Understanding dynamic changes in live cell adhesion with neutron reflectometry

Neutron reflectometry (NR) was used to examine various live cells' adhesion to quartz substrates under different environmental conditions, including flow stress. To the best of our knowledge, these measurements represent the first successful visualization and quantization of the interface between live cells and a substrate with sub-nanometer resolution. In our first experiments, we examined live mouse fibroblast cells as opposed to past experiments using supported lipids, proteins, or peptide layers with no associated cells. We continued the NR studies of cell adhesion by investigating endothelial monolayers and glioblastoma cells under dynamic flow conditions. We demonstrated that neutron reflectometry is a powerful tool to study the strength of cellular layer adhesion in living tissues, which is a key factor in understanding the physiology of cell interactions and conditions leading to abnormal or disease circumstances. Continuative measurements, such as investigating changes in tumor cell — surface contact of various glioblastomas, could impact advancements in tumor treatments. In principle, this can help us to identify changes that correlate with tumor invasiveness. Pursuit of these studies can have significant medical impact on the understanding of complex biological problems and their effective treatment, e.g. for the development of targeted anti-invasive therapies

The F-actin side binding activity of the Arp2/3 complex is essential for actin nucleation and lamellipod extension

Most eukaryotic cells rely on localized actin polymerization to generate and sustain the protrusion activity necessary for cell movement 1, 2. Such protrusions are often in the form of a flat lamellipod with a leading edge composed of a dense network of actin filaments 3, 4. The Arp2/3 complex localizes within that network in vivo 3, 4 and nucleates actin polymerization and generates a branched network of actin filaments in vitro 5, 6, 7. The complex has thus been proposed to generate the actin network at the leading edge of crawling cells in vivo 3, 4, 8. However, the relative contributions of nucleation and branching to protrusive force are still unknown. We prepared antibodies to the p34 subunit of the Arp2/3 complex that selectively inhibit side binding of the complex to F-actin. We demonstrate that side binding is required for efficient nucleation and branching by the Arp2/3 complex in vitro. However, microinjection of these antibodies into cells specifically inhibits lamellipod extension without affecting the EGF-stimulated appearance of free barbed ends in situ. These results indicate that while the side binding activity of the Arp2/3 complex is required for nucleation in vitro and for protrusive force in vivo, it is not required for EGF-stimulated increases in free barbed ends in vivo. This suggests that the branching activity of the Arp2/3 complex is essential for lamellipod extension, while the generation of nucleation sites for actin polymerization is not sufficient