h in culture, groups of mesenchyme cells condensed and formed cell contacts and specialized junctions. These condensations were the nodule primordia which gave rise to cartilage nodules. The cell contacts were lost as the mesenchyme cells in the primordia developed into cartilage nodules. The mature nodules contained chondrocytes isolated from one another by an extensive extracellular matrix consisting of cartilage type collagen fibrils and proteoglycan granules. The differentiation of the mesenchyme cells to chondrocytes was also characterized by the loss of a 240,000-MW cell surface glycoprotein and the appearance of an 80,000-MW surface protein. The addition of vitamin A to the medium on Day 1 inhibited chondrogenesis. The cells were closely packed together, and the limited extracellular space contained thick, banded collagen fibrils with no proteoglycan granules. The cells exhibited extensive areas of close membrane contact and specialized junctions. Vitamin A-treated cultures also retained the 240,000-MW surface glycoprotein and retarded the appearance of the 80,000-MW cell surface protein. The results of this study suggest that cell surface features normally present on mesenchyme cells are maintained and exaggerated by vitamin A.

Cellular recognition by rat liver cells of neuraminidase-treated erythrocytes: Demonstration and analysis of cell contacts

Freshly isolated rat liver cells adhere firmly to neuraminidase-treated rat or mouse erythrocytes but not to untreated erythrocytes. Binding between cells occurs only in the presence of calcium and is specially inhibited by d-galactose. We therefore suggest that cell adherence is mediated by a galactose-specific hepatic membrane receptor. Ultrastructural analysis of contact regions revealed point-like interactions between hepatic microvilli and erythrocytes and no broad areas of membrane contact. When liver cells are cultivated in vitro they lose their ability to bind erythrocytes within 24 h.

Neuroembryogenesis of ventral hypothalamus transplanted to kidney capsule.

Parts of the floor of the hypothalamus were microsurgically isolated from 12-day rat embryos and transplanted beneath the kidney capsule (KC) of adult hosts. The grafts became vascularized with extensive areas of neural tissue developing from 17 days through 33 days. Host animals were either hypophysectomized or intact, and no significant morphological differences could be detected in the transplants. Neural tissue developed with a high degree of organization, with neurons and glial cells, and an abundance of dendritic and axonal processes clustered among glial processes. Glial processes with junctional attachments formed a complete layer at the basal lamina of the neural tissue which prevented nerve endings from making direct contact with the basal lamina. Small clusters of synaptic vesicles were common in nerve endings and in addition some endings contained synaptic vesicles and 600–900A diameter dense-core granules. Junctional complexes ranged from well-formed synapses of the adult type to areas of membrane contact having minimal specialization. Synapses appeared increased in number, and to assume more mature features, with longer growth periods. The results indicate that the morphologically undifferentiated floor of the hypothalamus of the 12-day rat embryo can undergo morphological differentiation along lines similar to normal development even in an ectopic site such as the KC.

Membrane fusion during secretion. A hypothesis based on electron microscope observation of Phytophthora Palmivora zoospores during encystment.

Interpretation of freeze-fracture and thin-section results shows that fusion of the peripheral vesicle with the plasmalemma of a Phytophthora palmivora zoospore occurs at several discrete sites and results in the formation and expansion of a particle-free bilayer membrane diaphragm and in the appearance of a polymorphic network of membrane-bounded tunnels, the lumina of which are continuous with the cytoplasm. The outer half of the bilayer membrane diaphragm appears continuous with the outer half of the plasma membrane; the inner half of the bilayer membrane diaphragm with the inner half of the peripheral vesicle membrane; and the inner half of the plasmalemma with the outer half of the peripheral vesicle membrane. Interpretation of our results leads us to formulate a hypothesis for a sequence of several intermediate stages involved in membrane fusion. The initial fusion event is viewed as a local catastrophe (Thom, R. 1972. Stabilité Structurelle et Morphogenèse. W. A. Benjamin Inc., Reading, Mass.) involving the sudden reorganization of apposed elements of the inner half of the plasmalemma and the outer half of the peripheral vesicle membrane. Fusion of apposed components at the rim of the perimeter of fusion results in the formation of a toroid hemi-micelle which provides continuity between the inner half of the plasmalemma and the outer half of the peripheral vesicle membrane. Simultaneously, apposed components at the site of fusion may reorganize into an inverted membrane micelle. A bilayer membrane diaphragm is then formed by apposition and flowing of components form the outer half of the plasmalemma and the inner (exoplasmic) half of the peripheral vesicle membrane. The existence of large areas of membrane contact before fusion may lead to several fusion events and the formation of a polymorphic network of membrane-bound tunnels.

