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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.

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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.

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BACKGROUND: The labile nature of microtubules is critical for establishing cellular morphology and motility, yet the molecular basis of assembly remains unclear. Here we use optical tweezers to track microtubule polymerization against microfabricated barriers, permitting unprecedented spatial resolution. RESULTS: We find that microtubules exhibit extensive nanometer-scale variability in growth rate and often undergo shortening excursions, in some cases exceeding five tubulin layers, during periods of overall net growth. This result indicates that the guanosine triphosphate (GTP) cap does not exist as a single layer as previously proposed. We also find that length increments (over 100 ms time intervals, n = 16,762) are small, 0.81 +/- 6.60 nm (mean +/- standard deviation), and very rarely exceed 16 nm (about two dimer lengths), indicating that assembly occurs almost exclusively via single-subunit addition rather than via oligomers as was recently suggested. Finally, the assembly rate depends only weakly on load, with the average growth rate decreasing only 2-fold as the force increases 7-fold from 0.4 pN to 2.8 pN. CONCLUSIONS: The data are consistent with a mechanochemical model in which a spatially extended GTP cap allows substantial shortening on the nanoscale, while still preventing complete catastrophe in most cases.

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Studying the mechanics of nanometer-scale biomolecules presents many challenges; these include maintaining light microscopy image quality and avoiding interference with the laser used for mechanical manipulation, that is, optical tweezers. Studying the pushing forces of a polymerizing filament requires barriers that meet these requirements and that can impede and restrain nanoscale structures subject to rapid thermal movements. We present a flexible technique that meets these criteria, allowing complex barrier geometries with undercut sidewall profiles to be produced on #1 cover glass for the purpose of obstructing and constraining polymerizing filaments, particularly microtubules. Using a two-layer lithographic process we are able to separate the construction of the primary features from the construction of a depth and shape-controlled undercut. The process can also be extended to create a large uniform gap between an SU-8 photoresist layer and the glass substrate. This technique can be easily scaled to produce large quantities of shelf-stable, reusable microstructures that are generally applicable to microscale studies of the interaction of cellular structures with defined microscale features.

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Optical tweezers are an important tool for studying cellular and molecular biomechanics. We present a robust optical tweezers device with advanced features including: multiple optical traps, acousto-optic trap steering, and back focal plane interferometry position detection. We integrate these features into an upright microscope, with no compromise to its capabilities (differential interference contrast microscopy, fluorescence microscopy, etc.). Acousto-optic deflectors (AODs) steer each beam and can create multiple time-shared traps. Position detection, force calibrations and AOD performance are presented. The system can detect subnanometer displacements and forces below 0.1 pN.

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