Transforming growth factor beta (TGF-beta) signaling is important for many biological processes. Although the sequential events of this cascade are known, the dynamics remain speculative. Here, live-cell single-molecule total internal reflection fluorescence microscopy was used to monitor the dynamics of SMAD4, a TGF-beta downstream effector, in MDA-MB-231 breast cancer cells. Contrary to previous belief, SMAD4 was detectable at the cytoplasmic membrane, displaying two subpopulations with different membrane docking behaviors. These subpopulations were regulated by clathrin and caveolin-1, and had opposing roles in the nuclear shuttling of SMAD4 and the subsequent transcriptional regulation of genes associated with cell migration. The notion that membrane-docking behaviors of downstream molecules could predict the cellular response to growth factors may revolutionize the way we view cell signaling. Structured summary of protein interactions: SMAD4 and caveolin-1 colocalize by fluorescence microscopy (View interaction) Crown Copyright (C) 2013 Published by Elsevier B.V. on behalf of Federation of European Biochemical Society. All rights reserved.