Actin Cytoskeleton Rest Stops Regulate Anterograde Traffic of Connexin 43 Vesicles to the Plasma Membrane

Vascular hyperpermeability is a determinant factor in the pathophysiology of sepsis. While, AMP-activated protein kinase (AMPK) is known to play a role in maintaining endothelial barrier function in this condition. Therefore, we investigated the underlying molecular mechanisms of this protective effect. α1AMPK expression and/or activity was modulated in human dermal microvascular endothelial cells using either α1AMPK-targeting small interfering RNA or the direct pharmacological AMPK activator 991, prior to lipopolysaccharide (LPS) treatment. Western blotting was used to analyze the expression and/or phosphorylation of proteins that compose cellular junctions (zonula occludens-1 (ZO-1), vascular endothelial cadherin (VE-Cad), connexin 43 (Cx43)) or that regulate actin cytoskeleton (p38 MAPK; heat shock protein 27 (HSP27)). Functional endothelial permeability was assessed by in vitro Transwell assays, and quantification of cellular junctions in the plasma membrane was assessed by immunofluorescence. Actin cytoskeleton remodeling was evaluated through actin fluorescent staining. We consequently demonstrate that α1AMPK deficiency is associated with reduced expression of CX43, ZO-1, and VE-Cad, and that the drastic loss of CX43 is likely responsible for the subsequent decreased expression and localization of ZO-1 and VE-Cad in the plasma membrane. Moreover, α1AMPK activation by 991 protects against LPS-induced endothelial barrier disruption by reinforcing cortical actin cytoskeleton. This is due to a mechanism that involves the phosphorylation of p38 MAPK and HSP27, which is nonetheless independent of the small GTPase Rac1. This results in a drastic decrease of LPS-induced hyperpermeability. We conclude that α1AMPK activators that are suitable for clinical use may provide a specific therapeutic intervention that limits sepsis-induced vascular leakage.

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Counterregulation of clathrin-mediated endocytosis by the actin and microtubular cytoskeleton in human neutrophils

AJP Cell Physiology ◽

10.1152/ajpcell.00454.2008 ◽

2009 ◽

Vol 296 (4) ◽

pp. C857-C867 ◽

Cited By ~ 20

Author(s):

Silvia M. Uriarte ◽

Neelakshi R. Jog ◽

Gregory C. Luerman ◽

Samrath Bhimani ◽

Richard A. Ward ◽

...

Keyword(s):

Plasma Membrane ◽

Actin Cytoskeleton ◽

Human Neutrophils ◽

Functional Responses ◽

Membrane Expression ◽

Granule Exocytosis ◽

Plasma Membrane Expression ◽

Microtubular Cytoskeleton ◽

Latrunculin A ◽

Azurophil Granule

We have recently reported that disruption of the actin cytoskeleton enhanced N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated granule exocytosis in human neutrophils but decreased plasma membrane expression of complement receptor 1 (CR1), a marker of secretory vesicles. The present study was initiated to determine if reduced CR1 expression was due to fMLP-stimulated endocytosis, to determine the mechanism of this endocytosis, and to examine its impact on neutrophil functional responses. Stimulation of neutrophils with fMLP or ionomycin in the presence of latrunculin A resulted in the uptake of Alexa fluor 488-labeled albumin and transferrin and reduced plasma membrane expression of CR1. These effects were prevented by preincubation of the cells with sucrose, chlorpromazine, or monodansylcadaverine (MDC), inhibitors of clathrin-mediated endocytosis. Sucrose, chlorpromazine, and MDC also significantly inhibited fMLP- and ionomycin-stimulated specific and azurophil granule exocytosis. Disruption of microtubules with nocodazole inhibited endocytosis and azurophil granule exocytosis stimulated by fMLP in the presence of latrunculin A. Pharmacological inhibition of phosphatidylinositol 3-kinase, ERK1/2, and PKC significantly reduced fMLP-stimulated transferrin uptake in the presence of latrunculin A. Blockade of clathrin-mediated endocytosis had no significant effect on fMLP-stimulated phosphorylation of ERK1/2 in neutrophils pretreated with latrunculin A. From these data, we conclude that the actin cytoskeleton functions to limit microtubule-dependent, clathrin-mediated endocytosis in stimulated human neutrophils. The limitation of clathrin-mediated endocytosis by actin regulates the extent of both specific and azurophilic granule exocytosis.

