Quantitative 3D structured illumination microscopy of nuclear structures

Kraus, Felix; Miron, Ezequiel; Demmerle, Justin; Chitiashvili, Tsotne; Budco, A.; Alle, Quentin; Matsuda, Atsushi; Leonhardt, Heinrich; Schermelleh, Lothar; Markaki, Yolanda

doi:10.1038/nprot.2017.020

Cited by 80 publications

(67 citation statements)

References 50 publications

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“…The intracellular localization of GFP and mRFP fusion proteins was observed in living cells. The chromatic aberration and the geometry of the two EM‐CCD cameras were corrected using the in‐house “Chromagnon” software (Kraus et al., ), using transmission images of S. pombe cells simultaneously taken in both the green and red channels as a reference. The images are presented after deconvolution, using the built‐in SoftWoRx software.…”

Section: Methodsmentioning

confidence: 99%

Lem2 is retained at the nuclear envelope through its interaction with Bqt4 in fission yeast

et al. 2018

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Inner nuclear membrane (INM) proteins are thought to play important roles in modulating nuclear organization and function through their interactions with chromatin. However, these INM proteins share redundant functions in metazoans that pose difficulties for functional studies. The fission yeast Schizosaccharomyces pombe exhibits a relatively small number of INM proteins, and molecular genetic tools are available to separate their redundant functions. In S. pombe, it has been reported that among potentially redundant INM proteins, Lem2 displays a unique genetic interaction with another INM protein, Bqt4, which is involved in anchoring telomeres to the nuclear envelope. Double mutations in the lem2 and bqt4 genes confer synthetic lethality during vegetative growth. Here, we show that Lem2 is retained at the nuclear envelope through its interaction with Bqt4, as the loss of Bqt4 results in the exclusive accumulation of Lem2 to the spindle pole body (SPB). An N‐terminal nucleoplasmic region of Lem2 bears affinity to both Bqt4 and the SPB in a competitive manner. In contrast, the synthetic lethality of the lem2 bqt4 double mutant is suppressed by the C‐terminal region of Lem2. These results indicate that the N‐terminal and C‐terminal domains of Lem2 show independent functions with respect to Bqt4.

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Section: Methodsmentioning

confidence: 99%

Lem2 is retained at the nuclear envelope through its interaction with Bqt4 in fission yeast

et al. 2018

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“…Segmentation of Xist RNA foci was performed by using the TANGO plugin 88 on ImageJ according to the pipelines described in 75 . In brief, nuclear masks were created by using the nucleus processing chain.…”

