Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction.


Supplementary Results
Supplementary Methods: Plasmid construction. Full-length human Smad3 cDNA was amplified by use of the FastStart High Fidelity PCR system (Roche) from pCMV5B-Smad3 vector. The two PCR primers included the sequences of EcoRI and XhoI restrictive endonucleases. The product of PCR was digested with EcoRI and XhoI, and then ligated with pEGFP-C1, which had been digested with EcoRI and XhoI.
The E. coli strain BL21 was transformed with the ligation mixture. Then transformation colonies were chosen, and the recombinant plasmid (EGFP-Smad3) was extracted. After verification via PCR, restrictive endonuclease digestion and DNA sequencing, the recombinant plasmid was purified by using of the Endo Free Plasmid Maxi Kit (QIAGEN). The dominant-negative COOH-terminally truncated version of Smad3 (M-Smad3) was created by deleting the sequence coding for the COOHterminal SSXS motif of Smad3. The M-Smad3 sequence was ligated into the pEGFP-C1 construct with EcoRI and XhoI digestion sites. The constructed plasmid was verified and used to transfect cells to express the EGFP-labeled mutant Smad3, named EGFP-M-Smad3. Imaging analysis to identify single molecules. The central quarter of the EMCCD chip (256×256 pixel 2 ) was used for imaging analysis to ensure homogeneous illumination. The background fluorescence was first subtracted from each frame with the use of Image J software (NIH). Then every frame of each movie was used for selecting single-molecule fluorescent spots. The image was thresholded (5 times the mean intensity of an area without fluorescent spots) and filtered again with the use of a user-defined program in Matlab (Mathworks Corp.) to remove discrete signals. After the imaging process, the brightest pixel of each fluorescent spot within diffraction-limited size (3×3 pixels, 480480 nm) was determined as the central position and enclosed with green circles. The spot with a peak pixel close to that of another spot (<3 pixels) was excluded.
Potassium depletion and membrane protein extraction. Endocytosis inhibition by potassium depletion was achieved as previously described 1 . Briefly, cells were incubated in DMEM: water (1:1) for 5 min at 37ºC followed by incubation in a minimal media (20 mM Hepes at pH 7.5, 140 mM sodium chloride, 1 mM calcium chloride, 1 mM magnesium sulphate, 5.5 mM glucose and 0.5 % BSA) for 1 hour at 37 ºC. Control cells were incubated in the minimal media supplemented with 10 mM potassium chloride. Cells were then harvested and proteins from the membrane and cytosol were extracted using Eukaryotic Membrane Protein Extraction Reagent kit (Perice). Lysates were then provided for Western blot analysis. The fractions from membrane were concentrated as 1/5 volume using ultrafiltration tube (10 kD, Millipore). hours, and was validated by immunofluorescence assays.
3 Western blotting. For western blotting, the cells were lysed in the assay buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, protease inhibitors), and the protein concentration in the lysates was determined by a spectrophotometer. Equal amount of the lysates were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and the immunoblotting was performed with primary antibodies and secondary antibodies conjugated to horseradish peroxidase (Abcam). Proteins were visualized by chemiluminescence. The signal density of phosphorylated proteins was normalized to the corresponding unphosphorylated proteins.

Co-Immunoprecipitation.
HeLa cells were incubated with 10 ng/ml TGF-1 for different times (0, 1, 5, 10, 30, 60, 120 min) after starved with serum free medium overnight. Cells were harvested and proteins from the membrane and cytosol were extracted using Eukaryotic Membrane Protein Extraction Reagent kit. Immunoprecipitation was performed by standard methodologies. Briefly, 2 g rabbit polyclonal anti-TRI antibody (Santa Cruz Biotechnology) and 2 mg suspension were incubated for 4 hours, and then Agarose protein A/G beads were added for precipitation at 4 °C overnight.
Precipitates were harvested by centrifuge at 4000 g/s for 3 min. After extensive washing, precipitates were boiled in lysis buffer for 10 min and then subjected to Western blotting analyses for detection of potential interacting proteins. Normal rabbit Ig G served as a negative control.