The interplay between membrane and protein biomechanics and its effect on the formation of macromolecular structures
Das Zusammenspiel von Membran- und Proteinbiomechanik und seine Auswirkung auf die Bildung makromolekularer Strukturen
Creators: name: Stumpf, Bernd Henning
Publisher: name: Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU)
Year: 2023
Subjects: nascent adhesions
focal adhesions
protein-membrane interactions
phase separation

description: The organization of proteins in the cell membrane is crucial for many cellular functions. One such example is the organization of adhesion proteins in the cell membrane during the cell recognition process, when a cell binds to other cells, or when it binds to the extracellular matrix. Upon binding, these proteins typically cluster, thereby stabilizing the adhesion site. This allows for significant resistance to force and quick rebinding when one of the bonds in the agglomerate fails. However, the underlying processes controlling protein organization are still subject to debate. In this thesis, the focus lies on one possible mechanism, namely the coupling between proteins and the membrane itself, which has been implicated to be an important contributor to membrane-protein clustering, for example in nascent adhesions. Nascent adhesions are early cell attachments to the extracellular matrix which eventually mature into focal adhesions used by the cell for mechanosensing. They revolve around the integrin family of cell adhesion proteins and involve a palette of other ligands, proteins and signaling molecules, building a macromolecular assembly. Several studies suggested that integrins may interact with each other through the membrane (see publication P1), which helps the assembling process. Specifically, integrin binding can affect the membrane shape, which can influence the positioning of other integrins. Furthermore, the membrane experiences significant thermal and active stochastic fluctuations. These can bring adhesion proteins into the interaction range of its binding partners, on another cell or in the extracellular matrix, and hence promote the formation of bonds. Binding, nonetheless, locally restricts the membrane shape and its fluctuations, which in turn changes the probabilities of forming further adhesive contacts and induces effective interactions with other adhesion proteins. The potential acting between two proteins deforming the membrane was explicitly calculated using a Green’s function approach (see publication P2), a result published in Biophysical Journal as a part of a broader study on the effect of a bond on the membrane. I found that the correlation length for this interaction between two inclusions has a non-monotonic behavior as a function of the membrane tension. Due to their physical origin, membrane-mediated interactions are not unique to integrins. They will be present whenever proteins deform the membrane shape. It was demonstrated previously that forming adhesive bonds and introducing a protein with hydrophobic mismatch has the same effect, from a mathematical point of view. Hence interactions between proteins will also occur due to deformations of the membrane thickness profile. Furthermore, membrane-mediate interactions are not restricted to proteins of the same type but should also exist between proteins that have different biomechanical properties such as stiffness and size. Actually, this is a very common situation in the cell which hosts a plethora of different protein types. To explore the relation between the protein biomechanics and the membrane mediated adhesion-protein interactions, I generalized the calculations and showed, that if the two proteins deform the membrane similarly, the interaction is predominantly attractive (see publication P3). This will lead to the aggregation of proteins either due to the hydrophobic mismatch or due to the deformation upon forming an adhesive bond. If two types of proteins deforming the membrane are present, they could either mix or segregate into domains. I was able to characterize the onset of segregation by constructing an order parameter using virial coefficients associated with effective interaction potentials of proteins of same and different type. I was able to show that for fixed protein densities and affinities, it is the contrast in attractiveness (imposed for example by different protein lengths) that drives segregation, including the number and the characteristic size of proteins within the domains. I also showed that this contrast has to be significant for the separation to take place - it may not be sufficient that the size between two proteins differs even 10 nm, as was typically assumed in the field. This prediction could be verified in experiments (see publication P4). Collaborators constructed protein-mimics using DNA constructs, that despite significant length differences did not segregate up to length-scales typical for the optical resolution of the microscope. This unambiguously proved that the formation of domains in the membrane is not defined by the biomechanic properties of the proteins alone, but by the coupling of the protein and membrane biomechanics. The final part of this thesis is concerned with the fact that the membrane composition is not simple. Typical cell membranes are complex mixtures of different lipid types, each having a characteristic size of their hydrophobic core and elastic properties. Together with collaborators, I proposed that proteins, either forming adhesive bonds or presenting a hydrophobic mismatch, couple to the composition of the membrane and trigger adsorption of lipids which alleviates the elastic energy stored in the deformation. This idea was cast in an analytic model coupling the composition and the deformation field. The consequence is the formation of lipid domains around proteins which may further drive aggregation, even though the system is above the critical point, a result that could be verified through a collaborative work with experimental partners (see publication P5). This ensemble of work significantly extends the theoretical foundation for understanding the protein organization in the complex environment of a biological membrane and points to the importance of lipid-protein coupling in establishing protein aggregates. Importantly, this framework could be verified systematically with experiments performed in model systems, where the physical effects can be clearly delineated from the biochemical signaling. Additional effort, however, should be invested in the future to demonstrate the importance of these mechanisms in the environment of a living cell.