Biophotonic tools for the studying of vascular mechanobiology

The Botvinick BEAMS lab develops and deploys biophotonic tools in the study of mechanobiology. In my seminar I will focus on two of our projects. The first uses laser tweezers in the study of Notch-ligand interactions, and the second uses photonic tools to measure mechanical properties at the scale of single molecules and single cells within engineered tissues. The Notch pathway is a signaling system critical to life that allows cells to directly communicate with and respond to their neighbors during the development of multi-cellular organisms. In angiogenesis it plays a role in tip cell - stalk cell maintenance. Notch signaling induced by ligand involves a series of proteolytic cleavage events that release the Notch intracellular domain (NICD) from the plasma membrane, allowing Notch to move to the nucleus where it directly induces a transcriptional response. In collaboration with the Weinmaster lab (UCLA) we have used laser tweezers to test and support the hypotheses that mechanical force produced during endocytosis of ligand-bound Notch physically dissociates Notch to release the Notch extracellular domain (NECD) from the intact receptor thus exposing proteolytic cleavage sites. Furthermore, we have tested and cast doubt on the standing 'recycling' hypothesis that suggests inactive ligand must be endocytosed and returned to the membrane to generate an avid notch ligand. Together, our evidence supports a mechanical role of endocytosis in the Notch signaling pathway. Our lab is also studying the role of local mechanics in the guidance and control of new vessels during angiogenesis. In recent studies it has been demonstrated that the growth rate and maintenance of capillaries in engineered fibrin gels are correlated to changes in extracellular matrix (ECM) density and bulk substrate mechanical properties. However, because changes in matrix density simultaneously alter the number of integrin binding sites available to endothelial and stromal cells, the roles, if any, of changes in local ECM stiffness and architecture are yet to be understood. In 3D culture systems, an increase in fibrin concentration not only modulates local stiffness, but directly decreases the mean pore size, increases cell confinement and decreases matrix permeability to diffusing signals. Here we have constructed and utilized a laser based microrheology system to both characterize the microstructure of pores within fibrin matrices at physiologically relevant concentrations and to investigate the mechanics of the interaction between growing capillaries and the ECM. This passive and active microrheological method complements and adds to video based particle tracking techniques by providing both viscous and elastic measurements of the microenvironment around cells.