scholarly journals A synergy between mechanosensitive calcium- and membrane-binding mediates tension-sensing by C2-like domains

2021 ◽  
Vol 119 (1) ◽  
pp. e2112390119
Author(s):  
Zhouyang Shen ◽  
Kalina T. Belcheva ◽  
Mark Jelcic ◽  
King Lam Hui ◽  
Anushka Katikaneni ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Membrane Insertion ◽  
C2 Domain ◽  
Hydrophobic Membrane ◽  
Membrane Recruitment ◽  
Calcium Dependent ◽  
Nuclear Membranes ◽  
Cytosolic Phospholipase ◽  
Artificial Lipid Bilayers ◽  
Quantitative Basis

When nuclear membranes are stretched, the peripheral membrane enzyme cytosolic phospholipase A2 (cPLA2) binds via its calcium-dependent C2 domain (cPLA2-C2) and initiates bioactive lipid signaling and tissue inflammation. More than 150 C2-like domains are encoded in vertebrate genomes. How many of them are mechanosensors and quantitative relationships between tension and membrane recruitment remain unexplored, leaving a knowledge gap in the mechanotransduction field. In this study, we imaged the mechanosensitive adsorption of cPLA2 and its C2 domain to nuclear membranes and artificial lipid bilayers, comparing it to related C2-like motifs. Stretch increased the Ca2+ sensitivity of all tested domains, promoting half-maximal binding of cPLA2 at cytoplasmic resting-Ca2+ concentrations. cPLA2-C2 bound up to 50 times tighter to stretched than to unstretched membranes. Our data suggest that a synergy of mechanosensitive Ca2+ interactions and deep, hydrophobic membrane insertion enables cPLA2-C2 to detect stretched membranes with antibody-like affinity, providing a quantitative basis for understanding mechanotransduction by C2-like domains.

scholarly journals Formation of artificial lipid bilayers using droplet dielectrophoresis

Lab on a Chip ◽  
2008 ◽  
Vol 8 (10) ◽  
pp. 1617 ◽  
Author(s):  
Sara Aghdaei ◽  
Mairi E. Sandison ◽  
Michele Zagnoni ◽  
Nicolas G. Green ◽  
Hywel Morgan
Keyword(s):  
Lipid Bilayers ◽  
Artificial Lipid Bilayers

scholarly journals Role of Phosphorylation Sites and the C2 Domain in Regulation of Cytosolic Phospholipase A2

1999 ◽  
Vol 145 (6) ◽  
pp. 1219-1232 ◽  
Author(s):  
Miguel A. Gijón ◽  
Diane M. Spencer ◽  
Alan L. Kaiser ◽  
Christina C. Leslie
Keyword(s):  
Arachidonic Acid ◽  
Nuclear Envelope ◽  
Phospholipase A2 ◽  
Okadaic Acid ◽  
Cytosolic Phospholipase A2 ◽  
Phosphorylation Sites ◽  
C2 Domain ◽  
Arachidonic Acid Release ◽  
Cytosolic Phospholipase ◽  
Acid Release

Cytosolic phospholipase A2 (cPLA2) mediates agonist-induced arachidonic acid release, the first step in eicosanoid production. cPLA2 is regulated by phosphorylation and by calcium, which binds to a C2 domain and induces its translocation to membrane. The functional roles of phosphorylation sites and the C2 domain of cPLA2 were investigated. In Sf9 insect cells expressing cPLA2, okadaic acid, and the calcium-mobilizing agonists A23187 and CryIC toxin induce arachidonic acid release and translocation of green fluorescent protein (GFP)-cPLA2 to the nuclear envelope. cPLA2 is phosphorylated on multiple sites in Sf9 cells; however, only S505 phosphorylation partially contributes to cPLA2 activation. Although okadaic acid does not increase calcium, mutating the calcium-binding residues D43 and D93 prevents arachidonic acid release and translocation of cPLA2, demonstrating the requirement for a functional C2 domain. However, the D93N mutant is fully functional with A23187, whereas the D43N mutant is nearly inactive. The C2 domain of cPLA2 linked to GFP translocates to the nuclear envelope with calcium-mobilizing agonists but not with okadaic acid. Consequently, the C2 domain is necessary and sufficient for translocation of cPLA2 to the nuclear envelope when calcium is increased; however, it is required but not sufficient with okadaic acid.

scholarly journals Daptomycin Pore Formation and Stoichiometry Depend on Membrane Potential of Target Membrane

