Periodic Reporting for period 5 - MECHANICS (Mechanics of cells: the role of intermediate filaments)
Reporting period: 2023-05-01 to 2023-10-31
The mechanical properties of each of the more than 200 cell types in the human body are perfectly well adapted to their function. The wide variety of viscoelastic profiles, ranging from soft brain cells to stiff cartilage, and the temporal variability in the mechanical stress response as quiescent cells begin to migrate, e.g. during embryogenesis, wound healing, or cancer metastasis, are reflected in a surprisingly small number of molecular building blocks. Three distinct filament systems (actin filaments, microtubules, and intermediate filaments (IFs)) self-organize into a plethora of structural units, collectively referred to as the cytoskeleton. The major molecular players in this remarkable composite material are largely known. However, from a physical point of view, IFs, in particular, are less well understood, despite their importance in health and disease and astonishing mechanical properties, such as extreme extensibility and high flexibility. Moreover, it remains a challenge to characterize and quantify the interactions between the filaments, which play a major role within the composite intracellular network.
In this project our objective was to investigate how the remarkable mechanical properties of IFs are encoded in the molecular interactions of the protein monomers and how they are translated into the mechanical behavior of a whole cell. Thus, our research established a structure-mechanics-function relationship for this important component of the cytoskeleton. The genetic complexity of the IF protein family with 70 members that are expressed in a tissue-specific manner required a strategic approach with well-defined model systems and the combination of in vitro and cellular work. Direct mechanical testing by stress application was performed in a quantitative manner by optical tweezers.
Our work covers different length scales, from molecular interactions, which are investigated by numerical simulations, via single filament mechanics, interactions between filaments and within networks, to cellular mechanics.
In this project our objective was to investigate how the remarkable mechanical properties of IFs are encoded in the molecular interactions of the protein monomers and how they are translated into the mechanical behavior of a whole cell. Thus, our research established a structure-mechanics-function relationship for this important component of the cytoskeleton. The genetic complexity of the IF protein family with 70 members that are expressed in a tissue-specific manner required a strategic approach with well-defined model systems and the combination of in vitro and cellular work. Direct mechanical testing by stress application was performed in a quantitative manner by optical tweezers.
Our work covers different length scales, from molecular interactions, which are investigated by numerical simulations, via single filament mechanics, interactions between filaments and within networks, to cellular mechanics.
We have thorougly characterized vimentin and keratin intermediate filaments (IFs) concerning their force-strain behavior. Vimentin is the most abundant IF protein in humans and best characterized form an in vitro point of view. We were able to show that the filaments can be extended to at least 4 times their original length and the force-strain data show distinctly different regimes: an elastic extension at low strains is followed by a plateau-like region and eventually by strain stiffening. The extensibility is loading rate dependent, i.e. when pulled slowly, the filaments can be extended much further than when pulled fast (reminiscent of the behavior of a safety belt in the car). We further investigated the dissipative properties of the filaments and found pronounced hysteresis, tensile memory and softening upon repeated stretching. About 70-80% of the input energy is dissipated. By manipulating the charge interactions within the filaments (ionic strength and pH of the buffer, posttranslational modifications) the mechanical properties of the filament can be tuned very precisely.
We compared keratin and vimentin IFs concerning the mechanical properties and could - with the help of Monte Carlo simulations - reveal that differences in intra-filament coupling lead to intriguing differences between the two IF types. When stretched and relaxed in cycles, vimentin filaments become softer, but retains their length (like double-network gels). Keratin filaments, by contrast, become longer, but retains their stiffness (like metals).
We found that cytoskeletal filaments (vimentin-vimentin; vimentin-microtubules) interact directly with each other, due to electrostatic and hydrophobic interactions and that these forces are comparatively strong.
Our results have implications on biomedical research because the mechanical properties of cells play a major role in certain diseases, such as cancer, and in wound healing and embryogenesis. Furthermore the tunabilityof the properties is highly interesting for materials research and may serve a a "blueprint" for designing novel, sustainable matererials as functional as biomaterials.