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Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane.

The Journal of Cell Biology ◽

10.1083/jcb.125.2.381 ◽

1994 ◽

Vol 125 (2) ◽

pp. 381-391 ◽

Cited By ~ 245

Author(s):

J Mulholland ◽

D Preuss ◽

A Moon ◽

A Wong ◽

D Drubin ◽

...

Keyword(s):

Plasma Membrane ◽

Actin Cytoskeleton ◽

Binding Proteins ◽

Ultrastructural Level ◽

Actin Binding ◽

Cortical Actin ◽

Actin Binding Proteins ◽

Double Labeling ◽

Wall Growth ◽

Cortical Cytoskeleton

We characterized the yeast actin cytoskeleton at the ultrastructural level using immunoelectron microscopy. Anti-actin antibodies primarily labeled dense, patchlike cortical structures and cytoplasmic cables. This localization recapitulates results obtained with immunofluorescence light microscopy, but at much higher resolution. Immuno-EM double-labeling experiments were conducted with antibodies to actin together with antibodies to the actin binding proteins Abp1p and cofilin. As expected from immunofluorescence experiments, Abp1p, cofilin, and actin colocalized in immuno-EM to the dense patchlike structures but not to the cables. In this way, we can unambiguously identify the patches as the cortical actin cytoskeleton. The cortical actin patches were observed to be associated with the cell surface via an invagination of plasma membrane. This novel cortical cytoskeleton-plasma membrane interface appears to consist of a fingerlike invagination of plasma membrane around which actin filaments and actin binding proteins are organized. We propose a possible role for this unique cortical structure in wall growth and osmotic regulation.

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STED-FCS Reveals Diffusional Heterogeneity of Lipids and GPI-Anchored Proteins in the Plasma Membrane and Actin Cytoskeleton Free Plasma Membrane Vesicles

Biophysical Journal ◽

10.1016/j.bpj.2017.11.582 ◽

2018 ◽

Vol 114 (3) ◽

pp. 99a ◽

Cited By ~ 1

Author(s):

Falk Schneider ◽

Dominic Waithe ◽

Mathias Porsmose Clausen ◽

Silvia Galiani ◽

Thomas Koller ◽

...

Keyword(s):

Plasma Membrane ◽

Actin Cytoskeleton ◽

Membrane Vesicles ◽

Plasma Membrane Vesicles ◽

Free Plasma

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New interactions of invadopodia scaffold protein TKS5 with proteins that take part in actin cytoskeleton reorganization, endo-/exocytosis and membrane remodeling

Visnik ukrains'kogo tovaristva genetikiv i selekcioneriv ◽

10.7124/visnyk.utgis.16.2.1056 ◽

2019 ◽

Vol 16 (2) ◽

pp. 183-189

Author(s):

Y. M. Nemesh ◽

S. V. Kropyvko

Keyword(s):

Plasma Membrane ◽

Actin Cytoskeleton ◽

Scaffold Protein ◽

Scaffold Proteins ◽

Membrane Remodeling ◽

Sh3 Domains ◽

Protein Protein Interaction ◽

Cytoskeleton Reorganization ◽

New Interactions ◽

Cytoskeleton Rearrangement

Aim. TKS5 is a key scaffold protein of invadopodia. In its absence, the cells completely lose the ability to form invadopodia. This fact makes TKS5 a potential target for cancer cure and one of the central proteins in the investigation of cancer cell invasion. Additionally, the question remains about the function of TKS5 in normal cells. Therefore, in order to extend knowledge about TKS5 role in healthy and invasive cells, we tested the TKS5 interaction with the proteins involved in signal transduction: PLCγ1, SRC, CRK, CSK; the proteins involved in plasma membrane remodeling: AMPH1, BIN1, CIN85, ITSN1, ITSN2; the protein involved in the actin cytoskeleton rearrangement: CTTN. Methods. We used the GST Pull-down assay to identify the protein-protein interaction. Results. We revealed that TKS5 SH3 domains interact with CIN85. There were identified TKS5 interactions with SH3 domains of CTTN, ITSN1, ITSN2, AMPH1 and BIN1. Conclusions. TKS5 interacts with CIN85, CTTN, ITSN1, ITSN2, AMPH1 and BIN1, which take part in membrane remodeling, endo-/exocytosis and actin cytoskeleton rearrangement. Keywords: TKS5, scaffold proteins, actin cytoskeleton, plasma membrane.

