From Microscopy to Nanoscopy: Defining an Arabidopsis thaliana Meiotic Atlas at the Nanometer Scale

Abstract

Visualization of meiotic chromosomes and the proteins involved in meiotic recombination have become essential to study meiosis in many systems including the model plant Arabidopsis thaliana. Recent advances in super-resolution technologies changed how microscopic images are acquired and analyzed. New technologies enable observation of cells and nuclei at a nanometer scale and hold great promise to the field since they allow observing complex meiotic molecular processes with unprecedented detail. Here, we provide an overview of classical and advanced sample preparation and microscopy techniques with an updated Arabidopsis meiotic atlas based on super-resolution microscopy. We review different techniques, focusing on stimulated emission depletion (STED) nanoscopy, to offer researchers guidance for selecting the optimal protocol and equipment to address their scientific question.

Keywords: Arabidopsis; cytology; immunofluorescence; meiosis; super-resolution microscopy.

Figure 1

Acid-spread nuclei from pollen mother cells (PMCs) depicting the meiotic progression in wild-type, double-strand break (DSB)-deficient (spo11-2-3) and DNA-repair-deficient (com1-1) male meiocytes. The spreads were stained with DAPI and imaged with an epifluorescence microscope. See text for details. Meiotic stages are indicated. Scale Bar: 5 μm.

Figure 2

Detergent-spread nuclei from PMCs depicting the meiotic progression from leptotene to pachytene in the wild-type. The spreads were stained for the recombinase RAD51, the axial element protein ASY1, the transverse filament protein ZYP1, or the meiosis-specific cohesin subunit REC8. Images were acquired with an epifluorescence microscope. Meiotic stages are indicated. Scale Bar: 5 μm.

Figure 3

Acid-spread nuclei from pollen mother cells depicting the meiotic progression in wild-type male meiocytes in super-resolution. Chromatin was stained with SiR DNA and imaged with a stimulated emission depletion (STED) nanoscope. The meiotic stages are: (A) Leptotene; (B) Zygotene; (C) Pachytene; (D) Diplotene; (E) Diakinesis; (F) Metaphase I; (G) Anaphase I; (H) Telophase I; (I) Dyad; (J) Metaphase II; (K) Anaphase II; (L) Telophase II; (M) Tetrad. Scale Bar: 5 μm.

Figure 4

Graphical representation of homologous chromosomes with a fully formed synaptonemal complex (SC). The loops of the homologous chromosomes are depicted in gray, the axial elements in pink and dark gray, the SC transverse filaments in light gray and repair proteins in yellow. Chromosomal features, which can be assessed by STED nanoscopy, are highlighted. (A). Comparison between widefield and STED images of detergent-spread male meiotic nuclei, which were stained for the axis (ASY3) in magenta and the transverse filament (ZYP1) in green. (B). Magnification of meiotic chromosomes in zygotene and pachytene stage stained for ASY3 (magenta) and the recombinase RAD51 (green). White bars indicate the distance measurements of each focus from the center for the axis. (C) and (D) Examples of measurements of SC width and inter-axis distance in nuclei stained for ZYP1 (C) and ASY3 (D). (E) Measurement of DNA loop length on an acid spread pachytene nucleus. (F). Comparison between STED and confocal microscopy of an acid-spread nucleus at metaphase I stained with SYBR green (confocal) and SiR DNA (STED). Scale Bars: (A,E,F) 2 μm; (B,C,D) 100 nm.

Figure 5

Detergent-spread nuclei from pollen mother cells depicting the meiotic progression in the wild type. The spread nuclei were stained for the recombinase RAD51, the axial element protein ASY1, the transverse filament protein ZYP1, or the cohesin subunit REC8. Images were acquired with a STED nanoscope. Stages of meiotic prophase are indicated. Scale Bar: 2 μm.

Figure 6

Detergent-spread male meiotic nuclei were stained for the axial element protein ASY3 (A) or ASY3 and the ubiquitin ligase HEI10 (B) and imaged with a STED nanoscope. Scale Bar: 2 μm. Panel (C) shows a magnification of the highlighted region in panel (B). Scale Bar: 200 nm.

Figure 7

Detergent-spread male meiotic nuclei were stained for the axial element protein ASY3 and the recombinase RAD51 and imaged with a STED nanoscope. Scale Bar: 2 μm. Panels (B) and (C) show magnifications of the highlighted regions in panel (A). Scale Bars: 200 nm.