Molecular organization of mammalian meiotic chromosome axis revealed by expansion STORM microscopy

Xu, Huizhong; Tong, Zhisong; Ye, Qing; Sun, Tengqian; Hong, Zhenmin; Zhang, Lunfeng; Bortnick, Alexandra; Cho, Sunglim; Beuzer, Paolo; Axelrod, Joshua; Hu, Qiongzheng; Wang, Melissa; Evans, Sylvia M.; Murre, Cornelis; Lu, Li-Fan; Sun, Sha; Corbett, K.D.; Cang, Hu

doi:10.1073/pnas.1902440116

Cited by 100 publications

(100 citation statements)

References 54 publications

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“…By linking a protein of interest into a cross-linked network of a swellable polyelectrolyte hydrogel, biological specimens can be physically expanded allowing for sub-diffraction resolution imaging on conventional microscopes 1-10 . However, even in combination with super-resolution microscopy techniques, spatial resolutions below ~20 nm have so far proven to be very difficult to achieve by ExM 16 . Here, we have shown that re-embedding of expanded hydrogels enables the use of standard photoswitching buffers and dSTORM imaging of ~3.2x expanded samples.…”

Section: Discussionmentioning

confidence: 99%

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Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

Zwettler¹,

Reinhard²,

Gambarotto

et al. 2020

Preprint

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Expansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining expansion microscopy (ExM) with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.

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Section: Discussionmentioning

confidence: 99%

“…However, despite these apparent advantages, attempts to combine ExM with SMLM have remained rare and unoptimized due to several challenges 5,16 . There are two major determinants that control the resolution of SMLM, the localization precision and the localization density 11,12 .…”

Section: Introductionmentioning

confidence: 99%

Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

Zwettler¹,

Reinhard²,

Gambarotto

et al. 2020

Preprint

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“…By physically increasing the size of the specimen in an isotropic manner via expansion microscopy 12 (ExM), overall imaging throughput can be increased by opting for faster diffraction-limited microscopes 12–14 . Alternatively, combining ExM with existing super-resolution microscopies allows even further improvement in resolution 14–18 , since the resolvable scale is reduced by the expansion factor. However, expansion exacerbates limitations in imaging throughput since the effective FOV size is divided by the expansion factor along each dimension 19 .…”

Section: Introductionmentioning

confidence: 99%

Homogeneous multifocal excitation for high-throughput super-resolution imaging

Mahecic

Gambarotto

Douglass

et al. 2020

Preprint

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19Super-resolution microscopies, which allow features below the diffraction limit to be 20 resolved, have become an established tool in biological research. However, imaging 21 throughput remains a major bottleneck in using them for quantitative biology, which requires 22 large datasets to overcome the noise of the imaging itself and to capture the variability 23 inherent to biological processes. Here, we develop a multi-focal flat illumination for field 24 independent imaging (mfFIFI) module, and integrate it into an instant structured illumination 25 microscope (iSIM). Our instrument extends the field of view (FOV) to >100x100 µm 2 without 26 compromising image quality, and maintains high-speed (100 Hz), multi-color, volumetric 27 imaging at double the diffraction-limited resolution. We further extend the effective FOV by 28 stitching multiple adjacent images together to perform fast live-cell super-resolution imaging 29 of dozens of cells. Finally, we combine our flat-fielded iSIM setup with ultrastructure 30 expansion microscopy (U-ExM) to collect 3D images of hundreds of centrioles in human 31 cells, as well as of thousands of purified Chlamydomonas reinhardtii centrioles per hour at 32 an effective resolution of ~35 nm. We apply classification and particle averaging to these 33 large datasets, allowing us to map the 3D organization of post-translational modifications of 34 centriolar microtubules, revealing differences in their coverage and positioning. 35 (STED) 5 . Initial implementations of these methods were relatively slow, in part due to the 42 use of the time domain to separate single molecules (SMLM), the need to collect multiple 43 images to cover Fourier space (SIM), or the scanning of a reduced excitation volume 44 3 (STED). In both wide-field and patterned illumination, the imaging throughput -defined as 45 the area imaged per time -can be increased by parallelizing the acquisition, as long as the 46 necessary illumination can be maintained over a larger surface in the sample plane. 47Extending the array of patterned excitation has been used to increase the speed or FOV of 48 confocal 6 , STED 7,8 and SIM imaging 9 , but ensuring the quality of the extended pattern 49 across a larger FOV remains a limiting factor. For SMLM, flat-fielding approaches made it 50 possible to extend super-resolution imaging over ~100x100 µm 2 FOVs 10,11 , but their transfer 51 to patterned illumination remains limited. 52An entirely different approach to effectively achieve super-resolution modifies 53 sample preparation rather than image acquisition. By physically increasing the size of the 54 specimen in an isotropic manner via expansion microscopy 12 (ExM), overall imaging 55 throughput can be increased by opting for faster diffraction-limited microscopes 12-14 . 56 Alternatively, combining ExM with existing super-resolution microscopies allows even 57 further improvement in resolution [14][15][16][17][18] , since the resolvable scale is reduced by the 58 expansion factor. However, expansion exacerbates limita...

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“…Prdm9 loss-of-function in Mus musculus leads to sterility (Hayashi et al, 2005), and many wild hybrid mice with incompatible Prdm9 alleles are sterile (Davies et al, 2016), making Prdm9 the only known speciation gene so far identified in mammals (Mihola et al, 2009). Furthermore, single nucleotide polymorphisms in PRDM9 have been linked to non-obstructive azoospermia in humans (Irie et al, 2009;Miyamoto et al, 2008) During meiotic prophase I, chromatin re-organizes into condensed chromosomes, with DNA loops stemming from an axis composed of protein complexes (Cohen and Holloway, 2014;Xu et al, 2019). MR occurs simultaneously with homologous chromosome pairing and synapsis, and the relationship between these events is complex (Santos, 1999).…”

Section: Introductionmentioning

confidence: 99%

Dual Histone Methyl Reader ZCWPW1 Facilitates Repair of Meiotic Double Strand Breaks

Mahgoub

Paiano

Bruno

et al. 2019

Preprint

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Meiotic crossovers result from homology-directed repair of double strand breaks (DSBs). Unlike yeast and plants, where DSBs are generated near gene promoters, in many vertebrates, DSBs are enriched at hotspots determined by the DNA binding activity of the rapidly evolving zinc finger array of PRDM9 (PR domain zinc finger protein 9). PRDM9 subsequently catalyzes trimethylation of lysine 4 and lysine 36 of Histone H3 in nearby nucleosomes. Here, we identify the dual histone methylation reader ZCWPW1, which is tightly co-expressed during spermatogenesis with Prdm9 and co-evolved with Prdm9 in vertebrates, as an essential meiotic recombination factor required for efficient synapsis and repair of PRDM9-dependent DSBs. In sum, our results indicate that the evolution of a dual histone methylation writer/reader system in vertebrates facilitated a shift in genetic recombination away from a static pattern near genes towards a flexible pattern controlled by the rapidly evolving DNA binding activity of PRDM9.

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Molecular organization of mammalian meiotic chromosome axis revealed by expansion STORM microscopy

Cited by 100 publications

References 54 publications

Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

Homogeneous multifocal excitation for high-throughput super-resolution imaging

Dual Histone Methyl Reader ZCWPW1 Facilitates Repair of Meiotic Double Strand Breaks

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