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Role of Ezrin/Radixin/Moesin in the Surface Localization of Programmed Cell Death Ligand-1 in Human Colon Adenocarcinoma LS180 Cells

Pharmaceuticals ◽

10.3390/ph14090864 ◽

2021 ◽

Vol 14 (9) ◽

pp. 864

Author(s):

Takuro Kobori ◽

Chihiro Tanaka ◽

Mayuka Tameishi ◽

Yoko Urashima ◽

Takuya Ito ◽

...

Keyword(s):

Cell Death ◽

Plasma Membrane ◽

Programmed Cell Death ◽

Cell Surface ◽

Actin Cytoskeleton ◽

Mrna Level ◽

Scaffold Proteins ◽

Membrane Localization ◽

Erm Proteins ◽

Plasma Membrane Localization

Programmed cell death ligand-1 (PD-L1), an immune checkpoint protein highly expressed on the cell surface in various cancer cell types, binds to programmed cell death-1 (PD-1), leading to T-cell dysfunction and tumor survival. Despite clinical successes of PD-1/PD-L1 blockade therapies, patients with colorectal cancer (CRC) receive little benefit because most cases respond poorly. Because high PD-L1 expression is associated with immune evasion and poor prognosis in CRC patients, identifying potential modulators for the plasma membrane localization of PD-L1 may represent a novel therapeutic strategy for enhancing the efficacy of PD-1/PD-L1 blockade therapies. Here, we investigated whether PD-L1 expression in human colorectal adenocarcinoma cells (LS180) is affected by ezrin/radixin/moesin (ERM), functioning as scaffold proteins that crosslink plasma membrane proteins with the actin cytoskeleton. We observed colocalization of PD-L1 with all three ERM proteins in the plasma membrane and detected interactions involving PD-L1, the three ERM proteins, and the actin cytoskeleton. Furthermore, gene silencing of ezrin and radixin, but not of moesin, substantially decreased the expression of PD-L1 on the cell surface without affecting its mRNA level. Thus, in LS180 cells, ezrin and radixin may function as scaffold proteins mediating the plasma membrane localization of PD-L1, possibly by post-translational modification.

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N-cadherin in adult rat cardiomyocytes in culture. II. Spatio-temporal appearance of proteins involved in cell-cell contact and communication. Formation of two distinct N-cadherin/catenin complexes

Journal of Cell Science ◽

10.1242/jcs.109.1.11 ◽

1996 ◽

Vol 109 (1) ◽

pp. 11-20 ◽

Cited By ~ 7

Author(s):

C.M. Hertig ◽

S. Butz ◽

S. Koch ◽

M. Eppenberger-Eberhardt ◽

R. Kemler ◽

...

Keyword(s):

