In-section Click-iT detection and super-resolution CLEM
analysis of nucleolar ultrastructure and replication in plants

J 2024

In-section Click-iT detection and super-resolution CLEM analysis of nucleolar ultrastructure and replication in plants

FRANEK, Michal, Lenka KOPTASIKOVA, Jiri MIKSATKO, David LIEBL, Eliska MACICKOVA et. al.

Basic information

Original name

In-section Click-iT detection and super-resolution CLEM analysis of nucleolar ultrastructure and replication in plants

Authors

FRANEK, Michal, Lenka KOPTASIKOVA, Jiri MIKSATKO, David LIEBL, Eliska MACICKOVA, Jakub POSPÍŠIL, Milan EŠNER, Martina DVOŘÁČKOVÁ and Jiří FAJKUS

Edition

Nature Communications, Berlin, Nature, 2024, 2041-1723

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10400 1.4 Chemical sciences

Country of publisher

Germany

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

URL

Impact factor

Impact factor: 16.600 in 2022

Organization unit

Central European Institute of Technology

DOI

http://dx.doi.org/10.1038/s41467-024-46324-6

UT WoS

001187872900012

Keywords in English

FLUORESCENT PROTEINS; 45S RDNA; S-PHASE; CHEMISTRY; LOCALIZATION; ORGANIZATION; SELECTIVITY; FASCIATA1; CELLS; LIGHT

Abstract

V originále

Correlative light and electron microscopy (CLEM) is an important tool for the localisation of target molecule(s) and their spatial correlation with the ultrastructural map of subcellular features at the nanometre scale. Adoption of these advanced imaging methods has been limited in plant biology, due to challenges with plant tissue permeability, fluorescence labelling efficiency, indexing of features of interest throughout the complex 3D volume and their re-localization on micrographs of ultrathin cross-sections. Here, we demonstrate an imaging approach based on tissue processing and embedding into methacrylate resin followed by imaging of sections by both, single-molecule localization microscopy and transmission electron microscopy using consecutive CLEM and same-section CLEM correlative workflow. Importantly, we demonstrate that the use of a particular type of embedding resin is not only compatible with single-molecule localization microscopy but shows improvements in the fluorophore blinking behavior relative to the whole-mount approaches. Here, we use a commercially available Click-iT ethynyl-deoxyuridine cell proliferation kit to visualize the DNA replication sites of wild-type Arabidopsis thaliana seedlings, as well as fasciata1 and nucleolin1 plants and apply our in-section CLEM imaging workflow for the analysis of S-phase progression and nucleolar organization in mutant plants with aberrant nucleolar phenotypes.

Links

EF16_013/0001775, research and development project

Name: Modernizace a podpora výzkumných aktivit národní infrastruktury pro biologické a medicínské zobrazování Czech-BioImaging

EF18_046/0016045, research and development project

Name: Modernizace národní infrastruktury pro biologické a medicínské zobrazování Czech-BioImaging

GA23-06643S, research and development project

Name: Dispozice, výskyt a zpracování DNA lézí v genomu rostlin Arabidopsis thaliana defektních pro reparaci DNA a sestavování nukleozomů

Investor: Czech Science Foundation, Disposition, distribution, and processing of DNA lesions in the genome of Arabidopsis thaliana plants defective in DNA repair and nucleosome assembly

GX20-01331X, research and development project

Name: Biogeneze a evoluce telomerázy u rostlin

Investor: Czech Science Foundation

LM2023050, research and development project

Name: Národní infrastruktura pro biologické a medicínské zobrazování

Investor: Ministry of Education, Youth and Sports of the CR, Czech BioImaging: National research infrastructure for biological and medical imaging

Citovat

FRANEK, Michal, Lenka KOPTASIKOVA, Jiri MIKSATKO, David LIEBL, Eliska MACICKOVA, Jakub POSPÍŠIL, Milan EŠNER, Martina DVOŘÁČKOVÁ and Jiří FAJKUS. In-section Click-iT detection and super-resolution CLEM analysis of nucleolar ultrastructure and replication in plants. Nature Communications. Berlin: Nature, 2024, vol. 15, No 1, p. 1-13. ISSN 2041-1723. Available from: https://dx.doi.org/10.1038/s41467-024-46324-6.

