Femtotip

Adrenal chromaffin cells were injected with 5 μM of the GST-fusion peptides of cortactin SH3, SH3W525K or the N-WASP PRD by using an InjectMan system (Eppendorf, Hamburg, Germany) and 0.5 μm-diameter Femtotips (Eppendorf, Hamburg, Germany). All GST-peptides were injected in a buffer solution containing in mM: 139 K⁺- glutamate, 20 PIPES, 5 EGTA, 2 ATP-Mg²⁺, pH 6.6, in the presence of 4% Lucifer yellow, a fluorescent dye that allowed us to identify the injected cells. The injection time was 0.2 s at a pressure of 120 hPa. Then, ACCs were kept in the culture medium at 37°C for 30 min.
For transfections, ACCs were electroporated using an Amaxa Nucleofector 4D (Lonza, Cologne, Germany) according to the manufacturer’s instructions. After transfection, ACCs were cultured in Dulbecco’s modified F-12 medium supplemented with 10% fetal bovine serum and kept at 37°C in a 5% CO₂ atmosphere, for at least 48 h prior to experimentation.
To study the role of actin polymerization in exocytosis, ACCs were incubated with 2 μM Latrunculin A (LatA), or its vehicle dimethyl sulfoxide (DMSO) 10 min prior to experimentation and throughout the test. To evaluate the role of ERK1/2 signaling in exocytosis, ACCs were incubated with 10 μM of U0126, or its inactive analog U0124, 15 min prior to experimentation and throughout the test.

About 2 mM (proteo)liposomes and 10 µg/ml DAPI (4′,6-diamidino-2-phenylindole; Sigma) (injection marker) in HB150 were filled in Femtotips (Eppendorf); 1 × 10⁴ HeLa cells were plated on poly-l-lysine (Sigma-Aldrich)-coated 12 mm coverslips (Marienfeld GmbH) and then the coverslip was placed into a 35-mm petri dish (Becton Dickinson) filled with pre-warmed injection medium (F12 medium (Invitrogen), supplemented with 10% FCS, 10 mM HEPES (pH7.5), and 100 units/ml each of penicillin and streptomycin). Microinjection was performed using a Femtojet (Eppendorf) and Injectman micromanipulator (Eppendorf) under a Leica DMIL inverted microscope for 5 min per coverslip. After microinjection, the cells were incubated at 37 °C in the cell culture medium and then fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS for 10 min followed by immunocytochemistry using antibodies specific for organelles as indicated. To label Tfn-positive endosomes, microinjection was performed in 5 µg/ml Alexa488-, Alexa568-, or Alexa633-Tfn-containing injection medium for 5 min. After microinjection, the cells were incubated for 5 min at 37 °C in the cell culture medium, then were fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS for 10 min. To take time-lapse images, injected cells were quickly set on an LSM 780 confocal microscope (Carl Zeiss).

Dissociated SCG neurons were microinjected using a Zeiss Axiovert S100 microscope with an Eppendorf FemtoJet microinjector and Eppendorf TransferMan^® micromanipulator. Plasmids were diluted in 0.5× PBS (without CaCl₂ and MgCl₂) and filtered using a Spin-X filter (Costar). The mix was injected directly into the nuclei of SCG neurons using Eppendorf Femtotips. Approximately 100 neurons were injected per dish. Injected plasmids were allowed to express for 16 h before CCCP treatment. Plasmids were injected at the following concentrations: 30 ng/μl NMNAT2-EGFP, 30 ng/μl pDsRed2-N1. Time-lapse imaging of axonal transport was performed on an Olympus IX70 imaging system with 100×/1.35 Oil objective. During imaging, cell cultures were maintained at 37 °C and 5% CO₂ in an environment chamber. Images were captured at 4 frames per sec for 2 min. Three neurites per condition were imaged in every individual experiment.

To fabricate nanopipettes, borosilicate glass filaments (outer diameter 1.00 mm, inner diameter 0.58 mm) were pulled to an outer diameter of approx. 100 nm. The P2000 puller (Sutter Instruments) used the following program:
Line1: HEAT:350, FIL:3, VEL:30, DEL:220, PULL:0
Line2: HEAT:330, FIL:2, VEL:27, DEL:180, PULL:250
For comparison, microinjection pipettes (Femtotips, Eppendorf) with an inner diameter of 0.5 μm were purchased. Both pipette types were filled from the back with a microloader (Eppendorf).