The mechanical properties of cells fundamentally underlie all cellular behavior;the cell must support forces, must exert forces and must respond to forces (1-5). Moreover, while the genetic response within the cell ultimately provides the control mechanism, it is the mechanical response of the cell that dictates its primary function within a larger organism;without being able to withstand the forces of its environment, the cell would not be able to function at all. The mechanical properties of a cell are determined to a large extent by the three filamentous networks within the cytoskeleton, actin, microtubules and intermediate filaments (IF) (1). While actin networks and microtubules have been rather well studied, this is not the case for IF networks, whose study has significantly lagged that of the others (6, 7-9). Indeed, it has been proposed that networks of IF are essential in determining the mechanical properties of and mechanotransduction in virtually all vertebrate cells. However, there is little direct evidence supporting this proposed IF function. The IF networks within a cell are thought to be able to withstand very large strain;this can often be well in excess of 100% (6, 7, 10) In addition, the IF networks exhibit pronounced strain stiffening, effectively becoming much stiffer as they are stretched (6). However, the intracellular environment is highly heterogeneous and complex, making determination of the underlying mechanical properties of these networks extremely difficult. The IFs are remarkably dynamic, and are constantly being remodeled and reassembled, presumably driven in some fashion by the motors that run along either microtubules or actin filaments in the cell and these must guide the assembly of the VIF. There are also, presumably, associated proteins which regulate and control the VIF properties, and which provide crosslinking of the network to the surrounding networks within the cell, and within the VIF network itself (11-16). However, the complexity and richness of the behavior of the VIF within the cell, while controlling much of the function, also makes elucidating the fundamental properties much more difficult;moreover, it precludes measurement of the mechanical properties in a fashion that would allow determination of the underlying design principles of the network. The overarching goal of this section of the Program Project is therefore to measure the properties of VIF in a more controlled environment, thereby enabling us to elucidate their roles in establishing and regulating the mechanical properties of cells (17). The work proposed here will begin with a detailed study of the properties of networks of vimentin intermediate filament (VIF), which can be expressed in bacteria to enable us to produce sufficient quantifies to reconstitute the protein into networks and to make detailed measurements of the mechanical properties of these networks. These measurements will be performed using traditional bulk rheology (18). In addition, we will develop several new assays based on multi-particle tracking, measurements of the motion of small tracer particles embedded within the network and subject either to thermal agitation or to externally applied forces controlled by a magnetic field. The motion of these tracer particles will be interpreted using the formalism of microrheology to measure the elastic and viscous properties of the network. We will investigate the role of physiological concentrations of multivalent cations in regulating the network (6). In addition, we will work with the Goldman lab to investigate the role of phosphorylation in regulating VIF network elasticity (19, 20). We will also obtain constructs for vimentin mutants from our collaborator Harald Herrmann, and will use these to express the mutants in bacteria (21-23). This will enable us to elucidate fundamental design principles for the elasticity of these VIF networks. To complement these investigations of reconstituted networks, we will also form 'ghosts', where most of the cell proteins are washed away with detergent, leaving nearly the full IF network intact (24). By seeding these networks with probe particles, we will measure their elastic properties and compare to those of the reconstituted networks. This will provide a direct probe of the contribution of these VIF networks to cell elasticity. Importantly, these will also enable us to directly measure the response of the networks to shear;cells will be sheared prior to preparing the ghosts, allowing us to probe modifications in the structure and mechanics of the VIF networks due to the shear. We will, in addition, extend these particle tracking measurements to living cells: We will inject the cells with tracer particles and measure the motion of these particles due to both the internal molecular motors within the cell and to external forces, applied either with a magnetic field or with optical tweezers (8, 25 ). These studies will link with the others of this project program grant to elucidate the fundamental design principles of the elasticity of VIF networks.

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Lin, Ni-Hsuan; Huang, Yu-Shan; Opal, Puneet et al. (2016) The role of gigaxonin in the degradation of the glial-specific intermediate filament protein GFAP. Mol Biol Cell 27:3980-3990

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