Figure 1.
Detection of interactions between FMTRIP-hybridized mRNA and cytoskeletal elements using proximity ligation assay (PLA).
(A) Flag (dark green) bound to a neutravidin (yellow) with Cy3B-conjugated (red) oligonucleotides (red dash) formed an FMTRIP. (B) Streptolysin O created entrance for FMTRIP in the cell membrane, allowing access to the mRNA (gray) bound to the β-tubulin (blue), vimentin (magenta), and F-actin (green), via RNA-binding proteins (RBP, brown). (C) Antibodies against the flag (light blue) and the cytoskeletal element (light magenta) in addition to the proximity probes against the antibodies (dark blue and magenta) attached to the FMTRIP-bound mRNA (gray) and the cytoskeleton (green); the oligonucleotides (black dash) on the proximity probes join to synthesize a Cy5-equivalent hybridized DNA product (light green and black dash) via rolling circle amplification.
Figure 2.
Interactions between poly(A)+ mRNA and cytoskeletal elements in HDF post-depolymerization of microtubules using nocodazole, intermediate filaments using acrylamide, and actin using cytochalasin D.
(A) β-tubulin, vimentin, and phalloidin immunofluorescence (IF), poly(A)+ mRNA, and PLA between poly(A)+ mRNA and the cytoskeletal elements in HDF were imaged with a laser-scanning confocal microscope. Merged images of the cytoskeleton (white), poly(A)+ mRNA (red), PLA (green) and nuclei (blue) are shown. Single image plane is represented. Scale bar, 10 µm. (B) The mean FMTRIP volume decreased after 90 min exposure to cytochalasin D (Mann-Whitney rank sum test, β-tubulin, p<0.01; vimentin, p<0.01) in experiments quantifying interactions with β-tubulin (n=21, mean=254µm3, s.d.=110µm3) and vimentin (n=16, mean=253µm3, s.d.=188µm3). (C) The mean percentage of FMTRIP (PLA-FMTRIP) bound to β-tubulin increased after depolymerization (Mann-Whitney rank sum test, p<0.001; n=21, mean=16.3%, s.d.=9.5%) but decreased for vimentin (Mann-Whitney rank sum test, p<0.001; n=16, mean=1.5%, s.d.=1.9%). (D) The mean PLA frequency detecting interactions with β-tubulin also increased (Mann-Whitney rank sum test, p=0.015; n=21, mean=0.03µm-3, s.d.=0.02µm-3) and decreased for vimentin (Mann-Whitney rank sum test, p<0.001; n=16, mean=0.01µm-3, s.d.=0.01µm-3). Error bars, s.d.
Figure 3.
Interactions between poly(A)+ mRNA and cytoskeletal elements in HDF.
(A) β-tubulin, vimentin and phalloidin IF, poly(A)+ mRNA, and PLA between poly(A)+ mRNA and the cytoskeletal elements in HDF were imaged with a laser-scanning confocal microscope. Merged images of the cytoskeleton (white), poly(A)+ mRNA (red), PLA (green) and nuclei (blue) are shown. Single image plane is represented. Inset, images of boxed regions. Scale bar, 10 µm (2 µm in insets). (B) The mean FMTRIP volume was similar (Kruskal-Wallis One Way ANOVA on Ranks, p=0.5) in cells, where the interactions between poly(A)+ mRNA and β-tubulin (n=25, mean=706µm3, s.d.=427µm3), vimentin (n=33, mean=780µm3, s.d.=405µm3), or phalloidin (n=23, mean=916µm3, s.d.=346µm3) were quantified. (C) The mean percentage of FMTRIP colocalized with PLA (PLA-FMTRIP) was significantly different (Table S1) between the interactions of poly(A)+ mRNA with β-tubulin (n=25, mean=2.3%, s.d.=1.5%), vimentin (n=33, mean=11.5%, s.d.=9.7%), or phalloidin (n=23, mean=53.7%, s.d.=19.9%). (D) The mean PLA frequency was significantly different (Table S2) between the interactions of poly(A)+ mRNA with β-tubulin (n=25, mean=0.010µm-3, s.d.=0.007µm-3), vimentin (n=33, mean=0.019µm-3, s.d.=0.016µm-3), or phalloidin (n=23, mean=0.069µm-3, s.d.=0.039µm-3). Error bars, s.d.
Figure 4.
Interactions between poly(A)+ mRNA bound to MTRIP lacking flag tag and cytoskeletal elements in human dermal fibroblasts (HDF).
