Nuclear Transport and Accumulation of Smad Proteins Studied by Single-Molecule Microscopy

Abstract

Nuclear translocation of stimulated Smad heterocomplexes is a critical step in the signal transduction of transforming growth factor β (TGF-β) from transmembrane receptors into the nucleus. Specifically, normal nuclear accumulation of Smad2/Smad4 heterocomplexes induced by TGF-β1 is involved in carcinogenesis. However, the relationship between nuclear accumulation and the nucleocytoplasmic transport kinetics of Smad proteins in the presence of TGF-β1 remains obscure. By combining a high-speed single-molecule tracking microscopy and Förster resonance energy transfer technique, we tracked the entire TGF-β1-induced process of Smad2/Smad4 heterocomplex formation, as well as their transport through nuclear pore complexes in live cells, with a high single-molecule localization precision of 2 ms and <20 nm. Our single-molecule Förster resonance energy transfer data have revealed that in TGF-β1-treated cells, Smad2/Smad4 heterocomplexes formed in the cytoplasm, imported through the nuclear pore complexes as entireties, and finally dissociated in the nucleus. Moreover, we found that basal-state Smad2 or Smad4 cannot accumulate in the nucleus without the presence of TGF-β1, mainly because both of them have an approximately twofold higher nuclear export efficiency compared to their nuclear import. Remarkably and reversely, heterocomplexes of Smad2/Smad4 induced by TGF-β1 can rapidly concentrate in the nucleus because of their almost fourfold higher nuclear import rate in comparison with their nuclear export rate. Thus, we believe that the determined TGF-β1-dependent transport configurations and efficiencies for the basal-state Smad or stimulated Smad heterocomplexes elucidate the basic molecular mechanism to understand their nuclear transport and accumulation.

Mad dephosphorylation at the nuclear pore is essential for asymmetric stem cell division

“…Consistently with a published study (38), we assume p pMad in >p pMad out . The pMad binding to the nuclear matrix and DNA is approximated by a first-order reversible reaction with the rate k on ([pMad] nucl,i − K[pMad] bound nucl,i ), where k on is the on-rate constant in s −1 and K is the dimensionless dissociation constant.…”

“…Similarly, the rate of dephosphorylation is k 2 [pMad] nucl,i , with constant k 2 being the product of a dephosphorylation rate constant and the phosphatase surface density, assumed to be uniform over a nuclear envelope and the same for both envelopes, and index i = 1,2 denoting the nuclei of GSC (i = 1) and preCB (i = 2). p (p)Mad and K. The accumulation of pMad in cell nuclei is due to unequal permeabilities of a nuclear envelope for pMad import/export (38,39) are the respective permeabilities of a nuclear envelope and index i denotes the nuclei of GSC (i = 1) and CB (i = 2).…”

The BMP Pathway in Blood Vessel and Lymphatic Vessel Biology

“…Most SMAD1/5 functions seem to be critical during blood vessel development; conversely, the highly similar BMP effector SMAD8 (gene SMAD9) seems to function especially during pulmonary vascular homeostasis in adult mice [48] and humans [29,49], illustrating non-redundant roles between SMAD1/5 and SMAD8 in the endothelium. Target genes of SMAD1/5 in endothelial cells are, amongst others, Smad6/7, Atoh8 encoding atonal basic helix-loop-helix transcription factor 8, Tmem100 encoding transmembrane protein 100, EGFL7 encoding epidermal growth factor-like domain 7 and Id-genes encoding inhibitors of differentiation proteins [9,24,[50][51][52][53][54][55], whereas SMAD8 induces miR-21 and miR-27a and suppresses vascular endothelial growth [29]. The non-SMAD BMP pathway is less well understood in the blood vasculature, but BMP2-induced p38-heat shock protein 27 (HSP27)-dependent cell migration promotes tip cell competence and migration [15].…”

Mad dephosphorylation at the nuclear envelope is essential for asymmetric stem cell division

“…The accumulation of pMad in cell nuclei is due to unequal permeabilities of a nuclear envelope for pMad import/export (Li et al, 2018;Schmierer et al, 2008 , where on k is the on-rate constant in s -1 and K is the dimensionless dissociation constant. While Mad is also partially bound in the nuclei, no accumulation of Mad is observed in the nuclei of non-activated cells, likely because the effect of binding was counterbalanced by inequality of permeabilities in favor of export (Li et al, 2018;Schmierer et al, 2008).…”

“…The accumulation of pMad in cell nuclei is due to unequal permeabilities of a nuclear envelope for pMad import/export (Li et al, 2018;Schmierer et al, 2008 , where on k is the on-rate constant in s -1 and K is the dimensionless dissociation constant. While Mad is also partially bound in the nuclei, no accumulation of Mad is observed in the nuclei of non-activated cells, likely because the effect of binding was counterbalanced by inequality of permeabilities in favor of export (Li et al, 2018;Schmierer et al, 2008). In our model, we thus ignore, for simplicity, the binding of Mad inside the nuclei and describe the net Mad influx density as Parameter values were constrained by the data of Figure 1C, 2D, S1B and S1C, and by the estimate that pMad constitutes approximately 12.5% of total Mad; this estimate was obtained by comparing total Mad nuclear/cytoplasmic ratio between pMad positive (GSCs) and negative cells (CBs).…”