Cholesterol Slows down the Lateral Mobility of an Oxidized Phospholipid in a Supported Lipid Bilayer

We investigated the mobility and phase-partitioning of the fluorescent oxidized phospholipid analogue 1-palmitoyl-2-glutaroyl-sn-glycero-3-phospho-N-Alexa647-ethanolamine (PGPE-Alexa647) in supported lipid bilayers. Compared to the conventional phospholipid dihexadecanoylphosphoethanolamine (DHPE)-Bodipy we found consistently higher diffusion constants. The effect become dramatic when immobile obstacles were inserted into the bilayer. which essentially blocked the diffusion of DHPE-Bodipy but hardly influenced the movements of PGPE-Alexa647. In a supported lipid bilayer made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), the differences in probe mobility leveled off with increasing cholesterol content. Using coarse-grained molecular dynamics simulations, we could ascribe this effect to increased interactions between the oxidized phospholipid and the membrane matrix, concomitant with a translation in the headgroup position of the oxidized phospholipid: at zero cholesterol content, its headgroup is shifted to the outside of the DOPC headgroup region, whereas increasing cholesterol concentrations pulls the headgroup into the bilayer plane

Correlated Multimodal Imaging in Life Sciences:Expanding the Biomedical Horizon

International audienceThe frontiers of bioimaging are currently being pushed toward the integration and correlation of several modalities to tackle biomedical research questions holistically and across multiple scales. Correlated Multimodal Imaging (CMI) gathers information about exactly the same specimen with two or more complementary modalities that-in combination-create a composite and complementary view of the sample (including insights into structure, function, dynamics and molecular composition). CMI allows to describe biomedical processes within their overall spatio-temporal context and gain a mechanistic understanding of cells, tissues, diseases or organisms by untangling their molecular mechanisms within their native environment. The two best-established CMI implementations for small animals and model organisms are hardware-fused platforms in preclinical imaging (Hybrid Imaging) and Correlated Light and Electron Microscopy (CLEM) in biological imaging. Although the merits of Preclinical Hybrid Imaging (PHI) and CLEM are well-established, both approaches would benefit from standardization of protocols, ontologies and data handling, and the development of optimized and advanced implementations. Specifically, CMI pipelines that aim at bridging preclinical and biological imaging beyond CLEM and PHI are rare but bear great potential to substantially advance both bioimaging and biomedical research. CMI faces three mai

Temporal resolution of protein–protein interactions in the live-cell plasma membrane

We have recently devised a method to quantify interactions between a membrane protein (“bait”) and a fluorophore-labeled protein (“prey”) directly in the live-cell plasma membrane (Schwarzenbacher et al. Nature Methods 5:1053–1060 2008). The idea is to seed cells on surfaces containing micro-patterned antibodies against the exoplasmic domain of the bait, and monitor the co-patterning of the fluorescent prey via fluorescence microscopy. Here, we characterized the time course of bait and prey micropattern formation upon seeding the cells onto the micro-biochip. Patterns were formed immediately after contact of the cells with the surface. Cells were able to migrate over the chip surface without affecting the micropattern contrast, which remained constant over hours. On single cells, bait contrast may be subject to fluctuations, indicating that the bait can be released from and recaptured on the micropatterns. We conclude that interaction studies can be performed at any time-point ranging from 5 min to several hours post seeding. Monitoring interactions with time opens up the possibility for new assays, which are briefly sketched in the discussion section

