Checkpoint mechanism preventing errors in chromosome segregation revealed

When you travel from one country to another you must go through a “border checkpoint”, a place where you and your goods are inspected before you can go any further. The same control mechanism can also be found inside the cells. One of the most crucial checkpoints in life occurs during cell division (termed ‘mitosis’ in somatic cells and ‘meiosis’ in germ lines). Faithful segregation of the genetic material is so important that errors in the distribution of individual chromosomes can cause some of the most terrible human diseases. A single mistake in the segregation of chromosome 18 during meiosis is responsible for Edwards syndrome.

Kinetochores are mega-molecular assemblies formed at the centromeres of chromosomes at the onset of cell division. Successful completion of segregation requires that sister kinetochores become attached to spindle microtubules, which are responsible for chromosome movement into opposite poles of the cell. This controlled kinetochore-microtubule attachment step constitutes the Spindle Assembly Checkpoint (SAC), which relies on the kinetochore-localized protein kinase Mps1.

Until recently, a key unresolved question was how SAC prevents cell division to proceed until all kinetochores are attached to microtubules. In June 12th issue of Science, two independent studies were published which revealed that the key checkpoint protein Mps1 compete with microtubules for binding to Ndc80c^1,2, a major microtubule receptor complex localized at the kinetochore, thus monitoring its attachment to microtubules. Even though both papers conclusions overlap, they followed different analytical approaches, what makes them interesting to analyze and compare.

First, using MicroScale Thermophoresis (MST), both groups demonstrated a direct interaction between Mps1 and Ndc80c with µM binding affinities in perfect agreement, and also corroborated that this interaction occurs in cells.

In a more detailed analysis, Ji et al. showed that Mps1 interacts directly with Ndc80c through two distinct motifs: NTE and MR motifs on Mps1 bound to Hec1 and Nuf2 subunits of Ndc80c, both of which contain binding sites for microtubules.

Moreover, both studies revealed that this interaction is phosphorylation-dependent: using MST, Hiruma et al. showed that phosphorylation at the NTE domain of Msp1 increases the affinity of this interaction at least 20 times, while Ji et al. showed that phosphorylation at the middle region MR domain of Msp1 increases affinity by a factor of 4, as measured by Isothermal Titration Calorimetry.

The main conclusion of both studies, regarding the competition of Mps1 and microtubules for binding to Ndc80c, was reached by two different approaches. On one hand Ji et al. analyzed the release of Ndc80c protein bound to beads containing Mps1 fragments by microtubules, which was further resolved with SDS-PAGE and quantified by immunoblot. On the other hand, Hiruma et al. used MST and analyzed the resulting binding curves for Ndc80c titrated against fluorescent labeled Mps1 fragments alone or with the addition of microtubules. These results show that both, semi-quantitative and quantitative analytical procedures respectively are complementary.

Both studies revealed a mechanism for sensing kinetochore-microtubule attachment and how this interaction inhibit production of the anaphase inhibitor SAC. The proposed model below shows that there are two types of Mps1-Ndc80c interactions at kinetochores: a major one involving the NTE-Hec1 interface and a minor one involving the MR-Nuf2 interface. Binding of microtubules to Ndc80c releases both and inhibits Mps1 signaling, thus allowing cell division to proceed. Moreover, increasing phosphorylation of Hec1 by Aurora B, progressively weakens MT binding³ and enhances Mps1 binding.

An interesting feature is that the weak, multisite Mps1-Ndc80c interactions explain the transient nature of Mps1 at kinetochores and the inability to detect these interactions in human cell lysates. Since MST quantifies interactions and provides binding affinities with high precision, this technology is perfectly suited to shed light on complex mechanisms such as chromosome segregation.

1.         Hiruma et al. Science. 2015 Jun 12; 348(6240): 1264-7

2.         Ji et al. Science. 2015 Jun 12; 348(6240): 1260-4

3.         Zhu et al. J Biol Chem. 2013 Dec 13; 288(50): 36149-59