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

Nan Li,Yong Yang,Wei Liang,Jinghe Yuan,Kangmin He,Xiaohong Fang,Fayun Zhang,Libo Zhao,Wei Zhou

doi:10.1038/srep33469

Abstract

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.

Highlights

transforming growth factor β (TGF-β)/Smad signaling pathway plays a pivotal role in a variety of important biological processes, including cell growth, differentiation, apoptosis, embryonic development and extracellular matrix formation[1,2,3]
There are currently three different views on R-Smads activation: (i) The R-Smads bind to the activated TRI via SARA at plasma membrane, where the R-Smads are phosphorylated by TRI, dissociated from the membrane for nuclear translocation14. (ii) After the binding of R-Smads with TRI at the cell membrane, the whole signaling complex including TGF-βreceptors and R-Smads, is endocytosed into the early endosomes, followed by R-Smads phosphorylation, dissociation and nuclear translocation15,16. (iii) Upon ligand binding, the heteromeric
By calculating the docking frequency of enhanced green fluorescent protein (EGFP)-Smad[3] molecules appeared on cell membrane during 30 seconds, we found the value was increased from 0.12 ± 0.01 to 0.22 ± 0.01 events/μm2s−1 after TGF-β1 stimulation (Fig. 1c, ***p < 0.001)

Summary

Introduction

TGF-β/Smad signaling pathway plays a pivotal role in a variety of important biological processes, including cell growth, differentiation, apoptosis, embryonic development and extracellular matrix formation[1,2,3]. According to the current knowledge on TGF-βsignaling, R-Smads are predominantly localized in the cytoplasm of unstimulated cells and associated with the protein SARA (Smad Anchor for Receptor Activation). There are currently three different views on R-Smads activation: (i) The R-Smads bind to the activated TRI via SARA at plasma membrane, where the R-Smads are phosphorylated by TRI, dissociated from the membrane for nuclear translocation. (ii) After the binding of R-Smads with TRI at the cell membrane, the whole signaling complex including TGF-βreceptors and R-Smads, is endocytosed into the early endosomes, followed by R-Smads phosphorylation, dissociation and nuclear translocation. TGF-βreceptor complex is internalized into early endosomes, the receptor-binding, phosphorylation and dissociation of R-Smads all take place in early endosomes instead of at cell membrane[17]. We designed a new approach to directly monitor the dynamics of Smad[3] activation in living cells using single-molecule total internal reflection fluorescence microscopy (TIRFM)

Methods

Results

Conclusion