Turn it down a notch

In the developing vertebrate embryo, segmentation initiates through the formation of repeated segments, or somites, on either side of the posterior neural tube along the anterior to posterior axis. The periodicity of somitogenesis is regulated by a molecular oscillator, the segmentation clock, driving cyclic gene expression in the unsegmented paraxial mesoderm, from which somites derive. Three signaling pathways underlie the molecular mechanism of the oscillator: Wnt, FGF, and Notch. In particular, Notch has been demonstrated to be an essential piece in the intricate somitogenesis regulation puzzle. Notch is required to synchronize oscillations between neighboring cells, and is moreover necessary for somite formation and clock gene oscillations. Following ligand activation, the Notch receptor is cleaved to liberate the active intracellular domain (NICD) and during somitogenesis NICD itself is produced and degraded in a cyclical manner, requiring tightly regulated, and coordinated turnover. It was recently shown that the pace of the segmentation clock is exquisitely sensitive to levels/stability of NICD. In this review, we focus on what is known about the mechanisms regulating NICD turnover, crucial to the activity of the pathway in all developmental contexts. To date, the regulation of NICD stability has been attributed to phosphorylation of the PEST domain which serves to recruit the SCF/Sel10/FBXW7 E3 ubiquitin ligase complex involved in NICD turnover. We will describe the pathophysiological relevance of NICD-FBXW7 interaction, whose defects have been linked to leukemia and a variety of solid cancers

Confusing signals: Recent progress in CTLA-4 biology

The mechanism of action of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) remains surprisingly unclear. Regulatory T (Treg) cells can use CTLA-4 to elicit suppression; however, CTLA-4 also operates in conventional T cells, reputedly by triggering inhibitory signals. Recently, interactions mediated via the CTLA-4 cytoplasmic domain have been shown to preferentially affect Treg cells, yet other evidence suggests that the extracellular domain of CTLA-4 is sufficient to elicit suppression. Here, we discuss these paradoxical findings in the context of CTLA-4-mediated ligand regulation. We propose that the function of CTLA-4 cytoplasmic domain is not to transmit inhibitory signals but to precisely control the turnover, cellular location, and membrane delivery of CTLA-4 to facilitate its central function: regulating the access of CD28 to their shared ligands

Optical tweezers studies on Notch: single-molecule interaction strength is independent of ligand endocytosis.

Notch signaling controls diverse cellular processes critical to development and disease. Cell surface ligands bind Notch on neighboring cells but require endocytosis to activate signaling. The role ligand endocytosis plays in Notch activation has not been established. Here we integrate optical tweezers with cell biological and biochemical methods to test the prevailing model that ligand endocytosis facilitates recycling to enhance ligand interactions with Notch necessary to trigger signaling. Specifically, single-molecule measurements indicate that interference of ligand endocytosis and/or recycling does not alter the force required to rupture bonds formed between cells expressing the Notch ligand Delta-like1 (Dll1) and laser-trapped Notch1 beads. Together, our analyses eliminate roles for ligand endocytosis and recycling in Dll1-Notch1 interactions and indicate that recycling indirectly affects signaling by regulating the accumulation of cell surface ligand. Importantly, our study demonstrates the utility of optical tweezers to test a role for ligand endocytosis in generating cell-mediated mechanical force

Notch ligand endocytosis generates mechanical pulling force dependent on dynamin, epsins, and actin.

Notch signaling induced by cell surface ligands is critical to development and maintenance of many eukaryotic organisms. Notch and its ligands are integral membrane proteins that facilitate direct cell-cell interactions to activate Notch proteolysis and release the intracellular domain that directs Notch-specific cellular responses. Genetic studies suggest that Notch ligands require endocytosis, ubiquitylation, and epsin endocytic adaptors to activate signaling, but the exact role of ligand endocytosis remains unresolved. Here we characterize a molecularly distinct mode of clathrin-mediated endocytosis requiring ligand ubiquitylation, epsins, and actin for ligand cells to activate signaling in Notch cells. Using a cell-bead optical tweezers system, we obtained evidence for cell-mediated mechanical force dependent on this distinct mode of ligand endocytosis. We propose that the mechanical pulling force produced by endocytosis of Notch-bound ligand drives conformational changes in Notch that permit activating proteolysis

Notch ligand endocytosis generates mechanical pulling force dependent on dynamin, epsins, and actin.

Notch signaling induced by cell surface ligands is critical to development and maintenance of many eukaryotic organisms. Notch and its ligands are integral membrane proteins that facilitate direct cell-cell interactions to activate Notch proteolysis and release the intracellular domain that directs Notch-specific cellular responses. Genetic studies suggest that Notch ligands require endocytosis, ubiquitylation, and epsin endocytic adaptors to activate signaling, but the exact role of ligand endocytosis remains unresolved. Here we characterize a molecularly distinct mode of clathrin-mediated endocytosis requiring ligand ubiquitylation, epsins, and actin for ligand cells to activate signaling in Notch cells. Using a cell-bead optical tweezers system, we obtained evidence for cell-mediated mechanical force dependent on this distinct mode of ligand endocytosis. We propose that the mechanical pulling force produced by endocytosis of Notch-bound ligand drives conformational changes in Notch that permit activating proteolysis

Optical tweezers studies on Notch: single-molecule interaction strength is independent of ligand endocytosis.

Notch signaling controls diverse cellular processes critical to development and disease. Cell surface ligands bind Notch on neighboring cells but require endocytosis to activate signaling. The role ligand endocytosis plays in Notch activation has not been established. Here we integrate optical tweezers with cell biological and biochemical methods to test the prevailing model that ligand endocytosis facilitates recycling to enhance ligand interactions with Notch necessary to trigger signaling. Specifically, single-molecule measurements indicate that interference of ligand endocytosis and/or recycling does not alter the force required to rupture bonds formed between cells expressing the Notch ligand Delta-like1 (Dll1) and laser-trapped Notch1 beads. Together, our analyses eliminate roles for ligand endocytosis and recycling in Dll1-Notch1 interactions and indicate that recycling indirectly affects signaling by regulating the accumulation of cell surface ligand. Importantly, our study demonstrates the utility of optical tweezers to test a role for ligand endocytosis in generating cell-mediated mechanical force