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