Dynamic interplay between adhesive and lateral E-cadherin dimers

E-cadherin, an adhesive transmembrane protein of epithelial adherens junctions, forms two types of detergent-resistant dimers: adhesive dimers consisting of cadherin molecules derived from two neighboring cells and lateral dimers incorporating cadherins of the same cell. Both dimers depend on the integrity of the same residue, Trp156. While the relative amounts of these complexes are not certain, we show here that in epithelial A-431 cells, adhesive dimers may be a prevalent form. Inactivation of the calcium-binding sites, located between successive cadherin ectodomains, drastically reduced the amount of adhesive dimers and concomitantly increased the amount of lateral dimers. A similar interdependence of adhesive and lateral dimers was observed in digitonin-permeabilized cells. In these cells, adhesive dimers immediately disassembled after lowering the Ca2+ concentration below 0.1 mM. The disappearance of adhesive dimers was counterbalanced by an increase in Trp156-dependent lateral dimers. Increasing the calcium concentration to a normal level rapidly restored the original balance between adhesive and lateral dimers. We also present evidence that E-cadherin dimers in vivo have a short lifetime. These observations suggest that cadherin-mediated adhesion is based on the dynamic cycling of E-cadherin between monomeric and adhesive dimer states.     

Dynamics and stability of E-cadherin dimers

The extracellular domains of cadherins are known to play a major role in cell adhesion, although the structures involved in this process remain unclear. We have used molecular dynamics to characterize the conformational and thermodynamic properties of two of the dimer interfaces identified in E-cadherin crystals and involving the two outermost exodomains (EC1 and EC2): a dimer involving exchange of the N-terminal strand (referred to as the "swapped" dimer) and a "staggered" dimer involving an EC1-EC2 interface. The results show that the staggered dimer involves a much smaller interface area and is notably less stable than the swapped dimer. It is also found that, despite its stability, the swapped dimer undergoes a conformational transition leading to a structure closer to that experimentally observed for the homologous C-cadherin. Finally, comparing the simulated dimer structures with the sequences of E-, C-, and N-cadherins shows that the swapped dimer interface involves surprisingly few residues that vary from family to family and notably no changes between the E- and C-cadherin exodomains.     

Lipid-cell interactions: Liposome adsorption and cell-to-liposome lipid transfer are mediated by the same cell-surface sites

The competitive behavior of solid vs. fluid liposomes in liposome-to-cell adsorption and cell-to-liposome lipid transfer processes was investigated with L cells and FBT epithelial sheets. Binding, transfer and ³¹P-NMR experiments have demonstrated that: (i) solid liposomes adhere to the cell surface as integral vesicles retaining the entrapped substances; (ii) fluid liposomes are partly disintegrated at the cell surface with concomitant entry of entrapped substances into the cytoplasm, while their lipids remain on the cell surface; (iii) fluid liposomes that escape lysis dissociate from the cell, taking away cell lipid molecules. The latter process underlies the mechanism of cell-to-fluid liposome lipid transfer. In contrast, no lipid transfer occurs between the plasma membrane and solid liposomes. Cell-bound solid liposomes interfere with the transfer of cell lipids to fluid liposomes, while these in turn inhibit the binding of solid liposomes to the cell surface. Moreover, cell-induced aggregation of both fluid and solid freshly added liposomes is also inhibited by preincubation of the cells with either solid or fluid liposomes. Thus, different types of interaction of both fluid and solid liposomes with the cell are mediated by the same (or closely related) sites on the cell surface.     

Acetylcholinesterase H and T dimers are associated through the same contact. Mutations at this interface interfere with the C-terminal T peptide, inducing degradation rather than secretion.

Acetylcholinesterase (AChE) exists as AChE(H) and AChE(T) subunits, which differ by their C-terminal H or T peptides, generating glycophosphatidylinositol-anchored dimers and various oligomers, respectively. We introduced mutations in the four-helix bundle interface of glycophosphatidylinositol-anchored dimers, and analyzed their effect on the production and oligomerization of AChE(H), of AChE(T), and of truncated subunits, AChE(C) (without H or T peptide). Dimerization was reduced for all types of subunits, showing that they interact through the same contact zone; the formation of amphiphilic tetramers (Torpedo AChE(T)) and 13.5 S oligomers (rat AChE(T)) was also suppressed. Oligomerization appeared totally blocked by introduction of an N-linked glycan on the surface of helix alpha(7,8). Other point mutations did not affect the synthesis or the catalytic properties of AChE but reduced or blocked the secretion of AChE(T) subunits. Secretion of AChE(T) was partially restored by co-expression with Q(N), a secretable protein containing a proline-rich attachment domain (PRAD); Q(N) organized PRAD-linked tetramers, except for the N-glycosylated mutants. Thus, the simultaneous presence of an abnormal four-helix bundle zone and an exposed T peptide targeted the enzyme toward degradation, indicating a cross-talk between the catalytic and tetramerization domains.     

