Development of Biophysical Markers That Quantify ... - Cell Press

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Tuesday, February 14, 2017

(DCVs) from PC12 cells, an immortalized neuroendocrine cell line. Fusion assays based on bulk lipid mixing of DCVs with proteoliposomes containing a highly reactive SNARE acceptor complex were used to determine the overall calcium response of fusion. A fluorescently tagged secretory protein, neuropeptide Y (NPY), was used to specifically label the contents of the DCVs to monitor single vesicle docking and fusion events to a planar supported bilayer containing reconstituted acceptor SNARE complexes. The results show that docking and fusion both depend on calcium, but in different fashions, indicating that different molecular players contribute to each sub-reaction. Knock downs using shRNA were used to determine which resident DCV proteins are responsible for the calcium response of docking and fusion. Soluble accessory proteins known to modulate SNARE proteins were added to fusion assays to determine the molecular requirements to have a fully calcium dependent SNARE mediated fusion process. 1955-Pos Board B275 Fusion Pore Selectivity in Chromaffin Cells Joannalyn Delacruz1, Meng Huang2, Joan Lenz2, Manfred Lindau2, Shailendra Rathore2. 1 Field of Pharmacology, Cornell University, Ithaca, NY, USA, 2Applied Engineering Physics, Cornell University, Ithaca, NY, USA. Healthy physiological conditions depend on dynamic release of neurotransmitters and hormones by exocytosis, which is initiated by the fusion pore. Previous studies show that reduction of extracellular [NaCl] decreases the amperometric foot current, indicating no co-release of anions with transmitter. It is unknown if this is due to cation selectivity of the fusion pore or due to negatively charged ions, such as ATP, being bound to the granular matrix and unavailable for release. Such selectivity might be expected if the fusion pore is lined by negative charges from lipids and protein. If the initial fusion pore is selective for cations, then its selectivity will affect the rate of transmitter release via the reversal potential across the fusion pore. If the fusion pore is non-selective, then the flux of anions from extracellular space into the vesicle is expected to reduce the rate of release of positively charged catecholamines by making the vesicle lumen more negative. Using amperometry we investigate in chromaffin cells if the transmitter release rate through the fusion pore changes when extracellular chloride is exchanged by the less mobile D-glutamate anion. Electrodiffusion calculations predict that for a non-selective anion permeable fusion pore the average foot current amplitude should increase by ~30% when chloride is exchanged by D-glutamate with a mobility of 0.25 relative to chloride, whereas no change in foot current is expected for a cation selective fusion pore. Preliminary analysis of all amperometric events in chromaffin cells, results in a mean amperometric foot current of 1.1450.139 pA in the presence of chloride, and 1.0450.07 pA for D-glutamate. Further sample collections and analysis will reveal if there is a significant difference in foot current amplitude depending on anion mobility and reveal if the early narrow fusion pore is cation selective. 1956-Pos Board B276 Dissecting the Biomechanical Feedback Between Plasma Membrane Curvature and Endocytic Proteins in Mammalian Cells using Nanostructured Substrates Jessica R. Marks1, Guiseppe Calafiore2, Stefano Cabrini2, David Drubin1. 1 University of California Berkeley, Berkeley, CA, USA, 2Lawrence Berkeley National Laboratory, Berkeley, CA, USA. Plasma membrane curvature is the hallmark of endocytosis. A mechanistic feedback loop between endocytic proteins and the plasma membrane drives clathrin-mediated endocytosis from pit to vesicle; however, the macro-scale relationship between the endocytic protein complex and membrane curvature is still not understood. We previously used a novel quartz ‘‘nanostructured’’ substrate to induce stable plasma membrane curvature, showing that endocytic proteins are preferentially recruited to curvatures < 200 nm. Here we use a polymer molded on to a glass substrate and live genome edited cells to define the relationship between curvature-sensing endocytic proteins and the curved plasma membrane. MDA-MB231 and human fibroblasts, endogenously expressing the fluorescent fusion proteins AP2, clathrin, and/or dynamin2 were used to monitor endocytosis by time-lapse fluorescence microscopy. Cells serially depleted for the late stage endocytic BAR domain proteins SNX9, Bin1, and EndophilinA2 were imaged to determine the molecular requirements for clathrin-mediated endocytosis in cells with a pre-curved plasma membrane. We also monitored how actin destabilization, and the depletion of N-WASP or the Arp2/3 complex affected the outcome of clathrin-mediated endocytosis in the context of induced membrane curvature. Finally, the size of the curvature-generating substrates was varied to determine the relevant range of membrane curvatures for clathrin-mediated endocytosis under depletion conditions.

