Microtubules are dynamic polymers of the cytoskeleton, which play important roles in cell division, polarization, and intracellular transport. Self-assembly of microtubule polymer from αβ-tubulin heterodimers is highly variable, with stochastic switching between alternate states of net growth and net shortening, a phenomenon known as dynamic instability. Microtubule tip structures are also variable and directly influence the kinetics of assembly and vice versa. TipTracker, a semiautomated, image processing-based tool, permits high spatial and temporal resolution measurements from fluorescence microscopy images (~ 10-40 nm, or 1-5 dimer lengths, at 1-10 Hz) with simultaneous tip structure estimation. We provide a walkthrough of the TipTracker code to demonstrate methods used to (1) fit the coordinates of the microtubule backbone; (2) track microtubule tip position; and (3) estimate tip structure from the spatial decay of the tip fluorescence distribution, discuss possible sources of error, and include an example protocol for nanometer-scale tip tracking in living cells. Additionally, we evaluate TipTracker's accuracy on simulated digital images and fixed microtubules to estimate accuracy under realistic imaging conditions. In summary, this chapter demonstrates the use of TipTracker in making robust, high-resolution measurements of microtubule tip dynamics and structures, facilitating quantitative investigations into nanoscale/molecular control of microtubule assembly. Although our primary focus is on microtubules, these methods are, in principle, suitable for other polymer structures, such as F-actin.
|Original language||English (US)|
|Title of host publication||Reconstituting the Cytoskeleton|
|Publisher||Academic Press Inc.|
|Number of pages||18|
|State||Published - 2014|
|Name||Methods in Enzymology|
Bibliographical noteFunding Information:
We would like to thank Dr. Patricia Wadsworth (University of Massachusetts) for sharing the LLC-PK1α cells used to generate data for figures. L. S. P. is supported by a 3M Science and Technology Doctoral Fellowship and B. T. C. was supported by NIH fellowship T32 EB008389. This work was supported by NIH Grants R01 GM71522 and R01 GM76177 to D. J. O. and R01 GM103833 to M. K. G.
- Fluorescent proteins
- Image processing