The Nonbilayer Lipid MGDG and the Major Light-Harvesting Complex (LHCII) Promote Membrane Stacking in Supported Lipid Bilayers

The thylakoid membrane of algae and land plants is characterized by its intricate architecture, comprising tightly appressed membrane stacks termed grana. The contributions of individual components to grana stack formation are not yet fully elucidated. As an <i>in vitro</i> model, we use supported lipid bilayers made of thylakoid lipid mixtures to study the effect of major light-harvesting complex (LHCII), different lipids, and ions on membrane stacking, seen as elevated structures forming on top of the planar membrane surface in the presence of LHCII protein. These structures were examined by confocal laser scanning microscopy, atomic force microscopy, and fluorescence recovery after photobleaching, revealing multilamellar LHCII–membrane stacks composed of connected lipid bilayers. Both native-like and non-native interactions between the LHCII complexes may contribute to membrane appression in the supported bilayers. However, applying <i>in vivo</i>-like salt conditions to uncharged glycolipid membranes drastically increased the level of stack formation due to enforced LHCII–LHCII interactions, which is in line with recent crystallographic and cryo-electron microscopic data [Wan, T., et al. (2014) <i>Mol. Plant 7</i>, 916–919; Albanese, P., et al. (2017) <i>Sci. Rep. 7</i>, 10067–10083]. Furthermore, we observed the nonbilayer lipid MGDG to strongly promote membrane stacking, pointing to the long-term proposed function of MGDG in stabilizing the inner membrane leaflet of highly curved margins in the periphery of each grana disc because of its negative intrinsic curvature [Murphy, D. J. (1982) <i>FEBS Lett. 150</i>, 19–26]

Cell viability in the presence of PTX derivatives at different concentrations.

<p>(<b>A</b>) HeLa cells were incubated with PTX and caged PTX derivatives at concentrations between 0.01 and 10 µM. After 48 hours cultivation, cells were stained with PrestoBlue™ reagent. Metabolically active cells reduce the cell permeable dye resazurin into fluorescent resorufin, providing a quantitative measure of viable cells (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043657#s2" target="_blank">Materials and Methods</a>” section for details). (<b>B</b>) The viability of cells incubated with pre-irradiated solutions of 2′,7-bisNvoc-PTX.</p

Photolytic studies.

<p>(<b>A</b>) HPLC monitoring of the photoreaction and the changes in the composition of a 1 mM solution of 2′,7-bisNvoc-PTX after irradiation at 360 nm (2.7 mW/cm²) with increasing dose. (<b>B</b>) Time course of the quantified molar composition of the solution, as obtained from the HPLC data.</p

Effect of the presence of PTX and caged PTXs on the microtubule cytoskeleton of living cells.

<p>Fluorescence microscopy images of HeLa cells stably expressing GFP-tubulin imaged after 1 h (<b>A</b>) and 24 h (<b>B</b>) incubation in medium containing DMSO, PTX and caged derivatives at the indicated concentrations. White arrows indicate the cell edge deprived of microtubules. Scale bars: 20 µm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043657#pone.0043657.s009" target="_blank">Fig. S5</a> for images of assays with pre-irradiated compounds.</p

Water-Soluble NIR-Absorbing Rylene Chromophores for Selective Staining of Cellular Organelles

Biocompatible organic dyes emitting in the near-infrared are highly desirable in fluorescence imaging techniques. Herein we report a synthetic approach for building novel small <i>peri</i>-guanidine-fused naphthalene monoimide and perylene monoimide chromophores. The presented structures possess near-infrared absorption and emission, high photostability, and good water solubility. After a fast cellular uptake, they selectively stain mitochondria with a low background in live and fixed cells. They can be additionally modified in a one-step reaction with functional groups for covalent labeling of proteins. The low cytotoxicity allows a long time exposure of live cells to the dyes without the necessity of washing. Successful application in localization super-resolution microscopy was demonstrated in phosphate-buffered saline without any reducing or oxidizing additives

Ferrocenyl Glycidyl Ether: A Versatile Ferrocene Monomer for Copolymerization with Ethylene Oxide to Water-Soluble, Thermoresponsive Copolymers

