Rotation of Single Cell Surface Molecules Examined via Polarized FCS Measurements using Quantum Dot Probes

Sunday, February 12, 2017 molecule localization imaging [2]. In addition, fluorescence imaging of human cells and zebrafish has revealed rapid phosphatidylserine (PS) accumulation in the repair patch in a Dysf-mediated fashion. Single molecule trajectory analysis provides evidence that Dysf facilitates PS translocation from adjacent membrane regions to the site of lesion. [1] A. Lek et al., ‘‘Calpains, cleaved mini-dysferlinC72, and L-type channels underpin calcium-dependent muscle membrane repair’’, J. Neurosc., 2013, 33, 5085-94. [2] Middel, V., Zhou, L., Takamiya, M., Beil, T., Shahid, M., Roostalu, U., Grabher, C., Rastegar, S., Reischl, M., Nienhaus, G. U., & Str€ahle, U., ‘‘Dysferlin-Mediated Phosphatidylserine Sorting Engages Macrophages in Sarcolemma Repair’’, Nat. Commun., 2016, 7, 12875. 719-Pos Board B484 Rotation of Single Cell Surface Molecules Examined via Polarized FCS Measurements using Quantum Dot Probes Domgmei Zhang1, Peter W. Winter1, Deborah A. Roess2, B. George Barisas1. 1 Department of Chemistry, Colorado State University, Fort Collins, CO, USA, 2Department of Biomedical Sciences, Colorado State University, Fort Collins, CO, USA. Rotational motions of cell surface molecules are of particular interest given the sensitivity of such motions to molecular size and aggregation state. The time-autocorrelation function (TACF) of fluctuations in fluorescence polarization from asymmetric quantum dots (QD) such as Molecular Probes’ Qdot655 labeling cell surface molecules indicates the rotational correlation times (RCT) of the QD-containing complexes. Type I Fcε receptors (FcεRI) and insulin receptors (IR) on 2H3 cells, subjected to various treatments potentially affecting receptor rotation, including polyvalent DNP-BSA, methyl-b-cyclodextrin, cytochalasin D or paraformaldehyde, were labeled by Qdot655s. Over 700 individual QD were examined. Side-by-side vertically(v)- and horizontally(h)-polarized fluorescence images of QD-labeled cells were obtained simultaneously as image stacks at 10 ms per frame by means of a Princeton Instrument Dual-View. To minimize apparent polarization TACF arising from QD lateral diffusion, v- and h-substacks were aligned to subpixel accuracy. From fluorescence in identical regions around each QD in each stack, intensity and polarization fluctuation TACF were calculated. Given the blinking of QD, experimental parameters such as g-factor, camera background, etc. were carefully optimized for each QD to minimize cross-correlation between polarization and intensity fluctuations. This could otherwise appear as intensity fluctuation TACF contributing to the apparent polarization fluctuation TACF. Using these techniques, the initial polarization fluctuation TACF for FcεRI averages about 0.0015 and the geometricallyaveraged RCT is about 55-85 ms, both independent of cell treatment. More limited results on IR appear similar. Absence of treatment effects on magnitudes or decay rates of polarization TACF is puzzling. Previous time-resolved phosphorescence anisotropy studies on FcεRI suggest limited presence of orientational relaxations slower than 1 ms. However, the absence of treatment effects here suggests such slow reorientation may be a property of the membrane itself, perhaps reflecting large-scale fluctuations of mesoscale membrane regions. Supported by NSF grant MCB-1024668 and NIH grant CA175937 to BGB. 720-Pos Board B485 Super-Resolution Imaging Reveals Protein-Templated Patterns for Biosilica Formation Philip Gro¨ger, Nicole Poulsen, Jennifer Klemm, Nils Kro¨ger, Michael Schlierf. B CUBE – Center for Molecular Bioengineering, TU Dresden, Dresden, Germany. The intricate, genetically controlled biosilica nano- and micropatterns produced by diatoms are a testimony for biology’s ability to control mineral formation (biomineralization) at the nanoscale and regarded as paradigm for nanotechnology. Several recently discovered protein families involved in diatom biosilica formation remain tightly associated with the final biosilica structure. Determining the locations of biosilica-associated proteins with high precision is therefore expected to provide clues to their roles in biosilica morphogenesis. To achieve this, we introduce single-molecule localization microscopy to diatoms based on photo-activated light microscopy (PALM) to overcome the diffraction limit. We identified six photo-convertible fluorescent proteins (FPs) that can be utilized for PALM in the cytoplasm of Thalassiosira pseudonana. However, only three FPs that share a common molecular conversion-mechanism were also functional when embedded in diatom bio-

