Meanwhile, scientists at the University of Manchester in England had observed that misaligned microtubules were severed at junctions where a growing microtubule crosses an existing microtubule.
Zhang in the Dixit lab began with these observations and devised experiments to clarify the molecular mechanisms underlying them. He showed that they are insensitive to the geometry of the crossover or the stoutness of the underlying microtubule bundle. Instead, all that mattered was time.
How rapidly crossovers were cut determined what sort of array formed. In the transverse arrays, microtubules were cut, on average, within 41 seconds of a crossover event. In net-like arrays, on the other hand, cutting was three times slower and not as tightly controlled.
Plants have several severing proteins that might cut microtubules, but of these, katanin was the likeliest suspect. And in the katanin mutant, none of the crossovers were severed, demonstrating that this enzyme is solely responsible for cytoskeleton patterning.
To make sure, the scientists tagged katanin green and the microtubules red. When the cell was color coded in this way, they were able to see that katanin almost always localized to crossover sites before they were severed.
Coming in from the cold
These experiments showed that katanin was responsible for maintaining array patterning, but does it also create the pattern in the first place? To find out, Zhang used a trick he found in the literature, Dixit says.
Microtubules are cold sensitive and fall apart when they are chilled. "So Quan would put cells in the freezer for four or five minutes, take a slide out, run to the microscope, and watch to see what happened as the cells warmed up," Dixit says.
In the wild-type cells, the microtubules quickly re-ap
|Contact: Diana Lutz|
Washington University in St. Louis