Evidence that natural cytotoxicity and antibody-dependent cellular cytotoxicity are mediated in humans by the same effector cell populations.

The present study strongly suggests that, in humans, natural killer (NK) activity and antibody-dependent cell-mediated cytotoxicity (ADCC) are mediated by the same effector cell population. This is supported by two different experimental approaches. First, competition for NK effector cells was accompanied by simultaneous inhibition of ADCC activity. Target cells sensitive to NK activity were capable of inhibiting specifically an ADCC assay in cold target competition experiments. Second, specific removal of NK cells on monolayers formed by target cells sensitive to NK activity caused simultaneous depletion of ADCC effector cells. In association with the removal on the monolayers of effector cells for ADCC as well as NK activity, we also found a significant depletion of cells bearing Fc gamma receptors.     

Insulin action on adipocytes. Evidence that the anti-lipolytic and lipogenic effects of insulin are mediated by the same receptor.

1. The dose-response relationships of insulin stimulation of lipogenesis and inhibition of lipolysis were studied simultaneously by using rat adipocytes to determine whether these different effects of insulin are mediated through the same or different sets of receptors. 2. The sensitivity (defined as the concentration of insulin required to produce a half-maximal effect) of the stimulated lipogenic response to insulin was not significantly different from the sensitivity of the anti-lipolytic response to insulin. The addition of different adrenaline and glucose concentrations did not alter the half-maximal concentration of insulin required to inhibit lipolysis. 3. The specificities of the lipogenic and antilipolytic responses were studied by using insulin analogues. The sensitivities of the lipogenic and anti-lipolytic responses were the same for five chemically modified insulins and hagfish insulin, which have potencies compared with bovine insulin of between 3 and 90%. 4. Starving rats for 48h significantly increased the sensitivities of both the antilipolytic and lipogenic responses to insulin, but the changes in the sensitivities of both lipogenesis and anti-lipolysis returned to that of fed rats. 5. We conclude that insulin stimulates lipogenesis and inhibits lipolysis over the same concentration range. These observations provide powerful evidence that the different effects of insulin are mediated through the same set of receptors.     

Crown ether mediated cadmium halide dimers and polymers

The reactions of cadmium halides with the 15-membered macrocyclic crown ethers, 15-crown-5 and benzo-15-crown-5, have been carried out and six new complexes have been isolated and structurally characterized. Metal to ligand stoichiometries of 1:1, 2:1, 3:1 and 3:2 have been observed with a variety of different formulations. Examples of charge separated ion pairs ([(NH₄)(benzo-15-crown-5)₂]₂[Cd₂I₆]), halogen bridged monomers, dimers or polymers ([Cd(15-crown-5)(OHMe)(μ-Br)CdBr₃], [Cd(15-crown-5)(μ-Br)₂CdBr(μ-Br)]₂(isolated from the same reaction mixture) and [(CdCl₂)₂CdCl₂(15-crown-5)]_n), and hydrogen bonded finite chains or polymers ([(Cd(OH₂)₂(15-crown-5)][CdI₃(OH₂)]₂·2(15-crown-5)·2CH₃CN and [CdI₂(OH₂)₂(THF)]·benzo-15-crown-5) have been isolated. Three different types of 15-crown-5 coordination modes have been observed in these complexes. In-cavity coordination resulting in pentagonal bipyramidal geometries about Cd²⁺ was observed in [(CdCl₂)₂CdCl₂(15-crown-5)]_n, [Cd(15-crown-5)(OHMe)(μ-Br)CdBr₃], and [Cd(OH₂)₂(15-crown-5)][CdI₃(OH₂)]₂·2(15-crown-5)·2CH₃CN, [Cd(15-crown-5)(μ-Br)₂CdBr(μ-Br)]₂ displays out-of-cavity coordination with one etheric donor distorted into an axial position of a distorted pentagonal bipyramid. The third coordination mode is secondary sphere coordination via hydrogen bonding which is observed for [Cd(OH₂)₂(15-crown-5)][CdI₃(OH₂)]₂·2(15-crown-5)·2CH₃CN. The good fit of Cd²⁺ within the cavity of 15-crown-5 results in shorter bonding contacts and a more narrow distribution in Cd---O values (2.273(7)-2.344(6) Å) than observed for cadmium halide complexes of 18-crown-6 (Cd---O = 2.69(1)–2.81(1) Å).     

