Recent advances in fluorescent imaging and microscopy are allowing for better imaging of microtubule dynamics than ever before. Here, Dr. Ross and colleagues have collected the most effective techniques currently in use, and the major findings for each method to date. This comprehensive review was features as an Integrative Biology HOT article.
Microtubules are the stiffest of the three types of cytoskeletal filaments found in eukaryotic cells, and can be thought of as the “bones” of the cell. They are a crucial participant in cell division, and both push the chromosomes into alignment, and later pull them apart during anaphase. This involvement in cell division makes microtubules a target for several anti-cancer medications currently in use. In addition to being able to withstand and apply cellular forces, microtubules have been shown to undergo rapid switching between growth and shrinkage stages, called dynamic instability. The flexibility and structural properties of microtubules determines their functional roles in cells, and makes them a particularly compelling and challenging system to study. Imaging dynamic instability is therefore of paramount importance for elucidating the details of microtubule behavior in cells.
Transmitted light microscopy methods such as dark-field imaging and video-enhanced differential interference contrast (VE-DIC) led to the first observation of dynamic instability in 1986 by the Hotani group. They were able to observe individual microtubules growing and shrinking (at ~25 nm in diameter, microtubules are the smallest object that can be visualized).
Fluorescence microscopy can also be used both in living cells and in vitro, through different techniques and using different fluorescent labels. In cells, microtubules can be labeled with green fluorescent protein (GFP) and this has been used to image microtubule turnover, though there are limitations due to photobleaching under imaging conditions and spatial resolution in the crowded cellular environment.
For in vitro measurements, total internal reflection fluorescence (TIRF) microscopy is used. This technique provides better temporal resolution, and the ability to visualize microtubules and their associated proteins simultaneously with different fluorescent dyes. The determination that kinesin motors can either diffuse or walk towards the microtubule end was made using TIRF.
Another technique that has proven very useful is force microscopy. In this set-up the microtubule is fixed at one end and allowed to grow towards a barrier or trap. The force exerted by the microtubule during the growth or shrinkage stage can then be measured. This force has been determined to be on the pN scale, which is biologically relevant, and explains how microtubules can exert the necessary forces to align and then separate the chromosomes during cell division.
In future, techniques offering better resolution of microtubules both in living cells and in vitro could provide answers to many open questions, such as how the dynamics of microtubules are tuned in crowded living cells by both stabilizing and destabilizing associated proteins.
Modern methods to interrogate microtubule dynamics, Megan Bailey, Leslie Conway, Michael W. Gramlich, Taviare L. Hawkins and Jennifer L. Ross, Integr. Biol., 2013,5, 1324-1333. DOI: 10.1039/C3IB40124C