Abstract. We have developed video microscopy methods to visualize the assembly and disassembly of individual microtubules at 33-ms intervals. Porcine brain tubulin, free of microtubule-associated proteins, was assembled onto axoneme fragments at 37°C, and the dynamic behavior of the plus and minus ends of microtubules was analyzed for tubulin concentrations between 7 and 15.5 I.tM.Elongation and rapid shortening were distinctly different phases. At each end, the elongation phase was characterized by a second order association and a substantial first order dissociation reaction. Association rate constants were 8.9 and 4.3 ~tM -t s -t for the plus and minus ends, respectively; and the corresponding dissociation rate constants were 44 and 23 s -t. For both ends, the rate of tubulin dissociation equaled the rate of tubulin association at 5 lxM. The rate of rapid shortening was similar at the two ends (plus = 733 s-t; minus = 915 s-l), and did not vary with tubulin concentration.Transitions between phases were abrupt and stochastic. As the tubulin concentration was increased, catastrophe frequency decreased at both ends, and rescue frequency increased dramatically at the minus end. This resulted in fewer rapid shortening phases at higher tubulin concentrations for both ends and shorter rapid shortening phases at the minus end. At each concentration, the frequency of catastrophe was slightly greater at the plus end, and the frequency of rescue was greater at the minus end. Our data demonstrate that microtubules assembled from pure tubulin undergo dynamic instability over a twofold range of tubulin concentrations, and that the dynamic instability of the plus and minus ends of microtubules can be significantly different. Our analysis indicates that this difference could produce treadmilling, and establishes general limits on the effectiveness of length redistribution as a measure of dynamic instability. Our results are consistent with the existence of a GTP cap during elongation, but are not consistent with existing GTP cap models.T HE term "dynamic instability" describes microtubule assembly in which individual microtubules exhibit alternating phases of elongation and rapid shortening. Transitions between these phases are abrupt, stochastic, and infrequent in comparison to the rates of tubulin association and dissociation at the microtubule ends (17, 28). Substantial evidence has accumulated to indicate that dynamic instability is the basic mechanism of microtubule assembly in vitro (17, 28), and in both the mitotic spindle and the cyoplasmic microtubule complex (CMTC) l (10,11,34,35,37). Although microtubule dynamics within the cell may be regulated by various microtubule-associated proteins (MAPs) and other intracellular regulatory molecules, it is important 1. Abbreviations used in this paper: CMTC, cytoplasmic microtubule complex; DIC, differential interference contrast; MAP(s), microtubule-associated protein(s).first to understand the details of the inherent behavior of microtubules assembled from tubulin alone.Th...

Cancer cells are defined by their ability to invade through the basement membrane, a critical step during metastasis. While increased secretion of proteases, which facilitates degradation of the basement membrane, and alterations in the cytoskeletal architecture of cancer cells have been previously studied, the contribution of the mechanical properties of cells in invasion is unclear. Here we apply a magnetic tweezer system to establish that stiffness of patient tumor cells and cancer cell lines inversely correlates with migration and invasion through three-dimensional basement membranes, a correlation known as a power law. We found that cancer cells with the highest migratory and invasive potential are five times less stiff than cells with the lowest migration and invasion potential. Moreover, decreasing cell stiffness by pharmacological inhibition of myosin II increases invasiveness, while increasing cell stiffness by restoring expression of the metastasis suppressor TβRIII/betaglycan decreases invasiveness. These findings are the first demonstration of the power law relation between the stiffness and the invasiveness of cancer cells and show that mechanical phenotypes can be used to grade the metastatic potential of cell populations with the potential for single cell grading. The measurement of a mechanical phenotype, taking minutes rather than hours needed for invasion assays, is promising as a quantitative diagnostic method and as a discovery tool for therapeutics. By demonstrating that altering stiffness predictably alters invasiveness, our results indicate that pathways regulating these mechanical phenotypes are novel targets for molecular therapy of cancer.