Biopolymer and cytoskeletal mechanics
Mikkel Herholdt Jensen
The cytoskeleton is an intracellular mechanical network that determines cell shape and mechanics, facilitates intracellular transport and cell motility, and allows cells to sense and respond to external forces. Composed of the biopolymers actin, microtubules, and intermediate filaments, the cytoskeleton undergoes constant dynamic remodeling and is tightly regulated by dozens of other cellular proteins and binding partners. This non‑equilibrium nature and rich phase space of the cytoskeleton make for a complex system with many layers of emergent behavior. However, this complex environment also makes determination of the underlying mechanical properties of these networks difficult.
I study the biopolymers actin and vimentin, and aim to determine their mechanical properties and biophysical roles both in vitro and in the cell. Network mechanics and intracellular mechanics are quantified using bulk rheology and optical tweezer techniques, as well as optical imaging. I'm also interested in the binding factors that regulate cytoskeletal dynamics and mechanics, such as actin-binding proteins that induce bundling or alter the filament properties. In vitro model systems are used to address the physical properties and functional mechanisms of individual polymers and proteins.
Actin-vimentin network mechanics
To dissect the properties and functions of individual proteins and biopolymers, we use reconstituted networks of the biopolymers actin and vimentin as an intermediate step between single polymer networks and whole cytoskeletal extracts. This model system allows us to better understand the contributions of each polymer to the overall network mechanics of the cytoskeleton, as well as the interplay between the polymers, without additional cellular components.
Figure 1. As a first step, composite networks are made using a biotin-neutravidin crosslinker, and its mechanical properties are studied using bulk rheology. Even in this simplified passive system, the composite mechanical properties are greater than the sum of its parts.
At first glance, we might expect the properties of a composite network to roughly equal the sum of the parts, i.e., the two species of biopolymers. However, our initial results using bulk rheology show that adding vimentin to an actin network can have opposing effects on the tensile strength of that network depending on the experimental conditions. This differential behavior could have implications for cell mechanics in regions where both polymers are abundant, and is an example of unexpected emergent behavior in biological systems with even a small number of parameters. We are currently developing an understanding of this effect in an artificially crosslinked system (see Figure 1), and are also investigating more complicated model systems using biological crosslinkers.
This work is a collaboration with Eliza Morris, and Robert Goldman at Northwestern U.
Active optical tweezer microrheology in biopolymer networks and cells
We use optical tweezer microrheology to quantify local intracellular mechanics. A focused steerable laser beam is used to manipulate injected or endocytosed beads in the cell interior. By applying a driven oscillation and monitoring the phase shift and amplitude of the bead motion, the viscoelastic response of the local intracellular environment can be quantified in terms of the storage and loss shear moduli. In contrast to techniques like atomic force microscopy, which probes the mechanics of the cell cortex from the exterior of the cell, intracellular microrheology allows us to probe the cellular interior of a living cell.
Figure 2. Example data trace of a bead undergoing a 2 Hz driven oscillation in the interior of a cell. The storage and loss moduli can be determined from the amplitude and phase of the oscillations. By measuring several beads across a range of frequencies, we found that the cytoplasm is predominantly elastic, even as cytoplasmic beads and organelles exhibit diffusive-like motion.
Recent work has elucidated how the cell interior, despite being a largely elastic environment, can allow intracellular non-directional transport of objects much larger than the cytoskeletal network mesh size, and we were able to characterize intracellular forces resulting from motor activity in a novel force spectrum microscopy assay.
We are also applying active microrheology to cells and vimentin cell extracts to understand the role of the intermediate filament vimentin in intracellular mechanics. By combining optical tweezer microrheology and intracellular vesicle tracking techniques, we find that vimentin greatly contributes to the intracellular elasticity and helps localize intracellular components.
Mechanisms of actin-binding proteins
Actin is a highly dynamic cytoskeletal polymer with dozens of binding partners regulating polymerization, depolymerization, severing, bundling, and crosslinking. We are interested in the regulation of actin mechanics and dynamics by its actin-binding partners, both in cells and in vitro. To better understand the function of individual actin binders, we use mechanical and imaging techniques on in vitro model systems.
Figure 3. Bulk rheology stress‑strain curves of crosslinked actin networks with and without calponin, normalized by yield stress and strain. Fully percolated actin networks undergo a linear regime before stiffening and eventually failing. Calponin lowers the linear bulk modulus, delays the onset of strain stiffening, and increases the yield strain of the networks. These bulk data agree with direct measurements the persistence length of single actin filaments in the presence of calponin.
One actin‑binding protein, calponin, is known to stabilize actin structures under shear in cells, but the physical mechanism is unclear. We use bulk rheology on crosslinked in vitro actin networks to elucidate the physical mechanism of calponin stabilization in cells, and compare these data to flexural rigidity measurements of individual filaments. We find that calponin alone is sufficient to stabilize a crosslinked actin network in vitro (Figure 3), which suggests that calponin stabilization of actin networks doesn't rely on other cellular factors. Our data can be well understood within current models of affine semiflexible polymer networks under strain, and point to a potential mechanism by which calponin stabilizes actin networks by making individual filaments more flexible, allowing the networks to reach a higher bulk strain before failing.
We are also studying mutations in the actin-binding protein alpha-actinin. Using in vitro mechanical assays, we aim to characterize changes in actin binding and resulting mechanics by mutations eventually leading to disease phenotypes in kidneys.