Conformational adaptation of the microtubule lattice to mechanical forces and biochemical interactions.

• Applicant: Serapion Pyrpassopoulos
• Subject Area: Biophysics ; Cell Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 527934150

Microtubules are cytoskeletal filaments that span the cell and, among other functions, provide tracks for molecular motor proteins (motors). Since microtubules are considered stiff, they are thought to be passive substrates upon which motors move and microtubule-associated proteins (MAPs) bind to regulate intracellular trafficking and microtubule stability. Interestingly recent work shows that the microtubule lattice has remarkable structural plasticity with its conformation being modulated by the binding of MAPs and motors. Motor-induced changes can be felt microns away by MAPs and motors implying that the microtubule lattice adapts to binding interactions. However, adaptation to mechanical forces is poorly understood. In particular, whether and how mechanical forces can induce microtubule lattice changes that in turn modulate the binding interface and therefore the binding affinity of MAPs and motors, is unclear. I recently discovered that the interaction between kinesin-1 and microtubules under opposing external forces may be characterized as a slip, ideal, or catch bond. This surprisingly variable behavior depended on whether the opposing tensile force on the plus end of the microtubule is applied to the same protofilament that kinesin-1 is pulling on or a different protofilament. These data imply that tensile forces may deform the microtubule lattice and subsequently its binding interface with kinesin-1. In this proposal therefore I will develop experimental assays to apply tensile forces and mechanical perturbations on single microtubules using dual-beam optical tweezers combined with high-contrast fluorescence and label-free microscopy. Decoration of the microtubules with quantum dots will allow to measure with nanometer precision the induced mechanical distortions across the microtubule lattice. Furthermore, I will measure the lattice response time and its elasticity and plasticity as a function of force. The effect of induced distortions on the interaction with soluble mammalian and plant MAPs and motors will then be assessed by measuring the association and dissociation kinetics of fluorescent single kinesin-1 motors and MAPs. Finally, how saturating MAP concentrations affect the force sensing ability of the microtubule lattice and its structural conformation, e.g., expansion or compaction, will also be measured. Together, these experiments will provide novel insights in the ability of microtubules to function as sensors and transducers of mechanical and biochemical cues between distant parts of the cell. 2561 char, 363 words.

• DFG Programme: Research Grants