The diversity of microtubule functions is dependent on the status of tubulin C-termini. To address the physiological role of the C-terminal aromatic residue of α-tubulin, a tub1-Glu yeast strain expressing an α-tubulin devoid of its C-terminal amino acid was used to perform a genome-wide-lethality screen. The identified synthetic lethal genes suggested links with endocytosis and related processes. In the tub1-Glu strain, the routing of the v-SNARE Snc1 was strongly impaired, with a loss of its polarized distribution in the bud, and Abp1, an actin patch or endocytic marker, developed comet-tail structures. Snc1 trafficking required dynamic microtubules but not dynein and kinesin motors. Interestingly, deletion of the microtubule plus-end-tracking protein Bik1 (a CLIP170 ortholog), which is preferentially recruited to the C-terminal residue of α-tubulin, similarly resulted in Snc1 trafficking defects. Finally, constitutively active Rho1 rescued both Bik1 localization at the microtubule plus-ends in tub1-Glu strain and a correct Snc1 trafficking in a Bik1-dependent manner. Our results provide the first evidence for a role of microtubule plus-ends in membrane cargo trafficking in yeast, through Rho1- and Bik1-dependent mechanisms, and highlight the importance of the C-terminal α-tubulin amino acid in this process.

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.

Malformations of the human cerebral cortex can be caused by mutations in tubulins that associate to compose microtubules. Cerebral cortical folding relies on neuronal migration and on progenitor proliferation partly dictated by microtubule-dependent mitotic spindle positioning. A single amino acid change, F265L, in the conserved TUBB2B β-tubulin gene has been identified in patients with abnormal cortex formation. A caveat for studying this mutation in mammalian cells is that nine genes encode β-tubulin in human. Here, we generate a yeast strain expressing F265L tubulin mutant as the sole source of β-tubulin. The F265L mutation does not preclude expression of a stable β-tubulin protein which is incorporated into microtubules. However, impaired cell growth was observed at high temperatures along with altered microtubule dynamics and stability. In addition, F265L mutation produces a highly specific mitotic spindle positioning defect related to Bim1 (yeast EB1) dysfunction. Indeed, F265L cells display an abnormal Bim1 recruitment profile at microtubule plus-ends. These results indicate that the F265L β-tubulin mutation affects microtubule plus-end complexes known to be important for microtubule dynamics and for microtubule function during mitotic spindle positioning.

Emerging evidence indicates that microtubule-associated proteins (MAPs) are implicated in synaptic function; in particular, mice deficient for MAP6 exhibit striking deficits in plasticity and cognition. How MAP6 connects to plasticity mechanisms is unclear. Here, we address the possible role of this protein in dendritic spines. We find that in MAP6-deficient cortical and hippocampal neurons, maintenance of mature spines is impaired, and can be restored by expressing a stretch of the MAP6 sequence called Mc modules. Mc modules directly bind actin filaments and mediate activity-dependent stabilisation of F-actin in dendritic spines, a key event of synaptic plasticity. In vitro, Mc modules enhance actin filament nucleation and promote the formation of stable, highly ordered filament bundles. Activity-induced phosphorylation of MAP6 likely controls its transfer to the spine cytoskeleton. These results provide a molecular explanation for the role of MAP6 in cognition, enlightening the connection between cytoskeletal dysfunction, synaptic impairment and neuropsychiatric illnesses.

The posttranslational modification of carboxy-terminal tails of tubulin plays an important role in the regulation of the microtubule cytoskeleton. Enzymes responsible for deglutamylating tubulin have been discovered within a novel family of mammalian cytosolic carboxypeptidases. The discovery of these enzymes also revealed the existence of a range of other substrates that are enzymatically deglutamylated. Only four of six mammalian cytosolic carboxypeptidases had been enzymatically characterized. Here we complete the functional characterization of this protein family by demonstrating that CCP2 and CCP3 are deglutamylases, with CCP3 being able to hydrolyze aspartic acids with similar efficiency. Deaspartylation is a novel posttranslational modification that could, in conjunction with deglutamylation, broaden the range of potential substrates that undergo carboxy-terminal processing. In addition, we show that CCP2 and CCP3 are highly regulated proteins confined to ciliated tissues. The characterization of two novel enzymes for carboxy-terminal protein modification provides novel insights into the broadness of this barely studied process.

STOP (Stable Tubulin-Only Polypeptide) null mice show behavioral deficits, impaired synaptic plasticity, decrease in synaptic vesicular pools and disturbances in dopaminergic transmission, and are considered a neurodevelopmental model of schizophrenia. Olfactory neurons highly express STOP protein and are continually generated throughout life. Experimentally-induced loss of olfactory neurons leads to epithelial regeneration within two months, providing a useful model to evaluate the role played by STOP protein in adult olfactory neurogenesis.

