Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Abstract

Microtubules are polymers of αβ-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Figure 1:. Equipment for coverslip treatment and imaging chamber preparation

(A) slide-staining jar for 24×60 mm coverslips, (B) slide-washing racks for 18×18 mm coverslips, (C) vacuum set-up, (D) slide-drying rack, (E) hydration chamber, (F) coverslips, (G) imaging chamber, (H) slide holder

Figure 2:. Schematic for preparation of imaging chambers using double-sided tape (gray) and PEG/Biotin-PEG treated coverslips.

Created with BioRender.com.

Figure 3:. Schematic of addition of assay components to make and image fluorescently labeled bundles and single microtubules.

Biotinylated seeds are shown in blue, non-biotinylated seeds and soluble tubulin in red, PRC1 in black, protein of interest in cyan. Step numbers in figure correspond to those in Table 4. Panel corresponding to step 9 shows a pre-formed bundle (lower left); step 11 shows a newly formed bundle (upper left). Created with BioRender.com.

Figure 4:. Identification of single microtubules and crosslinked microtubules in the field of view.

Representative field of view showing 647 nm (left), 560 nm (center), and merged (right) channels. Single microtubules (yellow arrowheads) and bundles (white arrowheads) are indicated in the merged channel. Scale bar represents 2 μm.