scholarly journals KRAP tethers IP3 receptors to actin and licenses them to evoke cytosolic Ca2+ signals

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nagendra Babu Thillaiappan ◽  
Holly A. Smith ◽  
Peace Atakpa-Adaji ◽  
Colin W. Taylor
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Mobile Ip ◽  
Ip3 Receptors ◽  
Interacting Protein ◽  
Local Activation ◽  
Intense Stimulation

AbstractRegulation of IP3 receptors (IP3Rs) by IP3 and Ca2+ allows regenerative Ca2+ signals, the smallest being Ca2+ puffs, which arise from coordinated openings of a few clustered IP3Rs. Cells express thousands of mostly mobile IP3Rs, yet Ca2+ puffs occur at a few immobile IP3R clusters. By imaging cells with endogenous IP3Rs tagged with EGFP, we show that KRas-induced actin-interacting protein (KRAP) tethers IP3Rs to actin beneath the plasma membrane. Loss of KRAP abolishes Ca2+ puffs and the global increases in cytosolic Ca2+ concentration evoked by more intense stimulation. Over-expressing KRAP immobilizes additional IP3R clusters and results in more Ca2+ puffs and larger global Ca2+ signals. Endogenous KRAP determines which IP3Rs will respond: it tethers IP3R clusters to actin alongside sites where store-operated Ca2+ entry occurs, licenses IP3Rs to evoke Ca2+ puffs and global cytosolic Ca2+ signals, implicates the actin cytoskeleton in IP3R regulation and may allow local activation of Ca2+ entry.

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

2009 ◽  
Vol 296 (4) ◽  
pp. C857-C867 ◽  
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.

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

1994 ◽  
Vol 125 (2) ◽  
pp. 381-391 ◽  
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.

Association of the hepatic IP3 receptor with the plasma membrane: relevance to mode of action

1993 ◽  
Vol 265 (6) ◽  
pp. C1588-C1596 ◽  
Author(s):  
L. Feng ◽  
N. Kraus-Friedmann
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Ip3 Receptors ◽  
Ip3 Receptor ◽  
Plasma Membrane Fraction ◽  
T Lymphoma Cells ◽  
T Lymphoma ◽  
Triton X ◽  
Brain Receptor ◽  
The Brain

Studies were carried out to characterize the interaction between inositol 1,4,5-trisphosphate (IP3) receptors and the plasma membrane fraction. Extraction of the membranes with the nonionic detergents Nonidet P-40 and Triton X-100, followed by centrifugation at 100,000 g, resulted in the doubling of the IP3 receptor in the pellets, whereas no detectable binding was found in the supernatants. These data indicate that the detergents did not solubilize the receptor, that it remained associated with membrane particles, and that it is likely to be associated with the cytoskeleton. The cytoskeleton proteins actin, ankyrin, and spectrin were identified in the plasma membrane fraction. However, comparison of the amount of these proteins in different fractions of the detergent, or otherwise treated plasma membrane fractions, showed no direct correlation between the presence of any of these proteins in the plasma membrane fraction and their ability to bind [3H]IP3. This is in contrast to the brain and T-lymphoma cells in which the IP3 receptor is attached to ankyrin (L. Y. W. Bourguigon, H. Jin, N. Iida, N. R. Brandt, and S. H. Zhang. J. Biol. Chem. 268: 6477-6486, 1993; and S. K. Joseph and S. Samanta. J. Biol. Chem 268: 6477-6486, 1993). Thus the hepatic IP3 receptor, which is different from the brain receptor, might attach to the cytoskeleton by anchoring to a different protein. Because cytochalasin D treatment of livers diminishes the ability of IP3 to raise cytosolic free Ca2+ levels, the attachment of the IP3 receptor to the cytoskeleton seems to involve an association with microfilaments.

scholarly journals CD44 and β3 Integrin Organize Two Functionally Distinct Actin-based Domains in Osteoclasts

