Smooth muscle adherens junctions associated proteins are stable at the cell periphery during relaxation and activation

2005 ◽  
Vol 289 (6) ◽  
pp. C1379-C1387 ◽  
Author(s):  
Thomas J. Eddinger ◽  
Jessen D. Schiebout ◽  
Darl R. Swartz
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Adherens Junction ◽  
Rapid Freezing ◽  
Cellular Distribution ◽  
Cell Periphery ◽  
Tissue Sections ◽  
Associated Proteins ◽  
Punctate Pattern ◽  
The Stability

This study was performed to determine the stability of the adherens junction (AJ)-associated proteins at the smooth muscle cell (SMC) plasma membrane during relaxing and activating conditions. Dog stomach, ileum, colon, and trachea tissues were stored in Ca2+-free PSS or regular PSS or were activated in 10 μM carbachol in PSS before rapid freezing. The tissues were subsequently sectioned and immunoreacted using antibodies for vinculin, talin, fibronectin, and caveolin to determine their cellular distribution in these tissues under these conditions. In all four tissues and under all three conditions, the distribution of these four proteins remained localized to the periphery of the cell. In transverse tissue sections, the AJ-associated proteins formed a distinct punctate pattern around the periphery of the SMCs at the plasma membrane. These domains alternated with the caveolae (as identified by the presence of caveolin). In longitudinal tissue sections, the AJ-associated proteins formed continuous tracks or staves, while the caveolae remained punctate in this dimension as well. Caveolin is not present in the tapered ends of the SMCs, where the AJ-associated proteins appear continuous around the periphery. Densitometry of the fluorophore distribution of these proteins showed no shift in their localization from the SMC periphery when the tissues were relaxed or when they were activated before freezing. These results suggest that under physiologically relaxing and activating conditions, AJ-associated proteins remain stably localized at the plasma membrane.

scholarly journals The phagocytosis oxidase/Bem1p (PB1) domain-containing protein PB1CP negatively regulates the NADPH oxidase RBOHD in plant immunity

2020 ◽  
Author(s):  
Yukihisa Goto ◽  
Noriko Maki ◽  
Jan Sklenar ◽  
Paul Derbyshire ◽  
Frank L.H. Menke ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Stomatal Closure ◽  
Defense Responses ◽  
Mass Spectrometry Analysis ◽  
Regulation Mechanism ◽  
Cell Periphery ◽  
List Type ◽  
Colletotrichum Higginsianum ◽  
Associated Proteins ◽  
Pb1 Domain

SummaryPerception of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern-recognition receptors activates RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) through direct phosphorylation by BOTRYTIS-INDUCED KINASE 1 (BIK1) and induces the production of reactive oxygen species (ROS). ROS have direct antimicrobial properties but also serve as signaling molecules to activate additional defense responses such as stomatal closure. RBOHD activity must be tightly controlled to avoid the detrimental effects of ROS, but little is known about RBOHD downregulation.To better understand the regulation of RBOHD, we used co-immunoprecipitation of RBOHD coupled with mass spectrometry analysis to identify RBOHD-associated proteins.Among RBOHD-associated proteins, we identified PHAGOCYTOSIS OXIDASE/ BEM1P (PB1) DOMAIN-CONTAINING PROTEIN (PB1CP). We found that PB1CP negatively regulates RBOHD and the resistance against the fungal pathogen Colletotrichum higginsianum. PB1CP directly interacts with RBOHD in vitro, and PAMP treatment increases the interaction in vivo. PB1CP is localized at the cell periphery and in cytoplasm, but PAMP treatment induces PB1CP relocalization to small endomembrane compartments. PB1CP overexpression reduces plasma membrane localization of RBOHD, suggesting that PB1CP down-regulates RBOHD function by relocalizing it away from the plasma membrane.We reveal a novel negative regulation mechanism of ROS production through the relocalization of RBOHD by PB1CP.

scholarly journals Domain architecture of the smooth-muscle plasma membrane: regulation by annexins

2005 ◽  
Vol 387 (2) ◽  
pp. 309-314 ◽  
Author(s):  
Annette DRAEGER ◽  
Susan WRAY ◽  
Eduard B. BABIYCHUK
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Membrane Lipids ◽  
Domain Architecture ◽  
Focal Adhesions ◽  
Membrane Lipid ◽  
Dependent Manner ◽  
Detailed Structural Analysis ◽  
Specific Subset ◽  
Associated Proteins

