Cholesterol is an essential component of most biological membranes and serves important functions in controlling membrane integrity, organization, and signaling. However, probes to follow the dynamic distribution of cholesterol in live cells are scarce and so far show only limited applicability. Herein, we addressed this problem by synthesizing and characterizing a class of versatile and clickable cholesterol-based imidazolium salts. We show that these cholesterol analogs faithfully mimic the biophysical properties of natural cholesterol in phospholipid mono- and bilayers, and that they integrate into the plasma membrane of cultured and primary human cells. The membrane-incorporated cholesterol analogs can be specifically labeled by click chemistry and visualized in live-cell imaging experiments that show a distribution and behavior comparable with that of endogenous membrane cholesterol. These results indicate that the cholesterol analogs can be used to reveal the dynamic distribution of cholesterol in live cells.
The protein-mediated formation of membrane contacts is a crucial event in many cellular processes ranging from the establishment of organelle contacts to the docking of vesicles to a target membrane. Annexins are Ca2+ regulated membrane-binding proteins implicated in providing such membrane contacts; however, the molecular basis of membrane bridging by annexins is not fully understood. We addressed this central question using annexin A2 (AnxA2) that functions in secretory vesicle exocytosis possibly by providing membrane bridges. By quantitatively analyzing membrane contact formation using a novel assay based on quartz crystal microbalance recordings, we show that monomeric AnxA2 can bridge membrane surfaces Ca2+ dependently. However, this activity depends on an oxidative crosslink involving a cysteine residue in the N-terminal domain and thus formation of disulfide-linked dimers. Alkylated AnxA2 in which this cysteine residue has been modified and AnxA2 mutants lacking the N-terminal domain are not capable of bridging membrane surfaces. In contrast, a heterotetrameric complex comprising two membrane binding AnxA2 subunits linked by a S100A10 dimer can provide membrane contacts irrespective of oxidation status. Thus, monomeric AnxA2 only contains one lipid binding site and AnxA2-mediated linking of membrane surfaces under non-oxidative intracellular conditions most likely requires AnxA2-S100 complex formation.
Cholesterol is an essential component of cellular membranes regulating the structural integrity and fluidity of biological bilayers and cellular processes such as signal transduction and membrane trafficking. However, tools to investigate the role and dynamics of cholesterol in live cells are still scarce and often show limited applicability. To address this, we previously developed a class of imidazolium-based cholesterol analogs, CHIMs. Here we confirm that CHIM membrane integration characteristics largely mimic those of cholesterol. Computational studies in simulated phospholipid bilayers and biophysical analyses of model membranes reveal that in biologically relevant systems CHIMs behave similarly to natural cholesterol. Importantly, the analogs can functionally replace cholesterol in membranes, can be readily labeled by click chemistry and follow trafficking pathways of cholesterol in live cells. Thus, CHIMs represent chemically versatile cholesterol analogs that can serve as a flexible toolbox to study cholesterol behavior and function in live cells and organisms.
The use of Quantum Dots (QDs) as fluorescent probes for understanding biological functions has emerged as an advantageous alternative over application of conventional fluorescent dyes. Intracellular delivery of QDs is currently a specific field of research. When QDs are tracking a specific target in live cells, they are mostly applied for extracellular membrane labeling. In order to study intracellular molecules and structures it is necessary to deliver free QDs into the cell cytosol. In this work, we adapted the freeze and thaw method to encapsulate water dispersed carboxyl-coated CdTe QDs into liposomes of different compositions, including cationic liposomes with fusogenic properties. We showed that labeled liposomes were able to fuse with live human stem cells and red blood cells in an endocytic-independent way. We followed the interactions of liposomes containing QDs with the cells. The results were minutely discussed and showed that QDs were delivered, but they were not freely diffused in the cytosol of those cells. We believe that this approach has the potential to be applied as a general route for encapsulation and delivery of any membrane-impermeant material into living cells.
The specific transport of amphiphilic compounds such as fluorescently labeled phospholipids into cells is a prerequisite for the analysis of highly dynamic cellular processes involving these molecules, e.g., the intracellular distribution and metabolism of phospholipids. However, cellular delivery remains a challenge as it should not affect the physiological integrity and morphology of the cell membrane. To address this, polymer nanocontainers based on redox‐responsive cyclodextrin (CD) amphiphiles are prepared, and their potential to deliver fluorescently labeled phospholipids to intracellular membrane compartments is analyzed. It is shown that mixtures of reductively degradable cyclodextrin amphiphiles and different phospholipids form liposome‐like vesicles (CD–lipid vesicles, CSSLV) with a homogeneous distribution of each lipid. Host–guest‐mediated self‐assembly of a cystamine‐crosslinked polymer shell on these CSSLV produces polymer‐shelled liposomal vesicles (PSSCSSLV) with the unique feature of a redox‐sensitive CSSLV core and reductively degradable polymer shell. PSSCSSLV show high stability and a redox‐sensitive release of the amphiphilic cargo. Live cell experiments reveal that the novel PSSCSSLV are readily internalized by primary human endothelial cells and that the reductive microenvironment of the cells' endosomes triggers the release of the amphiphilic cargo into the cytosol. Thus, PSSCSSLV represent a highly efficient system to transport lipid‐like amphiphilic cargo into the intracellular environment.
Mechanisms keeping leukocytes distant of local inflammatory processes in a resting state despite systemic release of inflammatory triggers are a pivotal requirement for avoidance of overwhelming inflammation but are ill defined. Dimers of the alarmin S100A8/S100A9 activate Toll‐like receptor‐4 (TLR4) but extracellular calcium concentrations induce S100A8/S100A9‐tetramers preventing TLR4‐binding and limiting their inflammatory activity. So far, only antimicrobial functions of released S100A8/S100A9‐tetramers (calprotectin) are described. It is demonstrated that extracellular S100A8/S100A9 tetramers significantly dampen monocyte dynamics as adhesion, migration, and traction force generation in vitro and immigration of monocytes in a cutaneous granuloma model and inflammatory activity in a model of irritant contact dermatitis in vivo. Interestingly, these effects are not mediated by the well‐known binding of S100A8/S100A9‐dimers to TLR‐4 but specifically mediated by S100A8/S100A9‐tetramer interaction with CD69. Thus, the quaternary structure of these S100‐proteins determines distinct and even antagonistic effects mediated by different receptors. As S100A8/S100A9 are released primarily as dimers and subsequently associate to tetramers in the high extracellular calcium milieu, the same molecules promote inflammation locally (S100‐dimer/TLR4) but simultaneously protect the wider environment from overwhelming inflammation (S100‐tetramer/CD69).
Annexin A2 (AnxA2) is a cytosolic Ca2+ regulated membrane binding protein that can induce lipid domain formation and plays a role in exocytosis and endocytosis. To better understand the mode of annexin-membrane interaction, we analyzed membrane-bound AnxA2 assemblies by employing a novel 3-armed chemical crosslinker and specific AnxA2 mutant proteins. Our data show that AnxA2 forms crosslinkable oligomers upon binding to membranes containing negatively charged phospholipids. AnxA2 mutants with amino acid substitutions in residues predicted to be involved in lateral protein–protein interaction show compromised oligomer formation, albeit still being capable of binding to negatively charged membranes in the presence of Ca2+. These results suggest that lateral protein–protein interactions are involved in the formation of AnxA2 clusters on a biological membrane.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.