can occur at the plasma membrane prior to detaching of endocytic vesicles; yet, it shows that full activation of Smad1/5/8 is reliant on functional membrane dynamics. The nuclear translocation kinetics of Smad1/5/8 is regulated by dynamin-dependent endocytosis After phosphorylation, Smad proteins translocate into the nucleus to function as transcriptional co-regulators [7]. To further analyze the consequences of the altered Smad1/5/8 phosphory- lation kinetics on the propagation of BMP signaling, we examined the effect of inhibition of dynamin-dependent endocytosis on Smad nuclear translocation. C2C12 cells were seeded on glass coverslips and treated with dynasore prior to stimulation with BMP-2 for 30 min. The subcellular localization of endogenous Smad1 was analyzed by fluorescence microscopy. BMP-2 induced strong nuclear accumulation of Smad1, which was significantly decreased in the presence of dynasore (Figure 2A,B). To confirm these results we performed cell fractionation studies. C2C12 cells were pre-treated with dynasore prior to stimulation with BMP-2 for the indicated time periods; they were then subjected to cytoplasmic-nuclear fractionation (Figure 2C). In control cells, phosphorylation of Smad1/5/8 was detectable in the cytosol and nucleus, with a strong peak in the nucleus after 10 min of BMP-2 administration (Figure 2C, lanes 5 and 6). Intense signals for nuclear pSmad1/5/8 were detected also after 60 min of BMP-2 stimulation, whereas the amount of pSmad1/5/8 in the cytosolic fraction decreased from 10 min to 60 min (Figure 2C, lanes 5, 6, 9, 10, 13, 14). As demonstrated in Figure 1C, dynasore delayed the initial phosphorylation kinetics of Smad1/5/8 (Figure 2C, lanes 5 and 7). Moreover, cells treated with dynasore and stimulated for 10 min with BMP-2 did not display phosphorylated Smad1/5/8 in the nuclear fraction (Figure 2C, lanes 6 and 8). Interestingly, phosphorylation of Smad1/5/8 in dynasore-treated cells was detectable in the nucleus after 30 min and 60 min of BMP-2 administration with similar levels like control cells, but did not display a strong initial peak as compared to control cells after 10 min of BMP-2 stimulation. In addition, the level of pSmad1/5/ 8 in the cytosolic fraction was lower relative to control cells (Figure 2C, lanes 9–16). These results indicate that inhibition of dynamin-dependent endocytosis affects Smad phosphorylation kinetics and concomitantly the dynamics of nuclear translocation. After phosphorylation and nuclear translocation, Smads bind to specific motifs in promoter regions, recruit additional transcription factors and regulate transcription of target genes [7]. As already shown by us and by others, transcriptional activity is dependent on clathrin-mediated endocytosis [11]. To further investigate the effect of dynasore treatment on BMP-2 signaling, we examined its consequences on activation of the reportergene construct BRE- Luc after long and short term stimulation with BMP-2 [15]. As expected, the transcriptional activity was inhibited by dynasore after 6 h and 24 h of BMP-2-stimulation (Figure 2D,E). BMP-2 induced gene expression is differentially affected by inhibition of dynamin-dependent endocytosis As shown above, phosphorylation of Smad1/5/8 and its nuclear translocation are retarded and diminished when endocytosis is blocked. The altered signaling kinetics resulted in an inhibitory effect of dynasore on BMP-2 induced BRE-Luc activity (Figure 2D,E). These results suggest that the accurate timing and signal intensity of the initial steps in BMP signaling is important for the transcriptional activation of target genes and that the initial steps are reliant on endocytosis. To further investigate the dependence of BMP-2 induced transcriptional activity on Spatial Segregation of BMP/Smad Signaling dynamin-dependent endocytosis, we performed a whole genome gene expression profiling using the Illumina BeadChip system. Serum-starved C2C12 cells were pre-treated for 2 h with dynasore, followed by BMP-2 stimulation for 6 h. Isolated RNA was subjected to microarray analysis. Expression profiles were normalized and genes that were significantly detected in all the treatments and either up- or down-regulated by 1.4 fold relative to DMSO control treatment were considered for further analysis (detection P-value,0.05). This list consists of 2214 genes that were commonly or exclusively regulated. Distinct and overlapping gene regulations are depicted in a Venn diagram (Figure 3A; the entire gene list is depicted in Table S2). We identified genes that were exclusively regulated by BMP-2 in medium supplemented with vehicle (DMSO) only, (BMP- 2+DMSO; 254 genes), by BMP-2 in the presence of dynasore (BMP-2+Dynasore; 30 genes) or by dynasore alone (Dynasore) (39 genes). We also identified genes that were commonly regulated by BMP-2+DMSO or Dynasore (93 genes), by BMP-2+DMSO or by Dynasore (71 genes) or by BMP-2+Dynasore or by Dynasore (610 genes). 1117 genes showed a common regulation in all three treatments. In total, 925 genes were up-regulated by BMP-2+DMSO. These gene expression profiles were analyzed and those genes that showed nonspecific regulation by dynasore were not considered for further evaluation. Interestingly, we discovered genes that exhibited different expression patterns when the treatment BMP- 2+DMSO was compared to the treatment BMP-2+Dynasore. In this way we identified two classes of genes that were differentially affected by inhibition of dynamin-dependent endocytosis (Figure 3B,C). The first class included 483 genes that showed reduced BMP-2-induced gene expression following treatment with dynasore (BMP-2+Dynasore) as compared to BMP-2+DMSO (Figure 3B; the complete gene list is depicted in Table S3). We defined this class of genes as endocytosis-dependent, since their expression levels were reduced following treatment with dynasore. Among those, we found known BMP-2 target genes like Id1, Id3, Dlx2 and Hey1 (Figure 3D). The second class included 20 genes whose expression levels were affected similarly following treatment with BMP-2+DMSO and in the presence of dynasore (BMP- 2+Dynasore) (Figure 3C; the complete gene list is given in Table S4). We defined this class of genes as endocytosis-independent, since dynasore treatment did not affect their BMP-2 induced expression. Interestingly, this group also included known BMP-2 target genes such as Id2 and Dlx3, as well as the genes Krt16 and Zbtb2 (Figure 3D). Confirmation of BMP-2 induced genes that are differentially affected by dynamin-dependent endocytosis In the microarray analysis we identified genes that are differentially affected by inhibition of dynamin-dependent endocytosis. To validate these findings, we performed quantita- tive real-time PCR (qPCR) analysis with independent samples. Cells were pre-treated with dynasore for 2 h and were subsequently stimulated with BMP-2 for 6 h. These experiments confirmed the existence of two classes of genes, whose expression in response to BMP-2 is either dependent or independent of endocytosis (Figure 4A,B). Expression of Id1, Id3, Dlx2 and Hey1 was endocytosis-dependent, as shown by the dynasore-mediated inhibition of their BMP-2 induced expression (Figure 4A). On the other hand, expression of Id2, Dlx3, Krt16 and Zbtb2 was endocytosis-independent (i.e., no effect of dynasore) (Figure 4B). Id1, Id2 and Id3 (inhibitor of differentiation), which belong to the group of helix-loop-helix (HLH) transcription factors, are direct PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e25163