Individual signalling events are processed in distinct, spatially segregated domains of the plasma membrane. In a smooth muscle, the sarcolemma is divided into domains of focal adhesions alternating with caveolae-rich zones, both harbouring a specific subset of membrane-associated proteins. Recently, we have demonstrated that the sarcolemmal lipids are similarly segregated into domains of cholesterol-rich lipid rafts and glycerophospholipid-rich non-raft regions. In the present study, we provide a detailed structural analysis of the relationship between these proteinaceous and lipid domains. We demonstrate that the segregation of plasmalemmal protein constituents is intimately linked to that of the membrane lipids. Our results imply that lipid segregation is critical for the preservation of membrane protein architecture and essential for directional translocation of proteins to the sarcolemma. We show that the membrane lipid segregation is supported by the annexin protein family in a Ca2+-dependent manner. Eukaryotic cells harbour numerous, tissue-specific subsets of annexins. By examining the significance of this variety in a smooth muscle, we demonstrate that four different annexins target membrane sites of distinct lipid composition and that each annexin requires a different [Ca2+] for its translocation to the sarcolemma. Our results suggest that the interactions of annexins with distinct plasma membrane regions promote membrane segregation and, in combination with their individual Ca2+ sensitivity, might allow a spatially confined, graded response to a multitude of extra- or intracellular stimuli.

Adherens junction-associated protein distribution differs in smooth muscle tissue and acutely isolated cells

2007 ◽  
Vol 292 (2) ◽  
pp. G684-G697 ◽  
Author(s):  
Thomas J. Eddinger ◽  
Jessen D. Schiebout ◽  
Darl R. Swartz
Keyword(s):  
Smooth Muscle ◽  
Intracellular Signaling ◽  
Adherens Junction ◽  
Force Production ◽  
Enzymatic Digestion ◽  
Protein Distribution ◽  
Isolated Cells ◽  
Smooth Muscle Tissue ◽  
Associated Proteins ◽  
The Impact

This study was designed to examine how smooth muscle (SM) cell (SMC) isolation affects the distribution of some adherens junction (AJ) complex-associated proteins. Immunofluorescence procedures for identifying protein distribution were used on gastrointestinal and tracheal SM tissues and freshly isolated SMCs from dogs and rabbits. As confirmed by force measurements, relaxation, Ca2+ depletion, and cholinergic activation of SM tissues do not cause significant redistribution of the AJ-associated proteins vinculin, talin, or fibronectin away from the plasma membrane. Unlike SMCs in tissue, freshly isolated SMCs show a variable peripheral/cytoplasmic vinculin and talin distribution that is not altered by activation. Enzymatic treatment of SM tissues (as done for the first step of SMC isolation) results in loss of fibronectin immunoreactivity in SMCs still in the tissue but fails to cause redistribution of vinculin, talin, or caveolin away from the periphery. The loss of fibronectin immunofluorescence with enzymatic digestion correlates significantly with loss of tissue force production. These results confirm that the AJ-associated proteins vinculin and talin do not redistribute throughout SMCs in tissues when relaxed, when generating force, or after enzymatic digestion. In addition, in freshly isolated SMCs, the distribution of these proteins is significantly altered in ∼50% of the SMCs. The cause of this redistribution is currently unknown, as is the impact on intracellular signaling and mechanics of these cells. Use of these two systems (SMCs in tissues vs. freshly isolated SMCs) provides an ideal situation for studying the role of the AJ in SMC signaling and mechanics.

scholarly journals Mechanisms involved in AMPK-mediated deposition of tight junction components to the plasma membrane

2020 ◽  
Vol 318 (3) ◽  
pp. C486-C501
Author(s):  
Jingshing Wu ◽  
Pascal Rowart ◽  
Francois Jouret ◽  
Brandon M. Gassaway ◽  
Vanathy Rajendran ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Tight Junction ◽  
Adherens Junction ◽  
Bound State ◽  
Ampk Activation ◽  
Zonula Occludens ◽  
Adherens Junction Protein ◽  
Junction Protein ◽  
Associated Proteins ◽  
Amp Activated Protein Kinase

