2.1.1 Reagents
Table 2.1 List of reagents used in this study
Product Catalog
Number Company
Commercial membrane markers FM 1-43 70020 Biotium, Hayward, CA, USA
AM 1-43 70024 Biotium
FM 4-64 70021 Biotium
FM 4-64FX F34653 Molecular Probes, Life
Technologies, Darmstadt, Germany
FM 1-84 70047 Biotium
FM 3-25 70048 Biotium
5-Dodecanoylaminofluorescein
(DCF) D109 Molecular Probes, Life
Technologies
Di-2-ANEPEQ D6923 Molecular Probes, Life
Technologies
Development of new membrane-binding tools Transferrin – Alexa 594 T13343 Molecular Probes, Life Technologies
Insulin human I2643 Sigma-Aldrich, Munich, Germany
Atto 647N NHS ester AD 647N-31 ATTO-TEC GmbH, Siegen, Germany Palmitic acid N-hydroxysuccinimide
(PLL 1-5 kDa) P0879 Sigma-Aldrich
Poly-L-hydrobromide
4000-15000 Da (PLL 4-15 kDa) P6516 Sigma-Aldrich Disposable PD-10 desalting column
(PD Mini Trap G-10) 17-0851-01 GE Healthcare, Freiburg, Germany
34
mCLING Designed by Prof. Silvio O. Rizzoli.
Synthesized by Synaptic Systems,
Endocytosis reporters Low Density Lipoprotein from Human Plasma, DiI complex (DiI LDL)
L3482 Molecular Probes, Life
Technologies Transferrin From Human Serum,
Alexa Fluor 488 Conjugate T13342 Molecular Probes, Life
Technologies Transferrin From Human Serum,
Alexa Fluor 546 Conjugate T23364 Molecular Probes, Life
Technologies
Bromophenol blue B8026 Sigma-Aldrich
mCLING validation
Propidium Iodide P1304MP Molecular Probes, Life
Technologies
SynaptopHluorin (VAMP2) construct Kindly provided by Leon Lagnado, Medical Research Council, Cambridge, United Kingdom
Dynasore hydrate D7693 Sigma-Aldrich
Pitstop 2 ab120687 abcam, Cambridge, UK
Tetrodotoxin (TTX) T8024 Sigma-Aldrich
p-toluensulfonic acid monohydrate 402885 Sigma-Aldrich
Embedding media
2,4,6- Tris[bis(methoxymethyl)amino]-1,3,5-triazine (melamine)
T2059 TCI Europe, Zwijndrecht, Belgium
EpoFix kit (epoxy resin) 40200029 Struers A/S, Ballerup, Denkmark Mowiol 4-88 reagent 475904 Merck Millipore, Merck KGaA,
Darmstadt, Germany
2.1.2 Buffers and solutions
Table 2.2 List of buffers and solutions used in this study
Buffer Composition (concentrations in mM)
Tyrode’s buffer for neuronal cultures 124 NaCl, 5 KCl, 2 CaCl2,1 MgCl2, 30 glucose, 25 HEPES, pH 7.4
Ringer’s buffer for COS7 cells 130 NaCl, 4 KCl, 5 CaCl2, 1 MgCl2, 48 glucose, 10 HEPES, pH 7.4
Hank’s Balanced Salt Solution without calcium (HBSS without Ca2+), for OC dissection and resting condition of IHCs
141.7 NaCl, 5.36 KCl, 1 MgCl2, 0.5 MgSO4, 3.4 L-Glutamine, 6.3 glucose, 10 HEPES, pH 7.4. 295-300 mOsm
Hank’s Balanced Salt Solution with high 135.1 NaCl, 10 KCl, 2 CaCl2, 1 MgCl2, 0.5 MgSO4,
35 potassium (HBSS high K+ 10mM), for mild
stimulation of IHCs 3.4 L-Glutamine, 6.3 glucose, 10 HEPES, pH 7.4.
