Self-assembly of artificial microtubules

Understanding the complex self-assembly of biomacromolecules is a major outstanding question. Microtubules are one example of a biopolymer that possesses characteristics quite distinct from standard synthetic polymers that are derived from its hierarchical structure. In order to understand how to design and build artificial polymers that possess features similar to those of microtubules, we have initially studied the self-assembly of model monomers into a tubule geometry. Our model monomer has a wedge shape with lateral and vertical binding sites that are designed to form tubules. We used molecular dynamics simulations to study the assembly process for a range of binding site interaction strengths. In addition to determining the optimal regime for obtaining tubules, we have calculated a diagram of the structures that form over a wide range of interaction strengths. Unexpectedly, we find that the helical tubules form, even though the monomer geometry is designed for nonhelical tubules. We present the detailed dynamics of the tubule self-assembly process and show that the interaction strengths must be in a limited range to allow rearrangement within clusters. We extended previous theoretical methods to treat our system and to calculate the boundaries between different structures in the diagram.Comment: 15 pages, 11 figure

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

Assembly dynamics of microtubules at molecular resolution

Microtubules are highly dynamic protein polymers that form a crucial part of the cytoskeleton in all eukaryotic cells. Although microtubules are known to self-assemble from tubulin dimers, information on the assembly dynamics of microtubules has been limited, both in vitro and in vivo, to measurements of average growth and shrinkage rates over several thousands of tubulin subunits. As a result there is a lack of information on the sequence of molecular events that leads to the growth and shrinkage of microtubule ends. Here we use optical tweezers to observe the assembly dynamics of individual microtubules at molecular resolution. We find that microtubules can increase their overall length almost instantaneously by amounts exceeding the size of individual dimers (8 nm). When the microtubule-associated protein XMAP215 (ref. 6) is added, this effect is markedly enhanced and fast increases in length of about 40-60 nm are observed. These observations suggest that small tubulin oligomers are able to add directly to growing microtubules and that XMAP215 speeds up microtubule growth by facilitating the addition of long oligomers. The achievement of molecular resolution on the microtubule assembly process opens the way to direct studies of the molecular mechanism by which the many recently discovered microtubule end-binding proteins regulate microtubule dynamics in living cells