2.2 Methods
2.2.2 Experiments performed with cultured mammalian cells
18 mm diameter coverslips (thickness Nr. 1: 0,13 - 0,16 mm; Menzel Gläser, Braunschweig, Germany) were thoroughly washed before use:
- Incubation in 1M HCl overnight followed by 5 washes with Milli-Q-purified water - Wash with 1M NaOH for 1-2 hours followed by 5 or more washes with
Milli-Q-purified water until reaching a neutral pH - Wash and storage in 100% ethanol
Coverslips were flamed, placed on 12-well plates and coated with a PLL solution (0.1 mg/mL) for 1 hour. Excess of PLL was washed 3 times with distilled water. Plates were sterilized with UV light under a laminar flow hood and stored at 4°C until their use for cell culture.
2.2.2.2 Endocytosis assays in COS7 cells
COS7 cells (fibroblast-like cell line from monkey kidney) were cultured in Dulbbeco’s modified Eagle medium (DMEM with 4.5 g/L glucose; Lonza, Cologne, Germany) supplemented with 10% fetal calf serum (FCS; PAA Laboratories, Clöbe, Germany), 4 mM glutamine (Lonza) and 100 units/ml penicillin and streptomycin (Lonza).
One day before the experimental procedure, cells were briefly washed with PBS and treated with trypsin EDTA (Gibco, Life technologies, Darmstadt, Germany) for 5 minutes at 37°C.
After collection and wash, cells were resuspended in supplemented DMEM medium, plated on PLL-coated coverslips, and kept at 37°C and 5% CO2 culture conditions. Before performing the endocytosis assays, cells were washed with pre-warmed Ringer’s buffer, and then incubated for 5 minutes at 37°C with different membrane and/or endocytosis markers dissolved in pre-warmed Ringer’s buffer. A list of markers used and their concentrations is presented below:
- 0.2-0.4 µM mCLING, - 5 µM FM 1-43,
41 - 5 µM AM 1-43,
- 5 µM FM 4-64FX,
- 25 µg/ml Alexa 546-Transferrin, - 0.4 ng/ml tetramethylrhodamine-EGF, - or 15 µg/ml DiI-LDL
When necessary, membrane/endocytosis labeling was followed by fixation with 4% PFA + 0.2% glutaraldehyde for 20 minutes on ice and 20 minutes at RT. Excess of aldehyde fixatives was quenched during 30 min at RT using quenching buffer (100 mM NH4Cl and 100 mM glycine solution in PBS). Permeabilization was carried out in 3 rounds of 5 minutes with a 0.1% Triton X-100 + 2.5% BSA solution in PBS. Primary antibodies were incubated for 1 hour in permeabilization solution followed by 3 washes of 5 minutes with permeabilization solution. Secondary antibodies were incubated for 1 hour in permeabilization solution. After immunostaining, coverslips were washed 3 times for 5 minutes with high-salt PBS and 2 times for 5 minutes with standard PBS. Coverslips were mounted on glass slides using Mowiol as embedding medium.
For pictures in Figures 3.5, 3.7 and 3.8A, cells were imaged in live, fixed, or fixed and permeabilized conditions, in an Olympus IX 71 inverted microscope (60× 1.35 NA oil immersion objective, or 100× 1.45 NA TIRFM oil immersion objective) equipped with an F-View II CCD camera (Soft Imaging System GmbH, Münster, Germany).
For studying the effects of mCLING on membrane trafficking pathways, cells were first incubated for 5 minutes with mCLING and Alexa 546-Transferrin at 37°C, washed with Ringer’s buffer and incubated again at 37°C for 20 minutes in Ringer’s buffer. Cells were fixed and quenched as described above. For figures 3.6 and 3.8B-C, images were acquired in the confocal mode of a Leica TCS SP5 STED microscope using an HCX PL APO 63× 1.4 NA oil immersion objective.
