Organisation of the microtubule cytoskeleton
Microtubules make up the backbone of the cellular cytoskeleton. Tips of the microtubules work as molecular machines that can generate pushing and pulling forces when microtubules grow or shrink respectively. In the past, we have made important contributions to understanding the fundamental mechanisms of how “naked” microtubule tips generate pulling and pushing forces. However, in cells, lattices and tips of microtubules are covered with microtubule and tip binding proteins.
Some of these proteins can harness microtubule-generated forces and convert them into movement and, as we have also shown in the past, rearrange the microtubule networks itself. The mechanisms underlying force generation by microtubule tips free of associated proteins are now beginning to be well understood, but how these forces are transduced to perform intracellular rearrangements remains largely unknown. We are currently investigating molecular mechanisms underlying force-harnessing activities of MT binding proteins and the role that microtubule tip-generated forces play in the organisation of the microtubule networks and intracellular structures.
Tools for probing mechanical forces
Being able to measure and apply forces acting on specific molecules in cells is important for understanding how specific force generating and sensing mechanisms function. However, measuring forces applied to specific molecules in the crowded cellular environment is challenging. State-of-the-art methods rely on delivering micron-sized beads inside the cells, which can be manipulated using lasers or magnets. But large beads experience significant viscous forces and interact with many unknown molecules at the same time, making the disentangling effects of individual mechanisms difficult.
We are interested in developing new techniques that would allow for precise manipulation and force measurement inside living cells in order to measure force contributions generated by specific molecular mechanisms. To this end, we take a multidisciplinary approach by using ideas from nanotechnology, force-spectroscopy, physical modelling and simulations, and collaborate with the London Centre of Nanotechnology. Our vision is that the new tools that we develop in combination with recent advances in molecular biology and gene editing will transform our ability to interrogate and manipulate cellular mechanics.
Biophysics of the genome
The spatial organisation, expression, repair and segregation of DNA depends on the multisubunit cohesin complex. In dividing cells, cohesin keeps the sister chromatids together until anaphase. In interphase cells, cohesin physically organises DNA by generating chromatin loops and topologically associated domains. Cohesin is a multiprotein ring-like complex, whose function is thought to rely on its ability to encircle DNA topologically and move along DNA. However, how cohesin mechanistically interacts with DNA at the molecular level and how it generates forces necessary to keep together and rearrange DNA is poorly understood.
In collaboration with Frank Uhlmann’s laboratory at the Francis Crick Institute and Jan-Michael Peters laboratory (IMP, Vienna, Austria) we investigate the dynamics of cohesin loading on DNA at single molecule level in real time and analyse mechanical transients associated with cohesin-DNA interaction. To this end, we use optical trapping and single molecule fluorescence to monitor structural changes in both cohesin and DNA associated with cohesin-DNA interaction using different externally applied forces. Our aim is to gain a fundamental insight into the mechanistic principles underlying organisation and segregation of genomes.