Injectman

Undifferentiated Ax2 and erkB^- amoebae were harvested from K. aerogenes clearing plates and washed six times in KK₂-MC to remove bacteria. Cells were resuspended at 2x10⁷ cells/ml and shaken in suspension for 30 minutes before being washed again then diluted in KK₂-MC and placed in a modified 2-well chambered coverglass (Nunc Lab-tek) with cut-down sides. Cells were left to settle for 30 minutes before filming using a Zeiss Axiovert S100 inverted microscope with 20x phase contrast objective. A micropipette (Eppendorf Femtotip II) filled with 100 μM folate was placed in the centre of the field of view just above the plane of the coverglass using a micromanipulator (Eppendorf Injectman and Femtojet). Cell movement was imaged at 2 frames per minute for a 40 minutes period. Cells were tracked using the manual tracking plugin of FIJI ImageJ (https://fiji.sc/) and chemotaxis parameters extracted using the Ibidi chemotaxis tool software (Ibidi).

HeLa cells were grown on glass coverslips and RNA was microinjected using InjectMan coupled with FemtoJet (Eppendorf) as described previously^{30 (link),95} . For microinjection of denatured U2WT snRNA, RNA was incubated at 98 °C for 5 min and immediately microinjected into the HeLa cells. After 1 h incubation period, cells were rinsed twice with PBS and fixed for 20 min at room temperature in 4% PFA/PIPES (freshly prepared).

Microneedles were prepared from capillaries with an outer/inner diameter of 1.2/0.94 mm (Harvard Apparatus, Cambridge, UK) in Sutter P-2000 (Sutter instruments, Novato, CA, USA). The protein was centrifuged at 20000 × g for 1 h before injection to remove small aggregates. The protein solution was back loaded into the needle, and the needle was installed into the micromanipulator InjectMan (Eppendorf, Hamburg, Germany). The micromanipulator was installed on the microscope allowing for immediate fluorescence microscopy after the injection and was connected to the microinjector FemtoJet (Eppendorf, Germany). The microinjection was performed at 20–30 hPa injection pressure, 0.1 s injection time, and a compensatory pressure of 20–25 hPa. Preliminary experiments were performed to estimate the injection volume. The fluorescence intensity of the diluted protein solution was measured using the same microscopy settings (laser power, gain, objective) as with the injection experiments. At a dilution of ca. 50 times the fluorescence intensity was comparable to the median intensity in the cells after injection of the labeled MTS-SNAP-tag, thus the injected volume was estimated to be 1–4% of the cell volume, fluctuating between the cells due to variation in the injected volume and the cell volume.