Endothelial cell-cell junctions maintain endothelial integrity and regulate vascular morphogenesis homeostasis. Cell-cell are usually depicted with a linear morphology along the boundaries between adjacent cells in contact cortical F-actin. However, endothelium, highly dynamic morphologically heterogeneous. We report that can attach to ends of stress fibres instead F-actin, forming structures we name discontinuous adherens (AJ). Discontinuous AJ increased response tumour necrosis factor...

Brain tau protein is phosphorylated in vitro by cdc2 and MAP2 kinases, obtained through immunoaffinity purification from rat brain extracts. The phosphorylation sites are located on the molecule both upstream downstream of tubulin‐binding motifs. A synthetic peptide comprising residues 194–213 sequence, which contains epitope recognized monoclonal antibody tau‐1, also efficiently kinases. Phosphorylation this markedly reduces its interaction with as it has been described for Alzheimer's...

We have analyzed the in vitro phosphorylation of tau protein by Ca2+/calmodulin-dependent kinase, kinase C, CAMP-dependent casein 11, and proline-directed serinelthreonine kinase.These kinases phosphorylate sites localized different regions molecule, as determined peptide mapping analyses.Focusing on it was calculated an incorporation 4 mol phosphate/mol tau.Limited proteolysis assays suggest that could be located within tubulin-binding domain.Direct synthetic peptides corresponding to...

Peptides produced by mild chymotryptic digestion of human erythrocyte protein 4.1 mimic the ability intact to promote binding spectrin F-actin.This complex-promoting activity was found reside in an 8-kDa peptide which fully functional when dissociated from other 4.1-derived peptides, indicating that noncovalent complexes multiple peptides were not essential for activity.The incorporated into a ternary complex with and F-actin approximately stoichiometric amounts.Amino acid composition...

The interaction of actin with a synthetic peptide which corresponds to one the repeated tubulin-binding sites present in tau and MAP-2 (microtubule-associated protein 2) proteins has been analysed. analysis, uses affinity chromatography G-actin on column containing peptide, co-sedimentation co-localization F-actin (as determined by immunoelectron microscopy), indicates that part amino acid sequence involved binding tubulin is also binding.

We have conducted comparative analysis of nucleotide sequences encoding erythroid and lymphoid protein 4.1 isoforms. The isoforms exhibit several sequence motifs that appear to be either inserted into or deleted from the mRNA by alternative splicing a common precursor. One these motifs, located within spectrin-actin binding domain, is found only in cells specifically produced during cell maturation. selective expression alternatively spliced maturation implies existence lineage-specific...

T cell antigen receptor–proximal signaling components, Rho-family GTPases, and formin proteins DIA1 FMNL1 have been implicated in centrosome reorientation to the immunological synapse of lymphocytes. However, role these molecules process is not yet defined. Here we find that a subset microtubules became rapidly stabilized their α-tubulin subunit posttranslationally detyrosinated after engagement receptor. Formation stabilized, required INF2, which was also found be essential for...

The primary cilium is a membrane protrusion that crucial for vertebrate tissue homeostasis and development. Here, we investigated the uncharacterized process of ciliogenesis in polarized epithelial cells. We show after cytokinesis, midbody inherited by one daughter cells as remnant initially locates peripherally at apical surface then moves along and, once proximal to centrosome center surface, enables formation. physical removal greatly impairs ciliogenesis. developed probabilistic cell...

Endothelial barrier dysfunction underlies chronic inflammatory diseases. In searching for new proteins essential to the human endothelial response, we have found that endosomal GTPase RhoB is up-regulated in response cytokines and expressed endothelium of some chronically inflamed tissues. We show although related RhoA RhoC play additive redundant roles various aspects function, specifically inhibits restoration after acute cell contraction by preventing plasma membrane extension. During...

Transcytosis is used alone (e.g., hepatoma HepG2 cells) or in combination with a direct pathway from the Golgi epithelial MDCK as an indirect route for targeting proteins to apical surface. The raft-associated MAL protein essential element of machinery cells. Herein, we present functional characterization MAL2, member family, polarized MAL2 resided selectively rafts and predominantly distributed compartment localized beneath subapical F-actin cytoskeleton. greatly colocalized endosome...

The complete primary structure of the functional site erythrocyte protein 4.1 involved in spectrin-actin associations has been determined. sequence this domain, which contains 67 amino acids and a molecular mass 8045 daltons, obtained by NH2-terminal analysis an 8-kDa chymotryptic peptide, three endoproteinase lysine C-cleaved peptides two Staphylococcus aureus protease V8 cleavage. All including domain peptide were purified reverse-phase high performance liquid chromatography. Antibodies...

The MAL protein is an essential component of the specialized machinery for apical targeting in epithelial cells. src family kinase Lck plays a pivotal role T cell signaling. We show that required cells efficient expression at plasma membrane and activation IL-2 transcription. To investigate mechanism by which regulates targeting, we analyzed dynamics found it travels to specific transport carriers containing MAL. Coimmunoprecipitation experiments indicated association with Lck. Both carrier...

