Structural Changes And Dynamics Of Disassembling Microtubules Revealed By Label-Free Super-Resolution Microscopy

Microtubules are a key part of the eukaryotic cytoskeleton typically composed of linear chains of head-to-tail connected tubulin dimers denoted as protofilaments that are connected laterally to form a hollow cylindrical filament. Due to highly dynamic ends, microtubules undergo alternating periods of assembly and disassembly, also known as dynamic instability, which is essential for a number of microtubule functions. Electron micrographs provide still images of the disassembling microtubule suggesting a structural transition at the microtubule tip characterized by disintegration of the lateral bonds of the tubulin lattice before the longitudinal depolymerization. However, the dynamics of this process is not understood and no method has been able to capture the structural transition associated with the disassembly process with sufficient spatiotemporal resolution. Here, we introduce label-free optical microscopy capable of selective imaging of the transient structural changes of protofilaments at the tip of a disassembling microtubule. Our method utilizes interferometric detection of two orthogonal polarizations of scattered light to measure the optical anisotropy of the imaged structure with the sensitivity of few tubulin dimers and framerates up to 10 000 frames per second. Upon induced disassembly, we observe the transition of ordered protofilaments at the tip into a disordered conformation also known as ram's horns. Our data suggest a correlation between the ratio of the protofilaments found in the curved state and the disassembly rate. Furthermore, super-resolution imaging of the unlabelled individual tubulin oligomers unbinding from the microtubule tip reveals transient pauses and relapses in the disassembly, concurrent with enrichment of ordered protofilament segments at the microtubule tip. Our findings show that microtubule disassembly is a discrete process and suggest a mechanism of switching from the disassembly to the assembly phase.