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Exocytosis of Single Bovine Adrenal Chromaffin Cell: The Electrical and Morphological Studies

Exocytosis of Single Bovine Adrenal Chromaffin Cell: The Electrical and Morphological Studies. Chia-Chang Tsai ( 蔡佳璋 ) Institute of Atomic and Molecular Sciences, Academia Sinica. NCKU, October 9, 2008. Outline. Introduction Electrical studies-

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Exocytosis of Single Bovine Adrenal Chromaffin Cell: The Electrical and Morphological Studies

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  1. Exocytosis of Single Bovine Adrenal Chromaffin Cell: The Electrical and Morphological Studies Chia-Chang Tsai (蔡佳璋) Institute of Atomic and Molecular Sciences, Academia Sinica NCKU, October 9, 2008

  2. Outline • Introduction • Electrical studies- Single-walled carbon nanotube field effect transistor • Morphological studies- Atomic force microscopy • Summary

  3. Exocytosis of chromaffin cell Histamine binds to histamine receptor Activate the PLC to release IP3 IP3 binds to specific receptor on Endoplasmic Reticulum (ER) Open the Ca2+ channel of ER Increase the concentration of Ca2+ Exocytosis and release of CgA N. Engl. J. Med. 2003, 348, 1134-49

  4. Electrical studies Schematic Illustration of Biosensor:Single-Wall Carbon Nanotube (SWCNT)-FET Antigen Antibody Drain Source Al2O3 50 nm Cr 50 nm SWCNT Blocking Agent Linker SiO2 400 nm p-Si Backgate Ti/Au 10/40 nm

  5. nanotube/nanowire-based-FET drain SWCNT 2 m source 1.5 cm

  6. NT/NW-FET PDMS Microfluidic Channel50 m  500 m  6.25 mm Al wire Source/ Drain Chip Gate

  7. Experimental Setup GPIB Lock-in Amplifier NT/NW-FET LabView Program VSD Supply Cell Chemicals GPIB Syringe Pump DAQ Card Microfluidic Channel VG Supply

  8. Surface Modification of CgA-Ab 1-pyrenebutanoic acid succinimidyl ester Steric effect Tween 20 Hydrophobic- hydrophobic interaction -stacking R. J. Chen, Y. Zhang, D. Wang, H. Dai, J. Am. Chem. Soc., 123, 3838 (2001). R. J . Chen, H. Dai, Proc. Nat. Acad. Sci., 100, 4984 (2003).

  9. Successful Modification of CgA-Ab on SWCNT-FET Change of I-Vg Curves: • Hysteresis width •  Modification coating • 2. Threshold voltage (Vth) •  Q = C Vth • 3. Transconductance (dI/dVg)  Scattering effect Small, 3, 1350 (2007)

  10. Recognition of CgAP in PBS (Phosphate Buffer Saline)by CgA-Ab Modified SWCNT-FET 100 nM CgAP 10 nM CgAP 4 ﹪up 60  g/mL BSA 1 nM CgAP 0.7 ﹪up Small, 3, 1350 (2007)

  11. Sensing mechanism Chemical gating effect： Isoelectric point of CgA (pI ~ 4.5) CgA is negatively charged in PBS buffer at pH = 7.4. Accordingly, the bindings of CgA with the CgA-Ab modified SWCNT-FET have enhanced the conductivity in the p-type semiconductive device due to a negative gating effect. Thining the Schottky barrier ：

  12. Recognition of CgAP in FBS (Fetal Buffer Saline)by CgA-Ab Modified SWCNT-FET 1 nM CgAP 100 pM CgAP Selectivity to particular targeted molecules! Small, 3, 1350 (2007)

  13. (a) A schematic of CgA-Ab functionalized SWCNT-FET device for histamine-evoked exocytosis of single bovine adrenal chromaffin cell. (b) Real image of single chromaffin cell moved onto one of the SWCNT-FET devices by a glass micropipette.

  14. micromanipulator Probe

  15. Immunochemistry of a Bovine Adrenal Chromaffin Cell 10 mM blue: Nucleus (DNA) green: Chromogranin A (CgA) red: Actin

  16. Comparison 1: CgA released by a lot of chromaffin cells 200 mM histamine

  17. J Phys Chem B2008, 112, 9165

  18. Calcium image 實驗時, 將Fura-2 AM溶于無血清培養液中, 終濃度4 mmol/L, 37℃裝載細胞30 min。開始實驗前, 用細胞外液洗去細胞外Fura-2 AM, 將細胞在細胞外液中靜置10 min, 使其適應和穩定以及Fura-2的完全去酯化。實驗中用340 nm和380 nm兩個不同入射波長的紫外光交替照射細胞, 觀察相應波長下Fura-2的發射光強度。以340 nm和 380 nm下螢光強度的比值反映[Ca2＋]i 水平。入射波長下的螢光強度信號經數模信號轉換器轉換后輸入到計算機中, 用Axoscope8.2記錄兩個相應入射波長時的Fura-2螢光強度, 以二者的比值反映[Ca2+]i變化。 [Ca2+]i記錄所用細胞外液成分為Loading buffer (pH=7.4)。 Biol. Proced. Online (2002);3(1): 70-78.

  19. The tapping mode feedback loop maintains a constant oscillation amplitude by maintaining a constant RMS of the oscillation signal acquired by the split photodiode detector. F=-kx F: Force k: spring constant x: cantilever deflection • Morphological studies

  20. Morphological studies PNAS. 94, 316 (1997) Dia. of Depressions T=0 156.7±3.9 nm T=5 min after stimulation 211.23±5.6 nm 35% increase T=30 min after stimulation 171.5±4.9 nm 20% decrease from T=5 min

  21. Changes were observed only in the depressions following stimulation of secretion. PNAS. 94, 316 (1997) exocytotic fusion pores

  22. Cho etc., New York Acad. Sci. 971;254 (2002)

  23. Experimental results Living chromaffin cell (contact mode AFM) (diameter, height or depth) (D,H) ~ (15 um, 4 um)

  24. Morphological studies Gold nanoparticles labeled fusion pores (Synaptotagmin antibody, 30 nm-AuNPs labeled 2nd antibody) 190 nm

  25. Fusion pore of chromaffin cell: histamine stimulation

  26. Stimulation of High K+ buffer High [K+] outside membrane  Membrane is depolarized  [Ca2+] influx  exocytosis

  27. Dense core vesicle distribution of mouse chromaffin cell (Electron microscope) J Neurophysio. 94, 2093 (2005)

  28. Analysis of vesicle size

  29. Thank you for your attention ! Acknowledgments 中研院原分所 陳逸聰教授 臺大動物所 潘建源教授 中研院物理所 陳啟東博士

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