295-300 mOsm Hank’s Balanced Salt Solution with high
potassium (HBSS high K+ 25mM), for stimulation of IHCs
120.1 NaCl, 25 KCl, 2 CaCl2, 1 MgCl2, 0.5 MgSO4,
3.4 L-Glutamine, 6.3 glucose, 10 HEPES, pH 7.4.
295-300 mOsm Hank’s Balanced Salt Solution with high
potassium (HBSS high K+ 65mM), for strong stimulation of IHCs
79.7 NaCl, 65.36 KCl, 2 CaCl2, 1 MgCl2, 0.5 MgSO4, 3.4 L-Glutamine, 6.3 glucose, 10 HEPES, pH 7.4. 295-300 mOsm
Hank’s Balanced Salt Solution with calcium (HBSS with Ca2+) for recovery after stimulation of IHCs
139.7 NaCl, 5.36 KCl, 2 CaCl2, 1 MgCl2, 0.5 MgSO4, 3.4 L-Glutamine, 6.3 glucose, 10 HEPES, pH 7.4. 295-300 mOsm
Fluorescence quenching solutions (Figure
3.13) HBSS with Ca 2+ and HBSS high K+ 65 mM were
supplemented with 0.75 mM BPB Hank’s Balanced Salt Solution with high
potassium and no calcium (HBSS high K+ 65mM, without Ca2+), for strong stimulation in absence of calcium, used as control (Figure 3.13.)
81.7 NaCl, 65.36 KCl, 1 MgCl2, 0.5 MgSO4, 3.4 L-Glutamine, 6.3 glucose, 10 HEPES, pH 7.4. 295-300 mOsm, 5 EGTA, 0.75 BPB
Standard Drosophila stimulation buffer 130 NaCl, 36 sucrose, 5 KCl, 2 CaCl2, 2 MgCl2, 5 HEPES, pH 7.3 (Jan and Jan, 1976)
Yeast minimal medium 0.67 % Difco yeast nitrogen based w/o amino acids (Beckton, Dickison and company), 0.07 % complete supplement mix (MP biomedicals) and 2 % sucrose (Roth)
Phosphate buffer saline (PBS) 150 NaCl, 20 Na2HPO4, pH 7.4
High-salt PBS 500 NaCl, 20 Na2HPO4, pH 7.4
Permeabilization solution for cultured
neurons, mammalian cells and yeast cells 0.1% Triton X-100 and 2.5% bovine serum albumin (BSA) in PBS
Permeabilization solution for organs of Corti
and Drosophila preparations 0.5% Triton X-100 and 1.5% bovine serum albumin (BSA) in PBS
Quenching solution (after fixation) 100 NH4Cl and 100 Glycine in PBS
2.1.3 List of Antibodies
Table 2.3 List of antibodies used in this study
Antibody Catalog number, type Company/Source
β-tubulin single-chain recombinant
antibody (Nizak et al., 2003)
Bruchpilot nc82, mouse monoclonal Developmental Studies
Hybridoma Bank at University of Iowa, IA, USA
36
Calnexin ab22595, rabbit polyclonal abcam, Cambridge, UK Chromeo494-coupled goat
anti-Mouse IgG 15032 Active Motif, La Hulpe,
Belgium Chromeo494-coupled goat
anti-Rabbit IgG 15042 Active Motif
CtBP2 (also recognizing Ribeye
A domain) 612044, mouse monoclonal BD Biosciences, Heidelberg, Germany
Cy2-coupled goat anti-mouse
IgG 115-225-146 Dianova GmbH, Hamburg,
Germany Cy2-coupled goat anti-rabbit
IgG 111-225-144 Dianova GmbH
GM130 610822, mouse monoclonal BD Biosciences,
LAMP1 ab24170, rabbit polyclonal abcam
Otoferlin ab53233, mouse monoclonal abcam
Rab3A 610379, mouse monoclonal BD Biosciences. IHCs.
