Počet záznamů: 1

Prospects and limitations of expansion microscopy in chromatin ultrastructure determination

SYSNO ASEP	0538175
Druh ASEP	J - Článek v odborném periodiku
Zařazení RIV	J - Článek v odborném periodiku
Poddruh J	Článek ve WOS
Název	Prospects and limitations of expansion microscopy in chromatin ultrastructure determination
Tvůrce(i)	Kubalova, I. (DE) Schmidt Černohorská, Markéta (UMG-J) Huranová, Martina (UMG-J)_ORCID Weisshart, K. (DE) Houben, A. (DE) Schubert, V. (DE)
Celkový počet autorů	6
Zdroj.dok.	Chromosome Research. - : Springer - ISSN 0967-3849 Roč. 28, č. 3-4 (2020), s. 355-368
Poč.str.	14 s.
Forma vydání	Online - E
Jazyk dok.	eng - angličtina
Země vyd.	NL - Nizozemsko
Klíč. slova	Chromatin ; Expansion microscopy ; Nucleus ; Structured illumination microscopy ; Hordeum vulgare
Vědní obor RIV	EB - Genetika a molekulární biologie
Obor OECD	Immunology
CEP	GJ17-20613Y GA ČR - Grantová agentura ČR
Způsob publikování	Open access
Institucionální podpora	UMG-J - RVO:68378050
UT WOS	000571228600001
DOI	10.1007/s10577-020-09637-y
Anotace	Expansion microscopy (ExM) is a method to magnify physically a specimen with preserved ultrastructure. It has the potential to explore structural features beyond the diffraction limit of light. The procedure has been successfully used for different animal species, from isolated macromolecular complexes through cells to tissue slices. Expansion of plant-derived samples is still at the beginning, and little is known, whether the chromatin ultrastructure becomes altered by physical expansion. In this study, we expanded isolated barley nuclei and compared whether ExM can provide a structural view of chromatin comparable with super-resolution microscopy. Different fixation and denaturation/digestion conditions were tested to maintain the chromatin ultrastructure. We achieved up to similar to 4.2-times physically expanded nuclei corresponding to a maximal resolution of similar to 50-60 nm when imaged by wild-field (WF) microscopy. By applying structured illumination microscopy (SIM, super-resolution) doubling the WF resolution, the chromatin structures were observed at a resolution of similar to 25-35 nm. WF microscopy showed a preserved nucleus shape and nucleoli. Moreover, we were able to detect chromatin domains, invisible in unexpanded nuclei. However, by applying SIM, we observed that the preservation of the chromatin ultrastructure after the expansion was not complete and that the majority of the tested conditions failed to keep the ultrastructure. Nevertheless, using expanded nuclei, we localized successfully centromere repeats by fluorescence in situ hybridization (FISH) and the centromere-specific histone H3 variant CENH3 by indirect immunolabelling. However, although these repeats and proteins were localized at the correct position within the nuclei (indicating a Rabl orientation), their ultrastructural arrangement was impaired.
Pracoviště	Ústav molekulární genetiky
Kontakt	Nikol Škňouřilová, nikol.sknourilova@img.cas.cz, Tel.: 241 063 217
Rok sběru	2021
Elektronická adresa	https://link.springer.com/article/10.1007/s10577-020-09637-y

Počet záznamů: 1