Clathrin-mediated Endocytosis with Cell Confinement and during Neutrophil Polarization

Abstract

Clathrin-mediated endocytosis (CME) is one of the major pathway through which cells internalize nutrients and membrane proteins. It occurs on the membrane via clathrin-coated pits (CCPs). In this thesis, we studied CCPs' behavior when cells are under spatial constraints. In the first two projects, the constraint was applied using micro-contact printing. CCPs presented differential phenotypes on different-sized but the same shaped cells. In particular, CCPs were smaller with larger spreading area. We further showed that this might be due to the higher cortical tension associated with large spreading area. Seeding cells on anisotropic fibronectin patterns, we were able to manipulate where and how long CCPs appear on the cell. Together, these results showed that CCPs' distribution and behavior are regulated by mechanical cues in a cell.

In the last project, HL-60 differentiated neutrophils were used as the experimental system. They undergo rapid polarization in the presence of N-formylmethionyl-leucyl-phenylalanine (fMLP), during which cells not only present anisotropic morphology but also have asymetric distribution of cellular structures and signaling molecules. We found that CCPs did not have as polarized distribution upon the stimulation of fMLP, but they revealed differential interation with formyl peptitde recepter, actin, and β-arrestin with and without fMLP. Disruption of CME blocked effective neutrophil polarization as well as major signaling pathways. The results suggest a regulatory role of CME in neutrophil polarization.