Automated, Long-Distance Microtubule Tracking in Gliding Assays

Microtubules are microscopic tubular structures involved in a number of biological functions, such as cell division, intracellular transport, and mechanosensation. For particular functions, such as mechanosensation in C. elegans, microtubule diameter, or equivalently protofilament number, is tightly regulated. It is tempting to link microtubule diameter to microtubule bending rigidity, and hence to mechanosensation. However, even the link between microtubule diameter and rigidity remains unclear. Two particular challenges in measuring microtubule flexural rigidity as a function of diameter are the intrinsic heterogeneity in microtubule structures in in vitro preparations, and the intrinsic heterogeneity in microtubule rigidities measured for identically prepared microtubules.In order to address these heterogeneities, we simultaneously determine microtubule diameter and rigidity for single microtubules using statistical properties gathered from a microtubule gliding assay. In this poster, we report a technique we have developed to improve the precision of these measurements. The technique combines a microscope, integrated piezo-elements to control the sample stage, and software to automatically track a single microtubule and reposition the stage with nanometer precision to keep the microtubule centered within the field of view. We have been able to effectively double the field-of-view in each direction (from 30 micrometers to 60 micrometers), resulting in substantially longer microtubule gliding trajectories while maintaining a tracking precision on the order of 10 nm over the entire microtubule trajectory.