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Structured Illumination Microscopy Imaging Reveals Localization Of Yoon et al. Experimental & Molecular Medicine (2018) 50:112 DOI 10.1038/s12276-018-0139-5 Experimental & Molecular Medicine ARTICLE Open Access Structured illumination microscopy imaging reveals localization of replication protein A between chromosome lateral elements during mammalian meiosis Seobin Yoon1,Eui-HwanChoi1, Jung-Woong Kim1 and Keun P. Kim1 Abstract An important event enabling meiotic prophase I to proceed is the close juxtaposition of conjoined chromosome axes of homologs and their assembly via an array of transverse filaments and meiosis-specific axial elements into the synaptonemal complex (SC). During meiosis, recombination requires the establishment of a platform for recombinational interactions between the chromosome axes and their subsequent stabilization. This is essential for ensuring crossover recombination and proper segregation of homologous chromosomes. Thus, well-established SCs are essential for supporting these processes. The regulation of recombination intermediates on the chromosome axis/ SC and dynamic positioning of double-strand breaks are not well understood. Here, using super-resolution microscopy (structured illumination microscopy), we determined the localization of the replication protein A (RPA) complex on the chromosome axes in the early phase of leptonema/zygonema and within the CEs of SC in the pachynema during meiotic prophase in mouse spermatocytes. RPA, which marks the intermediate steps of pairing and recombination, 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; appears in large numbers and is positioned on the chromosome axes at the zygonema. In the pachynema, RPA foci are reduced but do not completely disappear; instead, they are placed between lateral elements. Our results reveal the precise structure of SC and localization dynamics of recombination intermediates on meiocyte chromosomes undergoing homolog pairing and meiotic recombination. Introduction recombination and search for homologous counterpart During meiosis, replicated chromosomes search for DNA; next, the paired homologs form SCs, prominent their homologous templates (also known as homologs) proteinaceous structures that assemble between homo- and form synapses to undergo meiotic recombination, a logous chromosomes during meiotic prophase2,4,7. process that is essential for producing crossover (CO) and Meiotic prophase I is categorized into four stages defined assuring correct chromosome separation during the first by chromosome structures and nucleation/polymerization meiotic division1,2. Throughout the first meiotic prophase, of the SCs: leptonema (development of axes, initiation of sister chromatids are held together by sister chromatid homolog pairing, followed by initiation of DSB); zygo- cohesion, which involves the formation of a chromosome- nema (synapsis of homologs and initiation of SC); – associated multi-subunit protein complex3 6. Pro- pachynema (completion of synapsis and formation of full- – grammed double-strand breaks (DSBs), the generation length SC); and diplonema (observable chiasmata)1,8 10. of which is catalyzed by Spo11, initiate meiotic The SC shows a general tripartite ladder-like structure with two parallel lateral elements (LEs) where the homolog chromatin is closed and a central element (CE) Correspondence: Keun P. Kim ([email protected]) that is in the gap between LEs. Ultrastructural analysis of 1Department of Life Sciences, Chung-Ang University, Seoul 06974, Korea © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Korean Society for Biochemistry and Molecular Biology Yoon et al. Experimental & Molecular Medicine (2018) 50:112 Page 2 of 12 the SC has revealed that the CE consists of transverse generates a so-called moiré pattern that gives rise to dark filaments (TFs), which are two interconnected LEs that and light stripes in the images. The moiré pattern is – are essential for crossing over11 13. The SC begins to form created by the interaction between the high frequency of during the leptonema stage of meiotic prophase I as the the stripe pattern and high-frequency organization of the first homologs become connected by a central region objects within the sample containing higher-frequency composed of TFs, which become visible between axial information. This causes the expansion frequency space elements. The axial element forms a proteinaceous to become visible, and the resolution in two or three structure related to the two sister chromatids of the dimensions is improved by approximately twofold homologs. During the zygonema-to-pachynema transi- because of the reduction in the size of the point-spread tion, the LEs and the CEs are completely assembled in a function. To restructure a final image, several raw images process known as synapsis. Synapsis is fully completed at (moiré pattern images) are collected, each of which is the mid-pachynema of meiosis I; subsequently, the captured from different angles of the structured homologs separate, and then the SCs disassemble in illumination20,21. diplonema3,8. Using super-resolution SIM, we investigated the In mammals, the core components required for forming dynamic architecture of SCs, which are assembled the SC have been identified as SC protein 1 (SYCP1), between homologous chromosomes. We also specified SYCP2, and SYCP3. SYCP1 is involved in the TF forma- the dynamic localization of RPA that binds to single- tion of SCs. Further, SYCP2 and SYCP3, known as LE stranded DNA at meiotic recombination sites associated – proteins, form the axial elements during leptonema14 16. with SCs during prophase I. We describe new features of When the homologs become synapsed during the zygo- the chromosome structure and reveal the RPA focus nema, the axial elements are joined by the TFs composed dynamics on the chromosome axes and SC nucleation of SYCP117. regions. Importantly, this information provides insight Replication protein A (RPA), a heterotrimeric protein into how the SC structure and DSBs/single-stranded complex consisting of the RPA1, RPA2, and RPA3 sub- DNA (ssDNA) are regulated in mammalian meiosis. units, tightly binds to single-stranded DNA during repli- cation and DNA repair in the eukaryotic cell cycle. RPA Materials and methods generally inhibits secondary structure of a DSB end until Mice recombinase displaces the RPA and initiates recombina- C57/BL6J mice were purchased from Jackson Labora- tion. Specifically, RPA is involved in chromosome axis- tory (Bar Harbor, ME, USA) via Orient Bio (Seongnam, bridge formation between homologs during meiotic pro- Korea) and were maintained under a 12-h light/dark cycle phase I7. Thus, high-resolution cytological imaging for with free access to food and water. The testes of 4-week- chromosome morphogenesis may be essential for old mice were dissected and rapidly isolated on an ice improving the understanding of how RPA is assembled on plate with phosphate-buffered saline (PBS). chromosome to induce morphological changes, in addi- tion to observing chromosome morphogenesis related to Chromosome spreads of mouse spermatocytes SC components and investigating the dynamics of how We slightly modified the “drying-down technique” to RPA plays crucial roles in meiotic recombination and prepare spermatocytes22. Seminiferous tubules were checkpoint signaling. However, the results of studies transferred to hypotonic buffer (30 mM Tris-HCl at pH using conventional microscopy are limited because the 8.2, 17 mM sodium citrate, 5 mM ethylenediaminete- diffraction limits the resolution, preventing precise traacetic acid, 50 mM sucrose, 5 mM dithiothreitol, and observation of subcellular localization and chromosome 0.5 mM phenylmethylsulfonyl fluoride) and incubated structures. at room temperature for 30 min. Swollen tubules were Various techniques have been developed to overcome fixed in 4% paraformaldehyde (pH 6.9) for 5 min at the limitations of conventional methods. The most fre- room temperature. Fixed tubules were washed with PBS quently used high-resolution techniques are structured for 10 min. Washed tubules were then transferred to illumination microscopy (SIM), stimulated emission fresh PBS and disrupted via vigorous pipetting to obtain depletion, and photo-activated localization microscopy; a cloudy suspension. Next, 100 mM sucrose was dropped these techniques have been used to analyze molecular onto a clean slide. The cell suspension was mixed with structures and their localization18,19. SIM imaging, an the sucrose drop on the clean slide and spread over the adapted wide-field imaging technique, uses patterned slide to dry more than 4 h. After drying, the slide was illumination to excite fluorescence samples. The emitted rinsed by immersion in 0.4% Photo-Flo (Eastman Kodak
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