Optical Tweezers Studies on Notch: Single-Molecule Interaction Strength Is Independent of Ligand Endocytosis

• Published in: Developmental cell 2012-06, Vol.22 (6), p.1313-1320
• Main Authors: Shergill, Bhupinder, Meloty-Kapella, Laurence, Musse, Abdiwahab A., Weinmaster, Gerry, Botvinick, Elliot
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Cell differentiation, maturation, development, hematopoiesis; Cell Line; Cell physiology; Endocytosis; Fundamental and applied biological sciences. Psychology; Intercellular Signaling Peptides and Proteins - chemistry; Intercellular Signaling Peptides and Proteins - metabolism; Ligands; Mice; Molecular and cellular biology; Optical Tweezers; Protein Binding; Receptor, Notch1 - chemistry; Receptor, Notch1 - metabolism; Signal Transduction
• ISSN: 1534-5807; 1878-1551
• DOI: 10.1016/j.devcel.2012.04.007

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. [Display omitted] [Display omitted] ► Optical tweezers measure single-molecule Notch (N1)-ligand (Dll1) bond strength ► Blockade of dynamin activity does not alter the strength of Dll1-N1 interactions ► Rab11-mediated Dll1 recycling does not alter single-molecule Dll1-N1 bond strength ► Dll1 recycling influences signal intensity by regulating ligand accumulation Why is Notch receptor activity dependent on ligand endocytosis? Does recycling modify the ligand? Do endocytic forces pull on ligand-bound receptor? Shergill et al. and Meloty-Kapella et al. show that endocytosis mediates pulling force on Notch, not ligand binding to Notch. Thus, endocytic biomechanics appear to drive Notch activation.