Conformational Changes as Driving Force for Phase Recognition: The Case of Laurdan

The development of a universal probe to assess the phase of a lipid membrane is one of the most ambitious goals for fluorescence spectroscopy. The ability of a well-known molecule as Laurdan to reach this aim is here exploited as the behavior of the probe is fully characterized in a dipalmitoyl-phosphatidylcholine (DPPC) solid gel (So) phase by means of molecular dynamics simulations. Laurdan can take two conformations, depending on whether the carbonyl oxygen points toward the beta-position of the naphthalene core (Conf-I) or to the alpha-position (Conf-II). We observe that Conf-I has an elongated form in this environment, whereas Conf-II takes an L-shape. Interestingly, our theoretical calculations show that these two conformations behave in an opposite way from what is reported in the literature for a DPPC membrane in a liquid disordered (Ld) phase, where Conf-I assumes an L-shape and Conf-II is elongated. Moreover, our results show that in DPPC (So) no intermixing between the conformations is present, whereas it has been seen in a fluid environment such as DOPC (Ld). Through a careful analysis of angle distributions and by means of the rotational autocorrelation function, we predict that the two conformers of Laurdan behave differently in different membrane environments.S.O. acknowledges the National Science Centre, Poland, grant UMO-2015/19/P/ST4/03636, for the funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement no. 665778. The Swedish Infrastructure Committee (SNIC) is acknowledged for the computational time granted through the medium allocations 1-87 and 1-415 (2016); 1-16, 1-102 (2017); and 3-397, 3-156, 3-396, 3-23 (2018). The Flemish Supercomputer Centre (VSC) (Flanders, Belgium) and the Herculesstichting (Flanders, Belgium) are acknowledged for the generously allocated computational time on the Tier-1 cluster Breniac as well as the Tier-2 cluster Thinking

Identification, evolution and functional characterization of two Zn CDF-family transporters of the ectomycorrhizal fungus Suillus luteus

Two genes, SlZnT1 and SlZnT2, encoding Cation Diffusion Facilitator (CDF) family transporters were isolated from Suillus luteus mycelium by genome walking. Both gene models are very similar and phylogenetic analysis indicates that they are most likely the result of a recent gene duplication event. Comparative sequence analysis of the deduced proteins predicts them to be Zn transporters. This function was confirmed by functional analysis in yeast for SlZnT1. SlZnT1 was able to restore growth of the highly Zn sensitive yeast mutant zrc1 and localized to the vacuolar membrane. Transformation of zrc1 yeast cells with SlZnT1 resulted in an increased accumulation of Zn compared to empty vector transformed zrc1 yeast cells and equals Zn accumulation in wild type yeast cells. We were not able to express functional SlZnT2 in yeast. In S. luteus, both SlZnT genes are constitutively expressed whatever the external Zn concentrations. A labile Zn pool was detected in the vacuoles of S. luteus free-living mycelium. Therefore we conclude that SlZnT1 is indispensable for maintenance of Zn homeostasis by transporting excess Zn into the vacuole

Fluorescent PCDTBT Nanoparticles with Tunable Size for Versatile Bioimaging

Conjugated polymer nanoparticles exhibit very interesting properties for use as bio-imaging agents. In this paper, we report the synthesis of PCDTBT (poly([9-(1’-octylnonyl)-9H-carbazole-2,7-diyl]-2,5-thiophenediyl-2,1,3-benzothiadiazole-4,7-diyl-2,5-thiophene-diyl)) nanoparticles of varying sizes using the mini-emulsion and emulsion/solvent evaporation approach. The effect of the size of the particles on the optical properties is investigated using UV-Vis absorption and fluorescence emission spectroscopy. It is shown that PCDTBT nanoparticles have a fluorescence emission maximum around 710 nm, within the biological near-infrared “optical window”. The photoluminescence quantum yield shows a characteristic trend as a function of size. The particles are not cytotoxic and are taken up successfully by human lung cancer carcinoma A549 cells. Irrespective of the size, all particles show excellent fluorescent brightness for bioimaging. The fidelity of the particles as fluorescent probes to study particle dynamics in situ is shown as a proof of concept by performing raster image correlation spectroscopy. Combined, these results show that PCDTBT is an excellent candidate to serve as a fluorescent probe for near-infrared bio-imaging

Diffusion of myelin oligodendrocyte glycoprotein in living OLN-93 Cells investigated by raster-scanning image correlation spectroscopy (RICS)

Many membrane proteins and lipids are partially confined in substructures ranging from tens of nanometers to micrometers in size. Evidence for heterogeneities in the membrane of oligodendrocytes, i.e. the myelin-producing cells of the central nervous system, is almost exclusively based on detergent methods. However, as application of detergents can alter the membrane phase behaviour, it is important to investigate membrane heterogeneities in living cells. Here, we report on the first investigations of the diffusion behavior of the myelin-specific protein MOG (myelin oligodendrocyte glycoprotein) in OLN-93 as studied by the recently developed RICS (raster-scanning image correlation spectroscopy) technique. We implemented RICS on a standard confocal laser-scanning microscope with one-photon excitation and analog detection. Measurements on FITC-dextran were used to evaluate the performance of the system and the data analysis procedure.status: publishe