MCAK is a member of the kinesin-13 family of microtubule (MT)-depolymerizing kinesins. We show that the potent MT depolymerizer MCAK tracks (treadmills) with the tips of polymerizing MTs in living cells. Tip tracking of MCAK is inhibited by phosphorylation and is dependent on the extreme COOH-terminal tail of MCAK. Tip tracking is not essential for MCAK's MT-depolymerizing activity. We propose that tip tracking is a mechanism by which MCAK is preferentially localized to regions of the cell that modulate the plus ends of MTs.

Regulation of microtubule dynamics is crucial for axon growth and guidance as well as for the establishment of synaptic connections. STOPs (Stable Tubule Only Polypeptides) are microtubule-associated proteins that regulate microtubule stabilization but are also able to interact with actin or Golgi membranes. Here, we have investigated the involvement of STOPs during the development of the olfactory system. We first describe the spatio-temporal expression patterns of N- and E-STOP, the two neuronal-specific isoforms of STOP. E- and N-STOP are expressed in the axonal compartment of olfactory sensory neurons, but are differentially regulated during development. Interestingly, each neuronal isoform displays a specific gradient distribution within the olfactory nerve layer. Then, we have examined the development of the olfactory system in the absence of STOPs. Olfactory axons display a normal outgrowth and targeting in STOP-null mice, but maturation of the synapses in the glomerular neuropil is altered.

In cells, stable microtubules (MTs) are covalently modified by a carboxypeptidase, which removes the C-terminal Tyr residue of alpha-tubulin. The significance of this selective detyrosination of MTs is not understood. In this study, we report that tubulin detyrosination in fibroblasts inhibits MT disassembly. This inhibition is relieved by overexpression of the depolymerizing motor mitotic centromere-associated kinesin (MCAK). Conversely, suppression of MCAK expression prevents disassembly of normal tyrosinated MTs in fibroblasts. Detyrosination of MTs suppresses the activity of MCAK in vitro, apparently as the result of a decreased affinity of the adenosine diphosphate (ADP)-inorganic phosphate- and ADP-bound forms of MCAK for the MT lattice. Detyrosination also impairs MT disassembly in neurons and inhibits the activity of the neuronal depolymerizing motor KIF2A in vitro. These results indicate that MT depolymerizing motors are directly inhibited by the detyrosination of tubulin, resulting in the stabilization of cellular MTs. Detyrosination of transiently stabilized MTs may give rise to persistent subpopulations of disassembly-resistant polymers to sustain subcellular cytoskeletal differentiation.

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.

Agents able to modify microtubule dynamics are important anticancer drugs. The absence of microtubules resulting from drug-induced depolymerization is easy to detect. However the detection of a stabilized microtubule network needs specific assays since there is not a significant visual difference between normal and stabilized microtubule networks. Here, we describe a quantitative cell-based assay, suitable for automation, which allows the detection of stabilized microtubules without the need of microscopic examination. The rationale of this assay is based on the drug-induced resistance of the microtubule network to the depolymerizing agent combretastatin A4 and the subsequent detection of the residual microtubules by immunoluminescence. Using this assay to screen a kinase inhibitor library allowed the selection of seven known kinase inhibitors: selonsertib, masatinib, intedanib, PF0477736, SNS-314 mesylate, MPI0479605, and ponatinib. The yet undescribed ability of these inhibitors to stabilize cellular microtubules was confirmed using additional markers of stable microtubules and time-lapse video-microscopy to track individual microtubules in living cells. None of the compounds interacted, however, directly with tubulin. By employing other inhibitors of the same kinases, which have structurally unrelated scaffolds, we determined if the microtubule stabilizing effect was due to the inhibition of the targeted kinase, or to an off-target effect. Many of these inhibitors are clinically approved or currently assayed in phase 2 or phase 3 clinical trials. Their microtubule-stabilizing effect may account for their therapeutic effect as well as for some of their adverse side effects. These results indicate also a possible repurposing of some of these drugs.

