Unveiling Functional Roles of Apical Surface Interactions Between Opposing Cell Layers

PROJECT SUMMARY Apical surface interactions (ASIs) arising between cells in opposing three dimensional architectures are relatively common in tissue structures, including vessels and tubes in a variety of different organs and stages, but little is known about the function or mechanisms of these interactions. The goal of this proposal is to illuminate the role of ASIs in tissue architecture and responses as an under-explored dimension of cell-cell interactions. Based on our data and the literature, we hypothesize that close-range ASIs (< 1 µm) are governed by electrostatic charge interactions between membrane glycoproteins, while long-range ASIs (1-20 µm) function through primary cilia which extend up from the cell surface to organize signaling pathways. To develop accurate methodologies to measure and characterize the forces arising between whole sheets of cells with geometrical separation, we have designed a novel method called Bilayer Intermolecular Force Microscopy (BIFM) to induce and measure ASIs between two opposite surfaces. BIFM will be applied to measure the force generated between two cell layers as they approach each other from opposite sides. For close-range ASIs, we predict that chemicals affecting electrostatic charge interactions will modulate force-response. Cell sheets with knockout mutations in PODXL, encoding an apical sialomucin (podocalyxin) with proposed anti-adhesive properties, will exhibit lower resistance force in proportion to reduced electrostatic charge repulsion. Antibodies targeting podocalyxin, in therapeutic development, will also be assessed. For long-range ASIs, we will determine the role of primary cilia, antenna-like organelles with sensory and signaling functions. Using our BIFM device, we will induce ciliary ASIs and assess their effects on signaling. As a negative control, we will employ cell lines that we have genetically engineered to ablate primary cilia (KIF3A-/- or KIF3B-/-). We will furthermore modify our device to enable microfluidic flow to perfuse between two sheets of cells within the BIFM at adjustable speed, to assess flow start/stop in a physiological context, and monitored for changes in signaling activity. These studies will reveal how cilia serve as ASI sensors. To validate findings in vivo, we will analyze physiological tissue structures exhibiting a range of apical surface interactions, focusing on arborized networks such as ductal trees and blood vessel plexi. Expression of podocalyxin or cilia will be correlated with geometric properties and differential gene expression patterns. In summary, our project will provide novel conceptual and technical advances for understanding ASIs as a novel dimension in tissue architecture and physiology. Cross-cutting impact includes (1) revealing functional roles for both close-range and long-range ASIs; (2) establishing a novel biophysical device to measure interactions between cell sheets; and (3) testing mechanisms of cell adhesion and signaling. Our prior experience in modeling and developing biophysical tools positions us well to succeed. Collectively these activities will establish an innovative new area for future investigation, with fundamental importance.