Isabelle Arnal

Activity-Dependent Tau Protein Translocation to Excitatory Synapse Is Disrupted by Exposure to Amyloid-Beta Oligomers

Tau is a microtubule-associated protein well known for its stabilization of microtubules in axons. Recently, it has emerged that tau participates in synaptic function as part of the molecular pathway leading to amyloid-beta (Aβ)-driven synaptotoxicity in the context of Alzheimers disease. Here, we report the implication of tau in the profound functional synaptic modification associated with synaptic plasticity. By exposing murine cultured cortical neurons to a pharmacological synaptic activation, we induced translocation of endogenous tau from the dendritic to the postsynaptic compartment. We observed similar tau translocation to the postsynaptic fraction in acute hippocampal slices subjected to long-term potentiation. When we performed live confocal microscopy on cortical neurons transfected with human-tau-GFP, we visualized an activity-dependent accumulation of tau in the postsynaptic density. Coprecipitation using phalloidin revealed that tau interacts with the most predominant cytoskeletal component present, filamentous actin. Finally, when we exposed cortical cultures to 100 nm human synthetic Aβ oligomers (Aβos) for 15 min, we induced mislocalization of tau into the spines under resting conditions and abrogated subsequent activity-dependent synaptic tau translocation. These changes in synaptic tau dynamics may rely on a difference between physiological and pathological phosphorylation of tau. Together, these results suggest that intense synaptic activity drives tau to the postsynaptic density of excitatory synapses and that Aβo-driven tau translocation to the spine deserves further investigation as a key event toward synaptotoxicity in neurodegenerative diseases.

Tau co-organizes dynamic microtubule and actin networks

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.

MAP65/Ase1 promote microtubule flexibility

Two microtubule cross-linkers of the major MAP65/PRC1/Ase1 family are found to modify the mechanical properties of dynamic microtubules (e.g., decrease the flexural rigidity of microtubules). This finding points to a role for these proteins in the formation of specific microtubule arrays in eukaryotic cells.

Tau regulates the localization and function of End‐binding proteins 1 and 3 in developing neuronal cells

The axonal microtubule‐associated protein tau is a well‐known regulator of microtubule stability in neurons. However, the putative interplay between tau and End‐binding proteins 1 and 3 (EB1/3), the core microtubule plus‐end tracking proteins, has not been elucidated yet. Here, we show that a cross‐talk between tau and EB1/3 exists in developing neuronal cells. Tau and EBs partially colocalize at extending neurites of N1E‐115 neuroblastoma cells and axons of primary hippocampal neurons, as shown by confocal immunofluorescence analyses. Tau down‐regulation leads to a reduction of EB1/3 comet length, as observed in shRNA‐stably depleted neuroblastoma cells and TAU−/− neurons. EB1/3 localization depends on the expression levels and localization of tau protein. Over‐expression of tau at high levels induces EBs relocalization to microtubule bundles at extending neurites of N1E‐115 cells. In differentiating primary neurons, tau is required for the proper accumulation of EBs at stretches of microtubule bundles at the medial and distal regions of the axon. Tau interacts with EB proteins, as shown by immunoprecipitation in different non‐neuronal and neuronal cells and in whole brain lysates. A tau/EB1 direct interaction was corroborated by in vitro pull‐down assays. Fluorescence recovery after photobleaching assays performed in neuroblastoma cells confirmed that tau modulates EB3 cellular mobility. In summary, we provide evidence of a new function of tau as a direct regulator of EB proteins in developing neuronal cells. This cross‐talk between a classical microtubule‐associated protein and a core microtubule plus‐end tracking protein may contribute to the fine‐tuned regulation of microtubule dynamics and stability during neuronal differentiation. We describe here a novel function for tau as a direct regulator of End binding (EB) proteins in differentiating neuronal cells. EB1/3 cellular mobility and localization in extending neurites and axons is modulated by tau levels and localization. We provide new evidence of the interplay between classical microtubule‐associated proteins (MAPs) and “core” microtubule plus‐end tracking proteins (+TIPs) during neuronal development.

MAP6-F is a temperature sensor that directly binds to and protects microtubules from cold-induced depolymerization

Background: Microtubules are intrinsically cold-sensitive polymers, but cold-stable microtubules are observed in cells. Results: Progressive temperature-dependent conformational change in MAP6-F coincides with its binding to microtubules and with its microtubule cold stabilization activity. Conclusion: MAP6-F is a temperature sensor that protects microtubules from cold-induced depolymerization at temperatures ranging from 4 to 20 °C. Significance: This work provides a better understanding of cellular microtubule stabilization under hypothermic stress. Microtubules are dynamic structures that present the peculiar characteristic to be ice-cold labile in vitro. In vivo, microtubules are protected from ice-cold induced depolymerization by the widely expressed MAP6/STOP family of proteins. However, the mechanism by which MAP6 stabilizes microtubules at 4 °C has not been identified. Moreover, the microtubule cold sensitivity and therefore the needs for microtubule stabilization in the wide range of temperatures between 4 and 37 °C are unknown. This is of importance as body temperatures of animals can drop during hibernation or torpor covering a large range of temperatures. Here, we show that in the absence of MAP6, microtubules in cells below 20 °C rapidly depolymerize in a temperature-dependent manner whereas they are stabilized in the presence of MAP6. We further show that in cells, MAP6-F binding to and stabilization of microtubules is temperature- dependent and very dynamic, suggesting a direct effect of the temperature on the formation of microtubule/MAP6 complex. We also demonstrate using purified proteins that MAP6-F binds directly to microtubules through its Mc domain. This binding is temperature-dependent and coincides with progressive conformational changes of the Mc domain as revealed by circular dichroism. Thus, MAP6 might serve as a temperature sensor adapting its conformation according to the temperature to maintain the cellular microtubule network in organisms exposed to temperature decrease.

