Design of nanostructured cell culture surfaces to explore the interplay between cells and nanotopgraphy

Abstract

In their environment, cells must cope with diverse biophysical stimuli. Among them, the nanotopography of the extracellular matrix plays a predominant role in regulating cell behavior. To mimic this native environment, we designed nanostructured cell culture surfaces using homemade fluorescent nanoparticles ranging from 100 nm to 500 nm. When cells are seeded on these nanostructured surfaces, their plasma membrane will be deformed, thereby influencing the underlying actin cytoskeleton organization. BAR domain proteins are attractive candidates since they can sense membrane deformations and induce downstream signaling. However, the recruitment and the dynamics of the different molecular actors are poorly understood. Since these events occur at an extremely small scale, they were observed in super-resolution using a STED microscope. First, we generated 18 genetic constructs to fuse a SNAP-tag or a Halo-tag to target BAR domain proteins, for their transient expression in cells. Second, we optimized conditions for three-color STED imaging. Then, we investigated various aspects of the recruitment of these proteins around membrane nanodeformations, such as recruitment along the z-axis in 3D, as well as their associated dynamics, using STED live-cell imaging. We demonstrated that certain BAR domain proteins are recruited at the base of nanoparticles (e.g. MIM), whereas others are recruited more uniformly. We also showed that CIP4 is punctually and dynamically recruitment to nanodeformations and involved in the polymerization of actin rings. Lastly, we studied the impact of overexpressing these proteins on actin polymerization around the nanodeformations. Among the target proteins, none appeared to significatively interfere with the formation of actin rings. However, the presence of these proteins seemed to correlate with the abundance of actin around nanodeformations, suggesting that it could be worth exploring further. Taken together, these results highlight the pivotal role of CIP4 in nanotopography induced actin polymerization, providing new insights into the dynamic relationship between cellular structures, actin dynamics, and nanodeformations.

Date of Award	22 Jan 2025
Original language	English
Awarding Institution	University of Namur
Supervisor	Henri François Renard (Jury) & Benjamin Ledoux (Jury)