2.2.2.3 mCLING toxicity assay in COS7 cells
A cell viability assay was performed to assess the concentration-dependent toxicity of mCLING labeling. COS7 cells were directly plated on a 24-well plastic plate. For the assay wells were first washed with pre-warmed Ringer’s buffer, incubated in Ringer’s buffer containing increasing concentrations of mCLING (in µM: 0, 0.21, 0.42, 0.85, 1.7, 3.4, 6.8) for 5 minutes, washed, and incubated in Ringer’s buffer containing propidium iodide (Sigma).
Cells were imaged after 5 minutes in an inverted epifluorescence Nikon Eclipse Ti-E
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microscope (CFI S Plan Fluor ELWD 40× 0.60 NA air objective). Propidium iodide was imaged using the Cy3 filter set (excitation: 545/25, dichroic mirror: 565, barrier filters:
605/70 BP). Imaging of mCLING was performed with the Cy5 filter set (excitation: 620/60, dichroic mirror: 660, barrier filter: 700/75 BP).
2.2.2.4 Culture methods for rat hippocampal neurons
The cultured hippocampal neurons were obtained from dissociated hippocampi of newborn rats (modified from (Banker and Cowan, 1977; Beaudoin et al., 2012)). Dissection procedures and culture methods were performed by our technical assistant Christina Schäffer. Briefly, brains were extracted from the skulls of P2 rat pups, and the hippocampi were isolated under a dissection microscope. The tissue was washed several times with HBSS (Invitrogen) to remove tissue debris and thereafter incubated for 1h in enzyme solution (10 ml DMEM, 2 mg cysteine, 100 mM CaCl2, 50 mM EDTA, and 25 units Papain sterile bubbled with carbogen for 10 minutes and sterile filtered). Neurons were washed thoroughly with HBSS and incubated for 15 min in inactivating solution (2 mg Albumin, 2 mg Trypsin-Inhibitor in 10 ml of FCS containing DMEM medium) followed by mechanical dissociation. The coverslips on which neurons were seeded were prepared as explained in section 2.2.2.1 but PLL coating was done at higher concentration (1mg/ml) overnight.
Neurons were seeded in plating medium (Eagle's Minimum Essential Medium, MEM, supplemented with 10% horse serum, 3.3 mM glucose, and 2 mM glutamine) and incubated for 1–4 hours at 37°C in a 5% CO2 humidified atmosphere to allow adhesion to the substrate.
After adhesion the medium was changed to Neurobasal-A medium containing: 500 ml Neurobasal-A (Gibco, Life technologies, Darmstadt, Germany), 10 ml B27 supplement (Gibco, Life Technologies), and 5 ml Glutamax I stock. To avoid glial proliferation 5-fluoro-2’-deoxyuridine (FUDR) was added to the culture after 2 DIV. The neurons were kept in culture at 37°C and 5% CO2 for 14 days before use.
2.2.2.5 Neuronal transfection with SynaptopHluorin construct
To study the effects of mCLING on synaptic vesicle recycling, neurons were transfected with a plasmid carrying SynaptopHluorin (VAMP2-pHluorin, see (Miesenböck et al., 1998;
Sankaranarayanan and Ryan, 2000; Granseth et al., 2006). The synaptopHluorin insert (kindly provided by Dr. Leon Lagnado, University of Sussex, UK) was subcloned into a pEGFP-N1 plasmid (CMV promoter, Clontech, Mountain View, CA, USA) by PCR by inclusion of a KpnI restriction site in the forward primer (AAT-GGTACC-GCCGGTCGCCACC) and a NotI
43 restriction site in the reverse primer (AAT-GCGGCCGC-TTTAACCGGTTTTGTATAG). Ligation was confirmed by sequencing. Transfection was carried out using the calcium phosphate-based ProFection Mammalian transfection system (E1200; Promega, Madison, WI, USA).