The first ferrocene-containing epoxide monomer, ferrocenyl glycidyl ether (fcGE), is introduced. The monomer has been copolymerized with ethylene oxide (EO). This leads to electroactive, water-soluble, and thermoresponsive poly­(ethylene glycol) (PEG) derived copolyethers. Anionic homo- and copolymerization of fcGE with EO was possible. Molecular weights could be varied from 2000 to 10 000 g mol^–1, resulting in polymers with narrow molecular weight distribution (<i>M</i>_w/<i>M</i>_n = 1.07–1.20). The ferrocene (fc) content was varied from 3 to 30 mol %, obtaining water-soluble materials up to 10 mol % incorporation of the apolar ferrocenyl comonomer. Despite the steric bulk of fcGE, random copolymers were obtained, as confirmed via detailed ¹H NMR kinetic measurements as well as ¹³C NMR studies of the polymer microstructure, including detailed triad characterization. In addition, the poly­(fcGE) homopolymer has been prepared. All water-soluble copolyethers with fc side chains exhibited a lower critical solution temperature (LCST) in the range 7.2–82.2 °C in aqueous solution, depending on the amount of fcGE incorporated. The LCST is further tunable by oxidation/reduction of ferrocene, as demonstrated by cyclic voltammetry. Investigation of the electrochemical properties by cyclovoltammetry revealed that the iron centers can be oxidized reversibly. Further, to evaluate the potential for biomedical application, cell viability tests of the fc-containing PEG copolymers were performed on a human cervical cancer cell line (HeLa), revealing good biocompatibility only in the case of low amounts of fcGE incorporated (below 5%). Significant cytotoxic behavior was observed with fcGE content exceeding 5%. The ferrocene-substituted copolyethers are promising for novel redox sensors and create new options for the field of organometallic (co)­polymers in general

Protein Corona of Nanoparticles: Distinct Proteins Regulate the Cellular Uptake

Understanding nanoparticle–protein interactions is a crucial issue in the development of targeted nanomaterial delivery. Besides unraveling the composition of the nanoparticle’s protein coronas, distinct proteins thereof could control nanoparticle uptake into specific cell types. Here we differentially analyzed the protein corona composition on four polymeric differently functionalized nanoparticles by label-free quantitative mass spectrometry. Next, we correlated the relative abundance of identified proteins in the corona with enhanced or decreased cellular uptake of nanoparticles into human cancer and bone marrow stem cells to identify key candidates. Finally, we verified these candidate proteins by artificially decorating nanoparticles with individual proteins showing that nanoparticles precoated with the apolipoproteins ApoA4 or ApoC3 significantly decreased the cellular uptake, whereas precoating with ApoH increased the cellular uptake

Protein Corona of Nanoparticles: Distinct Proteins Regulate the Cellular Uptake

Understanding nanoparticle–protein interactions is a crucial issue in the development of targeted nanomaterial delivery. Besides unraveling the composition of the nanoparticle’s protein coronas, distinct proteins thereof could control nanoparticle uptake into specific cell types. Here we differentially analyzed the protein corona composition on four polymeric differently functionalized nanoparticles by label-free quantitative mass spectrometry. Next, we correlated the relative abundance of identified proteins in the corona with enhanced or decreased cellular uptake of nanoparticles into human cancer and bone marrow stem cells to identify key candidates. Finally, we verified these candidate proteins by artificially decorating nanoparticles with individual proteins showing that nanoparticles precoated with the apolipoproteins ApoA4 or ApoC3 significantly decreased the cellular uptake, whereas precoating with ApoH increased the cellular uptake

Intrinsic Burst-Blinking Nanographenes for Super-Resolution Bioimaging

Single-molecule localization microscopy (SMLM) is a powerful technique to achieve super-resolution imaging beyond the diffraction limit. Although various types of blinking fluorophores are currently considered for SMLM, intrinsic blinking fluorophores remain rare at the single-molecule level. Here, we report the synthesis of nanographene-based intrinsic burst-blinking fluorophores for highly versatile SMLM. We image amyloid fibrils in air and in various pH solutions without any additive and lysosome dynamics in live mammalian cells under physiological conditions. In addition, the single-molecule labeling of nascent proteins in primary sensory neurons was achieved with azide-functionalized nanographenes via click chemistry. SMLM imaging reveals higher local translation at axonal branching with unprecedented detail, while the size of translation foci remained similar throughout the entire network. These various results demonstrate the potential of nanographene-based fluorophores to drastically expand the applicability of super-resolution imaging

Intrinsic Burst-Blinking Nanographenes for Super-Resolution Bioimaging

Single-molecule localization microscopy (SMLM) is a powerful technique to achieve super-resolution imaging beyond the diffraction limit. Although various types of blinking fluorophores are currently considered for SMLM, intrinsic blinking fluorophores remain rare at the single-molecule level. Here, we report the synthesis of nanographene-based intrinsic burst-blinking fluorophores for highly versatile SMLM. We image amyloid fibrils in air and in various pH solutions without any additive and lysosome dynamics in live mammalian cells under physiological conditions. In addition, the single-molecule labeling of nascent proteins in primary sensory neurons was achieved with azide-functionalized nanographenes via click chemistry. SMLM imaging reveals higher local translation at axonal branching with unprecedented detail, while the size of translation foci remained similar throughout the entire network. These various results demonstrate the potential of nanographene-based fluorophores to drastically expand the applicability of super-resolution imaging