Microtubule bending and breaking

Microtubules are linear filaments of the cytoskeleton that mediate such cellular processes as nerve growth and cell division and in general serve to organize the cytoplasm. In interphase cells microtubules tend to exhibit a radial pattern with the plus ends of microtubules pointing outward toward the cell periphery and the minus ends pointing inward toward the centrosome, near the nucleus. Distorting this archetypal pattern is the deformation of microtubules by cortical actin/myosin (Canman and Bement, 1997; Letourneau et al., 1987; Waterman-Storer and Salmon, 1997), manifested by varying degrees of microtubule curvature (Sammak and Borisy, 1988; Schulze and Kirschner, 1988; Tanaka et al., 1995). The presence of curvature indicates that the relatively stiff microtubules (Young’s modulus ~109 Pa; Gittes et al., 1993) have elastic strain energy stored in them. The elastic strain energy increase in the microtubule lattice could in turn alter the kinetics and thermodynamics of processes associated with microtubules and provide a basis for mechanochemical signal transduction. A variety of experimental results suggest that microtubules play a role in transducing mechanical stress into chemical responses. First, increasing tension on neurons promotes the assembly of microtubules (Zheng et al., 1993), consistent with thermodynamic models where reducing axial compression on microtubules promotes microtubule assembly over disassembly (Buxbaum and Heidemann, 1992; Hill, 1981). Similarly, stretching a monolayer of smooth muscle cells adherent to an equi-biaxially strained polymer substratum results in a net increase in microtubule assembly (Putnam et al., 1998). Second, analysis of Caenorhabditis elegans mutants implicates mec-7, a β-tubulin gene whose product forms an unusual 15 protofilament microtubule, as an essential component of the gentle-touch mechanosensory mechanism (Savage et al., 1989). Third, the sequence-specific transcription factor NF-κB, which mediates the fluid shear stress response in vascular endothelial cells (Khachigian et al., 1995), is activated by microtubule disassembly (Rosette and Karin, 1995). Together these results suggest that the chemical kinetics and thermodynamics of processes associated with microtubules are altered in response to mechanical deformation. However, there has not been any quantitative analysis of in vivo microtubule curvature, the associated elastic strain energies, and the impact of curvature on cellular processes. One microtubule-associated process that is likely affected by mechanical deformation is microtubule breaking. WatermanStorer and Salmon (1997) previously reported that a limited number of microtubule breaking events observed in rhodamine-tubulin injected newt lung cells occurred in microtubules with relatively high local curvature (average curvature = 1.7 rad/μm, n=7). However, the limited number of observations and lack of background curvature measurements (for microtubules that did not break) prevented the conclusive establishment of a relationship between bending and breaking. To quantitatively determine the degree of microtubule curvature in living cells and the quantitative relationship between microtubule bending and breaking we analyzed individual microtubules at the leading edge of rhodaminetubulin injected Swiss 3T3 fibroblasts. We found that the kinetics of microtubule breaking were accelerated when microtubules were curved to energy levels >1 kT/tubulin dimer. We also found that the amount of energy stored in the lattice as curvature-associated strain energy was ~2.5% of that associated with GTP hydrolysis, which occurs concomitantly 3283 Journal of Cell Science 112, 3283-3288 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 JCS0434