2007 ◽  
Vol 18 (12) ◽  
pp. 4899-4910 ◽  
Author(s):  
Anne Chabadel ◽  
Inmaculada Bañon-Rodríguez ◽  
David Cluet ◽  
Brian B. Rudkin ◽  
Bernhard Wehrle-Haller ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Actin Cytoskeleton ◽  
Interacting Protein ◽  
Wiskott Aldrich Syndrome ◽  
Diffuse Cloud ◽  
Sealing Zone ◽  
Β3 Integrin ◽  
Syndrome Protein

The actin cytoskeleton of mature osteoclasts (OCs) adhering to nonmineralized substrates is organized in a belt of podosomes reminiscent of the sealing zone (SZ) found in bone resorbing OCs. In this study, we demonstrate that the belt is composed of two functionally different actin-based domains: podosome cores linked with CD44, which are involved in cell adhesion, and a diffuse cloud associated with β3 integrin, which is involved in cell adhesion and contraction. Wiskott Aldrich Syndrome Protein (WASp) Interacting Protein (WIP)−/− OCs were devoid of podosomes, but they still exhibited actin clouds. Indeed, WIP−/− OCs show diminished expression of WASp, which is required for podosome formation. CD44 is a novel marker of OC podosome cores and the first nonintegrin receptor detected in these structures. The importance of CD44 is revealed by showing that its clustering restores podosome cores and WASp expression in WIP−/− OCs. However, although CD44 signals are sufficient to form a SZ, the presence of WIP is indispensable for the formation of a fully functional SZ.

scholarly journals Mammalian Homologue of the Caenorhabditis elegans UNC-76 Protein Involved in Axonal Outgrowth Is a Protein Kinase C ζ–interacting Protein

1999 ◽  
Vol 144 (3) ◽  
pp. 403-411 ◽  
Author(s):  
Shun'ichi Kuroda ◽  
Noritaka Nakagawa ◽  
Chiharu Tokunaga ◽  
Kenji Tatematsu ◽  
Katsuyuki Tanizawa
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Caenorhabditis Elegans ◽  
Protein Kinase ◽  
Rat Brain ◽  
Axonal Outgrowth ◽  
Yeast Two Hybrid ◽  
Interacting Protein ◽  
Kinase C ◽  
Two Hybrid

By the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C ζ (PKCζ) as a bait, we have cloned a gene coding for a novel PKCζ-interacting protein homologous to the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth and fasciculation. The protein designated FEZ1 (fasciculation and elongation protein zeta-1) consisting of 393 amino acid residues shows a high Asp/Glu content and contains several regions predicted to form amphipathic helices. Northern blot analysis has revealed that FEZ1 mRNA is abundantly expressed in adult rat brain and throughout the developmental stages of mouse embryo. By the yeast two-hybrid assay with various deletion mutants of PKC, FEZ1 was shown to interact with the NH2-terminal variable region (V1) of PKCζ and weakly with that of PKCε. In the COS-7 cells coexpressing FEZ1 and PKCζ, FEZ1 was present mainly in the plasma membrane, associating with PKCζ and being phosphorylated. These results indicate that FEZ1 is a novel substrate of PKCζ. When the constitutively active mutant of PKCζ was used, FEZ1 was found in the cytoplasm of COS-7 cells. Upon treatment of the cells with a PKC inhibitor, staurosporin, FEZ1 was translocated from the cytoplasm to the plasma membrane, suggesting that the cytoplasmic translocation of FEZ1 is directly regulated by the PKCζ activity. Although expression of FEZ1 alone had no effect on PC12 cells, coexpression of FEZ1 and constitutively active PKCζ stimulated the neuronal differentiation of PC12 cells. Combined with the recent finding that a human FEZ1 protein is able to complement the function of UNC-76 necessary for normal axonal bundling and elongation within axon bundles in the nematode, these results suggest that FEZ1 plays a crucial role in the axon guidance machinery in mammals by interacting with PKCζ.

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

2018 ◽  
Vol 114 (3) ◽  
pp. 99a ◽  
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

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

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.

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

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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