The quantification and regulation of microtubule dynamics in the mitotic spindle

Abstract

Microtubules play essential roles in cellular organization, cargo transport, and chromosome segregation during cell division. During mitosis microtubules form a macromolecular structure known as the mitotic spindle that is responsible for the accurate segregation of chromosomes between the two daughter cells. This is accomplished thanks to finely tuned control of microtubule dynamics. Even small changes in microtubule dynamics during spindle formation and/or operation may lead to chromosome mis-segregation, chromosome instability and aneuploidy. These three events are directly correlated with human diseases like cancer and developmental defects. Precise measurements of microtubule dynamics in the spindle will allow us to discover new molecules involved in regulating microtubule dynamics and enable a deeper understanding of the mechanisms that underlie mitosis and cancer emergence and development. Moreover, many chemotherapeutic agents for cancer treatment are targeted to microtubules, so continued investigation of their dynamics with utmost precision will facilitate the development of new drugs. Measuring microtubule dynamics in the spindle has been a difficult task until recently. With the development of new and gentler microscopic techniques, and new computer programs, we can perform better and more accurate measurements of microtubule dynamics during mitosis.

Figure 1:

Overview of spindle microtubule dynamics. (a) Diagram depicting dynamic microtubule behaviors in the spindle that contribute to microtubule turnover. (b) Diagram depicting a selection of MT regulators and their proposed region of action within the spindle (c) Mitotic cell showing microtubules (green) and chromosomes (magenta). (d) Live mitotic spindle expressing EB3-GFP. Projected temporal color overlay in which blue, green and red are successive one second time points.

Figure 2:

Quantification of Microtubule polymerization rates in mitotic spindles. (a) Screenshot of a mitotic HeLa cell transfected with EB3-GFP. (b) To measure MT polymerization rates using a kymograph the cell is rotated until both centrosomes are in the same horizontal line. A rectangle around the centrosomes is drawn to obtain a kymograph. (c) Kymograph of the rectangle shown in (b). The horizontal line represents distance (in μm) and the vertical line time (in seconds). Once we apply the “Directionality” plugin to the region between the centrosomes (blue square), we get a new window with the same kymograph with different color lines. The different colors represent the different angles of the kymograph lines. (d) Histogram with the frequency distribution of the angles obtained from (c).

Figure 3:

Symmetric and asymmetric monopolar spindles. HeLa cells treated with Monastrol for 5 hours, fixed and labeled for DNA (blue), Tubulin (green) and Pericentrin (red). Left: symmetric monopolar spindle with the centrosomes in the center of the cell. Right: asymmetric monopolar spindle with the centrosomes displaced from the center of the cell and closer to the cell membrane.