Endothelial cells provide a barrier between the blood and tissues, which is reduced during inflammation to allow selective passage of molecules cells. Adherens junctions (AJ) play central role in regulating this barrier. We aim investigate distinctive 3-dimensional reticular network AJ found endothelium.In endothelial AJ, vascular endothelial-cadherin recruits cytoplasmic proteins β-catenin p120-catenin. binds α-catenin, links actin filaments. are usually described as linear structures along...

We have fractionated tau isoforms by elution at increasing pH values using iron-chelated affinity chromatography, which discriminates between phosphorylated to different extents. Microtubule-associated elutes from the column a gradient narrower than that of total brain tau. Neither under-phosphorylated nor highly are found in microtubule-associated protein preparation. This indicates phosphorylation certain sites is needed for binding microtubules, whereas some other may prevent association....

In this work we have compared the human pancreatic polypeptide (hPP) response to a protein-rich meal (70 g smoked pork loin and 20 white bread) in 21 healthy obese [overweight by 53 ± 2.5% (SEM)] 28 control volunteers of similar ages. The mean fasting hPP concentration group was lower than that normal (32 7 vs. 55 pg/ml; P = 0.031). secretion evoked also smaller individuals, with differences between values both groups ranging from 74–140 pg/ml throughout experimental period. Furthermore,...

Membrane organization into condensed domains or rafts provides molecular platforms for selective recruitment of proteins. Cell migration is a general process that requires spatiotemporal targeting Rac1 to membrane rafts. The protein machinery responsible making competent recruit remains elusive. Some members the MAL family proteins are involved in specialized processes dependent on this type membrane. Because feature plasma all mammalian cells, we hypothesized with ubiquitous expression and...

Protein 4.1 is a major component of the erythrocyte membrane skeleton that promotes interaction spectrin with actin and links resulting complex network to integral proteins. Here we analyse distribution different proteins within nucleus mammalian cells. Nuclear matrices have been prepared from Madin-Darby canine kidney (MDCK) HeLa cells protein fractions isolated at each step purifications analysed by immunoblotting using characterized polyclonal antibodies against 4.1. Two polypeptides M(r)...

Although protein 4.1 was originally identified as an element of the erythrocyte membrane skeleton, its presence in most mammalian cell types is now well described. Antibodies raised against or synthetic peptides corresponding to spectrin-actin-binding domain react with plasma membranes and, unexpectedly, nuclei different types. Nuclear staining further confirmed isolated prepared from rat liver and human leukaemic lines. Immunoblot analysis subcellular fractions derived these cells revealed...

Abstract Analogues of the human erythroid membrane skeletal component protein 4.1 have been identified in perfused rat tissues and T B lymphocyte cell lines, olyclonal antibodies were used which are specific for all domains 4.1, spectrin‐actin‐promoting 8‐Kd peptide, membrane‐binding 30‐Kd domain, 50‐Kd domain. Antibody reactivity, by Western blotting tissue homogenates, shows reactivity with proteins varying molecular weight from 175 Kd to 30 Kd. Further, these analogues appear be expressed...

The endothelium maintains a barrier between blood and tissue that becomes more permeable during inflammation. Membrane rafts are ordered assemblies of cholesterol, glycolipids, proteins modulate proinflammatory cell signaling function. In epithelial cells, the MAL family members MAL, MAL2, myeloid-associated differentiation marker (MYADM) regulate function dynamics membrane domains. We analyzed expression these three in human endothelial cells found only MYADM is expressed. was confined...

The role of formins in microtubules is not well understood. In this study, we have investigated the mechanism by which INF2, a formin mutated degenerative renal and neurological hereditary disorders, controls microtubule acetylation. We found that silencing INF2 epithelial RPE-1 cells produced dramatic drop tubulin acetylation, increased G-actin/F-actin ratio, impaired myocardin-related transcription factor (MRTF)/serum response (SRF)-dependent transcription, known to be repressed levels...

Abstract The primary cilium is a single non-motile protrusion of the plasma membrane most types mammalian cell. structure, length and function must be tightly controlled because their dysfunction associated with disease. Caveolin 1 (Cav1), which best known as component invaginations called caveolae, also present in non-caveolar domains whose beginning to understood. We show that silencing α β Cav1 isoforms different cell lines increases ciliary regardless route ciliogenesis. sole expression...

ABSTRACT Protein 4.1 is a multifunctional polypeptide that links transmembrane proteins with the underlying spectrin/actin cytoskeleton. Recent studies have shown protein also present in nucleus, localized domains enriched splicing factors. Here we further analyze relationship between and components of machinery. Using HeLa nuclear extracts capable supporting pre-mRNAs vitro, show anti-4.1 antibodies specifically immunoprecipitate pre-mRNA intermediates. Immunodepletion from results...