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.

Phosphorylation by Cdk1 increases the binding of Eg5 to microtubules in vitro and in Xenopus egg extract spindles.

BACKGROUND: Motor proteins from the kinesin-5 subfamily play an essential role in spindle assembly during cell division of most organisms. These motors crosslink and slide microtubules in the spindle. Kinesin-5 motors are phosphorylated at a conserved site by Cyclin-dependent kinase 1 (Cdk1) during mitosis. Xenopus laevis kinesin-5 has also been reported to be phosphorylated by Aurora A in vitro. METHODOLOGY/PRINCIPAL FINDINGS: We investigate here the effect of these phosphorylations on kinesin-5 from Xenopus laevis, called Eg5. We find that phosphorylation at threonine 937 in the C-terminal tail of Eg5 by Cdk1 does not affect the velocity of Eg5, but strongly increases its binding to microtubules assembled in buffer. Likewise, this phosphorylation promotes binding of Eg5 to microtubules in Xenopus egg extract spindles. This enhancement of binding elevates the amount of Eg5 in spindles above a critical level required for bipolar spindle formation. We find furthermore that phosphorylation of Xenopus laevis Eg5 by Aurora A at serine 543 in the stalk is not required for spindle formation. CONCLUSIONS/SIGNIFICANCE: These results show that phosphorylation of Eg5 by Cdk1 has a direct effect on the interaction of this motor with microtubules. In egg extract, phosphorylation of Eg5 by Cdk1 ensures that the amount of Eg5 in the spindle is above a level that is required for spindle formation. This enhanced targeting to the spindle appears therefore to be, at least in part, a direct consequence of the enhanced binding of Eg5 to microtubules upon phosphorylation by Cdk1. These findings advance our understanding of the regulation of this essential mitotic motor protein.

Reconstitution of a microtubule plus-end tracking system in vitro.

The microtubule cytoskeleton is essential to cell morphogenesis. Growing microtubule plus ends have emerged as dynamic regulatory sites in which specialized proteins, called plus-end-binding proteins (+TIPs), bind and regulate the proper functioning of microtubules. However, the molecular mechanism of plus-end association by +TIPs and their ability to track the growing end are not well understood. Here we report the in vitro reconstitution of a minimal plus-end tracking system consisting of the three fission yeast proteins Mal3, Tip1 and the kinesin Tea2. Using time-lapse total internal reflection fluorescence microscopy, we show that the EB1 homologue Mal3 has an enhanced affinity for growing microtubule end structures as opposed to the microtubule lattice. This allows it to track growing microtubule ends autonomously by an end recognition mechanism. In addition, Mal3 acts as a factor that mediates loading of the processive motor Tea2 and its cargo, the Clip170 homologue Tip1, onto the microtubule lattice. The interaction of all three proteins is required for the selective tracking of growing microtubule plus ends by both Tea2 and Tip1. Our results dissect the collective interactions of the constituents of this plus-end tracking system and show how these interactions lead to the emergence of its dynamic behaviour. We expect that such in vitro reconstitutions will also be essential for the mechanistic dissection of other plus-end tracking systems.

Microtubule assembly dynamics at the nanoscale.

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.

Micropatterned structures for studying the mechanics of biological polymers.

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.

Advanced optical tweezers for the study of cellular and molecular biomechanics.

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.