Briefly, coverslips with neuronal cultures were transferred to a 12-well plate containing fresh DMEM (5 mL 1M MgCl2 and 2.5 mL 1M HEPES in 500 mL DMEM). The transfection reaction solution was prepared according to the amount of coverslips to be transfected, with the following amounts per well: 3.2 µL 2M CaCl2, 2 µg plasmidic DNA, Nuclease-free water to a final volume of 25 µL. This solution was mixed with 25 µL/well of HEPES-buffered saline and precipitates were allowed to form for 15-30 minutes at RT. 50µL of the transfection solution were applied drop-wise to every well and incubated for 15-30 minutes at 37°C.
Wells were washed 3 times with fresh DMEM and kept in the last wash for 15 minutes at 37°C. Coverslips were finally replaced to the original plate with the old medium and kept at 37°C 5% CO2 until imaging was performed.
2.2.2.6 SynaptopHluorin experiments in neuronal hippocampal cultures To test the innocuity of mCLING to neuronal cells and assess its possible effect on synaptic vesicle recycling, a fluorescent assay reporting synaptic vesicle release was designed. 8 days after SynaptopHluorin transfection coverslips with cultured neurons were placed in a stimulation chamber, labeled with 0.2 µM mCLING for 5 minutes and washed with Tyrode’s buffer. Spontaneous network activity was blocked after mCLING labeling using a solution containing 10 µM CNQX and 1 µM AP5 in Tyrode’s buffer. Neurons were stimulated with the same instruments described in section 2.2.2.7. 100-mA shocks were delivered initially in a short stimulus (60 AP, 3 seconds at 20 Hz) and 40 seconds later in a long stimulus (600 AP, 30 seconds at 20 Hz). Control neurons were directly treated with CNQX and AP5 and imaged in absence of mCLING. Imaging was performed in the same Nikon setup described in section 2.2.2.3. Synaptophluorin was imaged using the 60X oil immersion objective (plan apochromat, N.A. 1.4) and the filter set for EGFP (excitation: 470/40, dichroic mirror: 495, barrier filter: 525/50 BP).
2.2.2.7 mCLING applications to cultured rat hippocampal neurons
In order to label the recycling organelles and/or the plasma membrane of cultured hippocampal neurons, these were incubated in Tyrode’s buffer containing mCLING (0.68 µM). Three different types of labeling were performed:
- Selective labeling of the actively released pool of synaptic vesicles: neurons were
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prelabeled with mCLING for 5 minutes and then stimulated with 600 APs (30 seconds, 20 Hz) in presence of mCLING. For electric stimulation a custom-made chamber holding two platinum electrodes was fixed on top of the cells culture. 100 mA shock stimuli were delivered using an A385 stimulus isolator and an A310 Accupulser stimulator (both from World Precision Instruments, Saratosa, FL, USA).
- Selective labeling of the spontaneously released pool of synaptic vesicles: neurons were incubated in tetrodotoxin (1 µM) for 15 minutes in presence of mCLING.
- Surface labeling of the neuronal plasma membrane: cells were preincubated in cold mCLING-free buffer for 5 minutes on ice. Keeping the cells on ice, an mCLING solution was added and further incubated for 5 minutes.
After mCLING labeling neurons were fixed, permeabilized and immunostained, as described above for COS7 cells. After immunostaining, coverslips were mounted on glass slides using Mowiol as embedding medium. The following primary antibodies were used: VGLUT1/2, synaptophysin, synaptotagmin 1, VAMP2, synapsin, syntaxin 13, Vti1a, VAMP4, Rab3a, syntaxin, and SNAP-25. Chromeo494-coupled goat secondary antibodies were used accordingly.
2.2.2.8 Preparation of Mowiol embedding medium
24 g of glycerol and 9.6 g Mowiol 4-88 reagent were mixed with 62.4 mL distilled water and 9.6 mL 1M Tris buffer in a glass beaker. The mixture was stirred for 5 to 7 days and occasionally heated at 40-50°C to help Mowiol dissolving. The mixture was let to settle and only the supernatant was aliquoted. Aliquots were kept at -20°C for long-term storage and at 4°C for daily use.