Rapid microtubule self-assembly kinetics.

Microtubule assembly is vital for many fundamental cellular processes. Current models for microtubule assembly kinetics assume that the subunit dissociation rate from a microtubule tip is independent of free subunit concentration. Total-Internal-Reflection-Fluorescence (TIRF) microscopy experiments and data from a laser tweezers assay that measures in vitro microtubule assembly with nanometer resolution, provides evidence that the subunit dissociation rate from a microtubule tip increases as the free subunit concentration increases. These data are consistent with a two-dimensional model for microtubule assembly, and are explained by a shift in microtubule tip structure from a relatively blunt shape at low free concentrations to relatively tapered at high free concentrations. We find that because both the association and the dissociation rates increase at higher free subunit concentrations, the kinetics of microtubule assembly are an order-of-magnitude higher than currently estimated in the literature.

Spindle pole mechanics studied in mitotic asters: dynamic distribution of spindle forces through compliant linkages.

During cell division, chromosomes must faithfully segregate to maintain genome integrity, and this dynamic mechanical process is driven by the macromolecular machinery of the mitotic spindle. However, little is known about spindle mechanics. For example, spindle microtubules are organized by numerous cross-linking proteins yet the mechanical properties of those cross-links remain unexplored. To examine the mechanical properties of microtubule cross-links we applied optical trapping to mitotic asters that form in mammalian mitotic extracts. These asters are foci of microtubules, motors, and microtubule-associated proteins that reflect many of the functional properties of spindle poles and represent centrosome-independent spindle-pole analogs. We observed bidirectional motor-driven microtubule movements, showing that microtubule linkages within asters are remarkably compliant (mean stiffness 0.025 pN/nm) and mediated by only a handful of cross-links. Depleting the motor Eg5 reduced this stiffness, indicating that Eg5 contributes to the mechanical properties of microtubule asters in a manner consistent with its localization to spindle poles in cells. We propose that compliant linkages among microtubules provide a mechanical architecture capable of accommodating microtubule movements and distributing force among microtubules without loss of pole integrity-a mechanical paradigm that may be important throughout the spindle.

Nanometer-resolution microtubule polymerization assays using optical tweezers and microfabricated barriers.

Microtubule (MT) polymerization dynamics, which are crucial to eukaryotic life and are the target of important anticancer agents, result from the addition and loss of 8-nm-long tubulin-dimer subunits. Addition and loss of one or a few subunits cannot be observed at the spatiotemporal resolution of conventional microscopy, and requires development of approaches with higher resolution. Here we describe an assay in which one end of an MT abuts a barrier, and MT length changes are coupled to the movement of an optically trapped bead, the motion of which is tracked with high resolution. We detail assay execution, including preparation of the experimental chamber and orientation of the MT against the barrier. We describe design requirements for the experimental apparatus and barriers, and preparation of materials including stable, biotinylated MT seeds from which growth is initiated and NeutrAvidin-coated beads. Finally, we discuss advantages of moving the optical trap such that it applies a constant force (force clamping), detection limits, the importance of high temporal resolution, data analysis, and potential sources of experimental artifacts.