Structure and mechanics of strained membrane-bound F-actin/intermediate filament layers

• Applicants: Professorin Dr. Sarah Köster; Professorin Dr. Claudia Steinem
• Subject Area: Statistical Physics, Nonlinear Dynamics, Complex Systems, Soft and Fluid Matter, Biological Physics
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 430255655

Intermediate filaments (IFs) form, besides actin filaments and microtubules, one of the three cytoskeletal filament systems in eukaryotic cells and are believed to be pivotal determinants of cellular mechanics. The mechanical properties of IFs are complementary to those of actin filaments with much higher flexibility and an enormous extensibility up to at least 4.5 fold. Recently, it has been shown that apart from the well-known cytoplasm-spanning IF structures, a second, distinct structural entity of IFs exists in epithelia forming an "IF-Cortex" extending in parallel to the plasma membrane and interconnecting the desmosomes. In epithelia that line the intestine, this subplasmalemmal IF layer is closely positioned underneath the actin-rich terminal web. It was hypothesized that this circumferential IF-structure provides mechanical support to the plasma membrane and may play an important role in all cells that contain desmosomes or hemidesmosomes and that need mechanical enforcement of the plasma membrane. As yet, only very little information has been gathered about the IF organization at the plasma membrane as well as the influence of these protein structures on the mechanical properties of cells. Here we propose to combine in vitro experiments employing artificial solid supported and pore-spanning bilayers and purified proteins with studies on cell-membrane fragments and living cells. For the in vitro experiments, we will establish layered structures of lipid membranes and fluorescently-labeled actin and IF networks, i.e., vimentin and keratin 8/18, interconnected by linker molecules, and investigate their architecture by means of fluorescence microscopy. We will focus on the question, how IF-structures are influenced by coupling to an F-actin network and vice versa. We will further probe the mechanical properties of the protein networks by particle-based microrheology and atomic force microscopy. Most importantly, we will investigate how strain influences the organization of the membrane-associated layered F-actin/IF-structures by imaging the networks using high-resolution fluorescence microscopy and how their mechanical properties are altered. We will compare our results with those obtained on cell-membrane fragments lacking the cell interior including the nucleus, and on living cells. In particular, we will investigate cell lines with fluorescently tagged IFs and F-actin under strain with an emphasis on the mechanical properties in strained state. Our experiments will shed light on the question, whether the intriguing stress-strain behavior observed for individual IFs is of relevance for filament networks and living cells, and will improve our understanding of the role of IFs in cellular mechanics.

• DFG Programme: Research Grants