Csu x1

PCMs were seeded onto glass-bottom dishes (MatTek Corp). The cells were transfected with expression vectors for Flag-MEF2A and Myc-STAT3b. Before imaging the cells, the media was replaced for DMEM/F12 (Gibco). Hoechst 33342 (Sigma-Aldrich) was added to 2.5 μM into the media to stain nuclei. After 30 min, the stained cells were visualized using a Carl Zeiss Spinning disc system (Zeiss Observer Z1 with Yokogawa CSU-X1 and Axio- Cam MRm camera) in the environment chamber (37 °C, 5% CO₂). The raw images were processed using ZEN software (Carl Zeiss) to obtain pseudo-colored micrographs.

Fixed samples were imaged using an inverted Leica SP5 confocal microscope. For representative images, a 60 × /1.40 N.A oil immersion objective was used. Live cell imaging was performed on a UltraView Vox spinning disc confocal microscope (Perkin Elmer Nikon TiE; Yokogawa CSU-X1 spinning disc scan head) with 60 × /1.40 N.A oil objective and equipped with a Hamamatsu C9100-13 EMCCD camera, or a 3I spinning disc confocal microscope (Zeiss AxioObserver Z1; Yokogawa CSU-W1 spinning disc scan head) with 63 × /1.40 N.A objective and equipped with a photometrics prime 95B scientific CMOS camera. Both spinning disc microscopes are equipped with a temperature-controlled environment chamber set at 26C for the experiments. Airyscan pictures were taken on fixed and stained neuroblasts using an inverted LSM880 Multiphoton or on live samples using a LSM900 point scanning laser confocal with Airyscan 2 on an inverted Axio Observer Z1 microscope stand.

Laser ablation experiments were performed in an Olympus IX-81 inverted microscope equipped with a spinning disk confocal unit (Yokogawa CSU-X1), a 100× oil objective, a 355 nm pulsed laser, third-harmonic, solid-state UV laser, and an Evolve 512 EMCCD digital camera (Photometrics). To analyze tension in NCs and FCs, a pulse of 1,000 mJ energy and 20 msec. and 75 mJ energy and 4 msec. duration, respectively, was applied to sever plasma membranes of cells. In all cases, cell surfaces were visualized with the membrane marker Resille-GFP and a Cobolt Calypso state laser (l = 491 nm 50 mW) was used for excitation of the GFP. To minimize potential effects due to anisotropic distribution of forces in NCs and FCs, cuts were made perpendicular to the AP axis and to the dorsal ventral axis of the egg chamber, respectively. Images were taken 3 s before and 10 s after laser pulse, every 0,5 s. To analyze the vertex displacements of ablated cell bonds, the vertex distance increase from different ablation experiments (DL) was averaged using as L₀ the average of distance of the vertexes 3 s before ablation. The initial velocity was estimated as the velocity at the first time point (t1 = 0,5 sec). Standard deviation (SD) was determined.

Cells grown on 12-mm No. 1.5 coverslips (Carolina Biological Supply) were fixed with 4% paraformaldehyde (Electron Microscopy Sciences), washed with PBS, and permeabilized with either 0.1% TX100 or 0.1% saponin in a 3% BSA/PBS buffer. Subsequent primary and secondary antibody incubations were carried out in the permeabilization buffer. Coverslips were mounted in ProLong Gold reagent supplemented with DAPI (Invitrogen). Images were acquired with either a Zeiss LSM 800 laser scanning confocal microscope with a 63× Plan Apo (NA = 1.4) oil immersion objective and Zeiss Efficient Navigation software or an UltraVIEW VoX spinning disk confocal microscope (PerkinElmer) that consisted of a Nikon Ti-E Eclipse inverted microscope equipped with 60× CFI PlanApo VC, NA 1.4, oil immersion objective and a CSU-X1 (Yokogawa) scan head that was driven by Volocity (PerkinElmer) software. For live cell imaging analysis of microtubules and F-actin, the cells were labeled with SiR-tubulin and SiR-actin (Cytoskeleton Inc.) as per the manufacturer’s instructions. An automated analysis pipeline was developed with Cell Profiler (McQuin et al, 2018 (link)) for the quantification of nuclear versus cytoplasmic ratios of the NLS-td-Tomato-NES reporter (Zhang et al, 2015 (link)).

FRAP measurements were performed on an inverted LSM 780 microscope (Observer.Z1; Carl Zeiss, Oberkochen, Germany) with a Zeiss 100× oil immersion lens and a confocal spinning disk unit (CSU-X1; Yokogawa, Tokyo, Japan). A 488-nm laser [1 AU (Airy Unit)] with 100% laser power was used to bleach the droplets. Post-bleach images were collected at a rate of 1 s per frame for 100 s. Fluorescence intensity was normalized with pre-bleach as 100% and post-bleach as 0. Results (at least three FRAP curves) were analyzed by GraphPad prism 7.0.

All samples were visualized using a spinning disk confocal head (CSU-X1; Yokogawa Corporation of America) with Borealis modification (Spectral Applied Research) and a quad bandpass 405/491/561/642 dichroic mirror (Semrock). The confocal was mounted on a Ti inverted microscope (Nikon) equipped with a 60× Plan Apo NA 1.4 oil immersion objective or a 100x Plan Apo 100x NA 1.4 oil immersion objective and the Perfect Focus System for continuous maintenance of focus (Nikon). Green fluorescence images were collected using a 491-nm solid-state laser controlled with an AOTF (Spectral Applied Research) and ET525/50 emission filter (Chroma Technology Corp.). Red fluorescence images were collected using a 561-nm solid-state laser controlled with an AOTF (Spectral Applied Research) and ET620/60 emission filter (Chroma Technology Corp.). All images were acquired with a cooled CCD camera (ORCA AG; Hamamatsu Photonics) controlled with MetaMorph software (version 7.0; Molecular Devices) and archived using ImageJ (National Institutes of Health) and Photoshop CS5 (Adobe). In some cases, linear adjustments were applied to enhance the contrast of images using levels in the image adjustment function of ImageJ and Photoshop.