1957-Pos Board B277 Simulation-Guided Optimization of Electrode Arrays for Electrochemical Imaging of Quantal Exocytosis Seyedmehdi Orouji1, Kevin D. Gillis2. 1 Dalton Cardiovascular Research Center, University of Missouri - Columbia, Columbia, MO, USA, 2Dept. Bioengineering, University of Missouri Columbia, Columbia, MO, USA. Fluorescence imaging can provide important information about the mechanisms of exocytosis at the single-vesicle level, e.g., conformational changes in SNARE proteins reported via FRET. FRET signals are very small and therefore the time and location of the fusion events are not readily apparent to allow signal analyses. The Lindau lab has pioneered the use of ‘‘electrochemical imaging’’ to identify the time and location of quantal exocytosis by comparing the amperometric signal between 4 electrodes arranged around a cell. Since it is necessary to obtain a measurable signal in at least three electrodes in order to ‘‘triangulate’’ the position, a limitation of this method is that it can only localize exocytosis events that originate near the center of the electrode arrays. We therefore carried out finite-element-array (COMSOL) reaction-diffusion simulations to guide the design of electrode arrays that can maximize the area on the cell surface where a release event can be localized with high precision. The number and placement of electrodes around the cell were varied and the SD of localization precision was simulated. Our results indicate that as the number of electrodes arranged circumferentially around the cell increases, the detectable area further from the center of the array increases, however, the precision of localization in the center decreases because the amperometric charge is divided between more electrodes. Thus the optimal number of electrodes depends on the signal-to-noise ratio of the amperometric recordings and the acceptable location precision. Under our simulation conditions, if a localization precision (SD) less than 0.23 mm is needed, an array of 4 electrodes will give the largest detectable area. However, if slightly less precision is acceptable, 5 or 6 electrodes allows a larger detectable area than 4 or 3 electrodes. Supported by NIH R44MH096650. 1958-Pos Board B278 Development of Biophysical Markers That Quantify Metastatic Potentials of Prostate Cancer Cells using Tsunami Microscope Yen-Liang Liu1, Aaron M. Horning2, Evan P. Perillo1, Cong Liu1, Mirae Kim1, Rohan Vasisht1, Hannah Horng3, Andrew K. Dunn1, Chun-Liang Chen2, Hsin-Chih Yeh1. 1 Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX, USA, 2Department of Molecular Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA, 3Department of Bioengineering, University of Maryland, College Park, College Park, MD, USA. Dysregulated trafficking of receptor tyrosine kinases has been linked to oncogenesis. Here we study the trafficking patterns and dynamics of epidermal growth factor receptor (EGFR) trafficking of benign (BPH1), non-invasive malignant (LNCaP), and highly invasive malignant (PC-3) prostate cells using an advanced 3D single-particle tracking technique termed TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination). As a feedback-control tracking system, TSUNAMI is capable of tracking fluorescent nanoparticle-tagged EGFR for up to 10 minutes and in the z-direction for up to 550 microns. To analyze the long 3D trajectories generated by the TSUNAMI microscope, a trajectory analysis algorithm is developed to classify trajectories and extract the dynamic parameters, such as diffusivity, inward movement, and internalization duration. These parameters can be used to quantify the metastatic potentials of prostate cancer cell lines. For instance, the diffusivities of EGFRs on the highly invasive malignant PC-3 cells (0.010 5 0.014 mm2/s) are around one-quarter of those estimated from the benign BPH1 cells (0.036 5 0.058 mm2/s), possibly due to the abnormally high expression of EGFRs on the PC-3 cells. The highly invasive PC-3 cells also exhibit longer (2.83 5 0.23 mm vs. 1.45 5 0.16 mm) and faster (0.021 5 0.016 mm/s vs. 0.005 5 0.002 mm/s) inward movement as compared with the noninvasive LNCaP prostate cancer cells, which could be due to the high endocytotic activity associated with the invasive PC-3 cells. In addition, the dynamics parameters extracted from the EGFR trajectories are correlated with the expression levels of a number of epithelial-mesenchymal-transition (EMT)-related genes. The high EGFR expression is related to the decrease of EGFR diffusivity, and the increase of dynamins coheres with more active inward movement. After EMT induction, the non-invasive LNCaP prostate cancer cells also exhibit gene expression profiles and EGFR trafficking patterns similar to those shown in invasive PC-3 cells. Our work demonstrates that EGFR trajectory-derived dynamics parameters are linked to metastatic potentials. A new class of biophysical markers can be established based on the analysis of membrane protein trajectories.