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Nonradiative Excitation Fluorescence Microscopy (NEFM) is a promising technique allowing the observation of biological samples beyond the diraction limit. By coating a substrate with an homogeneous monolayer of quantum dots (QDs), NEFM is achieved through a nonradiative energy transfer from QDs (donors) to dye molecules located in the sample (acceptors). The excitation depth of the sample is then given by the Förster radius, which corresponds to few nanometers above the surface. The powerful axial resolution of NEFM is highlighted by observing the adhesion of Giant Unilamellar Vesicles (GUVs) on strong interaction with coated surfaces. In this paper, we demonstrate that the QD-quenching level is valuable to calculate and map the distance between the membrane and the surface with a high precision along the optical axis. By tuning the electrostatic interactions between the membrane and the substrate, we have been able to measure a height displacement of ≈ 1 nm of the lipid membrane. The experimental results were discussed according to simulations, which take into account all the common forces appearing between lipid membranes and surfaces. 1 Keywords Biophotonics, Förster Resonance Energy Transfer, Membrane, Nanoscopy Förster Resonance Energy Transfer (FRET) is now a standard technique, widely employed in biophysics and biophtonics. FRET relies on a nonradiative energy transfer from excited donor molecules to acceptor molecules in their ground state. This energy transfer is ecient as long as the distance between the two molecules is less than 10 nm. FRET is usually considered as a nanoscale ruler with a broad area of applications that includes structural biology, 1 biosensing, 2,3 binding measurement between molecules 4 or structure of intermembrane junction. 5 The signal obtained from FRET is typically examined by spectra, uorescence pictures or by time-resolved investigations. In this paper, we propose an original imaging method, based on donor quenching analysis, to measure, with a nanoscale accuracy, distances involved in the adhesion of membranes on a surface.

Amyloid fibrils are associated with many diseases; Amyloid-b in particular is related to Alzheimer's Disease. We present the structure of an Ab(1-42) fibril determined by cryo-EM to a resolution of 4.0 Å . The fibril consists of two intertwined protofilaments in which individual subunits form a 'LS'-shaped topology revealing a previously unknown dimer interface, different from previous solid-state NMR models. All 42 aminoacids are well resolved in the EM density map, including the N-terminal part. The high resolution structure is in agreement with solid-state NMR and X-ray diffraction experiments. 824-Pos Board B594Microtubules are dynamic, filamentous structures that are essential for many different cellular processes. These processes often rely on a number of microtubule associated proteins (MAPs) that either regulate or carry out these functions. Characterizing interactions between MAPs and microtubules is crucial to understanding of how these processes are carried out. Recent advances in cryoelectron microscopy have made observing interactions between microtubules and several different MAPs at near atomic resolution possible. However, the resolution of these structures is variable, depending on the MAP. Recent studies have shown that the microtubule lattice is flexible and different stabilizing drugs and MAPs affects this flexibility. These results suggest that distortions in the microtubule lattice are limiting overall resolution. Rather than aligning whole segments of microtubules (like in conventional microtubule reconstructions), we have developed a technique that aligns patches of tubulin. By subtracting the surrounding microtubule density and leaving a patch of tubulin dimers, each patch can be aligned independently from the bulk of the microtubule. We have tested this 'patch refinement' technique on a synthetically, distorted dataset. Using traditional reconstruction techniques yielded a low resolution structure, but upon application of the patch refinement technique, the resulting structure was nearly indistinguishable from an equivalent undistorted, synthetic dataset. The patch refinement method has also been applied to an experimental dataset that was resolved to >5Å using traditional methods. Following patch refinement, the resolution increased to <3.6Å . Using the alignment parameters following patch refinement, we were able to trace the path of individual protofilaments and observe distortions in the lattice. Further use and development of this technique will allow us to analyze microtubule distortions and work towards obtaining an atomic structure of a microtubule.

Brag2, which controls integrin endocytosis and cell adhesion and is impaired in cancer and developmental diseases. Brag2 activates Arf GTPases by stimulating the replacement of GDP by GTP, leading to the active, membraneattached form of Arf which recruits downstream effectors. We showed previously that, although Brag2 is highly active in solution, negatively charged membranes stimulate its efficiency by up to 3 orders of magnitude. We analyzed the determinants of this spectacular contribution of membranes by combining crystallography, coarse-grained molecular dynamics and reconstitution of Arf GTPases and Brag2 in artificial membranes. We found that the Arf/ Brag complex forms multiple interactions with charged lipids to organize a precise orientation of the complex with respect to the membrane that determines its activation kinetics. We discovered that this determinant can be harnessed by a new type of cell-active, allosteric inhibitors, that impair the orientation of the complex by binding at the GEF/lipid interface. 938-SympLipid Binding Specificity of the KRAS Membrane Anchor The KRAS membrane anchor exhibits exquisite specificity for phosphatidylserine (PtdSer) over other anionic phospholipids by virtue of defined structural dynamics of the C-terminal polybasic domain and farnesyl group. This specificity extends to PtdSer with specific combinations of lipid side chains. PtdSer binding is therefore essential for stable KRAS plasma membrane (PM) binding and the spatial organization of KRAS into nanoclusters that together are essential for signal transmission. In consequence depleting the PM of PtdSer would be expected to abrogate KRAS function. Two inhibitors of SM metabolism have recently been shown to reduce PM PtdSer levels and abrogate KRAS signaling. We therefore used RNAis against a further 18 C.elegans SM biosynthetic enzyme orthologs in a worm model of activated KRAS signaling to more broadly assess potential therapeutic possibilities. We found that knock down of many of these enzymes strongly or moderately suppress the multi-vulva phenotype induced by an oncogenic mutant of LET60, a KRAS ortholog. Concordantly pharmacological agents that alter the function of these enzymes in mammalian cells also cause mislocalization of KRAS from the PM, regardless of whether the net effect of the compound is to increase or decrease cellular SM. The common mechanism of action was depletion of PM PtdSer content. These compounds also potently inhibit the proliferation of oncogenic KRAStransformed pancreatic cancer cells. Thus normal SM metabolism is required to maintain PM PtdSer levels and hence KRAS function. Similar results were obtained by shRNA or CRIPSR knock down of components of the ER-PM PtdSer exchange machinery, which also abrogated KRAS PM binding and nanoclustering, again highlighting the key dependence of KRAS function on PM PtdSer content. 939-SympRole of the G-Domain in RAS Isoform Dependent Turmorigenisis Recent advances in fluorescence super-resolution microscopy have allowed subcellular features and ...