Multiple PV1 dimers reside in the same stomatal or fenestral diaphragm

Several of the endothelium-specific structures that have been involved in microvascular permeability [such as caveolae, transendothelial channels (TECs), vesiculovacuolar organelles (VVOs), and fenestrae] can be provided with either a stomatal or fenestral diaphragm. In the case of fenestrae, the diaphragm has the presumed function of creating a permselective barrier for solutes from blood plasma and interstitium. PV1 is an endothelium-specific integral membrane glycoprotein that is associated with both the stomatal diaphragms of caveolae, TECs, and VVOs as well as the diaphragms of endothelial fenestrae. The intimate structure of these diaphragms has been shown to consist of a meshwork formed by radial fibrils. We have recently shown that PV1 is a key structural element of both types of diaphragms, with its expression being sufficient to form de novo stomatal and fenestral diaphragms in both endothelial and nonendothelial cell types in culture. We have further tested the role of PV1 in the structure of the diaphragms and demonstrate here that multiple PV1 homodimers reside in close proximity within the same diaphragm. Our data bring further support to the paradigm by which PV1 dimers would form the fibrils of the diaphragms with a function in the microvascular permeability.     

Redistribution of soil water by lateral roots mediated by stem tissues

Evidence is increasing to suggest that a major activity of roots is to redistribute soil water. Roots in hydraulic contact with soil generally either absorb or lose water, depending on the direction of the gradient in water potential between root and soil. This leads to phenomena such as "hydraulic lift" where dry upper soil layers drive water transfer from deep moist layers to the shallow rhizosphere and, after rain or surface irrigation, an opposite, downward water transfer. These transport processes appear important in environments where rainfall is strongly seasonal (e.g. Mediterranean-type climates). Irrigation can also induce horizontal transfers of water between lateral roots. Compared with transpiration, the magnitudes, pathways, and resistances of these redistribution processes are poorly understood. Field evidence from semi-arid eucalyptus woodlands is presented to show: (i) water is rapidly exchanged among lateral roots following rain events, at rates much faster than previously described for other types of hydraulic redistribution using sap flow methods; (ii) large axial flows moving vertically up or down the stem are associated with the horizontal transfer of water between roots on opposite sides of the stem. It appears that considerable portions of the stem axis become involved in the redistribution of water between lateral roots because of partial sectoring of the xylem around the circumference of these trees.     

Antiport mediated rounding and endocytosis are enhanced by sulphate

Rapid cell detachment concomitant with the flat-to-round (FTR) change that is mediated by an upshifted Na+/H+ antiporter via HCO3(-)-dependent H+ pumping, is significantly enhanced by the addition of Na2SO4 (FTR + SO4): (1) a faster and greater reduction in cell surface area and perimeter, and (2) a higher level of macromolecular internalization which is also amiloride sensitive. At a fixed 1 mg/ml extracellular FITC-dextran (FDx) concentration, the intracellular FDx load is similar irrespective of the particle size, in the range from 4400 to 2 million mol.wt which is a 455-fold diversity. This is inconsistent with entry via limited sized portals which would discriminate against the larger molecular weight species, such as the 2 million mol.wt species that measures up to 5 microns in width. Two million mol.wt FDx loads linearly in direct proportion to the extracellular FDx concentration, simulating simple diffusion. Large-channel endocytosis is considered to be a characteristic of specialized cell types such as phagocytes and macrophages. However, the antiporter mediated endocytosis (AME) shown here is demonstrated in two different cell types which are not known for their endocytic prowess, viz. epitheloid human Chang liver cells (ATCC CCL 13) and human lung fibroblasts (ATCC CCL 202). The rounded cells with internalized FDx start reverting back to their flat and protracted form upon flooding with warm growth medium, a round-to-flat (RTF) change. However the cell surface reversion is not associated with efflux of FDx which are sorted out into 'granular patches', the later stage endosomes without membrane outlines in AME. FDx-loaded cells grow as well as trypsinized cells without FDx loaing and they maintain a significant FDx load even after nearly 4 cell divisions. Toad sperms internalized into Chang cells via antiporter activation are also sorted into granular patches. AME provides (a) distinctive access to large particles, simulating small ion influx, and (b) an alternate membrane recycling capability where granular patches are instrumental in sorting. It appears to be not a simple endocytosis-exocytosis pathway.     

Olfactory functions are mediated by parallel and hierarchical processing

How the human brain processes the perception, discrimination, and recognition of odors has not been systematically explored. Cerebral activations were therefore studied with PET during five different olfactory tasks: monorhinal smelling of odorless air (AS), single odors (OS), discrimination of odor intensity (OD-i), discrimination of odor quality (OD-q), and odor recognition memory (OM). OS activated amygdala-piriform, orbitofrontal, insular, and cingulate cortices and right thalamus. OD-i and OD-q both engaged left insula and right cerebellum. OD-q also involved other areas, including right caudate and subiculum. OM did not activate the insula, but instead, the piriform cortex. With the exception of caudate and subiculum, it shared the remaining activations with the OD-q, and engaged, in addition, the temporal and parietal cortices. These findings indicate that olfactory functions are organized in a parallel and hierarchical manner.     

