The Amot/Patj/Syx signaling complex spatially controls RhoA GTPase activity in migrating endothelial cells.

Controlled regulation of Rho GTPase activity is an essential component mediating growth factor-stimulated migration. We have previously shown that angiomotin (Amot), a membrane-associated scaffold protein, plays a critical role during vascular patterning and endothelial migration during embryogenesis. However, the signaling pathways by which Amot controls directional migration are not known. Here we have used peptide pull-down and yeast 2-hybrid (Y2H) screening to identify proteins that interact with the C-terminal PDZ-binding motifs of Amot and its related proteins AmotL1 and 2. We report that Amot and its related proteins bind to the RhoA GTPase exchange factor (RhoGEF) protein Syx. We show that Amot forms a ternary complex together with Patj (or its paralogue Mupp1) and Syx. Using FRET analysis, we provide evidence that Amot controls targeting of RhoA activity to lamellipodia in vitro. We also report that, similar to Amot, morpholino knockdown of Syx in zebrafish results in inhibition of migration of intersegmental arteries. Taken together, our results indicate that the directional migration of capillaries in the embryo is governed by the Amot:Patj/Mupp1:Syx signaling that controls local GTPase activity.

Effect of thickness of flowable resins on marginal leakage in class II composite restorations.

Despite limited scientific evaluation, there is an increased use of low elastic modulus flowable resin composite (FRC) as a stress-relieving gingival increment in Class II restorations. This study compared marginal leakage in preparations with gingival margins in enamel or dentin/cementum (sub-CEJ and supra-CEJ) after FRC was used as a gingival increment to hybrid resin composite used alone. In addition, the extent of leakage around restorations with or without the use of FRC gingival increments when light curing the resin composites from occlusal direction only or buccal, lingual and occlusal directions was compared. Sixty extracted human molars were prepared with two identical Class II (MO and OD) preparations (30 were 1 mm sub-CEJ and 30 were 1 mm supra-CEJ) and randomly assigned to six groups. After etching, dentin-bonding agent was applied to all prepared tooth surfaces according to the manufacturer's specifications. One of three different thicknesses of FRC (0.5 mm, 1 mm or 2 mm) was placed on the gingival floor, cured and a hybrid resin composite was placed occlusally to complete the restoration. The control preparation on each tooth was restored in the same manner, except that a hybrid resin composite was used for both the gingival and occlusal increments. The restored teeth were thermocycled (300 cycles), then immersed in 50% silver nitrate prior to the hemi-section and measured for leakage under a light microscope. The data were evaluated using paired measures analysis of variance (ANOVA). Most of the occlusal margins showed no leakage, while almost every gingival margin demonstrated some silver nitrate penetration regardless of whether it was located sub or supra-CEJ, although significantly less leakage was found in restorations with supra-CEJ margins (p=0.0001). Among supra-CEJ restorations, there was a pronounced reduction in leakage as FRC thickness increased (p=0.0005). In the teeth restored with the gingival-margin located supra-CEJ, the 2 mm thickness FRC gingival increment showed significantly less leakage (p<0.01) compared with the 0.5 mm thickness of FRC gingival increment. The direction of the curing light did not affect the extent of leakage (p>0.05). The use of FRC material as a gingival increment sub-CEJ in posterior hybrid resin restorations produced no significant difference in leakage (p>0.05). The results of this study indicated that restorations located supra-CEJ (with gingival margins in enamel) with 2 mm thick FRC gingival increments demonstrated significantly less leakage than did those with 0.5 mm FRC. When the margin of the restoration was located sub-CEJ (in dentin/cementum), neither the thickness nor the presence of FRC as a gingival increment significantly influenced the marginal leakage.