The kinetochore coordinates chromosome segregation at cell division, a process essential to cell viability. Accurate segregation depends on the kinetochore's ability to verify that all chromosomes are correctly attached to the spindle before it signals that their separation may begin. Mechanical cues are thought to relay global information to kinetochores about the attachment geometry of sister chromatids. However, we do not know what mechanical signatures the kinetochore listens for and how it processes mechanical signals. In large part, this is because we lack approaches to apply controlled forces on kinetochores inside cells. Here, we propose to develop approaches to rewire the cytoskeleton in real-time and hijack cellular mechanisms to generate force on kinetochores. We will exploit our recent findings on spindle self-organization to control cellular forces on a kinetochore in space, time, amplitude and direction, with the long-term goal of understanding how kinetochores integrate mechanical signals. We will use laser ablation to disengage and re- engage spindle forces where and when we want, and molecular engineering to recruit motors at the ablation site and select how strongly they pull and where they go. Using these tools, we will subject kinetochores to different mechanical input signatures, and measure kinetochore deformation and key signaling output responses in real time. We will thereby determine what mechanical signals inform the kinetochore's decision, and through which mechanistic intermediates these signals are processed. Finally, we will apply these approaches in different molecular backgrounds and cell lines to establish how robust mechanical signal processing is to kinetochore malfunctions associated with cancer. Together, this work will provide a basis for incorporating mechanical - not just biochemical - information in our understanding of signal processing at the kinetochore, and may as such inspire categorically new strategies and targets for cancer therapy. More broadly, the tools and framework we develop will make in vivo forces an experimentally tunable parameter, and empower us to probe how mechanical signals are processed for diverse cellular functions - and misprocessed in disease.
Chromosome segregation errors at cell division play a key role in diseases such as cancer. The work we propose will greatly enhance our understanding of how the cell listens for and processes mechanical signals to coordinate chromosome segregation - and how it fails to do so in disease. This will provide insight into how chromosome segregation errors occur, and provide new therapeutic targets and strategies.
