A dileucine motif targets E-cadherin to the basolateral cell surface in Madin-Darby canine kidney and LLC-PK1 epithelial cells

E-cadherin is a major adherens junction protein of epithelial cells, with a central role in cell-cell adhesion and cell polarity. Newly synthesized E-cadherin is targeted to the basolateral cell surface, We analyzed targeting information in the cytoplasmic tail of E-cadherin by utilizing chimeras of E-cadherin fused to the ectodo- main of the interleukin-2 alpha (IL-2 alpha) receptor expressed in Madin-Darby canine kidney and LLC-PK1 epithelial cells, Chimeras containing the full-length or membrane-proximal half of the E-cadherin cytoplasmic tail were correctly targeted to the basolateral domain. Sequence analysis of the membrane-proximal tail region revealed the presence of a highly conserved dileucine motif, which was analyzed as a putative targeting signal by mutagenesis. Elimination of this motif resulted in the loss of Tac/E-cadherin basolateral localization, pinpointing this dileucine signal as being both necessary and sufficient for basolateral targeting of E-cadherin, Truncation mutants unable to bind beta -catenin were correctly targeted, showing, contrary to current understanding, that beta -catenin is not required for basolateral trafficking. Our results also provide evidence that dileucine mediated targeting is maintained in UC-PK, cells despite the altered polarity of basolateral proteins with tyrosine-based signals in this cell line, These results provide the first direct insights into how E-cadherin is targeted to the basolateral membrane.

The surface accessibility of the glycine receptor M2-M3 loop is increased in the channel open state

Mutations in the extracellular M2-M3 loop of the glycine receptor (GlyR) alpha1 subunit have been shown previously to affect channel gating. In this study, the substituted cysteine accessibility method was used to investigate whether a structural rearrangement of the M2-M3 loop accompanies GlyR activation. All residues from R271C to V277C were covalently modified by both positively charged methanethiosulfonate ethyltrimethylammonium (MTSET) and negatively charged methanethiosulfonate ethylsulfonate (MTSES), implying that these residues form an irregular surface loop. The MTSET modification rate of all residues from R271C to K276C was faster in the glycine-bound state than in the unliganded state. MTSES modification of A272C, L274C, and V277C was also faster in the glycine-bound state. These results demonstrate that the surface accessibility of the M2-M3 loop is increased as the channel transitions from the closed to the open state, implying that either the loop itself or an overlying domain moves during channel activation.

Protein targeting to the plasma membrane of adult skeletal muscle fiber: An organized mosaic of functional domains

The plasma membrane of differentiated skeletal muscle fibers comprises the sarcolemma, the transverse (T) tubule network, and the neuromuscular and muscle-tendon junctions. We analyzed the organization of these domains in relation to defined surface markers, beta -dystroglycan, dystrophin, and caveolin-3, These markers were shown to exhibit highly organized arrays along the length of the fiber. Caveolin-3 and beta -dystroglycan/dystrophin showed distinct, but to some extent overlapping, labeling patterns and both markers left transverse tubule openings clear. This labeling pattern revealed microdomains over the entire plasma membrane with the exception of the neuromuscular and muscle-tendon junctions which formed distinct demarcated macrodomains. Our results suggest that the entire plasma membrane of mature muscle comprises a mosaic of T tubule domains together with sareolemmal caveolae and beta -dystroglycan domains. The domains identified with these markers were examined with respect to targeting of viral proteins and other expressed domain-specific markers, We found that each marker protein was targeted to distinct microdomains, The macrodomains were intensely labeled with all our markers. Replacing the cytoplasmic tail of the vesicular stomatitis virus glycoprotein with that of CD4 resulted in retargeting from one domain to another. The domain-specific protein distribution at the muscle cell surface may be generated by targeting pathways requiring specific sorting information but this trafficking is different from the conventional apical-basolateral division. (C) 2001 Academic Press.