Rab3A 107 003, rabbit polyclonal Synaptic Systems, Göttingen, Germany. Hippocampal neurons
Ribeye B domain 192 003, rabbit polyclonal Synaptic Systems SNAP-25 111 002, rabbit polyclonal Synaptic Systems Synapsin 106 002, rabbit polyclonal Synaptic Systems Synaptophysin G96, rabbit polyclonal raised
against synaptophysin Synaptotagmin 1 105 102, rabbit polyclonal Synaptic Systems
Syntaxin 1 110 011, mouse monoclonal Synaptic Systems Syntaxin 6 610636, mouse monoclonal BD Biosciences Syntaxin 13 110 131, mouse monoclonal Synaptic Systems Syntaxin 16 rabbit polyclonal Kindly provided by Prof.
Reinhard Jahn, Max Planck
VAMP2 104 211, mouse monoclonal Synaptic Systems
VAMP4 136 002, rabbit polyclonal Synaptic Systems
37 VGLUT1/2 135 503, rabbit polyclonal Synaptic Systems
VGLUT3 135 203, rabbit polyclonal Synaptic Systems
Vti1a 611220, mouse monoclonal BD Biosciences
2.1.4 Microscopes and equipment
Table 2.4 List of microscopes and equipment used in this study
Element Characteristics Company
Leica SP2 upright
confocal microscope - Water immersion objective (63×, 0.9 NA,
HCX APO L U-V-I) Leica Microsystems
GmbH, Wetzlar,
- HCX PL APO Oil immersion objective (63× 1.4 NA)
- Excitation: pulsed diode laser (18 mW, 80 MHz, 640 nm emission, PicoQuant, Germany)
- Detection: two ultra-sensitive avalanche photodiodes and high sensitivity, low
- Oil immersion objective (60× 1.35 NA) - TIRFM oil immersion objective (100×
1.45 NA.
- 100 W mercury lamp.
- F-View II CCD camera (Soft Imaging System GmbH, Münster, Germany)
- Plan apochromat oil immersion objective (60×, N.A. 1.4)
Spectrophotometer - Model ND1000 Wilmington, DE, USA
Leica Ultramicrotome - Model EM UC6 Leica Microsystems
GmbH
38
2.1.5 Software
Table 2.5 List of software used in this study
Software Application Provider
LAS AF Version 2.7.3.9723 Image acquisition software for
Leica TCS SP5 STED
microscope
Leica Microsystems GmbH, Wetzlar, Germany
Leica Confocal Software
Version 2.61 Image acquisition software for
Leica SP2 confocal microscope Leica Microsystems GmbH Cell^P Version 3.4 Image acquisition software for
Olympus IX 71 inverted
4.20 Image acquisition software for
Nikon Eclipse Ti-E microscope Nikon GmbH, Düsseldorf, Germany ACD/C ChemSketch (Freeware
version 12.01) Generation of chemical structures for mCLING and FM 1-43
Advanced Chemistry Development Inc., Toronto, Canada
PyMol (Version 1.6.0.0) Graphic rendering of molecular structures for mCLING and FM 1-43
Schrödinger, Portland, OR, USA
SigmaPlot Version 10.0 Statistical analysis, data plotting and graphs generation
Systat Software, Inc., Erkrath, Germany
Matlab Version 7.5.0.342
(R2007b) Image analysis The MathWorks Inc., Natick,
MA, USA
Huygens Essential 4.4 Image deconvolution Scientific Volume Imaging B.V., Hilversum, The Netherlands
Image J 1.47v Image processing and
preparation for figures Wayne Rasband, US National Institutes of Health,
http://rsb.info.nih.gov/ij/
Adobe Photoshop CS6 Version
13.0 Image processing and
preparation for figures Adobe Systems Inc., San Jose, CA, USA
Adobe Illustrator CS6 Version
16.0.0 Figures formatting and
graphic design Adobe Systems Inc., San Jose, CA, USA
39