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Gabriela Seydlová ◽  
Albert Sokol ◽  
Petra Lišková ◽  
Ivo Konopásek ◽  
Radovan Fišer
Keyword(s):  
Membrane Potential ◽  
Lipid Bilayers ◽  
Pore Formation ◽  
Content Type ◽  
Calcium Dependent ◽  
Screening Experiment ◽  
Serious Infections ◽  
Fluorescence Methods ◽  
Initial Membrane ◽  
Target Membrane

ABSTRACT Daptomycin is a calcium-dependent lipodepsipeptide antibiotic clinically used to treat serious infections caused by Gram-positive pathogens. Its precise mode of action is somewhat controversial; the biggest issue is daptomycin pore formation, which we directly investigated here. We first performed a screening experiment using propidium iodide (PI) entry to Bacillus subtilis cells and chose the optimum and therapeutically relevant conditions (10 µg/ml daptomycin and 1.25 mM CaCl2) for the subsequent analyses. Using conductance measurements on planar lipid bilayers, we show that daptomycin forms nonuniform oligomeric pores with conductance ranging from 120 pS to 14 nS. The smallest conductance unit is probably a dimer; however, tetramers and pentamers occur in the membrane most frequently. Moreover, daptomycin pore-forming activity is exponentially dependent on the applied membrane voltage. We further analyzed the membrane-permeabilizing activity in B. subtilis cells using fluorescence methods [PI and DiSC3(5)]. Daptomycin most rapidly permeabilizes cells with high initial membrane potential and dissipates it within a few minutes. Low initial membrane potential hinders daptomycin pore formation.

scholarly journals Cadherin Diffusion in Supported Lipid Bilayers Exhibits Calcium-Dependent Dynamic Heterogeneity

2016 ◽  
Vol 111 (12) ◽  
pp. 2658-2665 ◽  
Author(s):  
Yu Cai ◽  
Nitesh Shashikanth ◽  
Deborah E. Leckband ◽  
Daniel K. Schwartz
Keyword(s):  
Lipid Bilayers ◽  
Supported Lipid Bilayers ◽  
Dynamic Heterogeneity ◽  
Calcium Dependent

Functional reconstitution of receptors in artificial lipid bilayers

1987 ◽  
Vol 81 (1-2) ◽  
pp. 133-138 ◽  
Author(s):  
Vitaly Vodyanoy ◽  
Dominique Muller ◽  
Kathryn Kramer ◽  
Gary Lynch ◽  
Michel Baudry
Keyword(s):  
Lipid Bilayers ◽  
Artificial Lipid Bilayers

scholarly journals BAIAP3, a C2 domain–containing Munc13 protein, controls the fate of dense-core vesicles in neuroendocrine cells

2017 ◽  
Vol 216 (7) ◽  
pp. 2151-2166 ◽  
Author(s):  
Xingmin Zhang ◽  
Shan Jiang ◽  
Kelly A. Mitok ◽  
Lingjun Li ◽  
Alan D. Attie ◽  
...  
Keyword(s):  
Neuroendocrine Cells ◽  
Dense Core ◽  
Dense Core Vesicle ◽  
Protein Homeostasis ◽  
C2 Domain ◽  
Β Cells ◽  
Calcium Dependent ◽  
Trafficking Pathway ◽  
Protein Receptor ◽  
Golgi Network

Dense-core vesicle (DCV) exocytosis is a SNARE (soluble N-ethylmaleimide–sensitive fusion attachment protein receptor)-dependent anterograde trafficking pathway that requires multiple proteins for regulation. Several C2 domain–containing proteins are known to regulate Ca2+-dependent DCV exocytosis in neuroendocrine cells. In this study, we identified others by screening all (∼139) human C2 domain–containing proteins by RNA interference in neuroendocrine cells. 40 genes were identified, including several encoding proteins with known roles (CAPS [calcium-dependent activator protein for secretion 1], Munc13-2, RIM1, and SYT10) and many with unknown roles. One of the latter, BAIAP3, is a secretory cell–specific Munc13-4 paralog of unknown function. BAIAP3 knockdown caused accumulation of fusion-incompetent DCVs in BON neuroendocrine cells and lysosomal degradation (crinophagy) of insulin-containing DCVs in INS-1 β cells. BAIAP3 localized to endosomes was required for Golgi trans-Golgi network 46 (TGN46) recycling, exhibited Ca2+-stimulated interactions with TGN SNAREs, and underwent Ca2+-stimulated TGN recruitment. Thus, unlike other Munc13 proteins, BAIAP3 functions indirectly in DCV exocytosis by affecting DCV maturation through its role in DCV protein recycling. Ca2+ rises that stimulate DCV exocytosis may stimulate BAIAP3-dependent retrograde trafficking to maintain DCV protein homeostasis and DCV function.

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