A novel workflow for unbiased 3D quantification of autophagosomes in Arabidopsis thaliana roots
The result's identifiers
Result code in IS VaVaI
<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61389030%3A_____%2F24%3A00601977" target="_blank" >RIV/61389030:_____/24:00601977 - isvavai.cz</a>
Result on the web
<a href="https://doi.org/10.1093/jxb/erae084" target="_blank" >https://doi.org/10.1093/jxb/erae084</a>
DOI - Digital Object Identifier
<a href="http://dx.doi.org/10.1093/jxb/erae084" target="_blank" >10.1093/jxb/erae084</a>
Alternative languages
Result language
angličtina
Original language name
A novel workflow for unbiased 3D quantification of autophagosomes in Arabidopsis thaliana roots
Original language description
Macroautophagy is often quantified by live imaging of autophagosomes labeled with fluorescently tagged ATG8 protein (FP-ATG8) in Arabidopsis thaliana. The labeled particles are then counted in single focal planes. This approach may lead to inaccurate results as the actual 3D distribution of autophagosomes is not taken into account and appropriate sampling in the Z-direction is not performed. To overcome this issue, we developed a workflow consisting of immunolabeling of autophagosomes with an anti-ATG8 antibody followed by stereological image analysis using the optical disector and the Cavalieri principle. Our protocol specifically recognized autophagosomes in epidermal cells of Arabidopsis root. Since the anti-ATG8 antibody recognizes multiple AtATG8 isoforms, we were able to detect a higher number of immunolabeled autophagosomes than with the FP-AtATG8e marker, that most probably does not recognize all autophagosomes in a cell. The number of autophagosomes per tissue volume positively correlated with the intensity of autophagy induction. Compared with the quantification of autophagosomes in maximum intensity projections, stereological methods were able to detect the autophagosomes present in a given volume with higher accuracy. Our novel workflow provides a powerful toolkit for unbiased and reproducible quantification of autophagosomes and offers a convenient alternative to the standard of live imaging with FP-ATG8 markers.
Czech name
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Czech description
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Classification
Type
J<sub>imp</sub> - Article in a specialist periodical, which is included in the Web of Science database
CEP classification
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OECD FORD branch
10611 - Plant sciences, botany
Result continuities
Project
Result was created during the realization of more than one project. More information in the Projects tab.
Continuities
I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace
Others
Publication year
2024
Confidentiality
S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů
Data specific for result type
Name of the periodical
Journal of Experimental Botany
ISSN
0022-0957
e-ISSN
1460-2431
Volume of the periodical
75
Issue of the periodical within the volume
17
Country of publishing house
US - UNITED STATES
Number of pages
16
Pages from-to
5412-5427
UT code for WoS article
001189393200001
EID of the result in the Scopus database
2-s2.0-85204027049