Covalent Self-Labeling of Tagged Proteins with Chemical Fluorescent Dyes in BY-2 Cells and Arabidopsis Seedlings

Abstract

Synthetic chemical fluorescent dyes promise to be useful for many applications in biology. Covalent, targeted labeling, such as with a SNAP-tag, uses synthetic dyes to label specific proteins in vivo for studying processes such as endocytosis or for imaging via super-resolution microscopy. Despite its potential, such chemical tagging has not been used effectively in plants. A major drawback has been the limited knowledge regarding cell wall and membrane permeability of the available synthetic dyes. Of 31 synthetic dyes tested here, 23 were taken up into BY-2 cells, while eight were not. This creates sets of dyes that can serve to measure endocytosis. Three of the dyes that were able to enter the cells, SNAP-tag ligands of diethylaminocoumarin, tetramethylrhodamine, and silicon-rhodamine 647, were used to SNAP-tag α-tubulin. Successful tagging was verified by live cell imaging and visualization of microtubule arrays in interphase and during mitosis in Arabidopsis (Arabidopsis thaliana) seedlings. Fluorescence activation-coupled protein labeling with DRBG-488 was used to observe PIN-FORMED2 (PIN2) endocytosis and delivery to the vacuole as well as preferential delivery of newly synthesized PIN2 to the actively forming cell plate during mitosis. Together, the data demonstrate that specific self-labeling of proteins can be used effectively in plants to study a wide variety of cellular and biological processes.

Figure 1.

Representative Images of BY-2 Cells Exposed to Various Fluorescent Dyes under Various Conditions. (A) Representative images of four dyes that entered BY-2 cells within 1 min. Permeability was calculated as the ratio of fluorescence intensity inside/outside the cell. An intracellular/extracellular fluorescence intensity ratio of >0.4 indicated uptake. (B) Representative data for three dyes unable to enter BY-2 cells. Intracellular/extracellular fluorescence intensity ratios were <0.2. (C) Time-lapse analysis of 2MeRG and RG accumulation, measured by intracellular/extracellular signal ratios. Plots and error bands represent mean (n = 6 cells) and se, respectively. Arrow indicates when dyes were added. Initial positive ratio is in absence of dye and due to autofluorescence. (D) HMRG uptake at different pH values. (E) Confocal images of BY-2 cells incubated with SNAP-Surface Alexa Fluor 488, SNAP-Cell 430, SNAP-Cell TMR-Star, and SNAP-Cell 647SiR for 1 min. Images were taken with same settings at different time points. Bars = 50 µm. Experiments were repeated independently three times with comparable results.

Figure 2.

SNAP-Tagging Enabled in Vivo Imaging of Tubulin in BY-2 Cells and Arabidopsis. (A) Labeling mechanism of the SNAP-tag. (B) Cortical microtubules in BY-2 cells expressing SNAP-TUA5 with SNAP dyes denoted above the images. Images show max intensity projection of confocal z-stack slices taken in 0.5-µm steps. (C) Time-lapse imaging of mitotic microtubule dynamics via TMR-Star labeling of TUA5. Images were taken every 30 s, and elapsed time (minutes) is shown. (D) Fourteen-day-old pUBQ10:SNAP-TUA5 and Columbia-0 (wild-type) seedlings were stained with 500 nM SNAP-Cell TMR-Star for 3 h and lysed. The crude protein extract was analyzed by SDS-PAGE. Left, Fluorescence (FL). Right, Coomassie Brilliant Blue (CBB) staining. SNAP-Cell TMR-Star selectively labeled the SNAP-TUA5 in protein extracts. A red arrow indicates the expected molecular weight of SNAP-TUA5. (E) Confocal images of mitotic cells in the root epidermis of 4-d-old SNAP-TUA5 Arabidopsis seedlings stained with TMR-Star. Spindles and phragmoplasts were observed. (F) Confocal image of cortical microtubules of pavement cells and stomata in the cotyledon of 4-d-old SNAP-TUA5 Arabidopsis seedlings stained with SNAP-Cell TMR-Star. (G) Confocal image of cortical microtubules of etiolated hypocotyl epidermal cells stained with SNAP-Cell TMR-Star. Bars = 10 µm. Experiments were repeated independently three times with comparable results.

Figure 3.

