Dataset Information

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 ? (TGF-?) from transmembrane receptors into the nucleus. Specifically, normal nuclear accumulation of Smad2/Smad4 heterocomplexes induced by TGF-?1 is involved in carcinogenesis. However, the relationship between nuclear accumulation and the nucleocytoplasmic transport kinetics of Smad proteins in the presence of TGF-?1 remains obscure. By combining a high-speed single-molecule tracking microscopy and Förster resonance energy transfer technique, we tracked the entire TGF-?1-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-?1-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-?1, 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-?1 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-?1-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.

SUBMITTER: Li Y

PROVIDER: S-EPMC5961462 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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

Li Yichen Y Luo Wangxi W Yang Weidong W

Biophysical journal 20180501 9

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 si ...[more]

PMID: 29742417

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Project description:Essentials Factor XIII is a heterotetramer with 2 catalytic A subunits and 2 non-catalytic B subunits. Structure of active and inactive factor XIII was studied with atomic force microscopy. Inactive factor XIII is made of an A2 globule and 2 flexible B subunits extending from it. Activated factor XIII separates into a B2 homodimer and 2 monomeric active A subunits. SUMMARY: Background Factor XIII (FXIII) is a precursor of the blood plasma transglutaminase (FXIIIa) that is generated by thrombin and Ca2+ and covalently crosslinks fibrin to strengthen blood clots. Inactive plasma FXIII is a heterotetramer with two catalytic A subunits and two non-catalytic B subunits. Inactive A subunits have been characterized crystallographically, whereas the atomic structure of the entire FXIII and B subunits is unknown and the oligomerization state of activated A subunits remains controversial. Objectives Our goal was to characterize the (sub)molecular structure of inactive FXIII and changes upon activation. Methods Plasma FXIII, non-activated or activated with thrombin and Ca2+ , was studied by single-molecule atomic force microscopy. Additionally, recombinant separate A and B subunits were visualized and compared with their conformations and dimensions in FXIII and FXIIIa. Results and Conclusions We showed that heterotetrameric FXIII forms a globule composed of two catalytic A subunits with two flexible strands comprising individual non-catalytic B subunits that protrude on one side of the globule. Each strand corresponds to seven to eight out of 10 tandem repeats building each B subunit, called sushi domains. The remainder were not seen, presumably because they were tightly bound to the globular A2 dimer. Some FXIII molecules had one or no visible strands, suggesting dissociation of the B subunits from the globular core. After activation of FXIII with thrombin and Ca2+ , B subunits dissociated and formed B2 homodimers, whereas the activated globular A subunits dissociated into monomers. These results characterize the molecular organization of FXIII and changes with activation.

Dataset Information

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

Publications

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

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