AMP-activated protein kinase (AMPK) activation promotes early stages of epithelial junction assembly. AMPK activation in MDCK renal epithelial cells facilitates localization of the junction-associated proteins aPKCζ and Par3 to the plasma membrane and promotes conversion of Cdc42, a key regulator of epithelial polarization and junction assembly, to its active GTP bound state. Furthermore, Par3 is an important regulator of AMPK-mediated aPKCζ localization. Both aPKCζ and Par3 serve as intermediates in AMPK-mediated junction assembly, with inhibition of aPKCζ activity or Par3 knockdown disrupting AMPK’s ability to facilitate zonula occludens (ZO-1) localization. AMPK phosphorylates the adherens junction protein afadin and regulates its interaction with the tight-junction protein zonula occludens-1. Afadin is phosphorylated at two critical sites, S228 (residing within an aPKCζ consensus site) and S1102 (residing within an AMPK consensus site), that are differentially regulated during junction assembly and that exert different effects on the process. Expression of phospho-defective mutants (S228A and S1102A) perturbed ZO-1 localization to the plasma membrane during AMPK-induced junction assembly. Expression of S228A increased the ZO-1/afadin interaction, while S1102A reduced this interaction during extracellular calcium-induced junction assembly. Inhibition of aPKCζ activity also increased the ZO-1/afadin interaction. Taken together, these data suggest that aPKCζ phosphorylation of afadin terminates the ZO-1/afadin interaction and thus permits the later stages of junction assembly.

Ultrastructural and immunocytochemical studies of the rat hypothalamo-neurohypophysial system processed by rapid freezing and freeze-substitution fixation

1990 ◽  
Vol 48 (3) ◽  
pp. 884-885
Author(s):  
Seiji Shioda ◽  
Yasumitsu Nakai ◽  
Atsushi Ichikawa ◽  
Hidehiko Ochiai ◽  
Nobuko Naito
Keyword(s):  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Wistar Rat ◽  
Neurosecretory Cells ◽  
Rapid Freezing ◽  
Sodium Metaperiodate ◽  
Tissue Sections ◽  
Ultrathin Sections ◽  
Electron Microscopes

The ultrastructure of neurosecretory cells and glia cells in the supraoptic nucleus (SON) of the hypothalamus and the neurohypophysis (PN) was studied after rapid freezing followed by substituion fixation. Also, the ultrastructural localization of vasopressin (VP) or its carrier protein neurophys in II (NPII) in the SON and PN was demonstrated by using a post-embedding immunoco1loidal gold staining method on the tissue sections processed by rapid freezing and freeze-substitution fixation.Adult male Wistar rat hypothalamus and pituitary gland were quenched by smashing against a copper block surface precooled with liquid helium and freeze-substituted in 3% osmium tetroxide-acetone solutions kept at -80°C for 36-48h. After substituion fixation, the tissue blocks were warmed up to room temperature, washed in acetone and then embedded in an Epon-Araldite mixture. Ultrathin sections mounted on 200 mesh nickel grids were immersed in saturated sodium metaperiodate and then incubated in each of the following solutions: 1 % egg albumin in phosphate buffer, VP or NPII (1/1000-1/5000) antiserum 24h at 4°C, 3) colloidal gold solution (1/20) 1h at 20°C. The sections were washed with distilled waterand dried, then stained with uranylacetate and lead citrate and examined with Hitachi HU-12A and H-800 electron microscopes.

Mislocalization of Adherens Junction- Associated Proteins in a Patient with Darier Disease

2018 ◽  
Vol 2 (3) ◽  
pp. 184-201
Author(s):  
George D Glinos ◽  
Irena Pastar ◽  
Marjana Tomic-Canic ◽  
Rivka C Stone
Keyword(s):  
Adherens Junction ◽  
Cellular Adhesion ◽  
Calcium Flux ◽  
Keratinocyte Differentiation ◽  
Calcium Dependent ◽  
Endoplasmic Reticulum Calcium ◽  
Darier Disease ◽  
Associated Proteins ◽  
Attachment Proteins ◽  
E Cadherin