Morphometry characterization is an important procedure in describing neuronal cultures and identifying phenotypic differences. This task usually requires labor-intensive measurements and the classification of numerous neurites from large numbers of neurons in culture. To automate these measurements, we wrote AutoNeuriteJ, an imageJ/Fiji plugin that measures and classifies neurites from a very large number of neurons. We showed that AutoNeuriteJ is able to detect variations of neuritic growth induced by several compounds known to affect the neuronal growth. In these experiments measurement of more than 5000 mouse neurons per conditions was obtained within a few hours. Moreover, by analyzing mouse neurons deficient for the microtubule associated protein 6 (MAP6) and wild type neurons we illustrate that AutoNeuriteJ is capable to detect subtle phenotypic difference in axonal length. Overall the use of AutoNeuriteJ will provide rapid, unbiased and accurate measurement of neuron morphologies.

Microtubules (MTs) are an essential component of the neuronal cytoskeleton; they are involved in various aspects of neuron development, maintenance, and functions including polarization, synaptic plasticity, and transport. Neuronal MTs are highly heterogeneous due to the presence of multiple tubulin isotypes and extensive post-translational modifications (PTMs). These PTMs-most notably detyrosination, acetylation, and polyglutamylation-have emerged as important regulators of the neuronal microtubule cytoskeleton. With this review, we summarize what is currently known about the impact of tubulin PTMs on microtubule dynamics, neuronal differentiation, plasticity, and transport as well as on brain function in normal and pathological conditions, in particular during neuro-degeneration. The main therapeutic approaches to neuro-diseases based on the modulation of tubulin PTMs are also summarized. Overall, the review indicates how tubulin PTMs can generate a large number of functionally specialized microtubule sub-networks, each of which is crucial to specific neuronal features.

Paclitaxel is a microtubule stabilizing agent and a successful drug for cancer chemotherapy inducing, however, adverse effects. To reduce the effective dose of paclitaxel, we searched for pharmaceutics which could potentiate its therapeutic effect. We screened a chemical library and selected Carba1, a carbazole, which exerts synergistic cytotoxic effects on tumor cells grown in vitro, when co-administrated with a low dose of paclitaxel. Carba1 targets the colchicine binding-site of tubulin and is a microtubule-destabilizing agent. Catastrophe induction by Carba1 promotes paclitaxel binding to microtubule ends, providing a mechanistic explanation of the observed synergy. The synergistic effect of Carba1 with paclitaxel on tumor cell viability was also observed in vivo in xenografted mice. Thus, a new mechanism favoring paclitaxel binding to dynamic microtubules can be transposed to in vivo mouse cancer treatments, paving the way for new therapeutic strategies combining low doses of microtubule targeting agents with opposite mechanisms of action.

For a better translation from treatment designs of schizophrenia to clinical efficiency, there is a crucial need to refine preclinical animal models. In order to consider the multifactorial nature of the disorder, a new mouse model associating three factors (genetic susceptibility-partial deletion of the MAP6 gene, early-life stress-maternal separation, and pharmacological treatment-chronic Δ-9-tetrahydrocannabinol during adolescence) has recently been described. While this model depicts a schizophrenia-like phenotype, the neurobiological correlates remain unknown. Synaptic transmission and functional plasticity of the CA1 hippocampal region of male and female 3-hit mice were therefore investigated using electrophysiological recordings on the hippocampus slice. While basal excitatory transmission remained unaffected, NMDA receptor (NMDAr)-mediated long-term potentiation (LTP) triggered by theta-burst (TBS) but not by high-frequency (HFS) stimulation was impaired in 3-hit mice. Isolated NMDAr activation was not affected or even increased in female 3-hit mice, revealing a sexual dimorphism. Considering that the regulation of LTP is more prone to inhibitory tone if triggered by TBS than by HFS, the weaker potentiation in 3-hit mice suggests a deficiency of intrinsic GABA regulatory mechanisms. Indeed, NMDAr activation was increased by GABAA receptor blockade in wild-type but not in 3-hit mice. This electrophysiological study highlights dysregulations of functional properties and plasticity in hippocampal networks of 3-hit mice, one of the mechanisms suspected to contribute to the pathophysiology of schizophrenia. It also shows differences between males and females, supporting the sexual dimorphism observed in the disorder. Combined with the previously reported study, the present data reinforce the face validity of the 3-hit model that will help to consider new therapeutic strategies for psychosis.

