Measuring microtubule dynamics

Abstract

Microtubules are key players in cellular self-organization, acting as structural scaffolds, cellular highways, force generators and signalling platforms. Microtubules are polar filaments that undergo dynamic instability, i.e. transition between phases of growth and shrinkage. This allows microtubules to explore the inner space of the cell, generate pushing and pulling forces and remodel themselves into arrays with different geometry and function such as the mitotic spindle. To do this, eukaryotic cells employ an arsenal of regulatory proteins to control microtubule dynamics spatially and temporally. Plants and microorganisms have developed secondary metabolites that perturb microtubule dynamics, many of which are in active use as cancer chemotherapeutics and anti-inflammatory drugs. Here, we summarize the methods used to visualize microtubules and to measure the parameters of dynamic instability to study both microtubule regulatory proteins and the action of small molecules interfering with microtubule assembly and/or disassembly.

Keywords: +TIPs; EB3; dynamic instability; kymograph; microtubules; tubulin.

Figure 1. Microtubule dynamic instability

(A) Microtubules are tubular filaments assembled from αβ-tubulin heterodimers arranged head-to-tail in 13 helically arranged protofilaments. Microtubules grow by the addition of GTP-tubulin (green) to their ends. GTP hydrolysis occurs when β-tubulin is buried in the lattice. Catastrophe, i.e. transition to shrinkage occurs when the GTP cap is lost and GDP-tubulin is exposed at the microtubule end. The transition from shrinkage to growth is called rescue. Note that α-tubulin contains a non-exchangeable GTP trapped at the intradimer interface. (B) DIC images from a time series of microtubules growing from an axoneme (large object at bottom of image). Imaging data courtesy of Douglas Drummond and Rob Cross. Abbreviation: DIC, differential interference contrast.

Figure 2. Microtubule dynamics measurement in vitro

(A) Typical TIRF-based assay to reconstitute and measure microtubule dynamics in vitro in which a GMPCPP-stabilized microtubule seed is immobilized on a passivated glass surface that nucleates a microtubule. The seed and free tubulin typically contain 5–20% tubulin that is labelled with different fluorophores. Microtubule-binding proteins or small molecules can be added and their binding as well as effect on microtubule dynamics measured in this assay. (B) Montage and life history plot of a dynamic microtubule with seed (red), dynamic microtubule lattice (blue) and microtubule tip labelled with EB3-GFP (green). Phases are labelled as growth and shrinkage, transitions marked as catastrophe and rescue. (C) Workflow to measure microtubule dynamics parameters including the generation of kymographs (space–time plots), automatic or manual detection of the edge and extraction of speed and transition rates. The final violin plots are vertical histograms that show the full distribution of growth and shrinkage speeds that make up the median, which is also depicted by an inset boxplot.

Figure 3. Microtubule dynamics measurement in cells

(A) Widefield image of a human hTERT RPE1 cell expressing GFP-tubulin. Individual microtubules can be observed, especially close to the cell cortex (inset). (B) Widefield image of Ustilago maydis cell expressing YFP-tubulin (grey) and histone4-CFP (blue). Individual microtubules are visible throughout the cell, enabling measurement of four parameters of dynamics instability. Diamond graphs show growth speed (right), shrinkage speed (left), rescue frequency (top) and catastrophe frequency (bottom). Data are plotted for a temperature-sensitive dynein mutant Dyn2ts and the respective control strain at permissive (22°C) and restrictive temperature (29°C) [89]. (C) Microtubule labelling using the ensconsin microtubule domain fused to three tandem copies of GFP [49] results in higher signal noise than direct tubulin labelling in the same cell line as shown in (A). Measurement of microtubule dynamics by recording the length at different time points is indicated in the zoomed section for one growing microtubule. (D) Example of an hTERT RPE1 cell expressing EB3-tdTomato that has been analysed using plusTipTracker [79]. Tracks are presented colour-coded indicating average speed, and according to directionality relative to long axis of the cell [90].