Study of membrane curvature sensors/inducers (BAR domain proteins) on nanostructured substrates

Details

Supervisors

Morsomme, Pierre ; Dupont-Gillain, Christine C. ; Renard, Henri-François

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries

Abstract

Bin/Amphiphysin/Rvs (BAR) domain proteins are known for their membrane scaffolding capabilities, by acting as membrane curvature sensors and/or inducers. They have key roles in processes such as endocytosis, cell migration and actin regulation, in which they regulate membrane shapes. In particular, this ability to sense membrane deformations make them ideal candidates to translate these mechanical stimuli into biochemical responses. However, BAR proteins affinity for pre-existing curvatures is not well characterized in cells. This work aims to study this affinity in vivo by developing a tool that artificially induces plasma membrane deformations. The easiest way to produce such deformations is to grow cells on nanostructured substrates. However, nanostructures need to be well defined and of constant size and shape to precisely link BAR proteins affinity to specific membrane deformation geometries. To do so, nanostructured substrates were created by colloidal lithography, i.e. immobilisation of colloidal particles, as it is the most adapted technique to produce large and well-defined nanostructured areas in an efficient manner. The nanostructures were also fluorescently labelled to make them visible under confocal microscope. Moreover, the technique was adapted to create nanostructures directly into live microscopy dishes, showing its potential and versatility. Human HeLa cells were seeded on the nanostructured substrates to assess their ability to promote cell adhesion and to induce plasma membrane deformations. Cells adhered on 500 nm spherical nanostructures and their plasma membrane was deformed. Furthermore, the actin cytoskeleton was shown to encircle some nanoparticles. On the other hand, smaller nanostructures (100 nm) did not induce any visible cell response. Afterwards, the affinity of BAR domain proteins for membrane deformations, induced by 500 nm nanostructures, was studied. Five out of the 31 BAR proteins tested showed an increased affinity for curved compared to flat membranes. This work led to the creation of nanostructured substrates, featuring a homogeneous distribution of spherical particles, which are well suited to induce plasma membrane deformations and to further study subsequent cell responses. Moreover, the study of BAR domain proteins revealed that some of them have an affinity for curved membranes. This affinity could be related to their already known functions or to new ones. This work enlightens the mechanisms by which cells sense mechanical signals applied on their membranes. The developed substrates show great promises for further research on cell-material interactions, with perspectives for biomaterials science and tissue engineering.