The molecular machinery that propels cell division consists of parts that are, by now, fairly familiar. How the parts work together, generating and transmitting power as needed, is less clear. To see what really happens under the cell’s hood, researchers at Rockefeller University took a close look at a molecular motor called kinesin-5, as well as the microtubules with which kinesin-5 crosslinks. Kinesin-5 proteins and microtubules are important components of the mitotic spindle, which segregates chromosomes in dividing cells.
In a shop run by Tarun Kapoor, Ph.D., Rockefeller scientists essentially put the mitotic spindle up on the lift and ran some diagnostics. They attached microscopic plastic beads to a pair of microtubules linked by kinesin-5 molecules, then illuminated these spindle components with laser light. When they tracked the activity of these spindle components, the scientists discovered that when microtubule rods were aligned side by side but oriented in opposite directions, they were pushed so that they slid against each other, subject to a force that scaled with the length of microtubule overlap—the longer the overlap, the greater the force.
This result appeared September 28 in the journal Developmental Cell, in an article entitled, “Measuring Pushing and Braking Forces Generated by Ensembles of Kinesin-5 Crosslinking Two Microtubules.”
“Here, we employ an optical trap and total internal reflection fluorescence (TIRF)-based assay to show that ensembles of kinesin-5, a conserved mitotic motor protein, can push apart overlapping antiparallel microtubules to generate a force whose magnitude scales with filament overlap length,” wrote the authors. “We also find that kinesin-5 can produce overlap-length-dependent ‘brake-like’ resistance against relative microtubule sliding in both parallel and antiparallel geometries, an activity that has been suggested by cell biological studies but had not been directly measured.”
Ultimately, this work could have medical implications, since a better understanding of cell division could lead to new cancer therapies to hinder tumor cells' reproduction.
The mitotic spindle includes thousands of microtubules, rod-like structures with polarized tips, which biologists term “plus ends” and “minus ends.” Because microtubules exist in great numbers alongside each other, they tend to overlap and slide against each other. Around the spindle's center, they mostly exist in an anti-parallel configuration, in which the plus ends of neighboring rods point in opposite directions; toward its poles, the parallel configuration prevails, in which the plus ends of neighboring rods point in the same direction.
Curiously, the researchers found that kinesin-5 exerts different effects, depending on whether it links overlapping microtubules in antiparallel or parallel fashion. In the antiparallel case, pushing or resisting forces can be generated. In the parallel case, kinesin-5 generates a resisting force that can slow down the microtubules' motion. Here again, the force scales up with the length of overlap between the microtubules.
“We believe that kinesin-5 has the ability to coordinate the speed of microtubules and keep them from going too fast or too slow,” said the study’s co-first author Scott Forth, Ph.D., likening the protein to a gear box in a car. “It helps coordinate and govern the speed and location of the microtubules in the spindle.” As many kinesin-5 molecules work together directing microtubules, they become the governing force of the spindle formation.
“This work represents an important advance in our efforts to build, from the ground up, the dynamic spindle apparatus out of purified proteins,” concluded Dr. Kapoor, the study’s senior author. “It also helps reveal how nanometer-sized proteins work together to assemble complex cellular structures that are thousands of times larger than themselves.”