Mechanics of vimentin intermediate filament networks
The cytoskeleton is made up of networks of proteins that strongly contribute to the stiffness of cells as well as being involved in cellular force generation. The protein networks are comprised of filaments that fall into 3 classes: microfilaments (actin), microtubules, and intermediate filaments (IF). Of these, IFs have received much less attention than actin and microtubules due to their lack of associated motors and proteins. However, recent work has shown that they may be more important than previously thought. I am studying the mechanics of vimentin, a Type III IF, using bulk and microrheological techniques as well as traction force microscopy on single cells.
Bulk rheology of reconstituted vimentin networks
Vimentin can be bacterially-expressed, purified, and then reassembled into in vitro networks. This gives us a highly-controllable model system with which we can study vimentin mechanics and assembly. Bulk rheology is also made possible with the large sample volumes that are possible with reconstituted networks.
Mechanics of vimentin “ghost” networks
Collaborators: Ming Guo, Mikkel Jensen, Robert Goldman (Northwestern University)
While reconstituted networks are great to study as a pure, model system, they lack modifications that a cell would typically make. In the case of vimentin, the primary modification is phosphorylation, which induces reorganization or depolymerization and also affects assembly kinetics. Overall cell stiffness has been shown to decrease in cells lacking vimentin but the actual mechanics of the networks in cells is difficult to isolate. We have a method to remove most of the cell, leaving behind an intact intermediate filament network that retains all post-assembly modifications (Fig. 1). Using this, we can investigate the local mechanics with microrheology and further bridge the gap between in vitro network and live cell studies.
Figure 1. Fluorescence image of a ghost network (green) with tracer beads (red) for microrheology.
Vimentin and cell mechanosensitivity
Collaborators: Bomi Gweon (Fredberg Lab), Jennifer Shin (KAIST), Robert Goldman (Northwestern University)
The cytoskeleton is known to play a large part in how cells sense and interact with their environments – for example, integrins connect actin to the ECM and transmit force to the substrate. Although vimentin may not be directly connected to this force generation, we believe that it does play a role in mechanosensitivity. We study this through traction force measurements on fibroblasts derived from WT and vimentin -/- mic