Section: Segmentation Of Xist Rna Foci From 3d-sim Datasetsmentioning

confidence: 99%

AnXist-dependent protein assembly mediatesXistlocalization and gene silencing

Pandya-Jones

Markaki

Serizay

et al. 2020

Preprint

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Nuclear compartments play diverse roles in regulating gene expression, yet the molecular forces and components driving compartment formation are not well understood. Studying how the lncRNA Xist establishes the inactive-X-chromosome (Xi)-compartment, we found that the Xist RNA-binding-proteins PTBP1, MATR3, TDP43, and CELF1 form a condensate to create an Xidomain that can be sustained in the absence of Xist. The E-repeat-sequence of Xist serves a multivalent binding-platform for these proteins. Without the E-repeat, Xist initially coats the Xchromosome during XCI onset but subsequently disperses across the nucleus with loss of gene silencing. Recruitment of PTBP1, MATR3, TDP-43 or CELF1 to DE-Xist rescues these phenotypes, and requires both self-association of MATR3 and TDP-43 and a heterotypic PTBP1-MATR3-interaction. Together, our data reveal that Xist sequesters itself within the Xi-territory and perpetuates gene silencing by seeding a protein-condensate. Our findings uncover an unanticipated mechanism for epigenetic memory and elucidate the interplay between RNA and RNA-bindingproteins in creating compartments for gene regulation. Main textThe function of long non-coding RNAs (lncRNAs) and the mechanisms by which they act remain largely unknown. One of the best studied lncRNAs is Xist, which orchestrates X-chromosome inactivation (XCI) in placental female mammals 1-7 . By spreading across one X-chromosome and mediating chromosome-wide gene silencing, Xist equalizes X-linked gene expression with that of males 8-12 . XCI initiates when Xist is induced on one of the two X-chromosomes in pluripotent cells of the implanting blastocyst, or upon induction of differentiation in embryonic stem cells (ESCs) 4,13 , the latter providing a powerful model for the mechanistic dissection of XCI-initiation.Intriguingly, Xist shapes nuclear organization during XCI-initiation. Xist establishes a transcriptionally silent, intra-chromosomal domain (or compartment) by specifically localizing to the X-chromosome from which it is transcribed and inducing the compaction of the forming inactive X-chromosome (Xi), the enrichment of heterochromatin proteins, the repositioning of silenced genes into the center of the Xi, and the exclusion of active transcriptional regulators, such as RNA polymerase II 1,2,14-20 . Yet, the mechanisms that drive and maintain the Xist RNA within a spatially confined region to establish this Xi-domain remain unclear.6 the E-repeat occurred on the 129 allele, which also harbors 11 copies of an MS2-RNA tag within Xist 15 , yielding the X129 Xist DE, MS2 XCas Xist WT genotype (referred to as DE ESCs below) ( Fig. 2a and Extended Data Fig. 5a-d). We ensured that DE ESCs maintained two X-chromosomes, and differentiated normally, as judged by morphological changes and loss of NANOG expression (Extended Data Fig. 5e-g). When transcribed from the X129 Xist DE, MS2 allele, Xist exon 6 was spliced to a cryptic site within exon 7 to generate an RNA missing specifically the E-repeat (Extended Data Fig. 6).RNA FISH over ...

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“…15 SIM is typically performed by recording a series of images with striped illumination patterns in order to capture previously undetectable spatial frequencies; the image series is then used to computationally reconstruct a high resolution image. 16 Because of its generality, SIM is compatible with a wide range of fluorophores and is amenable to imaging of relatively thick, 17,18 multicolor specimens, such as those generated by the ExM protocol. We refer to the use of SIM to image expanded specimens as ExSIM.…”

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confidence: 99%

Hybrid Structured Illumination Expansion Microscopy Reveals Microbial Cytoskeleton Organization

et al. 2017

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Recently developed tissue-hydrogel methods for specimen expansion now enable researchers to perform super-resolution microscopy with ~65 nm lateral resolution using ordinary microscopes, standard fluorescent probes, and inexpensive reagents. Here we use the combination of specimen expansion and the optical super-resolution microscopy technique structured illumination microscopy (SIM) to extend the spatial resolution to ~30 nm. We apply this hybrid method, which we call ExSIM, to study the cytoskeleton of the important human pathogen Giardia lamblia including the adhesive disc and flagellar axonemes. We determined the localization of two recently identified disc-associated proteins, including DAP86676 which localizes to disc microribbons, and the functionally unknown DAP16263 which primarily localizes to dorsal microtubules of the disc overlap zone and the paraflagellar rod of ventral axonemes. Based on its strong performance in revealing known and unknown details of the ultrastructure of Giardia, we find that ExSIM is a simple, rapid, and powerful super-resolution method for the study of fixed specimens, and it should be broadly applicable to other biological systems of interest.

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Quantitative 3D structured illumination microscopy of nuclear structures

Cited by 80 publications

References 50 publications

Lem2 is retained at the nuclear envelope through its interaction with Bqt4 in fission yeast

Lem2 is retained at the nuclear envelope through its interaction with Bqt4 in fission yeast

AnXist-dependent protein assembly mediatesXistlocalization and gene silencing

Hybrid Structured Illumination Expansion Microscopy Reveals Microbial Cytoskeleton Organization

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