@article{2433818,
   author = {Franek, Michal and Koptasikova, Lenka and Miksatko, Jiri and Liebl, David and Macickova, Eliska and Pospíšil, Jakub and Ešner, Milan and Dvořáčková, Martina and Fajkus, Jiří},
   article_location = {Berlin},
   article_number = {1},
   doi = {http://dx.doi.org/10.1038/s41467-024-46324-6},
   keywords = {FLUORESCENT PROTEINS; 45S RDNA; S-PHASE; CHEMISTRY; LOCALIZATION; ORGANIZATION; SELECTIVITY; FASCIATA1; CELLS; LIGHT},
   language = {eng},
   issn = {2041-1723},
   journal = {Nature Communications},
   title = {In-section Click-iT detection and super-resolution CLEM analysis of nucleolar ultrastructure and replication in plants},
   url = {https://www.nature.com/articles/s41467-024-46324-6},
   volume = {15},
   year = {2024}
}

TY  - JOUR
ID  - 2433818
AU  - Franek, Michal - Koptasikova, Lenka - Miksatko, Jiri - Liebl, David - Macickova, Eliska - Pospíšil, Jakub - Ešner, Milan - Dvořáčková, Martina - Fajkus, Jiří
PY  - 2024
TI  - In-section Click-iT detection and super-resolution CLEM analysis of nucleolar ultrastructure and replication in plants
JF  - Nature Communications
VL  - 15
IS  - 1
SP  - 1-13
EP  - 1-13
PB  - Nature
SN  - 20411723
KW  - FLUORESCENT PROTEINS
KW  - 45S RDNA
KW  - S-PHASE
KW  - CHEMISTRY
KW  - LOCALIZATION
KW  - ORGANIZATION
KW  - SELECTIVITY
KW  - FASCIATA1
KW  - CELLS
KW  - LIGHT
UR  - https://www.nature.com/articles/s41467-024-46324-6
N2  - Correlative light and electron microscopy (CLEM) is an important tool for the localisation of target molecule(s) and their spatial correlation with the ultrastructural map of subcellular features at the nanometre scale. Adoption of these advanced imaging methods has been limited in plant biology, due to challenges with plant tissue permeability, fluorescence labelling efficiency, indexing of features of interest throughout the complex 3D volume and their re-localization on micrographs of ultrathin cross-sections. Here, we demonstrate an imaging approach based on tissue processing and embedding into methacrylate resin followed by imaging of sections by both, single-molecule localization microscopy and transmission electron microscopy using consecutive CLEM and same-section CLEM correlative workflow. Importantly, we demonstrate that the use of a particular type of embedding resin is not only compatible with single-molecule localization microscopy but shows improvements in the fluorophore blinking behavior relative to the whole-mount approaches. Here, we use a commercially available Click-iT ethynyl-deoxyuridine cell proliferation kit to visualize the DNA replication sites of wild-type Arabidopsis thaliana seedlings, as well as fasciata1 and nucleolin1 plants and apply our in-section CLEM imaging workflow for the analysis of S-phase progression and nucleolar organization in mutant plants with aberrant nucleolar phenotypes.
ER  -

FRANEK, Michal, Lenka KOPTASIKOVA, Jiri MIKSATKO, David LIEBL, Eliska MACICKOVA, Jakub POSPÍŠIL, Milan EŠNER, Martina DVOŘÁČKOVÁ and Jiří FAJKUS. In-section Click-iT detection and super-resolution CLEM analysis of nucleolar ultrastructure and replication in plants. \textit{Nature Communications}. Berlin: Nature, 2024, vol.~15, No~1, p.~1-13. ISSN~2041-1723. Available from: https://dx.doi.org/10.1038/s41467-024-46324-6.

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