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.

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.

BackgroundMotor 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 FindingsWe 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/SignificanceThese 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.

Bruschke, "The relation between QT intervals and heart rate in young healthy males using an incremental head-up tilt protocol," Comput.Abstract-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.

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.