Characterization of a distinct plasma membrane macrodomain in differentiated adipocytes

Caveolae are small invaginations of the cell surface that are abundant in mature adipocytes. A recent study (Kanzaki, M., and Pessin, J. E. (2002) J. Biol Chem 277, 25867-25869) described novel caveolin- and actin-containing structures associated with the adipocyte cell surface that contain specific signaling proteins. We have characterized these structures, here termed caves, using light and electron microscopy and observe that they represent surface-connected wide invaginations of the basal plasma membrane that are sometimes many micrometers in diameter. Rather than simply a caveolar domain, these structures contain all elements of the plasma membrane including clathrin-coated pits, lipid raft markers, and non-raft markers. GLUT4 is recruited to caves in response to insulin stimulation. Caves can occupy a significant proportion of the plasma membrane area and are surrounded by cortical actin. Caveolae density in caves is similar to that on the bulk plasma membrane, but because these structures protrude much deeper into the plane of focus of the light microscope molecules such as caveolin and other plasma membrane proteins appear more concentrated in caves. We conclude that the adipocyte surface membrane contains numerous wide invaginations that do not represent novel caveolar structures but rather large surface caves.

Caveolin interacts with the angiotensin II type 1 receptor during exocytic transport but not at the plasma membrane

The mechanisms involved in angiotensin II type 1 receptor (AT(1)-R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT(1)-R is not concentrated in caveolae but is clustered in cholesterol-independent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT(1)-R in caveolae, AT(1)-R. caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT(1)-R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT(1)-R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT(1)-R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT(1)-R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT(1)-R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT(1)-R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT(1)-R through the exocytic pathway, but this does not result in AT(1)-R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT(1)-R.

Direct visualization of Ras proteins in spatially distinct cell surface microdomains

Localization of signaling complexes to specific micro-domains coordinates signal transduction at the plasma membrane. Using immunogold electron microscopy of plasma membrane sheets coupled with spatial point pattern analysis, we have visualized morphologically featureless microdomains including lipid rafts, in situ and at high resolution. We find that an inner-plasma membrane lipid raft marker displays cholesterol-dependent clustering in microdomains with a mean diameter of 44 nm that occupy 35% of the cell surface. Cross-linking an outer-leaflet raft protein results in the redistribution of inner leaflet rafts, but they retain their modular structure. Analysis of Ras microlocalization shows that inactive H-ras is distributed between lipid rafts and a cholesterol-independent micro-domain. Conversely, activated H-ras and K-ras reside predominantly in nonoverlapping, cholesterol-independent microdomains. Galectin-1 stabilizes the association of activated H-ras with these nonraft microdomains, whereas K-ras clustering is supported by farnesylation, but not geranylgeranylation. These results illustrate that the inner plasma membrane comprises a complex mosaic of discrete microdomains. Differential spatial localization within this framework can likely account for the distinct signal outputs from the highly homologous Ras proteins.

Radio frequency plasma-induced hydrogen bonding on kaolinite

The radio frequency (RF) plasma-modified surfaces of kaolinite were investigated by diffuse reflectance infrared Fourier transform spectroscopy (DRIFT) and deuteration techniques to determine the nature of RF plasma-induced surface functional groups, the altered sites in the lattice, and interaction mechanism between RF plasma and the surface of the kaolinite. It has been concluded that the RF plasma-induced infrared (IR) vibration absorption bands at 2805, 3010, and 3100 cm(-1) are attributable to the stretching vibration of hydrogen-bonded hydroxyl groups, and the band at 1407 cm(-1) is attributable to the bending vibration of (HO-)Al-O or (HO-)Si-O groupings with hydrogen-bonded hydroxyl groups. Structural alteration occurred on both the surface and subsurface region of the kaolinite during RF plasma treatment. Further structural alteration or adjustment was also observed on well-modified and well-deuterated kaolinite. There are two types of OD bands visible in the DRIFT spectra of this kaolinite, one type which decreased rapidly as a function of time in moist air, and the other which remained unchanged during the measurement. Furthermore, the appearance of broad IR bands at 3500-3100 cm(-1) as a result of deuteration is evidence of structural disturbance by RF plasma treatment lattice deuteration. An RF plasma-induced hydrogen bonding model on the surface of the kaolinite is proposed.

InOx thin films deposited by plasma assisted evaporation: application in light shutters

