A new study has revealed insights into how new microtubules branch from the sides of existing ones. Researchers at Princeton University investigated proteins that control the formation of the thin, hollow tubes, which play an essential role in cellular structure and cell division. In a study published in the Journal of Cell Biology in March, the team found that one of these microtubule regulators—a protein called TPX2—controls the formation of new microtubule branches.
“TPX2 is often overexpressed in various cancers, and, in many cases, serves as a prognostic indicator,” said Raymundo Alfaro-Aco, a graduate student in the Department of Molecular Biology. Aco conducted the study with graduate student Akanksha Thawani in the Department of Chemical and Biological Engineering in the laboratory of Sabine Petry, assistant professor of molecular biology. “Therefore, elucidating the role of TPX2 in cell division in general can have important implications in our understanding of human diseases,” Alfaro-Aco said.
Microtubules are formed by the polymerization of two proteins, α- and ß-tubulin, but a third form of tubulin—γ-tubulin—helps to initiate (or “nucleate”) microtubule polymerization inside cells. γ-Tubulin combines with several other proteins to form γ-tubulin ring complexes (γ-TuRCs) that localize, for example, to the cell’s centrosomes, which nucleate and organize most of the microtubules that assemble into the mitotic spindle, the cellular structure that segregates chromosomes into newly forming daughter cells during cell division.
While a postdoc at the University of California-San Francisco, Petry demonstrated that spindles also contain microtubules that are nucleated from the sides of other microtubules (Petry et al., Cell. 152: 768-777, 2013). This “branching nucleation” process depends, in part, on a microtubule-binding protein called TPX2. Petry and Alfaro-Aco decided to investigate exactly how this protein stimulates branching microtubule nucleation.
To explore this question, the researchers used cell-free extracts prepared from frog eggs, which are capable of forming functional spindles in vitro, Alfaro-Aco said. “This powerful system allows us to easily add or remove factors, such as proteins or small molecules, to probe different aspects of spindle assembly,” he said. “Combining this extract system with a powerful imaging method — known as total internal reflection fluorescence microscopy — allows us to observe and measure microtubule events, such as nucleation, at the level of single microtubules.”
By adding different fragments of TPX2 to egg extracts and observing their effects on microtubules, Alfaro-Aco found that a fragment containing three of the protein’s seven alpha-helical domains was the smallest piece capable of stimulating branching microtubule nucleation.
This minimal fragment contained three short stretches of amino acids that are similar to sequences found in proteins that bind and activate γ-TuRC. The researchers found that deleting or mutating these sequences eliminated the TPX2 fragment’s capacity to stimulate microtubule branching, without affecting the protein’s ability to bind to microtubules.
The team also found that this region of TPX2 binds to γ-TuRC. Mutating the three sequences found in other γ-TuRC-binding proteins didn’t inhibit this interaction but, because these mutants no longer stimulate branching microtubule nucleation, Alfaro-Aco and colleagues think that the sequences are required to activate γ-TuRC. TPX2 may therefore bind to existing spindle microtubules and then bind and activate γ-TuRC to initiate the formation of a new microtubule branch. This process is crucial for spindle assembly and the accurate segregation of chromosomes.
This work was supported by the National Institutes of Health/National Institute of General Medical Sciences (grant # 4R00GM100013), the Pew Scholars Program in the Biomedical Sciences, the Sidney Kimmel Foundation, and the David and Lucile Packard Foundation. In addition, Alfaro-Aco received support from the Howard Hughes Medical Institute and the National Science Foundation.
Alfaro-Aco, R., A. Thawani, and S. Petry. Structural analysis of the role of TPX2 in branching microtubule nucleation. Journal of Cell Biology, 216: 983-997, 2017. DOI: 10.1083/jcb.201607060 | Published March 6, 2017.