Boulder Physicist Meredith Betterton Finds the “Gearshift” on Cellular Motor Proteins
A comparison of the kinesin-5 motor proteins' movements across microtubules in the cell. The lower set of kinesin proteins have been phosphorylated and move at a slower rate than their unmutated counterparts. Image credit: Kenna Hughes-Castleberry
In a newstudy, Boulder Physics Professor Meredith Betterton and her team explored a key motor protein in cells called kinesin-5, which helps organize the cell division machinery, and they discovered how a small chemical modification plays a big role in controlling its power.
In our cells, kinesins act like tiny “motors” to help move chromosomes, the packages of our genetic information, to opposite sides of the cell so it can split. Kinesins propel across a larger cellular structure—the mitotic spindle—made of long, thread-like filaments called microtubules. Kinesin-5 helps these filaments slide against each other, building a sturdy structure that pulls chromosomes into place.
“It’s a bit like having a crane that moves very heavy objects, despite being quite small itself,” Betterton explains.
For this process to work, kinesin-5 must apply just the right amount of force. Too little force and the spindle won’t form properly, but too much force can cause damage. It’s a balancing act, and this new study reveals how cells achieve this balance.
“Imagine a city full of moving parts—traffic lights, cars, pedestrians—all needing to be in sync to avoid chaos,” says Betterton. “Similarly, cells have systems in place to control these movements. This research helps us understand how those controls work to keep our cells, and us, healthy.”
A Deeper Dive into Phosphorylation
Previous research shows that one way that cells control kinesin-5’s activity is through a process called phosphorylation, where a small chemical group is added to the kinesin-5 protein. This modification acts like a “gear shift” for the protein, helping it adjust its activity levels.
“Think of it as putting the motor into the right gear, like a car,” adds Betterton.
For kinesin-5, scientists have found nine different areas on the “tail” of the kinesin-5 protein that can be phosphorylated. While one or two of the sites have been heavily studied, Betterton and her team decided to take a different approach, focusing on all nine sites at once, wondering if these modifications are the key to controlling the motor’s power.
“In some cases, proteins are affected by only a single phosphorylation site really dramatically, but that hadn't been seen in kinesin-5 motor proteins,” Betterton explains. “But in other cases, there can be multiple sites that somehow work together, which is called combinatorial phosphoregulation. So, we decided to kind of go for what would be the biggest change we could potentially make, to mutate all of them together. By altering all nine sites, we hoped to understand how much it would affect kinesin-5’s ability to move and organize the spindle.”
Modifications, Models, and Microscopy
The team created two types of mutations to investigate the role of phosphorylation in the kinesin-5 protein. The first mutation, known as the alanine or “9A” mutation, replaced nine phosphorylation sites with alanine, a non-phosphorylatable amino acid, effectively blocking phosphorylation at these locations.
The second mutation, called the aspartate or “9D” mutation, substituted the same sites with aspartate, an amino acid that partially mimics phosphorylation by carrying a similar negative charge. These two mutations allowed the team to compare the effects of blocking versus mimicking phosphorylation on kinesin-5's motor function and force generation during spindle assembly. By tracking these altered proteins with a green fluorescent protein (GFP) tag, the team watched their behavior in live cells under a microscope.
The team also built computer models and ran theoretical simulations to test whether these phosphorylation changes matched the effects they saw in the cells.
“The fact that we’re able to use not only fluorescent microscopy but also computer modeling and theoretical simulations is something that has developed over the course of my career,” Betterton adds. “When I started in the department more than 20 years ago, I only did simulations and theory work. I started this experimental part of my lab after I got tenure with support from the physics and MCDB [the Molecular, Cellular & Developmental Biology] departments. The fact we’ve been able to do work like this and have papers like this is exactly the new direction I wanted my lab to take.”
From their multidisciplinary work, the researchers saw that cells without properly phosphorylated kinesin-5 had a hard time forming the usual spindle structures. Normally, about 80% of cells show microtubule sliding in the spindle that can form protrusions, but when the phosphorylation was blocked, very few cells showed proper sliding.
“This dramatic drop really showed us how essential phosphorylation is for building a functional spindle,” said Betterton. “Without this fine-tuning, the whole system fails.”
The results confirmed that phosphorylation allows kinesin-5 to apply the right amount of force as the cell needs it, ensuring a stable, functioning spindle for cell division.
Looking at the Bigger Implications for Human Health
These findings have potential real-world implications for health. Problems in cell division are a hallmark of many diseases, including cancer, where cells divide uncontrollably.
“Understanding how motor proteins are controlled gives us insights into diseases where this regulation fails,” said Betterton.
This study may open up new strategies for cancer treatments that target proteins like kinesin-5 to interrupt abnormal cell division.