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silica and localized with a mean precision of 25 nm to resolve structural features. Further co-localization studies on proteins of the Cingulin family when extracted from the biosilica using a combined two-color PALMþSTORM approach revealed characteristic protein filaments with distinct protein specific patterns. The enhanced microscopy techniques introduced here for diatoms will aid in elucidating the molecular mechanism of silica biomineralization as well as other aspects of diatom cell biology. 721-Pos Board B486 Quantitative Super-Resolution Microscopy Detects HER2 Reorganization Following Meditope-Antibody Treatment Devin Wakefield, Raphael Jorand, Cindy Zer, John Williams, Tijana Jovanovic-Talisman. Beckman Research Institute at the City of Hope, Duarte, CA, USA. We combine quantitative super-resolution microscopy and meditope technology to investigate the effects of cross-linked antibodies on the organization of cancer receptor targets. A cyclic peptide, known as a meditope, has been recently discovered to bind within the Fab framework of Cetuximab and various meditope enabled monoclonal antibodies (memAbs). In this work, several divalent, Fc-based meditope ligands were generated with different linkers (linear sequences with 10, 20, or 30 amino acids). BT-474 cells were incubated with these meditopes, together with memAb Trastuzumab, and subsequently fixed for dSTORM imaging of human epidermal growth factor receptor 2 (HER2). The spatial organization of HER2 was characterized through pair-correlation (PC) analysis, using streamlined Matlab code with minimal user input and methods to reduce bias in selecting regions for analysis. According to our results, HER2 organization strongly depends on meditope linker length. Importantly, an increase in linker length was found to correlate with an increase in the proportion of HER2 oligomers. PC analysis further revealed that these HER2 oligomers organize within clusters ranging from approximately 14 to 25 nm in radius. Ultimately, exploring the molecular organization of receptors with our approach serves as an important step toward optimizing a diversity of ligands with potential therapeutic purposes. 722-Pos Board B487 3D Orbital Tracking of Single Gold Nanoparticles: A New Approach to Study Vesicle Trafficking in Chromaffin Cells Manuela Gabriel1, Jose Moya-Diaz2, Fernando D. Marengo2, Laura C. Estrada1. 1 Quantum Electronics Laboratory, Physics Department, University of Buenos Aires, Buenos Aires, Argentina, 2Institute of Physiology, Molecular Biology and Neuroscience, University of Buenos Aires, Buenos Aires, Argentina. Endocytosis and subsequent vesicle recycling serves to keep constant the pools of transmitter-containing vesicles ready for release in neurons and endocrine cells. The study of these processes has been carried out by using different experimental approaches, like electrophysiological measurements and single photon fluorescence microscopy. However, the diverse experimental limitations of these techniques restricted a detailed and high resolved study of the dynamics of vesicle trafficking after endocytosis in the whole cellular volume. Multiphoton microscopy provides optical sectioning for high-resolution imaging. In biological systems, most multiphoton microscopy studies have relied on two-photon excited fluorescence (TPEF) to produce images. Because of their strong brightness and imaging durability, metallic NPs have been recently introduced as labels in fluorescence microscopy. The use of TPEF and metallic nanoparticles in combination provides a noninvasive, spatially localized, in vivo characterization for biological samples. In this work, we tracked single gold nanoparticles after endocytotic internalization in mouse chromaffin cells stimulated with high potassium. We use an orbital-scanning tracking method in a two-photon absorption microscope. In the first place, we compare constitutive and active internalization of gold nanoparticles (AuNPs), assessing the number of internalized AuNPs and evaluating its dynamics in terms of velocity and displacement after stimulation. In the second place, we evaluate these parameters pharmacologically blocking proteins classically involved in the development of endocytotic process. Our results show that endocytosis of AuNPs was much more efficient when exocytosis was induced with high K in comparison with constitutive cycling. In addition, the dynamics of AuNPs had a strong dependence on clathrin dependent endocytosis as well as on cortical actin polymerization. This study shows that the combination of 3D orbital tracking and AuNPs is an interesting tool for the study of vesicle trafficking after endocytosis in live cells.