Deltavision elite system

For fluorescence microscopy, cells expressing GFP, pHluorin, or mCherry proteins were visualized using a DeltaVision Elite system (GE), equipped with a Photometrics CoolSnap HQ2/sCMOS Camera, a 100× objective, and a DeltaVision Elite Standard Filter Set (“FITC” for GFP/pHluorin fusion protein and “mCherry” for mCherry fusion proteins). Image acquisition and deconvolution were performed using Softworx.

MDA-MB-231 cells were seeded on coverslips at 50% confluency. At 24 hours after seeding, cells were transfected with FLAG-Dvl2 using LipoJet Transfection Reagent (Signagen). 72 hours after seeding cells were fixed and permeabilized using 4% paraformaldehyde (PFA) and immunofluorescence block buffer (PBS + 0.1% Triton-x + 10% FBS). Cells were then labeled with primary antibodies for 1 hour followed by a 1 hour incubation with Alexa Fluor conjugated secondary antibodies. Coverslips were mounted on slides using ProLong AntiFade mountant (Thermo). Microscopy images were acquired using a DeltaVision Elite system (GE Healthcare) and processed using SoftWoRx software (GE Healthcare, Chicago, IL).

To study actin dynamics in the inner territory (Figure 1B), we used the photoconvertible fluorescent protein Eos as a tag (Wiedenmann et al., 2004 (link)). For the generation of a pDEX-DdEos expression vector, the sequence of monomeric Eos (Wiedenmann et al., 2004 (link)) was adapted to the codon usage of D. discoideum and cloned into pDEX (bsr), enabling expression under control of an actin-15 promoter (Müller-Taubenberger, 2006 ). The full-length coding sequence of act16 (DDB_G0272248), one of 17 identical actin proteins encoded in D. discoideum, was cloned in frame into the EcoRI site of the pDEX-DdEos vector. The sequences of DdEos and actin 16 were separated by a linker sequence, GSGTAGPGSTGSGTEF. DdEos-­actin cells were cultivated in the presence of 10 µg/ml Blasticidin S.
For photoconversion, a 405-nm diode laser, 100 mW, was used on the GE DeltaVision Elite system. The laser was focused onto the actin cortex of the substrate-attached cell surface and a flash with an exposure time of 80 or 250 ms was applied. The cells were imaged in the TIRF mode using 488/525 nm for excitation/emission of the green version of Eos, and 568/584 nm of the photoconverted red version. Local fluorescence intensities were measured with point-scans of 36 × 36 pixels using a plug-in of Fiji-ImageJ.

Images of meiotic chromosomes were acquired with a Delta Vision ELITE system (GE) operated by the SoftWorx software using an Olympus IX71 microscope. To enhance the quality of images, about 10 serial optical sections were captured with 200-nm intervals and were subjected to deconvolution with default settings by SoftWorx. The best two sectioning images with the most signal–noise ratio were projected. Finally, only brightness-contrast adjustment was performed in Photoshop.
Images of somatic nuclei and metaphase chromosomes were acquired with FISHView 7.2.7 SP7 (Applied Spectral Imaging) using an Olympus BX61 microscope fitted with a cooled CCD camera (Cool-1300QS; VDS). One or more layers of the color images were adjusted using the curves and/or brightness-contrast functions of Photoshop CC 2015. Grayscale images were generated by converting the original, unadjusted red (probe) channel first to grayscale and then digitally increasing the signal strength to check for areas of nonspecific binding. For images in SI Appendix, Fig. S2, the conversion to grayscale was performed with FISHView, and then the brightest pixel was set to white using the levels eyedropper in Photoshop. For images in SI Appendix, Fig. S4, conversion was performed in Photoshop, and a pixel with a K (black) value of 70% was set to white.