Mechanism and Control of Microtubule Dynamic Instability Probed by in Vitro Reconstitutions and Microfluidics Approaches

Microtubules are non-covalent polymers that form an essential part of the cytoskeleton in eukaryotic cells. Alternating phases of growth and shortening are essential for space exploration, force generation and facilitate rearrangements of the microtubule cytoskeleton in response to various stimuli. Microtubule associated proteins regulate filament dynamics and can transport cargoes. The mechanism of how microtubules grow, what triggers the transition from a growing to a shrinking microtubule, and the interplay between the various microtubule-associated proteins is only poorly understood. In vitro reconstitution approaches from purified components in combination with microfluidics techniques and simultaneous multi-colour total internal reflection florescence microscopy were employed to shed new light on the mechanism of microtubule dynamics and the interplay of proteins that bind specifically to growing microtubule ends. Tubulin undergoes conformational changes during incorporation into the polymer. Using a conformation-sensitive designed ankyrin repeat protein probe, it has been found here that these conformational changes occur at much later steps during incorporation into the polymer than previously appreciated. Growing microtubules switch to a rapid shortening phase unless their ends contain a stabilizing structure whose nature is not fully understood. The decay of this stabilizing structure was directly measured by rapid tubulin dilutions and predictions from several theoretical models have been tested. The density of a particular tubulin conformation recognized by microtubule End Binding proteins (EB1/Mal3) could be linked to filament stability. Microtubule end tracking proteins form a dynamic protein interaction network. Here, the molecular mechanism of several main players of these proteins that lead to growing microtubule end accumulation of the motor protein dynein has been elucidated by in vitro reconstitutions. The bottom up approach applied in this thesis yielded new information about fundamental properties of microtubule dynamics and gained new insight into the interplay of an important class of microtubule associated proteins

Ensembles of Bidirectional Kinesin Cin8 Produce Additive Forces in Both Directions of Movement

Most kinesin motors move in only one direction along microtubules. Members of the kinesin-5 subfamily were initially described as unidirectional plus-end-directed motors and shown to produce piconewton forces. However, some fungal kinesin-5 motors are bidirectional. The force production of a bidirectional kinesin-5 has not yet been measured. Therefore, it remains unknown whether the mechanism of the unconventional minus-end-directed motility differs fundamentally from that of plus-end-directed stepping. Using force spectroscopy, we have measured here the forces that ensembles of purified budding yeast kinesin-5 Cin8 produce in microtubule gliding assays in both plus- and minus-end direction. Correlation analysis of pause forces demonstrated that individual Cin8 molecules produce additive forces in both directions of movement. In ensembles, Cin8 motors were able to produce single-motor forces up to a magnitude of ∼1.5 pN. Hence, these properties appear to be conserved within the kinesin-5 subfamily. Force production was largely independent of the directionality of movement, indicating similarities between the motility mechanisms for both directions. These results provide constraints for the development of models for the bidirectional motility mechanism of fission yeast kinesin-5 and provide insight into the function of this mitotic motor

A designed ankyrin repeat protein selected to bind to tubulin caps the microtubule plus end

International audienceMicrotubules are cytoskeleton filaments consisting of αβ-tubulin heterodimers. They switch between phases of growth and shrinkage. The underlying mechanism of this property, called dynamic instability, is not fully understood. Here, we identified a designed ankyrin repeat protein (DARPin) that interferes with microtubule assembly in a unique manner. The X-ray structure of its complex with GTP-tubulin shows that it binds to the β-tubulin surface exposed at microtubule (+) ends. The details of the structure provide insight into the role of GTP in microtubule polymerization and the conformational state of tubulin at the very microtubule end. They show in particular that GTP facilitates the tubulin structural switch that accompanies microtubule assembly but does not trigger it in unpolymerized tubulin. Total internal reflection fluorescence microscopy revealed that the DARPin specifically blocks growth at the microtubule (+) end by a selective end-capping mechanism, ultimately favoring microtubule disassembly from that end. DARPins promise to become designable tools for the dissection of microtubule dynamic properties selective for either of their two different ends

Mutations in Human Tubulin Proximal to the Kinesin-Binding Site Alter Dynamic Instability at Microtubule Plus- and Minus-Ends.

The assembly of microtubule-based cellular structures depends on regulated tubulin polymerization and directional transport. Here, we purify and characterize tubulin heterodimers that have human β-tubulin isotype III (TUBB3), as well as heterodimers with one of two β-tubulin mutations (D417H or R262H). Both point mutations are proximal to the kinesin-binding site and have been linked to an ocular motility disorder in humans. Compared to wild-type, microtubules with these mutations have decreased catastrophe frequencies and increased average lifetimes of plus- and minus-end-stabilizing caps. Importantly, the D417H mutation does not alter microtubule lattice structure or Mal3 binding to growing filaments. Instead, this mutation reduces the affinity of tubulin for TOG domains and colchicine, suggesting that the distribution of tubulin heterodimer conformations is changed. Together, our findings reveal how residues on the surface of microtubules, distal from the GTP-hydrolysis site and inter-subunit contacts, can alter polymerization dynamics at the plus- and minus-ends of microtubules

Mutations in Human Tubulin Proximal to the Kinesin-Binding Site Alter Dynamic Instability at Microtubule Plus- and Minus-Ends

The assembly of microtubule-based cellular structures depends on regulated tubulin polymerization and directional transport. Here, we purify and characterize tubulin heterodimers that have human β-tubulin isotype III (TUBB3), and heterodimers with one of two β-tubulin mutations (D417H or R262H). Both point mutations are proximal to the kinesin binding site and have been linked to an ocular motility disorder in humans. Compared to wild-type, microtubules with these mutations have decreased catastrophe frequencies and increased average lifetimes of plus- and minus-end stabilizing caps. Importantly, the D417H mutation does not alter microtubule lattice structure or Mal3 binding to growing filaments. Instead, this mutation reduces the affinity of tubulin for TOG domains and colchicine, suggesting that the distribution of tubulin heterodimer conformations is changed. Together, our findings reveal how residues on the surface of microtubules, distal from the GTP-hydrolysis site and inter-subunit contacts, can alter polymerization dynamics at the plus- and minus-ends of microtubules