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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44486/1/10544_2005_Article_6170.pd

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

Tracking microtubule polymerization under load with nanometer resolution: Methods, measurements, and implications for understanding microtubule dynamic instability.

The underlying mechanisms of microtubule (MT) dynamic instability have remained enigmatic largely because direct studies of events occurring at the tip have proven difficult. Studies that follow polymerization of individual dynamic microtubules face both severe temporal and spatial resolution limitations while biochemical studies can only determine the average behavior over a large population of microtubules. Cryo-electron-microscopy allows for detailed study of MT tip structure, but requires fixed samples that are no longer dynamic. These limitations are overcome by combining optical tweezers with a system of micropatterned barriers, allowing nanometer resolution tracking of events at the dynamic microtubule tip. An optical trapping device integrated with an upright microscope was developed to exert and measure forces at the MT tip. Barriers constructed by photolithography on a #1 cover glass were engineered to maintain all trapping, detection, and imaging capabilities while obstructing and constraining the polymerizing microtubule tip. A silica microsphere linked to a microtubule serves as a handle, which is held by the optical tweezers as the microtubule is allowed to polymerize and contact the barrier. Growth records with a stationary trap were able to detect pauses in microtubule growth. The use of a feedback system to maintain a constant force at the MT tip (force-clamp) allowed measurement of events at the tip with nanometer precision. These techniques were used to reveal several new features of microtubule growth that were undetectable with other methods. Microtubules that previously would be classified as growing are found to frequently undergo nanoscale shortening events. Furthermore, previously reported growth rate variability is seen to be the consequence of the frequency of short-scale shortening events. Most importantly microtubules often shorten 20--50 nm without entering into a phase of rapid shortening invalidating the canonical GTP-cap model for dynamic instability. As an alternative source of stability, we propose that the structure at the growing microtubule tip is able to stabilize the lattice by assuming a lower energy conformation. Additionally we propose that different structures will have different polymerization rates leading to the large variability in microtubule polymerization.Ph.D.Applied SciencesBiological SciencesBiomedical engineeringBiophysicsCellular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/125216/2/3186753.pd

Bulletin of the Technical Committee on Data Engineering (June, 1993 Vol. 16 No. 2)

In many real world applications (even in banking), imprecise data is a matter of fact. However, classic database management systems provide little if any help in the management of imprecise data. We are applying methods from interval arithmetic, epsilon serializability, and other related areas to help the application designers in the management of naturally imprecise data. Our approach includes operators on imprecise data that give bounds on the result imprecision and algorithms that constrain the imprecision propagation in the database. 1 Introduction Traditional database management systems provide support only for precise data, though in the physical world data is often imprecise. An important class of examples is the scientific data such as incomplete recording of data, instrument noise, measurement error, computational model imprecision, and data aggregation of one kind or another. Another example is the "fuzzy" data managed by Epsilon Serializability algorithms [PL91, DP93]. In t..

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