UCLA

The concept that form adapts to function has long been at the core of biological science. However, there are many cellular forms that we do not fully understand. Microridges are actin-based cell membrane protrusions that wind into labyrinthine patterns on the apical surfaces of mucosal epithelial cells, conferring a unique cellular morphology. Though the form of microridges has been studied in fixed tissues for many years, their morphogenesis is not understood. To investigate the formation of these enigmatic structures, I observed the process directly on the epidermis of developing zebrafish larvae using live fluorescence microscopy. In Chapter 2 of this dissertation I describe the mechanism of microridge formation and the role of contractile force in this process. Prior to microridge formation, epithelial cells are covered in “pegs”, short finger-like protrusions that resemble microvilli. Pegs are dynamic, appearing and disappearing within a matter of minutes, as well as fusing with one another to form short microridges. As epithelial cells constrict, short microridges and pegs gradually assemble into elongated microridges in a pattern that begins near cell borders and progresses toward the center of the cell surface. A biophysical model of this process demonstrated that contraction of the apical actomyosin cortex in a similar concentric pattern leads to cellular constriction and microridge formation by reducing membrane tension. Observation of myosin activity in live epithelial cells demonstrated that contraction progresses in a concentric manner and small molecule inhibition of contraction blocked both cellular constriction and microridge formation. Reduction of membrane tension through hyperosmolar shock and reorganization of the apical cortex through rapid cell elongation demonstrated that microridge formation is directly regulated by membrane tension and the organization of the cortical cytoskeleton. Chapter 3 describes the mechanics of microridge formation and pattern maturation with high spatial resolution. After formation, microridges elongate further, align with their neighbors, and become more regularly spaced. Microridge fusion and fission continue during this period of pattern maturation and these events correlate spatially and temporally with cortical myosin contraction. However, microridge rearrangements diminish after microridge formation and negatively correlate with the degree of microridge alignment. Inhibiting myosin contraction prevented microridge rearrangements and disrupted the microridge pattern. High-resolution Airyscan microscopy revealed individual myosin minifilaments in the cortex that connect microridges to one another and mediate microridge rearrangements through contraction. Overall, this work elucidates the mechanism of microridge formation. Pegs and microridges fuse, fission, and gradually rearrange, a process driven by the web-like network of myosin minifilaments in the apical cortex which contracts to reduce membrane tension. As myosin contraction diminishes, the input needed to overcome the energy barrier associated with the changes in membrane curvature during microridge rearrangement is decreased, and microridges slowly settle into their highly organized patterns.