The search for effective treatments for neuropsychiatric disorders is ongoing, with progress being made as brain structure and neuronal function become clearer. The central roles played by microtubules (MT) and actin in synaptic transmission and plasticity suggest that the cytoskeleton and its modulators could be relevant targets for the development of new molecules to treat psychiatric diseases. In this context, LIM Kinase - which regulates both the actin and MT cytoskeleton especially in dendritic spines, the post-synaptic compartment of the synapse - might be a good target. In this study, we analyzed the consequences of blocking LIMK1 pharmacologically using Pyr1. We investigated synaptic plasticity defects and behavioral disorders in MAP6 KO mice, an animal model useful for the study of psychiatric disorders, particularly schizophrenia. Our results show that Pyr1 can modulate MT dynamics in neurons. In MAP6 KO mice, chronic LIMK inhibition by long-term treatment with Pyr1 can restore normal dendritic spine density and also improves long-term potentiation, both of which are altered in these mice. Pyr1 treatment improved synaptic plasticity, and also reduced social withdrawal and depressive/anxiety-like behavior in MAP6 KO mice. Overall, the results of this study validate the hypothesis that modulation of LIMK activity could represent a new therapeutic strategy for neuropsychiatric diseases.

MAP6 proteins were first described as microtubule-stabilizing agents, whose properties were thought to be essential for neuronal development and maintenance of complex neuronal networks. However, deletion of all MAP6 isoforms in MAP6 KO mice does not lead to dramatic morphological aberrations of the brain but rather to alterations in multiple neurotransmissions and severe behavioural impairments. A search for protein partners of MAP6 proteins identified Tctex1 - a dynein light chain with multiple non-microtubule-related functions. The involvement of Tctex1 in calcium signalling led to investigate it in MAP6 KO neurons. In this study, we show that functional Cav 2.2/N-type calcium channels are deficient in MAP6 KO neurons, due to improper location. We also show that MAP6 proteins interact directly with both Tctex1 and the C-terminus of Cav 2.2/N-type calcium channels. A balance of these two interactions seems to be crucial for MAP6 to modulate calcium signalling in neurons.

Social and communication impairments are common features of psychiatric disorders. Animal models of schizophrenia display various social deficits due to difference in tests, mouse strains and drugs. Moreover, communication deficits have not been studied. Our objectives were to assess and compare three major features of social cognition in different mouse models of schizophrenia: interest for a social stimulus, organization and acceptance of social contact, and acoustic communication to question whether mouse models for schizophrenia with social dysfunction also exhibit vocal communication defects. To achieve these aims we treated acutely C57BL/6J mice either with MK-801 or ketamine and tested WT and microtubule-associated protein 6 -MAP6- KO mice in two complementary social tasks: the 3-chamber test which measures social motivation and the social interaction task -SIT- which relies on prefrontal cortex activity and measures the ability to organize and respond to a real interaction, and which promotes ultrasonic vocalizations. Our results reveal that schizophrenia models have intact interest for a social stimulus in the 3-chamber test. However, thanks to principal component analyses of social interaction data, we demonstrate that social motivation and the ability to act socially rely on distinct mechanisms in revealing a decrease in dominance and communication in pharmacological schizophrenia models along with social withdraw, classically observed in schizophrenia, in MK-801 model. In this latter model, some social parameters can be significantly improved by aripiprazole, an atypical antipsychotic. Our social protocol, combined with fine-tuned analysis, is expected to provide an innovative framework for testing future treatments in preclinical models. This article is part of the Special Issue entitled 'The neuropharmacology of social behavior: from bench to bedside'.

MAP6 proteins (MAP6s), which include MAP6-N (also called Stable Tubule Only Polypeptide, or STOP) and MAP6d1 (MAP6 domain-containing protein 1, also called STOP-Like protein 21 kD, or SL21), bind to and stabilize microtubules. MAP6 deletion in mice severely alters integrated brain functions and is associated with synaptic defects, suggesting that MAP6s may also have alternative cellular roles. MAP6s reportedly associate with the Golgi apparatus through palmitoylation of their N-terminal domain, and specific isoforms have been shown to bind actin. Here, we use heterologous systems to investigate several biochemical properties of MAP6 proteins. We demonstrate that the three N-terminal cysteines of MAP6d1 are palmitoylated by a subset of DHHC-type palmitoylating enzymes. Analysis of the subcellular localization of palmitoylated MAP6d1, including electron microscopic analysis, reveals possible localization to the Golgi and the plasma membrane but no association with the endoplasmic reticulum. Moreover, we observed localization of MAP6d1 to mitochondria, which requires the N-terminus of the protein but does not require palmitoylation. We show that endogenous MAP6d1 localized at mitochondria in mature mice neurons as well as at the outer membrane and in the intermembrane space of purified mouse mitochondria. Last, we found that MAP6d1 can multimerize via a microtubule-binding module. Interestingly, most of these properties of MAP6d1 are shared by MAP6-N. Together, these results describe several properties of MAP6 proteins, including their intercellular localization and multimerization activity, which may be relevant to neuronal differentiation and synaptic functions.

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.