Cells are dynamic structures that are subjected to a variety of mechanical stimuli and necessarily must respond to both intra- and extracellular forces [Janmey, 1998
]. For example, various mechanical stimuli play a role in normal physiological development in the heart [Kendrick-Jones et al., 1971
], vasculature [Skalak and Price, 1996
], and bone [Turner et al., 1995
]. Furthermore, exposure to abnormal mechanical forces often results in pathologic conditions such as cardiac hypertrophy, carpal tunnel syndrome, atherosclerosis and vascular smooth muscle apoptosis [Goldschmidt et al., 2001
; Katsumi et al., 2004
A characterization of the transmission of force from the external environment into the cell is crucial to our understanding the response of cells to forces. Specialized sensory cells, including skin mechanoreceptor cells and cochlear hair cells, display unique adaptations that sense their mechanical environment. Here, mechanical stimuli are directly coupled to ion channel opening and electrical signaling [Corey and Hudspeth, 1983
]. In other instances, distension may decrease the lateral spacing between cells thereby increasing extracellular ligand concentrations and activating signaling pathways [Tschumperlin et al., 2002
]. In some cases, a direct effect of stretch on signal transduction has been observed when the plasma membrane and its associated receptors have been removed [Sawada and Sheetz, 2002
]. Thus, direct stretching of the cytoskeleton can trigger mechanotransduction.
The cytoskeleton is a dynamic structure comprised of many proteins including actin. Actin filaments can be linked together via actin-binding proteins (ABP) to form a mesh-like network, which acts as the cytoskeleton scaffolding. Integrins, which attach to the actin matrix via additional scaffolding proteins such as talin and viniculin, often serve as force transducers between intracelluar actin filaments and the extracellular matrix [Wang et al. 1993
]. External forces applied to the cell will deform the cytoskeleton by differing amounts depending on the viscous and elastic properties of the mesh and the intrinsic mechanical properties of the actin filaments and their associated ABPs.
ABPs can affect the mechanical properties of the cytoskeleton by promoting filament assembly and disassembly or by structural reinforcement of the filament network. For example, mechanical stress on human umbilical cells promotes the expression of the cytoskeletal proteins caldesmon, calponin, and tropomyosin [Cevallos et al., 2006
], leading to filament elongation and stability. Similarly, repeated stress to smooth muscle tissue can cause dense body formation, expression of Erk1/2, and cytoskeletal remodeling [Kim and Hai, 2005
]. Formin binding can also affect actin polymerization directly by inducing torsional strain [Shemesh and Kozlov, 2007
; Shemesh et al., 2005
]. All of these effects come from the dynamic response of the cytoskeleton to applied mechanical stress, which is modulated by ABPs.
The ability of the cytoskeleton to sense and respond to forces also depends on the mechanical properties of the actin filaments themselves. ABPs can buttress the structural organization of actin filaments by reinforcing the interstrand interactions between opposite strands of the actin helix, the intrastrand interactions between successive monomers along filaments, or both. Furthermore, ABP binding could affect actin–actin interactions via an allosteric mechanism. These varied interactions can affect the torsional, flexural, and tensile properties of an actin filament differently. As a first step toward understanding the effects of ABP binding, we have undertaken a study to correlate structural information about actin-binding protein interactions with the mechanical effects of ABP binding on flexural rigidity.
We measured the persistence length, a parameter that is directly related to flexural rigidity by a simple linear transform (See Eq. 3
in the “Materials and Methods” section), of actin-ABP complexes by observing actin filaments vibrating under thermal fluctuations using fluorescence microscopy. We found that phalloidin increases the persistence length of actin by 1.9-fold. Consistent with kinetic [Lehrer and Morris, 1984
] and structural differences [Lehman et al., 2000
], the intrastrand reinforcement of tropomyosin increases persistence length, with differences between smooth and skeletal muscle isoforms, being 1.5- and 2-fold respectively. We also show that the interstrand crosslink of the C-terminal actin-binding fragment of caldesmon, H32K, increases persistence length by 1.6-fold while, consistent with the structure of phosphorylated H32K-actin complexes [Foster et al., 2004
], phosphorylation by ERK kinase reverses this effect. Lastly, we show that the effect of binding both smooth muscle tropomyosin and H32K is not additive.