SNAP-Tagging Did Not Have a Significant Impact on the Duration of Mitotic Cell Divisions and Enabled in Vivo Multicolor Imaging in Arabidopsis Seedlings. (A) Comparison of the duration from nuclear envelope breakdown to the phragmoplast initiation between mitotic cells in mCherry-TUA5 plants (n = 31) and SNAP-TUA5 plants incubated in 500 nM SNAP-Cell TMR-Star (n = 32). There was no significant difference (P = 0.2510 by Mann-Whitney U test). In the box plots, the boxes represent the range from the 25th to 75th percentiles, the horizontal lines represent the median value, and the whiskers span from the 5th to 95th percentiles. (B) Cotyledon epidermal cells of Arabidopsis coexpressing SPR2-GFP (SPR2) and pUBQ10:SNAP-TUA5 (MT). Four-day-old seedlings were incubated in 0.5× MS containing 500 nM SNAP-Cell TMR-Star. (Top) Representative confocal image of MTs and SPR2 in a pavement cell. (Bottom) Kymographs generated from the top panel (at the blue dotted line) showing SPR2 tracking the minus end of microtubule (yellow arrow). (C) Root tip of Arabidopsis coexpressing p35S:YFP-LTI6b, p35:H2B-RFP, and pUBQ10:SNAP-TUA5. Three-day-old seedlings were incubated in 0.5× MS containing 500 nM SNAP-Cell 647SiR for 30 min. Bars = 5 µm. Experiments were repeated independently two times ( [A] and [B]) and three times (C)) with comparable results.

Figure 4.

SNAP-Tagging of the PIN2 Auxin Transporter Enabled FAPL with DRBG-488. (A) Topological model of SNAP-PIN2-mCherry fusion protein. SNAP-tag and mCherry are fused at the N terminus and cytosolic loop of PIN2, respectively. (B) Conceptional illustration of labeling of SNAP-PIN2-mCherry by DRBG-488. DRBG-488 itself is impermeable to the cell membrane and is not fluorescent until reaction with a SNAP-tag. Once DRBG-488 binds to SNAP-tag, the quencher group is released and DRBG-488 becomes fluorescent. Since DRBG-488 reacts covalently with the SNAP-tag, it can be internalized with the chimeric SNAP-PIN2-mCherry. (C) Phenotype of wild-type Columbia-0, eir1-1, and eir1-1 harboring pPIN2:SNAP-PIN2-mCherry grown for 7 d on solid media on vertical plates. Arrow indicates a root detached from the medium. Bar = 10 mm. (D) Pulse-chase analysis of DRBG-488 labeling. Plants were incubated with 200 nM DRBG-488 for 30 min followed by washout and 30- or 240-min incubation in liquid medium. Arrows indicate signals in plasma membrane and intracellular punctate structures. Asterisks indicate vacuoles. Bars = 10 µm. (E) Ratio of fluorescence in the C/P of DRBG-488-SNAP-PIN2–derived fluorescence in the root gradually increased over time. In the box plots, the boxes represent the range from the 25th to 75th percentiles, the horizontal lines represent the median value, and the dots indicate outliers. Different letters above the plots represent significant differences among means (P < 0.005 by one-way ANOVA with Tukey’s post hoc test; exact P-values are described in the Supplemental Data Set). n = 86, 104, 104, 62, and 118 cells from three roots. Experiments were repeated independently three times.

Figure 5.

Evidence for Clathrin-Mediated Endocytosis of PIN2. (A) DRBG-488 labeling of SNAP-PIN2-mCherry after DMSO (mock) or 30 µM ES9-17 treatment for 60 min. Bars = 10 µm. (B) Number of DRBG-488–positive endosomes per cell in the presence or absence of ES9-17. Significance of difference was determined by unpaired two-tailed t test with Welch’s correction. n = 139 (DMSO) and 128 (ES9-17) cells from five roots. (C) C/P fluorescence ratio of DRBG-488 in the presence or absence of ES9-17. Box plots are centered at the data median and mark from the 25th to the 75th percentile and whiskers span from 5th to 95th percentile. Dots indicate outliers. Significance of difference was determined by unpaired two-tailed t test with Welch’s correction. n = 117 (DMSO) and 115 (ES9-17) cells from five roots. Data that show comparable effects for 120-min treatments are shown in Supplemental Figure 7.

Figure 6.

Preferential Delivery of Newly Produced PIN2 to the Newly Forming Cell Plate. (A) Time-lapse imaging of PIN2 in a dividing cell. Arrowheads indicate edges of the cell plate and connection sites between newly formed plasma membrane and the lateral plasma membrane. Bars = 10 µm. (B) Quantification of fluorescence intensities of DRBG-488 and mCherry along the line in the cell at 41 min after appearance of the cell plate. Orange and pink arrowheads indicate preexisting and newly formed plasma membrane, respectively. (C) Scan of relative fluorescence of DRBG-488 and mCherry in images from (B), with length representing the position from the top of the image. Colored arrowheads on peaks correspond to the scan position in (B). Experiments were repeated independently three times.