Plasma Membrane ◽

Gap Junctions ◽

Connexin 43 ◽

Cell Contact ◽

Beta Catenin ◽

Rat Cardiomyocytes ◽

Cell Contacts ◽

Adult Rat ◽

Spatio Temporal ◽

Cell Cell

The spatio-temporal appearance and distribution of proteins forming the intercalated disc were investigated in adult rat cardiomyocytes (ARC). The ‘redifferentiation model’ of ARC involves extensive remodelling of the plasma membrane and of the myofibrillar apparatus. It represents a valuable system to elucidate the formation of cell-cell contact between cardiomyocytes and to assess the mechanisms by which different proteins involved in the cell-cell adhesion process are sorted in a precise manner to the sites of function. Appearance of N-cadherin, the catenins and connexin43 within newly formed adherens and gap junctions was studied. Here first evidence is provided for a formation of two distinct and separable N-cadherin/catenin complexes in cardiomyocytes. Both complexes are composed of N-cadherin and alpha-catenin which bind to either beta-catenin or plakoglobin in a mutually exclusive manner. The two N-cadherin/catenin complexes are assumed to be functionally involved in the formation of cell-cell contacts in ARC; however, the differential appearance and localization of the two types of complexes may also point to a specific role during ARC differentiation. The newly synthesized beta-catenin containing complex is more abundant during the first stages in culture after ARC isolation, while the newly synthesized plakoglobin containing complex progressively accumulates during the morphological changes of ARC. ARC formed a tissue-like pattern in culture whereby the new cell-cell contacts could be dissolved through Ca2+ depletion. Presence of cAMP and replenishment of Ca2+ content in the culture medium not only allowed reformation of cell-cell contacts but also affected the relative protein ratio between the two N-cadherin/catenin complexes, increasing the relative amount of newly synthesized beta-catenin over plakoglobin at a particular stage of ARC differentiation. The clustered N-cadherin/catenin complexes at the plasma membrane appear to be a prerequisite for the following gap junction formation; a temporal sequence of the appearance of adherens junction proteins and of gap junctions forming connexin-43 is suggested.

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Endogenous plasma membrane t-SNARE syntaxin 4 is present in rab11 positive endosomal membranes and associates with cortical actin cytoskeleton

FEBS Letters ◽

10.1016/s0014-5793(02)03605-0 ◽

2002 ◽

Vol 531 (3) ◽

pp. 513-519 ◽

Cited By ~ 30

Author(s):

Arja M Band ◽

Heidi Ali ◽

Maria K Vartiainen ◽

Saara Welti ◽

Pekka Lappalainen ◽

...

Keyword(s):

Plasma Membrane ◽

Actin Cytoskeleton ◽

Cortical Actin ◽

Endosomal Membranes ◽

Syntaxin 4

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H-ras, K-ras, and inner plasma membrane raft proteins operate in nanoclusters with differential dependence on the actin cytoskeleton

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0504114102 ◽

2005 ◽

Vol 102 (43) ◽

pp. 15500-15505 ◽

Cited By ~ 323

Author(s):

S. J. Plowman ◽

C. Muncke ◽

R. G. Parton ◽

J. F. Hancock

Keyword(s):

Plasma Membrane ◽

Actin Cytoskeleton ◽

Membrane Raft

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Yeast actin cytoskeleton mutants accumulate a new class of Golgi-derived secretary vesicle.

Molecular Biology of the Cell ◽

10.1091/mbc.8.8.1481 ◽

1997 ◽

Vol 8 (8) ◽

pp. 1481-1499 ◽

Cited By ~ 90

Author(s):

J Mulholland ◽

A Wesp ◽

H Riezman ◽

D Botstein

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Actin Cytoskeleton ◽

Biochemical Analysis ◽

Small Gtpase ◽

Secretory Vesicles ◽

Cytoskeleton Organization ◽

New Class ◽

Actin Cytoskeleton Organization ◽

Vectorial Transport

Many yeast actin cytoskeleton mutants accumulate large secretory vesicles and exhibit phenotypes consistent with defects in polarized growth. This, together with actin's polarized organization, has suggested a role for the actin cytoskeleton in the vectorial transport of late secretory vesicles to the plasma membrane. By using ultrastructural and biochemical analysis, we have characterized defects manifested by mutations in the SLA2 gene (also known as the END4 gene), previously found to affect both the organization of the actin cytoskeleton and endocytosis in yeast. Defects in cell wall morphology, accumulated vesicles, and protein secretion kinetics were found in sla2 mutants similar to defects found in act1 mutants. Vesicles that accumulate in the sla2 and act1 mutants are immunoreactive with antibodies directed against the small GTPase Ypt1p but not with antibodies directed against the homologous Sec4p found on classical "late" secretory vesicles. In contrast, the late-acting secretory mutants sec1-1 and sec6-4 are shown to accumulate anti-Sec4p-positive secretory vesicles as well as vesicles that are immunoreactive with antibodies directed against Ypt1p. The late sec mutant sec4-8 is also shown to accumulate Ypt1p-containing vesicles and to exhibit defects in actin cytoskeleton organization. These results indicate the existence of at least two classes of morphologically similar, late secretory vesicles (associated with Ypt1p+ and Sec4p+, respectively), one of which appears to accumulate when the actin cytoskeleton is disorganized.

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