Surface properties of adult rat hepatocytes: Mechanism of increased concanavalin A-induced agglutinability following papain digestion

Adult rat hepatocytes, obtained by collagenase perfusion, exhibit a 4-fold increase in concanavalin A (ConA)-induced agglutination following papain digestion. Several parameters which may be responsible for this altered ConA-induced agglutination have been examined, namely the amount and topography of surface-bound ConA and the morphology of the cell-surface. Binding studies using [ 125I]ConA demonstrated that the amount of surface-bound ConA was the same for papain-digested and incubated control hepatocytes. Surface binding of ferritin-conjugated ConA revealed that both the incubated control and papain-digested hepatocytes possessed a similar clustered distribution of surface-bound ConA. The most significant difference between the incubated control and papain-digested hepatocytes was an alteration in the surface morphology of papain-digested cells. Cells incubated in the absence of papain maintained the surface morphology of the non-incubated hepatocyte, i.e., a surface characterized by numerous microvilli; whereas the papain-digested cells exhibited a smooth surface. These results indicate that the surface morphology of the papain-treated hepatocytes allows greater areas of membrane contact, thereby increasing their agglutinability by ConA.

Ultrastructure of cell-mediated immunity. I. Comparison between E and EA rosettes.

The ultrastructural comparison between E and EA rosettes showed that, in the former, the rosetting lymphocytes are mostly round in shape and their interaction with sheep erythrocytes only consists of limited areas of membrane contact, in the latter, rosetting lymphocytes are mostly in the shape of uropods and surrounding ox red cells show pseudopods protruding towards the lymphocyte and coming into contact with it.

A study of the ultrastructure of the small iris vessels in the vervet monkey (Ceropithecus aethiops).

Eyes of vervets were fixed by several methods, and the iris capillaries were studied by electron microscopy. The capillaries have a continuous endothelium without fenestrae. Tight junctions are always present in intercellular clefts of the endothelium, and marginal folds are frequent. A rather thick basement membrane is present, similar to what is found in the human iris. Pericytes are frequent, and specialized areas of membrane contact between endothelium and pericytes are described. Peculiar marginal vacuoles are found in the endothelium after perfusion with hypertonic fixative.

The fine structure of the mouse odontoblast

A study of the fine structure of the odontoblasts of young mice was undertaken in order to further elucidate the secretory role of this cell in the production of dentine. The observations suggested that fully differentiated odontoblasts possess all of the cytoplasmic characteristics of cells engaged in the production of proteins for external use. A large well developed granular endoplasmic reticulum occupied the greater portion of the cytoplasm. Transitional vesicles originating from the cisternae of the endoplasmic reticulum appeared to give rise to the forming face of the Golgi apparatus by a process of fusion. There was evidence that coated vesicles derived from the mature face of the Golgi apparatus fused to produce dense granules and multivesicular bodies in the vicinity of the Golgi complex, while other coated vesicles migrated to and accumulated in the secretory pole of the cell, i.e. the odontoblastic process. Dense bodies containing distinct crystalloid inclusions were also observed in the area of the Golgi complex. Fusion of coated vesicles with the cell membrane of the odontoblastic process in the region of the predentine matrix was a frequent finding. Observations suggest that coated vesicles play a role in segregation of material by the Golgi complex and in secretion of dentinal collagen and ground substance. The presence of specialized areas of membrane contact (tight junctions) between adjacent odontoblasts were regularly observed.