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本帖最后由 细胞海洋 于 2013-5-6 09:12 编辑 * d9 c) T4 @; O: o& N9 r4 x
: s$ k$ g( C, K8 C. yNanoCellBiology of Secretion% w7 y6 B4 ]1 \2 F% U& H
Imaging Its Cellular and Molecular Underpinnings
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2 s. D% U- p$ r; YNanoCellBiology of Secretion ....................................................................... 1: V- M& p4 r8 D( r
Introduction ...................................................................................................... 1
# k$ H3 {* f* K$ U8 Q. AMaterials and Methods ..................................................................................... 5: Z6 `' q0 @; I) p# x* D
Pancreatic Acini Isolation ............................................................................ 5
. T% s. X- Q' |9 y! VZymogen Granule Isolation ......................................................................... 5
8 J H6 n7 t |/ e# G$ rIsolation of Synaptosomes, Synaptosomal Membrane,
1 C6 ~7 Y; P: U9 pand Synaptic Vesicles ................................................................................... 6
+ W9 w; e* I% S N0 g3 HPorosome Isolation ....................................................................................... 6
- b4 {* D% F1 I! w2 O# oAtomic Force Microscopy ........................................................................... 7
4 \, H$ T* Q( W \0 z7 h5 Q6 oMeasurement of Secretion ........................................................................... 7. l( w0 Z |7 V. _! [
Transmission Electron Microscopy.............................................................. 8
% W! o# z: |# s" ^: p+ oDynamic Light Scattering (DLS) to Determine Vesicle
7 {8 U. @: @3 G& {0 b( h% g, jVolume Change ............................................................................................ 8$ G1 I) @2 h+ V; t( r5 {
Photon Correlation Spectroscopy ................................................................ 8
6 d' J0 `0 D; z0 M+ yPorosome Functional Reconstitution Assay ................................................ 9* W: [& R% E7 W+ v# N
Preparation of Liposomes and SNARE Reconstitutions .............................. 9
8 H, b) r& F& S) a6 _, XCircular Dichroism Spectroscopy ................................................................ 10
- G2 Q) O6 b& I0 d. ^$ D4 gWide-Angle X-ray Diffraction ..................................................................... 10
/ G, Z; N8 k$ Q" V* d8 LPorosome Discovery ........................................................................................ 11
& z( k; i3 `2 s+ {Calcium and SNARE-Induced Membrane Fusion ........................................... 235 O* h1 t2 Y& }
v- and t-SNAREs Need to Reside in Opposing Membrane: l& k$ S4 H& u$ h
for Appropriate Interactions and the Establishment
: r9 O9 X |0 s& k+ }/ Iof Continuity Between Opposing Membranes ............................................. 26$ x0 v2 |- J& s! C5 j7 I5 g- C
Membrane Curvature Dictates the Size of the SNARE
3 u( Q# W1 W0 R4 W$ N3 M: c4 VRing Complex .............................................................................................. 31
% y/ h6 x N9 X' s& xDisassembly of the t-/v-SNARE Complex .................................................. 32
, A; }$ C+ ^0 Z4 ], s, u2 `6 YCD Spectroscopy Confi rms Membrane Requirement( ~$ ~" Y# _+ o- r! }; x% R
for Appropriate t-/v-SNARE Assembly and Suffi ciency
1 T y7 ]/ J* H1 ~. sof NSF–ATP for Its Disassembly ................................................................. 38' f3 g& d3 }, h% r' k. K
SNAREs Bring Opposing Bilayers into Close Approximation,
" j% Z6 x' k. p/ n, R! v7 KEnabling Calcium Bridging and Membrane Fusion .................................... 40
# `8 P6 |! E, u+ _" a/ |9 R( \Membrane Lipids Infl uence SNARE Complex
/ A) k* t% Q/ nand Assembly–Disassembly ........................................................................ 42
2 [0 Q: `) F0 P7 u7 PSecretion Involves Vesicle Swelling and Content Expulsion........................... 47
! `( k' u* `# A) a7 ], V h6 L1 a, `Secretory Vesicle Swelling Is Required for the Expulsion
$ q$ @: X. O* a; u* ?# p1 Aof Intravesicular Contents During Cell Secretion ........................................ 477 Y5 F# h" Y* O- M9 \, A$ N
Molecular Mechanism of Secretory Vesicle Swelling ................................. 53
" _; o8 x U" t: D; P, TPresence of Functional b-Adrenergic Receptors at the Synaptic
% e L1 j+ n9 v0 HVesicle Membrane ........................................................................................ 58
# V8 ?1 \ Y/ [* L" z% v) fConclusion ....................................................................................................... 626 J5 u0 F4 ~6 P$ L
References ........................................................................................................ 63
. R' u6 c7 D: Q$ T# |8 mIndex ................................................................................................................ 69% i0 ^( k7 {. r7 p
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发表于 2013-5-5 21:52
