Session R1: Cytoskeletal Dynamics and Mechanics

Structural organization and dynamics of the cytoskeletal network

Actin cytoskeleton is the major player in mechanisms driving multiple forms of cell motility. Actin filaments cooperating with numerous actin-binding proteins are able to form distinct types of higher order structures: networks and bundles, which are designed for carrying out various functions. One of important actin functions is generation of pushing force for protrusion of a leading edge of motile cells. Lamellipodia and filopodia are the two major protrusive organelles utilized by different cells for leading edge protrusion. Even though both are driven by actin polymerization, lamellipodia and filopodia have strikingly different structural design and use different sets of actin-binding proteins. Lamellipodia, which are broad, flat protrusions are filled with a branched network of actin filaments, which propagates through cycles of dendritic nucleation, elongation, capping, and depolymerization of actin filaments. Filopodia, which are thin cellular processes, contain a tight bundle of parallel actin filaments, which elongates at the tip and depolymerizes from the rear. Although basic models for the leading edge protrusion have been formulated, many questions remain about the molecular design of the protrusive machinery and specific roles of individual molecules. Our approach is to analyze molecular architecture of actin cytoskeleton during protrusion and correlate these data with live cell behavior. Recent progress in understanding the molecular mechanisms of actin-based protrusion will be presented.   

Elasticity, adhesion and actin based propulsion

When a cells crawls, its shape re-organizes via polymerization and depolymerization of actin filaments. The growing ends of the filaments are oriented towards the outside of the cell, and their polymerization pushes the cell membrane forwards. The same mechanism comes into play when the bacterial pathogen Listeria monocytogenes infects a cell. The bacterium hijacks the host cell's actin machinery to create an actin network (the actin comet tail) that propels the bacterium through cells and into neighboring cells. We propose a mechanism for how polymerization gives rise to motility that incorporates the effects of inhomogeneous polymerization. We treat the actin comet tail as an elastic continuum tethered to the rear of the bacterium. The interplay of polymerization and tethering gives rise to inhomogeneous stresses calculated with a finite element analysis. We quantitatively reproduce many distinctive features of actin propulsion that have been observed experimentally, including stepped motion, hopping, tail shape and the propulsion of flat surfaces.   

Fluorescent Speckle Microrheology

The actin cortex is the dense shell of actin filaments between the cell membrane and the cytoplasm maintaining and regulating cell shape. It is one of the principal determinants of cell mechanical properties, whose spatiotemporal modulations play a central role in processes that involve architectural dynamics of a cell, such as cell migration, division and morphogenesis. However, the exact mechanism of cortical actin elasticity regulation \textit{in vivo} is still unresolved. We present a high-resolution and molecularly specific assay of \textit{in vivo} cortical actin elasticity, fluorescent speckle microrheology. Speckles originate when fluorescent actin is randomly incorporated into the network along with abundant endogeneous non-fluorescent actin, leading to high spatial variations of the local fluorophore density; high-density areas appear as diffraction-limited spots (speckles) upon high-resolution imaging. Speckles act as fiduciary marks of the network and can be used to directly image strain fluctuations, in contrast to classical microrheology techniques using imbedded probes. When tracking positional fluctuations of actin speckles in cells without convective network flow with subpixel precision, we find that the displacements of neighboring speckles are spatially correlated. Their correlation function decays as 1/r with interspeckle distance r, which is consistent with theoretical predictions for strain field decay in a 3D continuous viscoelastic medium. On the basis of these results, we use the amplitude of the correlation function to measure viscoelastic properties of the actin network. Due to high intracellular speckle densities and their homogeneous distribution throughout the cell, this approach yields much higher spatial resolution than other microrheology techniques, which is validated using \textit{in vitro} actin networks. Thus, this assay allows us to map intracellular actin cortex elasticity with micron resolution, and to relate intracellular heterogeneities of elasticity to heterogeneities in other dynamic cellular parameters.   

Nonlinear Elasticity in Biological Gels

The mechanical properties of soft biological tissues are essential to their physiologic function and cannot easily be duplicated by synthetic materials. Unlike simple polymer gels, many biological materials including blood vessels, mesentery tissue, lung parenchyma, cornea and blood clots, stiffen as they are deformed, or strained. Stiffening under deformation allows tissues to be compliant at small strains and strengthen at larger deformations that could threaten tissue integrity. The molecular structures and design principles responsible for this non-linear elasticity are unknown. I will outline a molecular theory that accounts for strain-stiffening in a range of molecularly distinct gels formed from cytoskeletal and extracellular proteins and reveals universal stress-strain relations at low to intermediate strains. The input to this theory is the force-extension curve for individual semi- flexible filaments and the assumptions that networks composed of them are isotropic and that their elastic response is affine. The theory shows that systems of filamentous proteins arranged in an open crosslinked meshwork invariably stiffen at low strains without requiring a specific architecture or multiple elements with different intrinsic stiffness.