We compared keratin and vimentin IFs concerning the mechanical properties and could - with the help of Monte Carlo simulations - reveal that differences in intra-filament coupling lead to intriguing differences between the two IF types. When stretched and relaxed in cycles, vimentin filaments become softer, but retains their length (like double-network gels). Keratin filaments, by contrast, become longer, but retains their stiffness (like metals).
We found that cytoskeletal filaments (vimentin-vimentin; vimentin-microtubules) interact directly with each other, due to electrostatic and hydrophobic interactions and that these forces are comparatively strong.
Our results have implications on biomedical research because the mechanical properties of cells play a major role in certain diseases, such as cancer, and in wound healing and embryogenesis. Furthermore the tunabilityof the properties is highly interesting for materials research and may serve a a "blueprint" for designing novel, sustainable matererials as functional as biomaterials.
The primary goal of this research program was to establish a convincing structure-mechanics-function relationship for IFs and thus provide the missing link between IF protein mechanics and cell mechanics. The large variety of IF proteins that are expressed in different cells of the human body on the one hand and the versatile viscoelastic properties of the cells on the other hand, lead to the hypothesis that the type (or combination) of IF protein(s) in a cell is, at least in parts, responsible for the mechanical properties of the cell. Furthermore, we assume that the mechanical properties are directly encoded in the hierarchical structure of the filaments. Both aspects are accessible using biophysical methods. The importance lies in at least three reasons:
1. A thorough understanding of the physical principles underlying the processes in a healthy cell is the necessary prerequisite for investigating situations in disease. Long alpha-helices are abundant in mechanically relevant proteins and our findings may be generalized in this respect.
2. Apart from the importance in biomedicine, I also expect this work to open up new opportunities for materials research. Apparently, the special hierarchical architecture of IFs leads to astonishing viscoelastic properties. We are now able to understand how these structural elements work individually and in concert (e.g. the high stretchability), mimicking the mechanical properties and development of equally remarkable materials might be possible.
3. As much as physics can help biology (see aspect 1), the other way around, highly complex biological systems, like IFs, provide a wealth of intriguing soft condensed matter physics problems, which can be studied on accessible time, length and force scales.
During the this project, we have thoroughly characterized the force-strain behavior of IFs, underlined with both analytical and numerical modeling. As a consequence, we suggested an alternative (three-state) model to the previous "alpha-to-beta transitions" within the protein monomers, which can now explain all experimental findings by others and us. We were furthermore able to explain the differences in mechanical behavior between vimentin and keratin IFs based on the amino acid sequences of the two proteins. We were able to measure the interactions between two vimentin IFs as well as between one vimentin IF and one microtubule and could show that the presence of vimentin stabilizes microtubules from rapid depolymerization (catastrophe). We also established methods to investigate cell mechanics, such as traction force microscopy, atomic force microscopy and cell stretching.
1. A thorough understanding of the physical principles underlying the processes in a healthy cell is the necessary prerequisite for investigating situations in disease. Long alpha-helices are abundant in mechanically relevant proteins and our findings may be generalized in this respect.
2. Apart from the importance in biomedicine, I also expect this work to open up new opportunities for materials research. Apparently, the special hierarchical architecture of IFs leads to astonishing viscoelastic properties. We are now able to understand how these structural elements work individually and in concert (e.g. the high stretchability), mimicking the mechanical properties and development of equally remarkable materials might be possible.
3. As much as physics can help biology (see aspect 1), the other way around, highly complex biological systems, like IFs, provide a wealth of intriguing soft condensed matter physics problems, which can be studied on accessible time, length and force scales.
During the this project, we have thoroughly characterized the force-strain behavior of IFs, underlined with both analytical and numerical modeling. As a consequence, we suggested an alternative (three-state) model to the previous "alpha-to-beta transitions" within the protein monomers, which can now explain all experimental findings by others and us. We were furthermore able to explain the differences in mechanical behavior between vimentin and keratin IFs based on the amino acid sequences of the two proteins. We were able to measure the interactions between two vimentin IFs as well as between one vimentin IF and one microtubule and could show that the presence of vimentin stabilizes microtubules from rapid depolymerization (catastrophe). We also established methods to investigate cell mechanics, such as traction force microscopy, atomic force microscopy and cell stretching.