An integration of undoped InOx and commercial ITO thin films into laboratory assembled light shutter devices is made. Accordingly, undoped transparent conductive InOx thin films, about 100 nm thick, are deposited by radiofrequency plasma enhanced reactive thermal evaporation (rf-PERTE) of indium teardrops with no intentional heating of the glass substrates. The process of deposition occurs at very low deposition rates (0.1-0.3 nm/s) to establish an optimized reaction between the oxygen plasma and the metal vapor. These films show the following main characteristics: transparency of 87% (wavelength, lambda = 632.8 nm) and sheet resistance of 52 Omega/sq; while on commercial ITO films the transparency was of 92% and sheet resistance of 83 Omega/sq. The InOx thin film surface characterized by AFM shows a uniform grain texture with a root mean square surface roughness of Rq similar to 2.276 nm. In contrast, commercial ITO topography is characterized by two regions: one smoother with Rq similar to 0.973 nm and one with big grains (Rq similar to 3.617 nm). For the shutters assembled using commercial ITO, the light transmission coefficient (Tr) reaches the highest value (Tr-max) of 89% and the lowest (Tr-min) of 1.3% [13], while for the InOx shutters these values are 80.1% and 3.2%, respectively. Regarding the electric field required to achieve 90% of the maximum transmission in the ON state (E-on), the one presented by the devices assembled with commercial ITO coated glasses is 2.41 V/mu m while the one presented by the devices assembled with InOx coated glasses is smaller, 1.77 V/mu m. These results corroborate the device quality that depends on the base materials and fabrication process used. (C) 2014 Elsevier Ltd. All rights reserved.

Chlorella sp. coagulation‐flocculation by inducing a modification on the pH broth medium

Dissertação apresentada à Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para a obtenção do grau de Mestre em Bioenergia

Carbon key-properties for microcystin adsorption in drinking water treatment: structure or surface chemistry?

Dissertação para Obtenção de Grau de Mestre em Engenharia Química e Bioquímica

Characterization of physical, mechanical and chemical properties of quiscal fibres : the influence of atmospheric DBD plasma treatment

This paper reports the first attempt of characterizing various physical, mechanical and chemical properties of Quiscal fibres, used by the native communities in Chile and investigating the influence of atmospheric dielectric barrier discharge plasma treatment on various properties such as diameter and linear density, fat, wax and impurity%, moisture regain, chemical elements and groups, thermal degradation, surface morphology, etc. According to the experimental observations, Quiscal fibre has lower tenacity than most of the technical grade natural fibres such as sisal, hemp, flax, etc., and plasma treatment at optimum dose improved its tenacity to the level of sisal fibres. Plasma treatment also reduced the amount of fat, wax and other foreign impurities present in Quiscal fibres as well as removed lignin and hemicellulose partially from the fibre structure. Plasma treatment led to functionalization of Quiscal fibre surface with chemical groups, as revealed from attenuated total reflection spectroscopy and also confirmed from the elemental analysis using energy dispersive Xray technique and pH and conductivity measurements of fibre aqueous extract. The wetting behavior of Quiscal fibre also improved considerably through plasma treatment. However, untreated and plasma treated Quiscal fibres showed similar thermal degradation behavior, except the final degradation stage, in which plasma treated fibres showed higher stability and incomplete degradation unlike the untreated fibres. The experimental results suggested that the plasma treated Quiscal fibres, like other technical grade natural fibres, can find potential application as reinforcement of composite materials for various industrial applications.

Characterization of physical, mechanical and chemical properties of quiscal fibres: the influence of atmospheric dbd plasma treatment

This paper reports the first attempt of characterizing several physical, mechanical and chemical properties of Quiscal fibres, usually used by the native communities in Chile and on investigations concerning the influence of atmospheric dielectric barrier discharge (DBD) plasma treatment on various properties such as diameter and linear density, percent of impurity, moisture regain, chemical elements and groups, thermal degradation, surface morphology, among others.

O estudo da aplicação de acabamentos funcionais de barreira UV em fibras previamente ativadas por plasma

Tese de Doutoramento Engenharia Têxtil

In vitro phosphorylation as tool for modification of silk and keratin fibrous materials

An overview is given of the recent work on in vitro enzymatic phosphorylation of silk fibroin and human hair keratin. Opposing to many chemical "conventional" approaches, enzymatic phosphorylation is in fact a mild reaction and the treatment falls within "green chemistry" approach. Silk and keratin are not phosphorylated in vivo, but in vitro. This enzyme-driven modification is a major technological breakthrough. Harsh chemical chemicals are avoided, and mild conditions make enzymatic phosphorylation a real "green chemistry" approach. The current communication presents a novel approach stating that enzyme phosphorylation may be used as a tool to modify the surface charge of biocompatible materials such as keratin and silk.

The cell surface of Trypanosoma cruzi

The cell surface of trypanosomatids is formed by the plasma membrane and a layer of sub-pellicular microtubules which are connected to the plasma membrane. The plasma membrane is composed by proteins, lipids and carbohydrates which form the glycocalix. In this paper we will review briefly aspects related to the organization of the cell surface of Trypanosoma cruzi.