Linking Cellular and In Vitro Microtubule Behavior: Reconstitution Assays to the Rescue

The cytoskeleton is composed of intermediate filaments, the actin network and microtubules (MTs); it is a dynamic cellular web that serves multiple purposes which go beyond a mere structural function. The cytoskeleton allows complex morphological changes in cells, which are critical for cell specialization and functions.
This thesis describes how MT behavior can be influenced by subtle changes in its building block, the tubulin protein, and by a subset of MT-associated proteins (the so-called “+end-tracking proteins”, or +TIPs), which associate to the dynamic, fast-growing ends of MTs. The studies presented in this thesis widen our knowledge on MTs, tubulin and +TIPs.
Three major issues are discussed:
1) the discovery of novel tubulin-associated proteins (TAPs) as well as the effects of some tubulin mutations involved in neurodevelopmental disorders on the MT network stability in cells and on TAP binding;
2) the effects of these same mutations on tubulin structure within a protofilament, investigated in silico with molecular modelling and molecular dynamics simulations;
3) the regulation of MTs by +TIPs, in particular the cytoplasmic linker proteins (CLIPs).

Chapter 1 provides an overview of MT function, structure and behavior, together with the structural features, isotypes and post-translational modifications of its building block, the tubulin dimer. Since regulation of MTs is carried out by +TIPs, a description of the structure and role of the main ones presented in this thesis is also provided. Finally, a brief introduction to molecular dynamics simulations is given to guide the reading of Chapter 3.
Chapter 2 describes our strategy and studies centered on the identification of novel TAPs via purification of isotypes of α- and β-tubulin. We also report on the binding of TAPs to two tubulin mutants found in patients with neurodevelopmental disorders, and the effects of these mutations on the MT network in cells.
Chapter 3 describes the effects of the tubulin mutations (discussed in Chapter 2) on tubulin and protofilament structure at the atomic level, by using molecular modelling and molecular dynamics simulations. The model proposed helps in understanding possible mechanisms leading to tubulin/MT dysfunction in cells (Chapter 2).
Chapter 4 focuses on the roles in MT dynamics of the +TIP CLIP-115 compared to those of its close relative CLIP-170. These two proteins have always been considered redundant in function, but our studies suggest that they also have distinct properties.
Finally, Chapter 5 discusses the major findings presented in the other chapters, and some aspects of current methodologies commonly used in the field, such as in vitro reconstitution assays; the implications of the studies presented in this thesis and future prospects are addressed.