Figure 3.
FIT was present in homodimeric protein complexes in NBs, dependent on Ser271/272 site. Anisotropy (or homo-FRET) measurements of FIT-GFP and FITmSS271AA-GFP to determine homodimerization strength. (A) Schematic illustration of the anisotropy principle. Energy transfer between the same kind of fluorescently tagged proteins leads to depolarization of the emitted light. The extent of the depolarization gives a hint of dimerization and oligomerization of a protein as the fluorescence anisotropy (FA) value decreases. (B) Representative images showing color-coded FA values of FIT-GFP and FITmSS271AA-GFP at t = 0 and t = 5/15 min. (C and D) Box plots representing quantification of FA values. FA was measured at t = 0 within the whole nucleus and at t = 5/15 min within the whole nucleus, in NBs and residual NP. Free GFP and GFP-GFP served as references for mono- and dimerization. FA values for C, FIT-GFP, and D, FITmSS271AA-GFP. In C and D, FA values were calculated from 10 to 15 nuclei from a transformed plant (n = 10–15). Two experiments were conducted, and one representative result is shown. C and D show the same free GFP and GFP-GFP references because both measurements were performed on the same day. FA values decreased for FIT-GFP, but not FITmSS271AA-GFP, in the whole nucleus (compare t = 0 with t = 5/15 min). FA values were also lowered in NBs versus NP in the case of FIT-GFP but not FITmSS271AA-GFP (compare t = 5/15 min of NBs and NP). This indicates stronger homodimerization of FIT than FITmSS271AA-GFP in the whole nucleus and in NBs. IDRSer271/272 may therefore be relevant for FIT NB formation and FIT homodimerization (Fig. S2). Box plots show 25–75 percentile with min-max whiskers, mean as small square, and median as the line. Statistical analysis was performed with one-way ANOVA and Tukey post-hoc test. Different letters indicate statistically significant differences (P < 0.05). Scale bar: 2 µm. Arrowheads indicate NBs. G = GFP. Fluorescence protein analysis was conducted in transiently transformed N. benthamiana leaf epidermis cells, following the standardized FIT NB analysis procedure. Figure A has been created with the help of https://BioRender.com.

FIT was present in homodimeric protein complexes in NBs, dependent on Ser271/272 site. Anisotropy (or homo-FRET) measurements of FIT-GFP and FITmSS271AA-GFP to determine homodimerization strength. (A) Schematic illustration of the anisotropy principle. Energy transfer between the same kind of fluorescently tagged proteins leads to depolarization of the emitted light. The extent of the depolarization gives a hint of dimerization and oligomerization of a protein as the fluorescence anisotropy (FA) value decreases. (B) Representative images showing color-coded FA values of FIT-GFP and FITmSS271AA-GFP at t = 0 and t = 5/15 min. (C and D) Box plots representing quantification of FA values. FA was measured at t = 0 within the whole nucleus and at t = 5/15 min within the whole nucleus, in NBs and residual NP. Free GFP and GFP-GFP served as references for mono- and dimerization. FA values for C, FIT-GFP, and D, FITmSS271AA-GFP. In C and D, FA values were calculated from 10 to 15 nuclei from a transformed plant (n = 10–15). Two experiments were conducted, and one representative result is shown. C and D show the same free GFP and GFP-GFP references because both measurements were performed on the same day. FA values decreased for FIT-GFP, but not FITmSS271AA-GFP, in the whole nucleus (compare t = 0 with t = 5/15 min). FA values were also lowered in NBs versus NP in the case of FIT-GFP but not FITmSS271AA-GFP (compare t = 5/15 min of NBs and NP). This indicates stronger homodimerization of FIT than FITmSS271AA-GFP in the whole nucleus and in NBs. IDRSer271/272 may therefore be relevant for FIT NB formation and FIT homodimerization (Fig. S2). Box plots show 25–75 percentile with min-max whiskers, mean as small square, and median as the line. Statistical analysis was performed with one-way ANOVA and Tukey post-hoc test. Different letters indicate statistically significant differences (P < 0.05). Scale bar: 2 µm. Arrowheads indicate NBs. G = GFP. Fluorescence protein analysis was conducted in transiently transformed N. benthamiana leaf epidermis cells, following the standardized FIT NB analysis procedure. Figure A has been created with the help of https://BioRender.com.

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