Networks of filamentous polymers are a common feature of cells and biological tissues. From the crosslinked gels providing strength to blood clots and extracellular matrices to the dynamic cytoskeleton that undergoes gel-sol transitions as cells move and the more obscure matrix within the cell nucleus, nearly all cellular surfaces are interconnected by a series of fluctuating but continuous meshworks. In part the function of these networks is mechanical, and their viscoelasticity, together with new methods to observe the dynamics of single polymer strands within the network, has made biological polymers, especially those derived from the cytoskeleton, attractive materials by which to test theories of polymer physics. The unusual viscoelasticity of these networks is crucial to their biological function to provide mechanical continuity throughout cells and tissues. In addition, formation and disassembly of networks composed of the polyanionic filaments comprising the cytoskeleton has many consequences for sequestration of proteins and metabolites and in providing directionality and spatial segregation to biochemical reactions and signal transduction pathways. Concepts related to network formation may transform the way intracellular biochemistry and signaling are understood.

References:

1. Eichinger, L., Koppel, B., Noegel, A. A., Schleicher, M., Schliwa, M., Weijer, K., Witke, W. and Janmey, P. A. (1996). Mechanical perturbation elicits a phenotypic difference between Dictyostelium wild-type cells and cytoskeletal mutants. Biophys J. 70, 1054-60.

To determine the specific contribution of cytoskeletal proteins to cellular viscoelasticity we performed rheological experiments with Dictyostelium discoideum wild-type cells (AX2) and mutant cells altered by homologous recombination to lack alpha-actinin (AHR), the ABP120 gelation factor (GHR), or both of these F-actin cross-linking proteins (AGHR). Oscillatory and steady flow measurements of Dictyostelium wild-type cells in a torsion pendulum showed that there is a large elastic component to the viscoelasticity of the cell pellet. Quantitative rheological measurements were performed with an electronic plate-and-cone rheometer, which allowed determination of G', the storage shear modulus, and G", the viscous loss modulus, as a function of time, frequency, and strain, respectively. Whole cell viscoelasticity depends strongly on all three parameters, and comparison of wild-type and mutant strains under identical conditions generally produced significant differences. Especially stress relaxation experiments consistently revealed a clear difference between cells that lacked alpha-actinin as compared with wild-type cells or transformants without ABP120 gelation factor, indicating that alpha-actinin plays an important role in cell elasticity. Direct observation of cells undergoing shear deformation was done by incorporating a small number of AX2 cells expressing the green fluorescent protein of Aequorea victoria and visualizing the strained cell pellet by fluorescence and phase contrast microscopy. These observations confirmed that the shear strain imposed by the rheometer does not injure the cells and that the viscoelastic response of the cell pellet is due to deformation of individual cells.

2. Janmey, P. (1994). Phosphoinositides and calcium as regulators of cellular actin assembly and disassembly. Annual Review of Physiology. 56, 169-191.

3. Janmey, P. A. (1991). Mechanical properties of cytoskeletal polymers. Curr Opin Cell Biol. 3, 4-11.

The mechanical properties of cytoplasm are dominated by microfilaments, microtubules, and intermediate filaments, collectively termed the cytoskeleton. This review discusses how the physical properties of these biopolymer systems are related to their molecular structures and interactions, and how remodelling of these biopolymers in vivo affects cell shape and motility.

4. Leterrier, J. F., Kas, J., Hartwig, J., Vegners, R. and Janmey, P. A. (1996). Mechanical effects of neurofilament cross-bridges. Modulation by phosphorylation, lipids, and interactions with F-actin. J Biol Chem. 271, 15687-94.

The structure of gels formed by bovine spinal cord neurofilaments was determined by fluorescence and electron microscopy and compared to mechanical properties measured by their elastic and viscous response to shear forces. Neurofilaments formed gels of high elastic modulus (>100 Pa) after addition of millimolar Mg2+. Gelation caused a slow increase in shear moduli to levels similar to those of vimentin intermediate filament networks, followed by a rapid rise due to formation of links between neurofilaments, mediated by cross-bridging structures that vimentin filaments lack. Neurofilament gels are more resistant to large deformations than are vimentin networks, suggesting the importance of cross-bridges for neurofilament mechanical properties. Fluorescence imaging of single neurofilaments showed flexible filaments that became straighter when they adhered to glass or were incorporated into filament bundles. Electron microscopy of neurofilament gels showed a system of bundles intertwined within a more isotropic network of individual filaments. Neurofilament gel formation was stimulated in vitro by acid phosphatase treatment or by inositol phospholipids. In contrast, addition of actin filaments reduced the resistance of neurofilament gels to large stresses. These results suggest that dynamic and regulated interactions occur between neurofilaments to form viscoelastic networks with properties distinct from other cytoskeletal structures.

5. Tang, J. X. and Janmey, P. A. (1996). The polyelectrolyte nature of F-actin and the mechanism of actin bundle formation. J Biol Chem. 271, 8556-63.

Polymerized (F-)actin is induced to form bundles by a number of polycations including divalent metal ions, Co(NH3)63+, and basic polypeptides. The general features of bundle formation are largely independent of the specific structure of the bundling agent used. A threshold concentration of polycation is required to form lateral aggregates of actin filaments. The threshold concentration varies strongly with the valence of the cation and increases with the ionic strength of the solution. Polyanions such as nucleoside phosphates or oligomers of acidic amino acids disaggregate actin bundles into single filaments. These features are similar to the phenomenon of DNA condensation and can be explained analogously by polyelectrolyte theories. Similar results were found when F-actin was bundled by the peptide corresponding to the actin binding site of myristoylated alanine-rich protein kinase C substrate protein (MARCKS) or by smooth muscle calponin, suggesting that a broad class of actin bundling factors may function in a common manner. Physiologic concentrations of both small ions and large proteins can induce actin interfilament association independent of a requirement for specific binding sites.