(A) β-tubulin, vimentin and phalloidin IF, poly(A)+ mRNA, and PLA between poly(A)+ mRNA, and the cytoskeletal elements in HDF were imaged with a laser-scanning confocal microscope. Merged images of the cytoskeleton (white), poly(A)+ mRNA (red), PLA (green), and nuclei (blue) are shown. Single image plane is represented. Scale bar, 10 µm. (B) The mean MTRIP volume was similar (Kruskal-Wallis One Way ANOVA on Ranks, p=0.08) in cells, where the interactions between β-actin mRNA and β-tubulin (n=18, mean=404µm3, s.d.=317µm3), vimentin (n=19, mean=518µm3, s.d.=362µm3), or phalloidin (n=15, mean=653µm3, s.d.=211µm3) were quantified. (C) The mean percentage of MTRIP colocalized with PLA (PLA-MTRIP) was similarly minimal (Kruskal-Wallis One Way ANOVA on Ranks, p=0.15) in β-tubulin (n=18, mean=0.01%, s.d.=0.03%), vimentin (n=19, mean=0.04%, s.d.=0.1%), or phalloidin (n=15, mean=0.20%, s.d.=0.41%). (D) The mean PLA frequency was also minimal (Kruskal-Wallis One Way ANOVA on Ranks, p=0.23) in β-tubulin (n=18, mean=0.0004µm-3, s.d.=0.001µm-3), vimentin (n=19, mean=0.0008µm-3, s.d.=0.0001µm-3), or phalloidin (n=15, mean=0.001µm-3, s.d.=0.002µm-3). Error bars, s.d.
Figure 5.
Interactions between β-actin mRNA and cytoskeletal elements in HDF.
(A) β-tubulin, vimentin and phalloidin IF, β-actin mRNA, and PLA between β-actin mRNA and the cytoskeletal elements in HDF were imaged with a laser-scanning confocal microscope. Merged images of the cytoskeleton (white), β-actin mRNA (red), PLA (green), and nuclei (blue) are shown. Single image plane is represented. Inset, images of boxed regions. Scale bar, 10 µm (2 µm in insets). (B) The mean FMTRIP volume was similar (Kruskal-Wallis One Way ANOVA on Ranks, p=0.5) in cells, where the interactions between β-actin mRNA and β-tubulin (n=40, mean=567µm3, s.d.=355µm3), vimentin (n=36, mean=607µm3, s.d.=385µm3), or phalloidin (n=30, mean=660µm3, s.d.=389µm3) were quantified. (C) The mean percentage of FMTRIP colocalized with PLA (PLA-FMTRIP) was significantly different (Table S5) between the interactions of β-actin mRNA with β-tubulin (n=40, mean=3.9%, s.d.=3.2%), vimentin (n=36, mean=12.7%, s.d.=7.9%), or phalloidin (n=30, mean=71.5%, s.d.=20.1%). (D) The mean PLA frequency was significantly different (Table S6) between the interactions of β-actin mRNA with β-tubulin (n=40, mean=0.012µm-3, s.d.=0.011µm-3), vimentin (n=36, mean=0.027µm-3, s.d.=0.023µm-3), or phalloidin (n=30, mean=0.33µm-3, s.d.=0.17µm-3). Error bars, s.d.
Figure 6.
Arsenite-induced oxidative stress reduced poly(A)+ mRNA binding to F-actin and vimentin, while increasing interactions with β-tubulin.
(A) β-tubulin, vimentin and phalloidin IF, poly(A)+ mRNA, and PLA between poly(A)+ mRNA and the cytoskeletal elements in HDF were imaged at 0 min (or no) and 10 min exposure to arsenite with a laser-scanning confocal microscope. Merged images of the cytoskeleton (white), poly(A)+ mRNA (red), PLA (green), and nuclei (blue) are shown. All image planes are represented. Scale bar, 10 µm. (B) The mean percentage of FMTRIP (PLA-FMTRIP) bound to β-tubulin increased significantly from 0 min exposure (n=28, mean=2.5%, s.d.=1.6%) to 5 min exposure (Kruskal-Wallis One Way ANOVA on Ranks, p<0.001; n=31, mean=12.8%,s.d.=14.6%); remained high for 10 min exposure (n=26, mean=11.3%, s.d.=9.1%); and decreased to the pre-exposure level at 20 (Kruskal-Wallis One Way ANOVA on Ranks, p<0.05; n=22, mean=2.6%, s.d.=3.8%) and 40 min (n=20, mean=2.2%, s.d.=3.0%) (Table S9). (C) The mean percentage of FMTRIP (PLA-FMTRIP) bound to vimentin remained similar to 0 min exposure (n=42, mean=11.1%, s.d.=8.8%) for 5 (n=23, mean=13.9%,s.d.=6.5%),10 (n=19, mean=18.7%, s.d.=7.5%), and 20 min (n=13, mean=13.3%, s.d.=7.1%) exposure; it decreased at 40 min (n=17, mean=5.9%, s.d.=4.4%) (Table S10). (D) The mean percentage of FMTRIP (PLA-FMTRIP) bound to F-actin decreased significantly from 0 min exposure (n=38, mean=49.3%, s.d.=19.4%) to 5 min exposure (Kruskal-Wallis One Way ANOVA on Ranks, p<0.001; n=25, mean=6.0%,s.d.=5.2%); remained low for 10 (n=21, mean=9.6%, s.d.=6.4%), 20 (n=19, mean=15.4%, s.d.=9.4%), and 40 min (n=16, mean=6.8%, s.d.=4.4%) exposure (Table S11). Error bars, s.d.