Tau antagonizes end-binding protein tracking at microtubule ends through a phosphorylation-dependent mechanism

Tau antagonizes tracking of end-binding proteins (EBs) at microtubule ends, a process requiring the C-terminal part of EBs and the microtubule-binding sites of tau. The inhibiting activity of tau on EB properties is regulated by tau phosphorylation. The interplay between EBs and tau proteins results in modulation of microtubule dynamics.

α-Synuclein is a Novel Microtubule Dynamase

α-Synuclein is a presynaptic protein associated to Parkinson’s disease, which is unstructured when free in the cytoplasm and adopts α helical conformation when bound to vesicles. After decades of intense studies, α-Synuclein physiology is still difficult to clear up due to its interaction with multiple partners and its involvement in a pletora of neuronal functions. Here, we looked at the remarkably neglected interplay between α-Synuclein and microtubules, which potentially impacts on synaptic functionality. In order to identify the mechanisms underlying these actions, we investigated the interaction between purified α-Synuclein and tubulin. We demonstrated that α-Synuclein binds to microtubules and tubulin α2β2 tetramer; the latter interaction inducing the formation of helical segment(s) in the α-Synuclein polypeptide. This structural change seems to enable α-Synuclein to promote microtubule nucleation and to enhance microtubule growth rate and catastrophe frequency, both in vitro and in cell. We also showed that Parkinson’s disease-linked α-Synuclein variants do not undergo tubulin-induced folding and cause tubulin aggregation rather than polymerization. Our data enable us to propose α-Synuclein as a novel, foldable, microtubule-dynamase, which influences microtubule organisation through its binding to tubulin and its regulating effects on microtubule nucleation and dynamics.

GDP-tubulin incorporation into growing microtubules modulates polymer stability

Microtubule growth proceeds through the endwise addition of nucleotide-bound tubulin dimers. The microtubule wall is composed of GDP-tubulin subunits, which are thought to come exclusively from the incorporation of GTP-tubulin complexes at microtubule ends followed by GTP hydrolysis within the polymer. The possibility of a direct GDP-tubulin incorporation into growing polymers is regarded as hardly compatible with recent structural data. Here, we have examined GTP-tubulin and GDP-tubulin incorporation into polymerizing microtubules using a minimal assembly system comprised of nucleotide-bound tubulin dimers, in the absence of free nucleotide. We find that GDP-tubulin complexes can efficiently co-polymerize with GTP-tubulin complexes during microtubule assembly. GDP-tubulin incorporation into microtubules occurs with similar efficiency during bulk microtubule assembly as during microtubule growth from seeds or centrosomes. Microtubules formed from GTP-tubulin/GDP-tubulin mixtures display altered microtubule dynamics, in particular a decreased shrinkage rate, apparently due to intrinsic modifications of the polymer disassembly properties. Thus, although microtubules polymerized from GTP-tubulin/GDP-tubulin mixtures or from homogeneous GTP-tubulin solutions are both composed of GDP-tubulin subunits, they have different dynamic properties, and this may reveal a novel form of microtubule “structural plasticity.”

Negative regulation of EB1 turnover at microtubule plus ends by interaction with microtubule-associated protein ATIP3

The regulation of microtubule dynamics is critical to ensure essential cell functions. End binding protein 1 (EB1) is a master regulator of microtubule dynamics that autonomously binds an extended GTP/GDP-Pi structure at growing microtubule ends and recruits regulatory proteins at this location. However, negative regulation of EB1 association with growing microtubule ends remains poorly understood. We show here that microtubule-associated tumor suppressor ATIP3 interacts with EB1 through direct binding of a non-canonical proline-rich motif. Results indicate that ATIP3 does not localize at growing microtubule ends and that in situ ATIP3-EB1 molecular complexes are mostly detected in the cytosol. We present evidence that a minimal EB1-interacting sequence of ATIP3 is both necessary and sufficient to prevent EB1 accumulation at growing microtubule ends in living cells and that EB1-interaction is involved in reducing cell polarity. By fluorescence recovery of EB1-GFP after photobleaching, we show that ATIP3 silencing accelerates EB1 turnover at microtubule ends with no modification of EB1 diffusion in the cytosol. We propose a novel mechanism by which ATIP3-EB1 interaction indirectly reduces the kinetics of EB1 exchange on its recognition site, thereby accounting for negative regulation of microtubule dynamic instability. Our findings provide a unique example of decreased EB1 turnover at growing microtubule ends by cytosolic interaction with a tumor suppressor.

A TIRF microscopy assay to decode how tau regulates EB’s tracking at microtubule ends

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.