The cell-cycle transition from G1 to S phase has been difficult to visualize. We have harnessed antiphase oscillating proteins that mark cell-cycle transitions in order to develop genetically encoded fluorescent probes for this purpose. These probes effectively label individual G1 phase nuclei red and those in S/G2/M phases green. We were able to generate cultured cells and transgenic mice constitutively expressing the cell-cycle probes, in which every cell nucleus exhibits either red or green fluorescence. We performed time-lapse imaging to explore the spatiotemporal patterns of cell-cycle dynamics during the epithelial-mesenchymal transition of cultured cells, the migration and differentiation of neural progenitors in brain slices, and the development of tumors across blood vessels in live mice. These mice and cell lines will serve as model systems permitting unprecedented spatial and temporal resolution to help us better understand how the cell cycle is coordinated with various biological events.

Smad family members are newly identified essential intracellular signalling components of the transforming growth factor‐β (TGF‐β) superfamily. Smad2 and Smad3 are structurally highly similar and mediate TGF‐β signals. Smad4 is distantly related to Smads 2 and 3, and forms a heteromeric complex with Smad2 after TGF‐β or activin stimulation. Here we show that Smad2 and Smad3 interacted with the kinase‐deficient TGF‐β type I receptor (TβR)‐I after it was phosphorylated by TβR‐II kinase. TGF‐β1 induced phosphorylation of Smad2 and Smad3 in Mv1Lu mink lung epithelial cells. Smad4 was found to be constitutively phosphorylated in Mv1Lu cells, the phosphorylation level remaining unchanged upon TGF‐β1 stimulation. Similar results were obtained using HSC4 cells, which are also growth‐inhibited by TGF‐β. Smads 2 and 3 interacted with Smad4 after TβR activation in transfected COS cells. In addition, we observed TβR‐activation‐dependent interaction between Smad2 and Smad3. Smads 2, 3 and 4 accumulated in the nucleus upon TGF‐β1 treatment in Mv1Lu cells, and showed a synergistic effect in a transcriptional reporter assay using the TGF‐β‐inducible plasminogen activator inhibitor‐1 promoter. Dominant‐negative Smad3 inhibited the transcriptional synergistic response by Smad2 and Smad4. These data suggest that TGF‐β induces heteromeric complexes of Smads 2, 3 and 4, and their concomitant translocation to the nucleus, which is required for efficient TGF‐β signal transduction.

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Smad7 is an inhibitory Smad that acts as a negative regulator of signaling by the transforming growth factor-␤ (TGF-␤) superfamily proteins. Smad7 is induced by TGF-␤, stably interacts with activated TGF-␤ type I receptor (T␤R-I), and interferes with the phosphorylation of receptor-regulated Smads. Here we show that Smurf1, an E3 ubiquitin ligase for bone morphogenetic proteinspecific Smads, also interacts with Smad7 and induces Smad7 ubiquitination and translocation into the cytoplasm. In addition, Smurf1 associates with T␤R-I via Smad7, with subsequent enhancement of turnover of T␤R-I and Smad7. These results thus reveal a novel function of Smad7, i.e. induction of degradation of T␤R-I through recruitment of an E3 ligase to the receptor.

SMAD proteins have been identified as signalling mediators of the TGF-beta superfamily, which is involved in a range of biological activities including cell growth, morphogenesis, development and immune responses. Smad1, Smad2, Smad3 and Smad5 are ligand-specific: Smadl and Smad5 transduce signals from bone morphogenetic proteins, and Smad2 and Smad3 mediate signalling by TGF-beta and activin, whereas Smad4 acts as a common signalling component. For example, Smad2 is phosphorylated by the TGF-beta type I receptor upon ligand binding, forms a heteromer with Smad4, and then translocates into the nucleus where it activates transcription. Here we report the isolation of Smad6 in the mouse. Smad6 is quite different in structure from the other SMAD proteins, and forms stable associations with type I receptors. Smad6 interferes with the phosphorylation of Smad2 and the subsequent heteromerization with Smad4, but does not inhibit the activity of Smad3. Smad6 also inhibits the phosphorylation of Smad1 that is induced by the bone morphogenetic protein type IB receptor. These data indicate that signals of the TGF-beta superfamily are regulated both positively and negatively by members of the SMAD family.

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Bone morphogenetic proteins (BMPs) are members of the transforming growth factor ,B superfamily. Bone morphogenetic proteins (BMPs) are a family of proteins that induce bone formation at extraskeletal sites in vivo (reviewed in refs. 1-3). BMPs act on osteoblasts and chondrocytes (4) as well as other cell types, including neural cells (5, 6), and they play important roles in the embryonal development (3). More than a dozen proteins belong to the BMP family, including BMP-2 to -6, osteogenic protein (OP)-1 (also termed BMP-7), OP-2 (BMP-8), and growth/differentiation factors 5 to 7.BMPs belong to the transforming growth factor ,B (TGF-f3) superfamily, which includes TGF-,3s, activins/inhibins, Miillerian inhibiting substance (MIS), and glial cell line-derived neurotrophic factor (GDNF) (7). TGF-13s and activins transduce their signals through the formation of heteromeric complexes of two different types of serine(threonine) kinase