In neurons, microtubule networks alternate between single filaments and bundled arrays under the influence of effectors controlling their dynamics and organization. Tau is a microtubule bundler that stabilizes microtubules by stimulating growth and inhibiting shrinkage. The mechanisms by which tau organizes microtubule networks remain poorly understood. Here, we studied the self-organization of microtubules growing in the presence of tau isoforms and mutants. The results show that tau's ability to induce stable microtubule bundles requires two hexapeptides located in its microtubule-binding domain and is modulated by its projection domain. Site-specific pseudophosphorylation of tau promotes distinct microtubule organizations: stable single microtubules, stable bundles, or dynamic bundles. Disease-related tau mutations increase the formation of highly dynamic bundles. Finally, cryo-electron microscopy experiments indicate that tau and its variants similarly change the microtubule lattice structure by increasing both the protofilament number and lattice defects. Overall, our results uncover novel phosphodependent mechanisms governing tau's ability to trigger microtubule organization and reveal that disease-related modifications of tau promote specific microtubule organizations that may have a deleterious impact during neurodegeneration.

Microtubule and actin cytoskeletons are key players in vital processes in cells. Although the importance of microtubule-actin interaction for cell development and function has been highlighted for years, the properties of these two cytoskeletons have been mostly studied separately. Thus we now need procedures to simultaneously assess actin and microtubule properties to decipher the basic mechanisms underlying microtubule-actin crosstalk. Here we describe an in vitro assay that allows the coassembly of both filaments and the real-time observation of their interaction by TIRF microscopy. We show how this assay can be used to demonstrate that tau, a neuronal microtubule-associated protein, is a bona fide actin-microtubule cross-linker. The procedure relies on the use of highly purified proteins and chemically passivated perfusion chambers. We present a step-by-step protocol to obtain actin and microtubule coassembly and discuss the major pitfalls. An ImageJ macro to quantify actin and microtubule interaction is also provided.

Tau is a major microtubule-associated protein (MAP) mainly expressed in the brain. Tau binds the lattice of microtubules and favors their elongation and bundling. Recent studies have shown that tau is also a partner of end-binding proteins (EBs) in neurons. EBs belong to the protein family of the plus-end tracking proteins that preferentially associate with the growing plus-ends of microtubules and control microtubule end behavior and anchorage to intracellular organelles. Reconstituted cell-free systems using purified proteins are required to understand the precise mechanisms by which tau influences EB localization on microtubules and how the concerted activity of these two MAPs modulates microtubule dynamics. We developed an in vitro assay combining TIRF microscopy and site-directed mutagenesis to dissect the interaction of tau with EBs and to study how this interaction affects microtubule dynamics. Here, we describe the detailed procedures to purify proteins (tubulin, tau, and EBs), prepare the samples for TIRF microscopy, and analyze microtubule dynamics, and EB binding at microtubule ends in the presence of tau.

Proper regulation of microtubule dynamics is essential for cell functions and involves various microtubule-associated proteins (MAPs). Among them, end-binding proteins (EBs) accumulate at microtubule plus ends, whereas structural MAPs bind along the microtubule lattice. Recent data indicate that the structural MAP tau modulates EB subcellular localization in neurons. However, the molecular determinants of EB/tau interaction remain unknown, as is the effect of this interplay on microtubule dynamics. Here we investigate the mechanisms governing EB/tau interaction in cell-free systems and cellular models. We find that tau inhibits EB tracking at microtubule ends. Tau and EBs form a complex via the C-terminal region of EBs and the microtubule-binding sites of tau. These two domains are required for the inhibitory activity of tau on EB localization to microtubule ends. Moreover, the phosphomimetic mutation S262E within tau microtubule-binding sites impairs EB/tau interaction and prevents the inhibitory effect of tau on EB comets. We further show that microtubule dynamic parameters vary, depending on the combined activities of EBs and tau proteins. Overall our results demonstrate that tau directly antagonizes EB function through a phosphorylation-dependent mechanism. This study highlights a novel role for tau in EB regulation, which might be impaired in neurodegenerative disorders.

The crosstalk between microtubules and actin is essential for cellular functions. However, mechanisms underlying the microtubule-actin organization by cross-linkers remain largely unexplored. Here, we report that tau, a neuronal microtubule-associated protein, binds to microtubules and actin simultaneously, promoting in vitro co-organization and coupled growth of both networks. By developing an original assay to visualize concomitant microtubule and actin assembly, we show that tau can induce guided polymerization of actin filaments along microtubule tracks and growth of single microtubules along actin filament bundles. Importantly, tau mediates microtubule-actin co-alignment without changing polymer growth properties. Mutagenesis studies further reveal that at least two of the four tau repeated motifs, primarily identified as tubulin-binding sites, are required to connect microtubules and actin. Tau thus represents a molecular linker between microtubule and actin networks, enabling a coordination of the two cytoskeletons that might be essential in various neuronal contexts.

Les microtubules sont des éléments clés du cytosquelette impliqué dans de nombreux processus cellulaires. Ce sont des structures dynamiques qui alternent continuellement entre polymérisation et dépolymérisation, un comportement appelé instabilité dynamique. Les microtubules sont particulièrement abondants dans les neurones et sont organisés sous formes de faisceaux dans les axones et les dendrites. Cette organisation particulière leur permet de maintenir la forme de ses cellules hautement spécialisées et d’assurer le transport intracellulaire d’éléments essentiels dans l’ensemble des compartiments neuronaux. De nombreux facteurs participe à la régulation de l’arrangement des microtubules dans les neurones. Parmi ces facteurs, la protéine tau fait partie de la famille des protéines associées aux microtubules (ou MAPs) et est majoritairement neuronale. Tau est un agent pontant majeur des microtubules et est également connue pour stabiliser les microtubules en stimulant leur polymérisation et en inhibant leur dépolymérisation. Malgré de nombreuses études sur l’interaction de tau avec les microtubules, les mécanismes par lesquels cette MAP contrôle leur organisation spatiale restent élusifs. Pour répondre à cette question, nous avons reconstitué in vitro des réseaux de microtubules en présence de divers isoformes, fragments et mutants de tau. La capacité de tau à induire des faisceaux stables de microtubules dépend de deux hexa-peptides localisés dans son domaine de liaison aux microtubules, et est régulée par son domaine de projection N-terminal. Nos résultats montrent que la phosphorylation spécifique de certains sites de tau inhibe soit la formation de faisceaux soit la stabilisation des microtubules, produisant des populations composées de microtubules individuels stable ou de faisceaux dynamiques. De plus, des mutations de tau impliquées dans des démences apparentées à la maladie d’Alzheimer augmentent drastiquement la capacité de tau à former des faisceaux composés de microtubules très dynamiques. Pour finir, des expériences de cryo-microscopie électroniques indiquent que tau génèrent des défauts dans la paroi des microtubules. Ces défauts sont connus pour assouplir les microtubules et pourraient donc constituer un mécanisme structural primaire permettant leur déformation au cours de la formation de faisceaux. En conclusion, nos résultats mettent en évidence un nouveau mécanisme phospho-dépendant par lequel tau régule l’organisation de réseaux de microtubules. De plus, ce travail révèle comment des modifications anormales de tau, telles que des phosphorylations anormales ou des mutations, peuvent altérer l’organisation du cytosquelette dans les maladies neurodégénératives.