Cell-cell contacts mediated by E-cadherin (uvomorulin) restrict invasive behavior of L-cells

L-cells were cotransfected with plasmids coding for mouse E-cadherin (uvomorulin) and the neophosphotransferase gene, and stable transfectants expressing E-cadherin at the cell surface were selected and cloned. Control transfection was done with the neophosphotransferase gene alone. The invasive migration of transfected and untransfected L-cells into three-dimensional collagen gels was then analyzed. L-cells not expressing E-cadherin migrated efficiently into the gels, whereas invasion of the E-cadherin-expressing L-cells was restricted in a cell density dependent manner. At sparse density, when the cells exhibited little cell-cell contacts, no difference was observed between the level of invasion of the cadherin-expressing cells and the control cells. However, with increasing cell density, decreasing amounts of the cadherin-expressing cells but increasing amounts of the control cells migrated into the gels. At confluent density hardly any cadherin-expressing cells were able to migrate into the gels. The inhibition of the invasion of the cadherin-expressing cells could be reverted if confluent cells were cultured in the presence of monoclonal antibodies against E-cadherin. Since the expression of E-cadherin did not influence the invasive mobility of single cells, these results indicate that E-cadherin-mediated cell-cell contacts inhibited invasive cellular migration. Time-lapse videoscopy and studies of cell migration from a monolayer into a cell-free area demonstrated that the restricted invasion could be explained by contact inhibition of cell movement of the cadherin-expressing cells.     

Adhesive But Not Lateral E-cadherin Complexes Require Calcium and Catenins for Their Formation

We examined intercadherin interactions in epithelial A-431 cells producing endogenous E-cadherin and recombinant forms of E-cadherin tagged either by myc or by flag epitopes. Three distinct E-cadherin complexes were found. The first is a conventional E-cadherin–catenin complex consisting of one E-cadherin molecule linked either to β-catenin/α-catenin or to plakoglobin/α-catenin dimers. The second is a lateral E-cadherin complex incorporating two E-cadherin– catenin conventional complexes combined in parallel fashion via dimerization of the NH₂-terminal extracellular domain of E-cadherin. The third complex is likely to contain two E-cadherin–catenin conventional complexes derived from two opposing cells and arranged in an antiparallel fashion. Formation of the antiparallel but not lateral complex strictly depends on extracellular calcium and E-cadherin binding to catenins. Double amino acid substitution Trp156Ala/Val157Gly within the extracellular NH₂-terminal E-cadherin domain completely abolished both lateral and antiparallel inter–E-cadherin association. These data support an idea that the antiparallel complex has the adhesion function. Furthermore, they allow us to suggest that antiparallel complexes derive from lateral dimers and this complex process requires catenins and calcium ions.     

Antiparallel dimers of the small multidrug resistance protein EmrE are more stable than parallel dimers

The bacterial multidrug transporter EmrE is a dual-topology membrane protein and as such is able to insert into the membrane in two opposite orientations. The functional form of EmrE is a homodimer; however, the relative orientation of the subunits in the dimer is under debate. Using EmrE variants with fixed, opposite orientations in the membrane, we now show that, although the proteins are able to form parallel dimers, an antiparallel organization of the subunits in the dimer is preferred. Blue-native PAGE analyses of intact oligomers and disulfide cross-linking demonstrate that in membranes, the proteins form parallel dimers only if no oppositely orientated partner is present. Co-expression of oppositely orientated proteins almost exclusively yields antiparallel dimers. Finally, parallel dimers can be disrupted and converted into antiparallel dimers by heating of detergent-solubilized protein. Importantly, in vivo function is correlated clearly to the presence of antiparallel dimers. Our results suggest that an antiparallel arrangement of the subunits in the dimer is more stable than a parallel organization and likely corresponds to the functional form of the protein.     

Glycoprotein tertiary and quaternary structures are monitored by the same quality control mechanism

Folding of glycoproteins entering the secretory pathway is strictly surveyed in the endoplasmic reticulum by a quality control system. Folding intermediates and proteins irreparably misfolded are marked via glucosylation by the UDPglucose:glycoprotein glucosyltransferase, an enzyme that acts as a folding sensor by exclusively labeling glycoproteins not displaying their native structures. Here we show that this sensing mechanism also applies to the oligomerization of protein complexes, as the glucosyltransferase appeared to be able to glucosylate folded complex subunits lacking the full complement of oligomer components.