Inferring the Forces Controlling Metaphase Kinetochore Oscillations by Reverse Engineering System Dynamics.

Kinetochores are multi-protein complexes that mediate the physical coupling of sister chromatids to spindle microtubule bundles (called kinetochore (K)-fibres) from respective poles. These kinetochore-attached K-fibres generate pushing and pulling forces, which combine with polar ejection forces (PEF) and elastic inter-sister chromatin to govern chromosome movements. Classic experiments in meiotic cells using calibrated micro-needles measured an approximate stall force for a chromosome, but methods that allow the systematic determination of forces acting on a kinetochore in living cells are lacking. Here we report the development of mathematical models that can be fitted (reverse engineered) to high-resolution kinetochore tracking data, thereby estimating the model parameters and allowing us to indirectly compute the (relative) force components (K-fibre, spring force and PEF) acting on individual sister kinetochores in vivo. We applied our methodology to thousands of human kinetochore pair trajectories and report distinct signatures in temporal force profiles during directional switches. We found the K-fibre force to be the dominant force throughout oscillations, and the centromeric spring the smallest although it has the strongest directional switching signature. There is also structure throughout the metaphase plate, with a steeper PEF potential well towards the periphery and a concomitant reduction in plate thickness and oscillation amplitude. This data driven reverse engineering approach is sufficiently flexible to allow fitting of more complex mechanistic models; mathematical models of kinetochore dynamics can therefore be thoroughly tested on experimental data for the first time. Future work will now be able to map out how individual proteins contribute to kinetochore-based force generation and sensing.

Specific removal of TACC3-ch-TOG-clathrin at metaphase deregulates kinetochore fiber tension.

Microtubule-associated proteins of the mitotic spindle are thought to be important for the initial assembly and the maintenance of spindle structure and function. However, distinguishing assembly and maintenance roles for a given protein is difficult. Most experimental methods for protein inactivation are slow and therefore affect both assembly and maintenance. Here, we have used 'knocksideways' to rapidly (∼5 minutes) and specifically remove TACC3-ch-TOG-clathrin non-motor complexes from kinetochore fibers (K-fibers). This method allows the complex to be inactivated at defined stages of mitosis. Removal of TACC3-ch-TOG-clathrin after nuclear envelope breakdown caused severe delays in chromosome alignment. Inactivation at metaphase, following a normal prometaphase, significantly delayed progression to anaphase. In these cells, K-fiber tension was reduced and the spindle checkpoint was not satisfied. Surprisingly, there was no significant loss of K-fiber microtubules, even after prolonged removal. TACC3-ch-TOG-clathrin removal during metaphase also resulted in a decrease in spindle length and significant alteration in kinetochore dynamics. Our results indicate that TACC3-ch-TOG-clathrin complexes are important for the maintenance of spindle structure and function as well as for initial spindle assembly.

Super-resolution kinetochore tracking reveals the mechanisms of human sister kinetochore directional switching.

The congression of chromosomes to the spindle equator involves the directed motility of bi-orientated sister kinetochores. Sister kinetochores bind bundles of dynamic microtubules and are physically connected through centromeric chromatin. A crucial question is to understand how sister kinetochores are coordinated to generate motility and directional switches. Here, we combine super-resolution tracking of kinetochores with automated switching-point detection to analyse sister switching dynamics over thousands of events. We discover that switching is initiated by both the leading (microtubules depolymerising) or trailing (microtubules polymerising) kinetochore. Surprisingly, trail-driven switching generates an overstretch of the chromatin that relaxes over the following half-period. This rules out the involvement of a tension sensor, the central premise of the long-standing tension-model. Instead, our data support a model in which clocks set the intrinsic-switching time of the two kinetochore-attached microtubule fibres, with the centromeric spring tension operating as a feedback to slow or accelerate the clocks.