Darier disease (DD) is an autosomal dominant keratinizing genodermatosis that manifests clinically with red-brown pruritic papules in a seborrheic distribution often in association with palmoplantar pits and dystrophic nail changes. It is caused by mutation in ATP2A2 which encodes a sarco/endoplasmic reticulum calcium ATPase isoform 2 (SERCA2) pump that regulates calcium flux. Consequent alteration of intracellular calcium homeostasis is thought to impair trafficking of cellular adhesion proteins and to lead to aberrant keratinocyte differentiation, contributing to the characteristic histopathologic features of acantholysis and dyskeratosis in DD, though the precise mechanisms are incompletely understood. Previous studies have identified defective localization of desmosomal attachment proteins in skin biopsies and cultured keratinocytes from DD patients, but reports of effects on adherens junction proteins (including calcium-dependent E-cadherin) are conflicting. Here we describe a case of DD presenting with characteristic clinical and histologic features in which we performed immunofluorescence staining of four adherens junction-associated proteins (E-cadherin, α-catenin, β-catenin, and vinculin). In lesional (acantholytic) DD skin, we identified loss of distinctive bright membranous staining that was present at the periphery of keratinocytes throughout the epidermis in the healthy skin of a matched donor. Perilesional (non-acantholytic) portions of DD skin partially recapitulated the normal phenotype. Our findings support a role for SERCA2 dysfunction in impaired assembly of adherens junctions, which together with defective desmosomes contribute to acantholysis in DD.

Biotinylation and Purification of Plasma Membrane-associated Proteins from Rodent Cultured Neurons

BIO-PROTOCOL ◽  
2016 ◽  
Vol 6 (10) ◽  
Author(s):  
Margarida Caldeira ◽  
Joana Ferreira ◽  
Ana Carvalho ◽  
Carlos Duarte
Keyword(s):  
Plasma Membrane ◽  
Cultured Neurons ◽  
Associated Proteins

scholarly journals TRPC6 regulates phenotypic switching of vascular smooth muscle cells through plasma membrane potential‐dependent coupling with PTEN

2019 ◽  
Vol 33 (9) ◽  
pp. 9785-9796 ◽  
Author(s):  
Takuro Numaga‐Tomita ◽  
Tsukasa Shimauchi ◽  
Sayaka Oda ◽  
Tomohiro Tanaka ◽  
Kazuhiro Nishiyama ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Membrane Potential ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Phenotypic Switching ◽  
Plasma Membrane Potential

Digital RNA-seq transcriptome plus tissue anatomy analyses reveal the developmental mechanism of the calabash-shaped root in Tetrastigma hemsleyanum

2021 ◽  
Author(s):  
Taihe Xiang ◽  
Jiangshan Li ◽  
Shuying Bao ◽  
Zhengxian Xu ◽  
Leizhen Wang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Chinese Herb ◽  
Acc Oxidase ◽  
Mapk Signaling ◽  
Cell Periphery ◽  
Vascular Bundles ◽  
Rna Seq ◽  
Short Root ◽  
Tetrastigma Hemsleyanum ◽  
Intrinsic Component

Abstract Tetrastigma hemsleyanum is a liana plant with promising medicinal and ornamental values. Its calabash-shaped roots (CRs) are served as a traditional Chinese herb. However, it takes a long growth period to form CRs. In the present study, three types of architectural roots, including fine roots (FRs), bar-shaped roots (BRs) and CRs, were employed as materials, and the characteristics of histo-anatomy and digital RNA-seq transcriptome profiles were analyzed. Among the three types of roots, the vascular bundles in FRs were intact, while some of the vascular bundles degenerated in BRs, and only few traces of vascular bundles existed in CRs. Meanwhile, no obvious cell inclusions were found in the cytoplasm of FRs, while a few inclusions were found in BRs, and abundant inclusions were detected in CRs, which might be the main source of medicinal components in roots. The transcriptome profiles and qRT-PCR validation indicated that seven up-regulated genes encoding xyloglucan glycosyltransferase, ACC oxidase, CYP711A1, SHORT-ROOT transcript factor, galacturonosyltransferas, WAT1 and WRKY, and two down-regulated genes encoded LRR receptor-like serine/threonine-protein kinase and CYP83B1, were probably involved in the formation and development of CRs. Besides, GO terms of intrinsic component of membrane, integral component of membrane, cell periphery, membrane part, plasma membrane, membrane, intrinsic component of plasma membrane, cellular chemical homeostasis, and plasma membrane part were probably related to the formation of CRs. KEGG pathways related to the development of CRs probably included MAPK signaling pathway-plant, plant hormone signal transduction, and circadian rhythm-plant. Our finding suggested a probable mode for the formation of CRs.

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