Dual-color 3D-dSTORM colocalization and quantification of ROXY1 and RNAPII variants throughout the transcription cycle in root meristem nuclei

Autor(en): Mass, Lucia
Holtmannspoetter, Michael
Zachgo, Sabine 
Stichwörter: ARABIDOPSIS-THALIANA; ARCHITECTURE; colocalization; dual-color 3D-dSTORM; ELONGATION; GROWTH; MICROSCOPY; NETWORKS; PETAL DEVELOPMENT; Plant Sciences; POLYMERASE-II; REDOX REGULATION; RNAPII; root meristem; ROXY1; super-resolution microscopy; technical advance; transcription cycle; TRANSITION
Erscheinungsdatum: 2020
Herausgeber: WILEY
Volumen: 104
Ausgabe: 5
Startseite: 1423
Seitenende: 1436
To unravel the function of a protein of interest, it is crucial to asses to what extent it associates via direct interactions or by overlapping expression with other proteins. ROXY1, a land plant-specific glutaredoxin, exerts a function in Arabidopsis flower development and interacts with TGA transcription factors in the nucleus. We detected a novelROXY1function in the root meristem. Root cells that lack chlorophyll reducing plant-specific background problems that can hamper colocalization 3D microscopy. Thus far, a super-resolution three-dimensional stochastic optical reconstruction microscopy (3D-dSTORM) approach has mainly been applied in animal studies. We established 3D-dSTORM using theroxy1mutant complemented with green fluorescence protein-ROXY1 and investigated its colocalization with three distinct RNAPII isoforms. To quantify the colocalization results, 3D-dSTORM was coupled with the coordinate-based colocalization method. Interestingly, ROXY1 proteins colocalize with different RNA polymerase II (RNAPII) isoforms that are active at distinct transcription cycle steps. Our colocalization data provide new insights on nuclear glutaredoxin activities suggesting that ROXY1 is not only required in early transcription initiation events via interaction with transcription factors but likely also participates throughout further transcription processes until late termination steps. Furthermore, we showed the applicability of the combined approaches to detect and quantify responses to altered growth conditions, exemplified by analysis of H(2)O(2)treatment, causing a dissociation of ROXY1 and RNAPII isoforms. We envisage that the powerful dual-color 3D-dSTORM/coordinate-based colocalization combination offers plant cell biologists the opportunity to colocalize and quantify root meristem proteins at an increased, unprecedented resolution level <50 nm, which will enable the detection of novel subcellular protein associations and functions.
ISSN: 09607412
DOI: 10.1111/tpj.14986

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