"So we use plant cells as a model system instead," Dixit says. They don't have centrosomes; instead the microtubules nucleate at dispersed sites in the cell cortex, a layer of cytoplasm on the inner side of the plasma membrane. And when the cells are not dividing, the microtubules are plastered to the inside of the plasma membrane, where they're easily accessible and easy to image.
The cells Dixit's lab use are from a lineage of Arabidopsis plants created by Erica Fishel, PhD, then a WUSTL graduate student in biology, that express two fluorescent tags, or marker proteins. One colors the entire microtubule fluorescent green and the other marks its growing tip cherry red.
Quan Zhang, PhD, who was a postdoctoral research associate in the Dixit lab, crossed this marker line with an Arabidopsis mutant that does not produce the katanin enzyme.
While a WUSTL undergraduate, Tyler Bertroche (AB '11), generated a plant lineage where katanin is tagged with green fluorescent protein. Thanks to their efforts, the Dixit lab now has several different lineages of color-coded wild-type and katanin-mutant Arabidopsis.
Time is of the essence
A short movie clip shows a single severing event. The microtubule array is fluorescent green and the microtubules' growing tips are marked red. The white arrow follows the growing tip of one microtubule as it crosses over existing microtubules. A pink arrow appears when a crossover is cut. The microtubule disintegrates behind the cut and disappears.
"So how do cells pattern a microtubule array and how do different cells do it differently?" Dixit asks.
Microtubules originate in the cell cortex but once a cytoskeleton is established, they also sprout from existing microtubules in what is called branching nucleation.
Dixit and other scientists had shown
|Contact: Diana Lutz|
Washington University in St. Louis