The dynamic interplay between

DNA recombination and repair

Meiosis is a dynamic developmental process that occurs in germ cells. Meiosis is extensively studied and several specialized events that happen during meiosis can be characterized. These include, for instance, DNA double-strand break formation and repair, recombination, synaptonemal complex assembly and organization.

Mice are often used as a model organism to study meiosis in mammals and the availability of mutant mice in key proteins involved in the process comes to our aid in the comprehension of crucial meiotic developmental steps.

An approach widely used to study meiosis is the chromosome spread, a technique of spreading meiotic chromatin on a microscope slide (for further details see Barchi et al., Methods Mol Biol 2009). The slide containing surface-spread nuclei can be subsequently immunostained with antibodies against proteins of interest and then analyzed via microscopy.

Here, we focus on the dynamic localization of various markers in mouse spermatocytes undergoing prophase I (Zygotene stage), and processed by chromosome spread. In particular, we show SYCP3 protein (red), a synaptonemal complex protein forming axial elements which colocalize to the sister-chromatid cohesion axes during early prophase; IHO1 (green; IHO1 antibody is a kind gift from Dr. Attila Toth), which associates with chromatin during prophase and is required for DNA double-strand breaks formation in unsynapsed regions during meiotic recombination; DMC1 (grey), a DNA repair protein localized at DNA double-strand break repair sites in meiotic prophase; lastly, DNA is shown in blue.

We performed acquisition on a Nikon Ti2 inverted microscope equipped with CrestOptics confocal spinning disk and structured illumination device by using a 100x objective (Nikon Plan Apo Lambda oil immersion, 1.45 NA). We compared widefield and structured illumination super-resolved images and, in the latter, it is evident how gain in resolution helps clarifying the dynamic interplay between double-strand break and recombination mechanisms in meiosis at the level of synaptonemal scaffold (Figure A). Noteworthy, super-resolution images allow to reveal previously hidden features of protein complex rearrangements (see fluorescence intensity profile of a single SYCP3 filament (Figure B.1) and possibility to resolve the separation between two SYCP3 filaments (Figure B.2) occurring in specific windows of meiotic progression and in particular chromosome areas.

Figure A:
Maximum Intensity Projection
WIDEFIELD
STRUCTURED ILLUMINATION

Figure B(1): Resolution measurements

WIDEFIELD
STRUCTURED ILLUMINATION

Figure B(2)

The application note has been prepared in collaboration with Alvaro Crevenna and Marco Barchi

EUROPEAN MOLECULAR BIOLOGY LABORATORY (EMBL), ROME

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