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Updated: Aug 8, 2026
Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy
Published on: March 6, 2018
Multi-color dSTORM microscopy in Hormad1-/- spermatocytes reveals alterations in meiotic recombination intermediates
Lieke Koornneef1,2, Johan A Slotman3, Esther Sleddens-Linkels1
1Department of Developmental Biology, Erasmus MC, Rotterdam, The Netherlands.
Abstract:
Recombinases RAD51 and its meiosis-specific paralog DMC1 accumulate on single-stranded DNA (ssDNA) of programmed DNA double strand breaks (DSBs) in meiosis. Here we used three-color dSTORM microscopy, and a mouse model with severe defects in meiotic DSB formation and synapsis (Hormad1-/-) to obtain more insight in the recombinase accumulation patterns in relation to repair progression. First, we used the known reduction in meiotic DSB frequency in Hormad1-/- spermatocytes to be able to conclude that the RAD51/DMC1 nanofoci that preferentially localize at distances of ~300 nm form within a single DSB site, whereas a second preferred distance of ~900 nm, observed only in wild type, represents inter-DSB distance. Next, we asked whether the proposed role of HORMAD1 in repair inhibition affects the RAD51/DMC1 accumulation patterns. We observed that the two most frequent recombinase configurations (1 DMC1 and 1 RAD51 nanofocus (D1R1), and D2R1) display coupled frequency dynamics over time in wild type, but were constant in the Hormad1-/- model, indicating that the lifetime of these intermediates was altered. Recombinase nanofoci were also smaller in Hormad1-/- spermatocytes, consistent with changes in ssDNA length or protein accumulation. Furthermore, we established that upon synapsis, recombinase nanofoci localized closer to the synaptonemal complex (SYCP3), in both wild type and Hormad1-/- spermatocytes. Finally, the data also revealed a hitherto unknown function of HORMAD1 in inhibiting coil formation in the synaptonemal complex. SPO11 plays a similar but weaker role in coiling and SYCP1 had the opposite effect. Using this large super-resolution dataset, we propose models with the D1R1 configuration representing one DSB end containing recombinases, and the other end bound by other ssDNA binding proteins, or both ends loaded by the two recombinases, but in below-resolution proximity. This may then often evolve into D2R1, then D1R2, and finally back to D1R1, when DNA synthesis has commenced.
Insights
RAD51 and DMC1 recombinase nanofoci form within single DNA double-strand break sites during meiosis. The HORMAD1 protein influences recombinase accumulation patterns and synaptonemal complex coiling, impacting DNA repair progression.
Area of Science:
- Meiosis
- DNA repair
- Super-resolution microscopy
Background:
- RAD51 and DMC1 recombinases are crucial for homologous recombination repair of DNA double-strand breaks (DSBs) during meiosis.
- HORMAD1 is a meiosis-specific protein implicated in DSB repair and synapsis, but its precise role in recombinase accumulation dynamics is unclear.
Purpose of the Study:
- To investigate the spatial organization and dynamics of RAD51 and DMC1 recombinase accumulation at meiotic DSBs.
- To elucidate the role of HORMAD1 in regulating recombinase loading, DNA repair intermediate lifetimes, and synaptonemal complex structure.
Main Methods:
- Three-color direct stochastic optical reconstruction microscopy (dSTORM) was employed to visualize recombinase nanofoci with high resolution.
- A mouse model deficient in HORMAD1 (Hormad1-/-) was used to study the impact of HORMAD1 absence on DSB formation, synapsis, and recombinase patterns.
Main Results:
- RAD51/DMC1 nanofoci at ~300 nm distances represent single DSB sites, while ~900 nm distances indicate inter-DSB spacing in wild-type meiosis.
- HORMAD1 deficiency altered the dynamics and reduced the size of RAD51/DMC1 nanofoci, suggesting changes in ssDNA length or protein accumulation.
- HORMAD1 was found to inhibit coil formation within the synaptonemal complex, a function distinct from SPO11 and SYCP1.
Conclusions:
- HORMAD1 plays a critical role in regulating the spatiotemporal dynamics of RAD51/DMC1 accumulation at meiotic DSBs.
- The D1R1 and D2R1 recombinase configurations represent distinct stages of DSB repair, with altered lifetimes in Hormad1-/- mutants.
- HORMAD1 has a novel function in controlling synaptonemal complex architecture, impacting meiotic chromosome organization.
