PDF电子书：NanoCellBiology of Secretion

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NanoCellBiology of Secretion. n- C' ?5 K& G4 h& V
Imaging Its Cellular and Molecular Underpinnings+ P# l: B) e& K
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NanoCellBiology of Secretion ....................................................................... 12 s4 \+ V8 `+ B! u* q
Introduction ...................................................................................................... 1
- Q) O1 F5 d) ?) n3 zMaterials and Methods ..................................................................................... 5  y* X% j6 f+ R; j6 F* v  L
Pancreatic Acini Isolation ............................................................................ 5* C' v7 ?6 C' M- d  k, B) x! Y
Zymogen Granule Isolation ......................................................................... 5
1 e" T+ I. F6 M% xIsolation of Synaptosomes, Synaptosomal Membrane,/ b& G1 I, i7 p5 s- [4 c
and Synaptic Vesicles ................................................................................... 6
$ s$ u& m5 x$ d2 Y+ o/ G1 Y* iPorosome Isolation ....................................................................................... 6
4 i1 d( }6 \9 u! vAtomic Force Microscopy ........................................................................... 7
1 `( o) v$ D$ K# B) SMeasurement of Secretion ........................................................................... 7
8 r, G" _! F+ D4 U# {  oTransmission Electron Microscopy.............................................................. 8
  h5 s3 N+ z8 }1 z) ?Dynamic Light Scattering (DLS) to Determine Vesicle
; {# w: X. X0 L& A: J" a# r! pVolume Change ............................................................................................ 8- N5 n, s/ C# @
Photon Correlation Spectroscopy ................................................................ 8: h# }6 \  E, l( [' g4 B
Porosome Functional Reconstitution Assay ................................................ 9
9 {6 c# r5 m4 O% X, OPreparation of Liposomes and SNARE Reconstitutions .............................. 9
3 e) }1 n8 ^) S( n; MCircular Dichroism Spectroscopy ................................................................ 10
3 Q7 r) Z! [, b% @0 Q' t" B2 _Wide-Angle X-ray Diffraction ..................................................................... 10
0 e2 i7 q( p6 O/ `2 OPorosome Discovery ........................................................................................ 118 K$ Z: R; P7 q5 W6 R  V- ~7 m
Calcium and SNARE-Induced Membrane Fusion ........................................... 23, s* y0 e+ c! c* `) B  [$ ~2 S
v- and t-SNAREs Need to Reside in Opposing Membrane' i1 h) x$ L. s& A- N- w
for Appropriate Interactions and the Establishment
- ?1 d1 D" x' _8 ^# M3 Jof Continuity Between Opposing Membranes ............................................. 26; I1 _- e& {% o+ p/ v) i* _
Membrane Curvature Dictates the Size of the SNARE
& L1 {7 }$ h5 n" \% v1 ORing Complex .............................................................................................. 31" `1 j  ?) `& u* m3 r( ?. \$ W& F
Disassembly of the t-/v-SNARE Complex .................................................. 32
7 B( T2 E# o' J5 J/ _' nCD Spectroscopy Confi rms Membrane Requirement
! ^3 y  H# y# G0 t. q# t! Efor Appropriate t-/v-SNARE Assembly and Suffi ciency. L9 M+ d/ ~# O  s4 H
of NSF–ATP for Its Disassembly ................................................................. 38
$ h8 g. s: _% J. s% Q' gSNAREs Bring Opposing Bilayers into Close Approximation," C, p' y% p9 e& U5 ^
Enabling Calcium Bridging and Membrane Fusion .................................... 40
9 W  [7 K8 E: V. }$ Z6 S/ ]$ p& b0 DMembrane Lipids Infl uence SNARE Complex3 u* Z. W. d/ b
and Assembly–Disassembly ........................................................................ 421 z, g8 M2 S- z* |$ H" H
Secretion Involves Vesicle Swelling and Content Expulsion........................... 47
1 F6 q9 D+ A  X, J" ^Secretory Vesicle Swelling Is Required for the Expulsion
) n) u" E# z# o& x0 R- qof Intravesicular Contents During Cell Secretion ........................................ 47& J+ K. h; l2 u, O
Molecular Mechanism of Secretory Vesicle Swelling ................................. 53
0 z( G/ Q/ }1 t& aPresence of Functional b-Adrenergic Receptors at the Synaptic: d) v. G% l' \
Vesicle Membrane ........................................................................................ 58
2 [" G+ V2 W. F% ?Conclusion ....................................................................................................... 62
+ f$ X$ _) [, X, g2 GReferences ........................................................................................................ 637 I- ?9 z* }7 g/ I
Index ................................................................................................................ 69
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