A1rsi microscope

The RNA fluorescence in situ hybridization (FISH) was performed with the use of DNA/LNA probe (CAG)₆-CA, labeled at the 5′-end with Cy-3 as previously described (31 (link)). Microscopic slides were mounted using medium containing 2% propyl gallate (Sigma), 10% glycerol and 4′,6-diamidino-2-phenylindole (DAPI), and then sealed with fingernail polish. All FISH images were acquired on the Nikon A1Rsi microscope equipped with the Nikon DigitalSight DS-Fi1c camera and processed with Nikon NIS Elements AR software. The quantification of RNA foci was performed using fluorescence part of microscope, filter DA/FI/TR-A-NTE (Semrock; range of excitation 387/478/555–25; emission range 433/517/613–25) and 60x oil immersion objective (Plan Apo VC 60x/1.4 Oil DIC N2). Quantification was performed by visual inspection of fluorescence spots present in cell nuclei. The number of foci was counted in at least 200 nuclei and the same experiment was repeated twice. Foci volume was estimated by Imaris software using images captured by confocal part of Nicon’ microscope in the following excitation conditions: diode lasers 405 and 561 nm, dichroic mirror 405/488/561, emission filters 450/50 for DAPI and 595/50 for Cy-3. All images were acquired with sequential scanning to avoid spectral bleed-through. Eighteen to twenty five optical sections were acquired.

Staining results were scored using light (for AB) and fluorescent microscopes (for CMA3 and FISH) (Zeiss D1 AxioImager) with an oil immersed 100× objective fitted with a proper filter set (FITC/SpO/DEAC/Triple/DAPI). The images were acquired using a CCD camera and analyzed with CellB (Olympus) or ISIS (MetaSystems, Germany) software. For meiotic segregation, 3,400 sperm cells of the sSMC carrier were evaluated. To investigate the aneuploidy level, at least 5,000 sperm cells were evaluated for each male (sSMC carrier; control males, n = 7; RF-group, n = 7) and chromosome. When two FISH signals in one colour were observed in a sperm cell, the criterion of the space between them (=minimum the size of the signal) was applied. The efficiency of FISH was estimated at 99%. For confocal analysis, a Nikon A1Rsi microscope (Japan) equipped with an appropriate range of lasers (405–641 nm) was used, followed by further image analyzes with Imaris software (Bitplane, Switzerland). To obtain 3D images, a series of image stacks were acquired for each spermatozoa (7 per μm of the nucleus depth along to the z-axis; an example of these stacks is presented in Suppl. Fig. S6).

Images were acquired using a dual point-scanning Nikon A1R-si microscope equipped with a PInano Piezo stage (MCL), using a 60× PlanApo VC oil objective numerical aperture (NA)=1.40.

The coding sequence of ER luminal marker gene ER–YFP included the signal peptide of WALL‐ASSOCIATED KINASE 2 (WAK2), YFP and HDEL ER retention signal (Nelson et al., 2007 (link)). ER–YFP was subcloned into the pYL436 binary vector, which contained the Cauliflower Mosaic Virus 35S promoter sequence. Following introduction of this binary vector construct into Agrobacterium tumefaciens strain GV3101, the resulting strain was used to transform WT and jazD plants using the floral dip method (Clough & Bent, 1998 (link)). Seedlings (T1 generation) of transformed lines were screened on LS plates containing gentamycin (100 μg ml⁻¹) and resistant plants were transferred into soil. Homozygous lines were selected by testing the T3 progeny for resistance to gentamycin. A jazD mycT line overexpressing ER–YFP was obtained by crossing jazD mycT to jazD plants harbouring the 35S:ER–YFP transgene. A jazD mycT line containing the transgene was confirmed by PCR genotyping to be homozygous for all three myc mutations. The fifth rosette leaves of 30‐d‐old homozygous lines were inspected by confocal laser scanning microscopy with a Nikon (Tokyo, Japan) A1Rsi microscope. NIS‐Elements Advanced Research (Nikon) and Photoshop (Adobe, San Jose, CA, USA) software were used for image processing.

For live cell imaging we used four-well microscope glass-bottom plates (IBIDI) or Cellview cell culture dish (Greiner Bio One). Plates were coated with Concanavalin A (Sigma) for live cell imaging of yeast. Confocal images and movies were acquired using a dual point-scanning Nikon A1R-si microscope equipped with a PInano Piezo stage (MCL), temperature and CO₂ incubator, using a ×60 PlanApo VC oil objective NA 1.40. We used 406, 488, 561, and 640 nm laser (Coherent, OBIS). Movies for kymographs were acquired in resonant-scanning mode. Image processing was performed using NIS-Elements software.

Cells were fixed with 4% paraformaldehyde in PBS for 30 min, permeabilized with 0.2% (v/v) Triton X-100/PBS for 5 min, and blocked with 1% bovine serum albumin/PBS for 30 min at room temperature. Subsequently, cells were incubated with anti-Flag (1:300, #F1804; Sigma-Aldrich), anti-Tom20 (1:300, #612278; BD Biosciences), anti-LC3B (1:200, NB100-2220; Novus Biologicals), anti-LAMP1 (1:300, ab24170; Abcam), or anti-Hsp70 (1:400, ADI-SPA-810; Enzo Life Sciences) antibodies for 2 h, and further incubated with Alexa Fluor 488/546-conjugated goat anti-mouse IgG or Alexa Fluor 488-conjugated goat anti-rabbit IgG antibodies (Molecular Probes) for 30 min at room temperature. Mitochondria were stained with MitoTracker Red CMXRos (Cell Signaling Technology) for 30 min at 37 °C before fixation. 4ʹ,6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. Confocal images were taken using a Nikon A1Rsi microscope, equipped with oil-immersion objectives (×60 and ×100). Images were acquired using Nikon NIS-Elements imaging software.