Cytoskeleton and Cell Motility

• V.6.iv Subproject 4 - Experiments on the Actin Cytoskeleton
• V.6.iv.a Introduction
• V.6.iv.b Background and Significance
• V.6.iv.c Aims
• V.6.iv.d Biochemical Assembly of F-actin: Thermodynamics
  • V.6.iv.d.1 Introduction
  • V.6.iv.d.2 Preliminary Results
  • V.6.iv.d.3 Methodology
  • V.6.iv.d.4 Significance
• V.6.iv.e Construction of a Composite Actin Network to Mimic Cytoskeletal Mechanics
  • V.6.iv.e.1 Introduction
  • V.6.iv.e.2 Preliminary Results
  • V.6.iv.e.3 Hypothesis
  • V.6.iv.e.4 Methodology
  • V.6.iv.e.5 Model
  • V.6.iv.e.6 Significance

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Our primary hypothesis concerning the mechanism of the extraordinary elasticity of the filamin-actin gel is that the two floppy arms of the filamin dimer enable the actin filaments to remain crosslinked even when the filaments rotate greatly with respect to each other. The known structure of nonmuscle human filamin (which the older literature refers to as ABP-280 to distinguish it from muscle filamin, which lacks a 24 amino acid hinge domain) strongly supports this hypothesis. We will explore the factors that can reduce the network strength by diminishing the crosslinking activity of non-muscle filamin. Phosphorylation of filamin reduces its affinity for F-actin, but its effect on crosslinking remains untested. The exposed hinge 1 of non-muscle filamin between repeats 15 and 16 contains a calpain cleavage site. We expect the effect of cleavage on rheological properties to partially reveal the structural basis of the unusually strong and resilient gelation activity of intact non-muscle filamin.

We will compare the mechanical strength and response to deformation of actin networks formed by filamin and alpha-actinin, using biophysical techniques including mechanical rheology, fluorescence microscopy and atomic force microscopy (AFM). We will use two types of rheometers to test the mechanical properties. A commercial constant strain rheometer will provide a quantitative and systematic characterization of the reconstituted gels. In addition, we will also use a home-built torsion pendulum rheometer which allows diffusion of external chemicals during measurement. The torsion pendulum will include an optical microscope, to visualize the network structure while mechanically deforming the network.

A dynamic network of actin at the cell leading edge is essential and perhaps even sufficient for the shape change and motility of most cells. The actin network crosslinked by filamin therefore defines a minimal model for the mechanical properties of the cytoskeleton. In cellular environments, local strain fields on the order of 100% are common. For instance, macrophages undergo large shape changes in order to crawl through the narrow extra-cellular space to reach a site of infection. The epithelial cells in blood vessels typically align with the blood flow in order to withstand large shear stresses. Constructing a crosslinked actin gel that is resilient to large deformation is a valuable step toward understanding the mechanical properties of cells. Methods to manipulate such mechanical properties may affect related cellular properties and may even lead to intervention for diseases related to unregulated cell motility.