Image provided by Dr. Matthew Miller.
Cell division is one of the most basic processes of life, generating and extending the lifespan of the tissues and organs in your body and creating specialized cells for reproduction. In a typical cell division, a process called mitosis, the genetic material, DNA, is replicated and folded into structures called chromosomes that are equally distributed between two 'daughter' cells. Chromosomes are physically pulled into the daughter cells by dynamic polymers called microtubules. An array of microtubules emanates from each daughter cell, forming what is known as the mitotic spindle. A large protein complex called the kinetochore connects microtubules of the spindle to a single spot on each chromosome. Kinetochores must stay attached to microtubules, which are constantly moving, lengthening, and shortening. If the connection between the kinetochore and the microtubule is lost, the chromosome can get left behind, leading to cells with missing or extra chromosomes. Abnormal chromosome number is a hallmark of many human cancers. Thus, understanding how kinetochores function may lead to better therapeutics that can target the abnormal cell division machinery of cancer cells.
In a recent investigation published in Cell, postdoctoral researcher Dr. Matthew Miller in the Biggins Laboratory (Basic Sciences Division) identified a microtubule-binding protein that associates with kinetochores and strengthens their attachment to microtubules. The protein is called ch-TOG (colonic and hepatic tumor overexpressed gene) in human cells and is known as Stu2 (suppressor of tubulin 2) in budding yeast, a model organism used to study chromosome segregation.
Researchers in the Biggins Laboratory have developed methods to isolate kinetochores from yeast cells and study their properties in vitro. Dr. Miller found that the microtubule-associated protein Stu2 binds to the major microtubule-binding protein complex of the 'outer' kinetochore, Ndc80. Next, he wanted to test whether Stu2 contributed to the microtubule-binding function of the kinetochore. To accomplish this, he added a small tag called a degron onto the end of Stu2 that allows inducible degradation of the protein upon the addition of a drug. Indeed, he found that kinetochores lacking Stu2 could be purified and that the loss of Stu2 did not cause any changes to the levels of all of the other standard components of the kinetochore.
In collaboration with researchers in the laboratory of Dr. Charles Asbury at University of Washington Department of Biophysics, purified kinetochores can be attached to tiny glass beads and manipulated with a laser beam using a microscopic manipulation tool called an optical trap. The kinetochores, held by a laser beam, can be positioned along microtubules attached to a slide and then brought to the tip of the microtubule where they attach and can be pulled until the attachment is broken. Previously, researchers in the Biggins and Asbury Labs have measured that the average yeast kinetochore-microtubule connection can withstand up to 9 piconewtons (pN) of force before breaking. Dr. Miller found that kinetochores lacking Stu2 could only withstand 4 pNs of force before rupture. This demonstrates that Stu2 contributes significantly to the strength of the kinetochore connection with the microtubule.
Previous research uncovered that at a constantly applied high force, the lifetime of an attachment is actually significantly longer than the lifetime of an attachment held under constant low force. The researchers hypothesized that Stu2 may be responsible for this 'tension-selectivity' of kinetochores. Dr. Miller performed experiments measuring the lifetime of kinetochore attachments to microtubules at fixed forces ranging from 1-9pN. Strikingly, he found that at the constant low force of 1pN, kinetochores lacking Stu2 actually held on longer than wild-type kinetochores which have Stu2. Furthermore, at higher forces of 2-3pN, kinetochores lacking Stu2 had shorter microtubule-binding lifetimes than wild-type. Therefore, Stu2 appears to be responsible for the intrinsic preference of kinetochores for higher tension states by selectively destabilizing low-tension attachments and stabilizing high-tension attachments. It is widely thought that this tension selectivity stabilizes productive chromosome attachments where each copy of a chromosome is bound and held in a 'high tension' state by microtubules from the two opposing spindle poles. Conversely, when the two copies of each chromosome ('sisters') instead both bind microtubules from only one pole, these tensionless attachments need to be destabilized or missegregation will result.
"This is the first molecule identified that is involved in sensing tension to ensure cells inherit the right chromosomes so there is a lot of potential to exploit its functions for therapeutic benefit in the future," said Dr. Miller.
This research was supported by a Damon Runyon Cancer Research Fellowship, a Packard Fellowship, the National Institutes of Health, and Howard Hughes Medical Institute.
Miller MP, Asbury CLA, Biggins S. 2016. "A TOG Protein Confers Tension Selectivity to Kinetochore-Microtubule Attachments." Cell. 165, 1-12.