Plasma Membrane Lipids: An Important Binding Site for All Lipoprotein Classes

Cholesterol is one of the main constituents of plasma membranes; thus, its supply is of utmost importance. This review covers the known mechanisms of cholesterol transfer from circulating lipoprotein particles to the plasma membrane, and vice versa. To achieve homeostasis, the human body utilizes cellular de novo synthesis and extracellular transport particles for supply of cholesterol and other lipids via the blood stream. These lipoprotein particles can be classified according to their density: chylomicrons, very low, low, and high-density lipoprotein (VLDL, LDL, and HDL, respectively). They deliver and receive their lipid loads, most importantly cholesterol, to and from cells by several redundant routes. Defects in one of these pathways (e.g., due to mutations in receptors) usually are not immediately fatal. Several redundant pathways, at least temporarily, compensate for the loss of one or more of them, but the defects trigger systemic diseases, such as atherosclerosis later on. Recently, intracellular membrane–membrane contact sites were shown to be involved in intracellular cholesterol transfer and the plasma membrane itself has been proposed to act as a binding site for lipoprotein-mediated cargo unloading

Serum and Lipoprotein Particle miRNA Profile in Uremia Patients

microRNAs (miRNAs) are post-transcriptional regulators of messenger RNA (mRNA), and transported through the whole organism by—but not limited to—lipoprotein particles. Here, we address the miRNA profile in serum and lipoprotein particles of healthy individuals in comparison with patients with uremia. Moreover, we quantitatively determined the cellular lipoprotein-particle-uptake dependence on the density of lipoprotein particle receptors and present a method for enhancement of the transfer efficiency. We observed a significant increase of the cellular miRNA level using reconstituted high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yielded no measurable effect. Thus, we conclude that no relevant effect of lipoprotein-particle-mediated miRNA-transfer exists under in vivo conditions though the miRNA profile of lipoprotein particles can be used as a diagnostic marker

Enzymatic synthesis of hyaluronic acid vinyl esters for two-photon microfabrication of biocompatible and biodegradable hydrogel constructs

The final publication is available via https://doi.org/10.1039/c4py00792a.Two-photon polymerization (2PP) allows 3D microfabrication of biomaterial scaffolds with user-defined geometry. This technique is highly promising for 3D cell culture and tissue engineering. However, biological applications of 2PP require photopolymerizable hydrogels with high reactivity and low cytotoxicity. This paper describes a novel hydrogel system based on hyaluronic acid vinyl esters (HA-VE), which enabled fast 2PP-fabrication of 3D hydrogel constructs with μm-scale accuracy. A series of HA-VE macromers with tunable degrees of substitution were synthesized by lipasecatalyzed transesterification. HA-VE gels were proved to be injectable, photocurable, enzymatically degradable and mechanically comparable to various soft tissues. Owing to the unique molecular design, degradation products of HA-VE gels through hydrolysis are non-toxic polyvinyl alcohol and adipic acid. Furthermore, HA-VE gels were systematically characterized and compared to HA-acrylates (HA-AC) and HA-methacrylates (HA-MA) gels including macromer cytotoxicity, photoreactivity, swelling, and gel stiffness. Cytotoxicity assay with L929 fibroblasts revealed that HA-VE was significantly less toxic than HA-AC (P<0.01) and HA-MA (P<0.05). Crosslinking efficiency of HA-VE was comparable to HA-AC and much higher than HA-MA. Although the reactivity of HA-VE for homopolymerization was insufficient for 2PP, it was demonstrated that thiol-ene chemistry could substantially improve its reactivity. This optimization led to 2PP-fabrication of a HA-VE hydrogel construct with μm-scale accuracy. Low cytotoxicity, high reactivity and good biodegradability makes HA-VE promising candidates for biological applications in cell culture and tissue engineering

Localization Microscopy of Actin Cytoskeleton in Human Platelets

Here, we measure the actin cytoskeleton arrangement of different morphological states of human platelets using a new protocol for photo-switching of rhodamine class fluorophores. A new medium composition was established for imaging the cytoskeleton using Alexa Fluor 488 conjugated to phalloidin. Morphological states of platelets bound to a glass substrate are visualized and quantified by two-dimensional localization microscopy at nanoscopic resolution. Marker-less drift correction yields localization of individual Alexa 488 conjugated to phalloidin with a positional accuracy of 12 nm