Stress granules (SGs) are cytoplasmic aggregates of stalled translational preinitiation complexes that accumulate during stress. GW bodies/processing bodies (PBs) are distinct cytoplasmic sites of mRNA degradation. In this study, we show that SGs and PBs are spatially, compositionally, and functionally linked. SGs and PBs are induced by stress, but SG assembly requires eIF2α phosphorylation, whereas PB assembly does not. They are also dispersed by inhibitors of translational elongation and share several protein components, including Fas-activated serine/threonine phosphoprotein, XRN1, eIF4E, and tristetraprolin (TTP). In contrast, eIF3, G3BP, eIF4G, and PABP-1 are restricted to SGs, whereas DCP1a and 2 are confined to PBs. SGs and PBs also can harbor the same species of mRNA and physically associate with one another in vivo, an interaction that is promoted by the related mRNA decay factors TTP and BRF1. We propose that mRNA released from disassembled polysomes is sorted and remodeled at SGs, from which selected transcripts are delivered to PBs for degradation.

Mammalian stress granules (SGs) harbor untranslated mRNAs that accumulate in cells exposed to environmental stress. Drugs that stabilize polysomes (emetine) inhibit the assembly of SGs, whereas drugs that destabilize polysomes (puromycin) promote the assembly of SGs. Moreover, emetine dissolves preformed SGs as it promotes the assembly of polysomes, suggesting that these mRNP species (i.e., SGs and polysomes) exist in equilibrium. We used green flourescent protein–tagged SG-associated RNA-binding proteins (specifically, TIA-1 and poly[A] binding protein [PABP-I]) to monitor SG assembly, disassembly, and turnover in live cells. Fluorescence recovery after photobleaching shows that both TIA-1 and PABP-I rapidly and continuously shuttle in and out of SGs, indicating that the assembly of SGs is a highly dynamic process. This unexpected result leads us to propose that mammalian SGs are sites at which untranslated mRNAs are sorted and processed for either reinitiation, degradation, or packaging into stable nonpolysomal mRNP complexes. A truncation mutant of TIA-1 (TIA-1ΔRRM), which acts as a transdominant inhibitor of SG assembly, promotes the expression of cotransfected reporter genes in COS transfectants, suggesting that this process of mRNA triage might, directly or indirectly, influence protein expression.

Vascular endothelial-cadherin (VE-cad) is localized to adherens junctions at endo-thelial cell borders and forms a complex with- ,- ,-, and p120-catenins (p120). We previously showed that the VE-cad complex disassociates to form short-lived "gaps" during leukocyte transendo-thelial migration (TEM); however, whether these gaps are required for leukocyte TEM is not clear. Recently p120 has been shown to control VE-cad surface expression through endocytosis. We hypothesized that p120 regulates VE-cad surface expression, which would in turn have functional consequences for leukocyte transmigration. Here we show that endo-thelial cells transduced with an adenovi-rus expressing p120GFP fusion protein significantly increase VE-cad expression. Moreover, endothelial junctions with high p120GFP expression largely prevent VE-cad gap formation and neutrophil leuko-cyte TEM; if TEM occurs, the length of time required is prolonged. We find no evidence that VE-cad endocytosis plays a role in VE-cad gap formation and instead show that this process is regulated by changes in VE-cad phosphorylation. In fact, a nonphosphorylatable VE-cad mutant prevented TEM. In summary, our studies provide compelling evidence that VE-cad gap formation is required for leukocyte transmigration and identify p120 as a critical intracellular mediator of this process through its regulation of VE-cad expression at junctions. (Blood. 2008;112:2770-2779) Introduction Vascular endothelial-cadherin (VE-cad) is a transmembrane protein expressed in the vascular endothelium 1 that participates in endothelial barrier function, angiogenesis, signaling, and endothelial cell survival (reviewed in Dejana et al 2). Surface-expressed VE-cad localizes to cell-cell junctions and associates with-catenin,-catenin, plakoglobin (-catenin), and p120-catenin (p120) through its cytoplasmic tail, and with the actin cytoskeleton 3,4 in combination with vinculin and-actinin, which is thought to be critical for VE-cad adhesive interactions (reviewed in Vestweber 5). p120 is a substrate for Src family kinases and other receptor tyrosine kinases 6,7 and regulates cadherin-dependent adhesion positively and negatively, depending on the cell system under study (reviewed in Alemà and Salvatore 8). p120 associates with the juxtamembrane cytoplasmic region of VE-cad, 9 and this is crucial to maintain cadherin surface expression. 10 Overexpres-sion of VE-cad mutants that competed for p120 binding, or siRNA knockdown of p120 in endothelium, resulted in dramatically decreased surface-expressed VE-cad and concomitant increased VE-cad degradation by an endocytic pathway. In contrast, overexpression of wild-type p120 augments VE-cad surface expression and diminishes its endocytosis. 11 The precise mechanism(s) by which p120 controls the turnover and endocy-tosis of junctional VE-cad is not completely understood, 8,11 but it is conclusive that cytosolic levels of p120 regulate VE-cad surface expression in endothelial cells, and the level of E-cadherin in epithelial cells. 12 The idea that VE-ca...