Cooperativity and forces in molecular interactions governing chromosome stability
Lead Research Organisation:
Queen Mary University of London
Department Name: Sch of Biological & Behavioural Sciences
Abstract
Living cells constantly rearrange themselves; this way they can adapt to the changing environment, and eventually divide and transfer their genetic material to daughter cells. These processes all require mechanical force to be applied by the cells to their contents in a precise manner. Some of the intracellular movements are fuelled by microtubules, protein polymers that can grow and shorten, and pull on parts of the cell with their ends. Pulling forces provided by microtubules are especially important for division of the cells, when two copies of the genetic material, DNA compacted into chromosomes, need to be physically separated in space. How chromosomes keep attached, or coupled, to microtubule ends which are falling apart as they shorten, is poorly understood. Protein components that are important for this 'coupling' are present in multiple copies in a kinetochore, a structure that binds chromosomes to microtubules. Keeping the copy number of kinetochore proteins in balance prevents chromosome loss, while deletions and mutations in these proteins are associated with cancer. We have evidence that identical proteins in the kinetochore interact with each other, but the mechanisms of these interactions are challenging to study in living cells.
To understand how kinetochore proteins team up to properly attach chromosomes to microtubules, we will recreate these attachments using purified components in vitro. We will focus on two components of human kinetochore: Ndc80 complex, which cross-links kinetochores and microtubule ends, and Ska complex that dynamically accumulates at the Ndc80-microtubule interface and stabilizes it. Both Ska and Ndc80 are essential for cell viability, and both of them are present at kinetochore in multiple copies. Using light and electron microscopy, we will determine domains of the Ska complex that are important for interactions between neighbouring Ska molecules. By mutating these domains, we will distinguish Ska:Ska interactions from Ska-microtubule and Ska:Ndc80 interactions, leading to a better understanding how accumulation of Ska is specifically happening at properly formed chromosome-microtubule attachments.
Once we have identified the interactions that control Ska accumulation, we will study how Ska and Ndc80 interact with each other and with themselves as microtubules pull on kinetochores. Using advanced light microscopy techniques, we will study accumulation of Ska and Ndc80 at the site of force generation. We will also study Ska with impaired self-interactions to further understand the mechanism of Ska-mediated stabilization of chromosome-microtubule attachments.
In this work, we focus on two components of the human kinetochore. However, many kinetochore-microtubule interactions are conserved in other species. The resulting data will allow us to start investigating how force influences the composition and performance of kinetochores. Detailed understanding of the mechanism of cell division will help researchers to design more specific treatments that disrupt cell division, for example drugs that stop cancer cells from proliferating.
To understand how kinetochore proteins team up to properly attach chromosomes to microtubules, we will recreate these attachments using purified components in vitro. We will focus on two components of human kinetochore: Ndc80 complex, which cross-links kinetochores and microtubule ends, and Ska complex that dynamically accumulates at the Ndc80-microtubule interface and stabilizes it. Both Ska and Ndc80 are essential for cell viability, and both of them are present at kinetochore in multiple copies. Using light and electron microscopy, we will determine domains of the Ska complex that are important for interactions between neighbouring Ska molecules. By mutating these domains, we will distinguish Ska:Ska interactions from Ska-microtubule and Ska:Ndc80 interactions, leading to a better understanding how accumulation of Ska is specifically happening at properly formed chromosome-microtubule attachments.
Once we have identified the interactions that control Ska accumulation, we will study how Ska and Ndc80 interact with each other and with themselves as microtubules pull on kinetochores. Using advanced light microscopy techniques, we will study accumulation of Ska and Ndc80 at the site of force generation. We will also study Ska with impaired self-interactions to further understand the mechanism of Ska-mediated stabilization of chromosome-microtubule attachments.
In this work, we focus on two components of the human kinetochore. However, many kinetochore-microtubule interactions are conserved in other species. The resulting data will allow us to start investigating how force influences the composition and performance of kinetochores. Detailed understanding of the mechanism of cell division will help researchers to design more specific treatments that disrupt cell division, for example drugs that stop cancer cells from proliferating.
Technical Summary
Forces generated by dynamic microtubules are crucial for proper inheritance of genetic material during cell division. These forces are transmitted to chromosomes by multi-protein complexes, kinetochores. In humans, the two principal force-transmitting protein complexes are Ndc80 and Ska. Both are present at kinetochore at multiple copies, but it is unclear how does the force transmission and multivalency influence each other, and whether cooperative interactions play a physiologically important role.
We will use a combination of in vitro reconstitution, single-molecule force measurement, electron cryo-tomography, and cell biology to understand how cooperative binding of Ska to multiple microtubule end-bound Ndc80 molecules affects the force that microtubules generate, and the shapes of their ends. One of the important experimental tools that we will use is DNA origami-based single-molecule force sensor we have recently developed to simultaneously probe microtubule-generated force, and the oligomeric state of the protein complexes at the site of force transmission. Functional relevance of Ska mutants with reduced cooperativity will be validated in vivo, focusing on efficiency of formation of end-on kinetochore-microtubule attachments, and sensitivity of Ska-mediated cell cycle arrest to inhibitors of regulatory kinases. Finally, effects of Ska cooperativity on the shapes of dynamic microtubule ends will be studied using a combination of cryoET and neural network-driven denoising methods that we recently employed to segment unique flexible protein structures.
We will use a combination of in vitro reconstitution, single-molecule force measurement, electron cryo-tomography, and cell biology to understand how cooperative binding of Ska to multiple microtubule end-bound Ndc80 molecules affects the force that microtubules generate, and the shapes of their ends. One of the important experimental tools that we will use is DNA origami-based single-molecule force sensor we have recently developed to simultaneously probe microtubule-generated force, and the oligomeric state of the protein complexes at the site of force transmission. Functional relevance of Ska mutants with reduced cooperativity will be validated in vivo, focusing on efficiency of formation of end-on kinetochore-microtubule attachments, and sensitivity of Ska-mediated cell cycle arrest to inhibitors of regulatory kinases. Finally, effects of Ska cooperativity on the shapes of dynamic microtubule ends will be studied using a combination of cryoET and neural network-driven denoising methods that we recently employed to segment unique flexible protein structures.
