Visualize and Analyze “Force” at Kinetochores

Project 1

Accurate cell division, which transmits replicated chromosomes equally into daughter cells, is an essential process to maintain life in all organisms. In order to achieve faithful chromosome segregation, microtubules must be properly attached to the kinetochore. The kinetochore is a macro-molecular protein architecture on centromere chromatin and serves as a platform for the microtubule assembly (Figure 1). The kinetochore structure is built by at least 26 different kinds of core-kinetochore proteins, with their multiple copies.

Important kinetochore functions are: 1) serving as a platform for microtubule binding on chromosome 2) force production and transmission, 3) mitotic checkpoint (SAC: Spindle Assembly Checkpoint) control, and 4) mitotic error correction. Force at kinetochore is critical for dynamic chromosome movements as well as kinetochore deformation in chromosome segregation process, but it requires technical advance to detect cellular tension when studying functions of force at kinetochores. We have recently succeeded in developing a sensitive device to detect tension at Ndc80 complex in budding yeast kinetochores.

Ndc80 complex, a highly conserved kinetochore MAP (microtubule associated proteins), is a hetero-tetramer protein complex (Ndc80 (also known as Hec1 (Highly Expressed in Cancer 1)), Nuf2, Spc24, and Scp25) and directly binds to microtubules at kinetochore through its N-terminal tail and CH domains. Ndc80 complex is thought to be a primary force coupler at kinetochores. A well-characterized FRET (Fluorescent Resonance Energy Transfer) probe was inserted into endogenous Ndc80 protein (Figure 2). Tension at Ndc80 complex was measured as a value of FRET emission ratio recorded by a calibrated light microscope. We found Ndc80 tension is highest in metaphase and lowest in late anaphase. In addition, tension at Ndc80 complex is changed depending on microtubule dynamic instability. A budding yeast kinetochore is not a simple force coupler, rather it acts as a complex force coupler by sharing a critical load between Ndc80 complex and other kinetochore MAPs, such as Dam1 complex and Stu2. Our next question is how human kinetochore generates and transmits forces for faithful chromosome segregation. We are trying to build a human Ndc80 tension biosensor in order to study functions of “force” at human kinetochores.

Related Publications:

Suzuki et al., Current Biology, 2018
Suzuki et al., Nature Cell Biology, 2016

Figure 1. Schematic and FRET images of Cell Cycle, Mitosis, and Kinetochore architecture.


Figure 2. Schematic of Ndc80 FRET tension biosensor.