Neuronal microtubule stabilization by Microtubule Inner Proteins (MIPs): deciphering the role of the universal microtubule-binding Mn motif – NEUROMIP

In neurons, stable microtubules (MTs) are essential for neuronal architecture and functions. Proteins located in the MT lumen, known as MT Inner Proteins (MIPs), are thought to contribute to neuronal MT stability, by analogy with the MIP-controlled extreme stability of MT doublets in cilia/flagella required to withstand high beating frequency. The presence of MIPs inside neuronal MTs has long been established, but their identities were unknown until we discovered the first as MAP6. MAP6 binds MTs via several copies of the Mn motif, which recently emerged as an evolutionarily conserved signature crucial for the binding of several MIPs inside the ciliary/flagellar MT lumen. The number, spacing and distribution of Mn motifs are observed to vary within this MIP family, but the functional role(s) of this versatility remain unknown.
With NEUROMIP, our objective is to determine how neuronal MIPs use the universal Mn motif to regulate MT stability and organization. To this end, we will draw on our unique expertise from working with MAP6 and MAP6D1, the only two Mn motif-containing MIPs identified so far in neurons. MAP6 and MAP6D1 display distinct Mn motif arrangements, and our recent results indicate that the two proteins stabilize and organize MTs in very different ways. Thus, MAP6 induces slow growth of helical MTs, whereas MAP6D1 promotes the assembly of highly stable MT doublets. We postulate that these distinct MT-regulatory activities are controlled by the different Mn motif arrangements in the two proteins, likely producing different MT-binding modes.
Using cell-free system reconstitutions and high-resolution cryo-electron microscopy and tomography approaches (cryo-EM & cryo-ET), we will determine how the Mn motif arrangements in MAP6 and MAP6D1 drive their luminal MT binding mode (e.g. recognition of a given subset of tubulin dimers and/or protofilaments) and regulatory functions (e.g. MT helices, MT doublets). In parallel, we will characterize the different MT assemblies induced by MAP6 and MAP6D1 in various neuronal compartments including axons, dendrites and primary cilia, using in situ cryo-ET applied to MAP6- and MAP6D1-deficient neurons, and comparing them to wild-type neurons. The ultrastructural information will be correlated with MT stability, neurite length/branching and cilia formation. Rescue experiments using relevant Mn motif-mutated MAP6/MAP6D1 will be used to determine whether specific Mn motif arrangements induce specific phenotypes. Finally, we will search for novel neuronal Mn motif-containing MIPs using proteomic and structural approaches applied to MTs extracted from neurons. Our results should thus contribute to the ongoing efforts of the scientific community to identify the full repertoire of MIPs in neurons.
To achieve this ambitious program, we have gathered a consortium of two teams with highly complementary and internationally-recognized expertise in cytoskeleton structure and dynamic properties (I Arnal team, GIN, Grenoble) and state-of-the-art cryo-EM and structural biology (G Schoehn team, IBS, Grenoble).
Overall, NEUROMIP will unravel how the universal Mn motif contributes to the MT-stabilizing functions of neuronal MIPs, and provide new insights into the as yet little-explored mechanisms through which MIPs control neuronal MT stability from the lumen. Combining results from this program with published works from cilia and flagella should improve our understanding of how various cell types use different Mn motif arrangements to produce distinct MT assemblies and stable networks. In the longer term, the structural characteristics of the Mn motifs bound to MTs may help guide the design of innovative drugs targeting the MT lumen (e.g., Mn-mimicking peptides or small compounds). Such molecules might be useful to preserve stable MTs and/or restore them as part of treatment for neurodegenerative and psychiatric diseases, or ciliopathies.