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Most eukaryotic cells have cilia that serve vital functions in sensing, signalling, motility. The core architecture of cilia is an array of microtubule doublets, which consist of a complete A-tubule and an incomplete B-tubule. How these structures assemble remains poorly understood. Using total internal reflection fluorescence microscopy and cryo-electron tomography, we investigate the role of MAP6d1, a brain-specific protein containing microtubule lumen-targeting Mn-motifs. We show that MAP6d1 assembles stable microtubule doublets by recruiting tubulin dimers onto the A-tubule lattice to initiate B-tubule nucleation. MAP6d1 also promotes the formation of luminal protofilaments in singlet and doublet microtubules, a previously undescribed phenomenon that likely enhances microtubule stability. In neurons, MAP6d1 localises to the proximal part of primary cilia via its Mn-motif, with its loss resulting in shortened cilia, a characteristic of ciliopathies. MAP6d1 is thus a neuronal Mn-motif protein with a specific role in assembling microtubule doublets and regulating ciliary length. Cilia are composed of microtubule doublets, but how they assemble is unclear. Here, the authors show that the brain-specific MAP6d1, found in neuronal primary cilia, assembles doublet microtubules and forms protofilaments inside microtubules.

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.

The development and function of the central nervous system rely on the microtubule (MT) and actin cytoskeletons and their respective effectors. Although the structural role of the cytoskeleton has long been acknowledged in neuronal morphology and activity, it was recently recognized to play the role of a signaling platform. Following this recognition, research into Microtubule Associated Proteins (MAPs) diversified. Indeed, historically, structural MAPs—including MAP1B, MAP2, Tau, and MAP6 (also known as STOP);—were identified and described as MT-binding and -stabilizing proteins. Extensive data obtained over the last 20 years indicated that these structural MAPs could also contribute to a variety of other molecular roles. Among multi-role MAPs, MAP6 provides a striking example illustrating the diverse molecular and cellular properties of MAPs and showing how their functional versatility contributes to the central nervous system. In this review, in addition to MAP6’s effect on microtubules, we describe its impact on the actin cytoskeleton, on neuroreceptor homeostasis, and its involvement in signaling pathways governing neuron development and maturation. We also discuss its roles in synaptic plasticity, brain connectivity, and cognitive abilities, as well as the potential relationships between the integrated brain functions of MAP6 and its molecular activities. In parallel, the Collapsin Response Mediator Proteins (CRMPs) are presented as examples of how other proteins, not initially identified as MAPs, fall into the broader MAP family. These proteins bind MTs as well as exhibiting molecular and cellular properties very similar to MAP6. Finally, we briefly summarize the multiple similarities between other classical structural MAPs and MAP6 or CRMPs.In summary, this review revisits the molecular properties and the cellular and neuronal roles of the classical MAPs, broadening our definition of what constitutes a MAP.

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.

The MAP6 (microtubule-associated protein 6) KO mouse is a microtubule-deficient model of schizophrenia that exhibits severe behavioral disorders that are associated with synaptic plasticity anomalies. These defects are alleviated not only by neuroleptics, which are the gold standard molecules for the treatment of schizophrenia, but also by Epothilone D (Epo D), which is a microtubule-stabilizing molecule. To compare the neuronal transport between MAP6 KO and wild-type mice and to measure the effect of Epo D treatment on neuronal transport in KO mice, MnCl2 was injected in the primary somatosensory cortex. Then, using manganese-enhanced magnetic resonance imaging (MEMRI), we followed the propagation of Mn2+ through axonal tracts and brain regions that are connected to the somatosensory cortex. In MAP6 KO mice, the measure of the MRI relative signal intensity over 24h revealed that the Mn2+ transport rate was affected with a stronger effect on long-range and polysynaptic connections than in short-range and monosynaptic tracts. The chronic treatment of MAP6 KO mice with Epo D strongly increased Mn2+ propagation within both mono- and polysynaptic connections. Our results clearly indicate an in vivo deficit in neuronal Mn2+ transport in KO MAP6 mice, which might be due to both axonal transport defects and synaptic transmission impairments. Epo D treatment alleviated the axonal transport defects, and this improvement most likely contributes to the positive effect of Epo D on behavioral defects in KO MAP6 mice. •The microtubular drug Epothilone D restores normal neuronal transport in MAP6 KO mice.•MAP6 knockout mouse, a model of schizophrenia, exhibits altered neuronal transport.•Neuronal transport can be directly measured in vivo by MEMRI technique.

Neurodevelopmental axonal pathfinding plays a central role in correct brain wiring and subsequent cognitive abilities. Within the growth cone, various intracellular effectors transduce axonal guidance signals by remodeling the cytoskeleton. Semaphorin-3E (Sema3E) is a guidance cue implicated in development of the fornix, a neuronal tract connecting the hippocampus to the hypothalamus. Microtubule-associated protein 6 (MAP6) has been shown to be involved in the Sema3E growth-promoting signaling pathway. In this study, we identified the collapsin response mediator protein 4 (CRMP4) as a MAP6 partner and a crucial effector in Sema3E growth-promoting activity. CRMP4-KO mice displayed abnormal fornix development reminiscent of that observed in Sema3E-KO mice. CRMP4 was shown to interact with the Sema3E tripartite receptor complex within detergent- resistant membrane (DRM) domains, and DRM domain integrity was required to transduce Sema3E signaling through the Akt/GSK3 pathway. Finally, we showed that the cytoskeleton-binding domain of CRMP4 is required for Sema3E’s growth-promoting activity, suggesting that CRMP4 plays a role at the interface between Sema3E receptors, located in DRM domains, and the cytoskeleton network. As the fornix is affected in many psychiatric diseases, such as schizophrenia, our results provide new insights to better understand the neurodevelopmental components of these diseases.

Neuronal differentiation is under the tight control of both biochemical and physical information arising from neighboring cells and micro-environment. Here we wished to assay how external geometrical constraints applied to the cell body and/or the neurites of hippocampal neurons may modulate axonal polarization in vitro. Through the use of a panel of non-specific poly-L-lysine micropatterns, we manipulated the neuronal shape. By applying geometrical constraints on the cell body we provided evidence that centrosome location was not predictive of axonal polarization but rather follows axonal fate. When the geometrical constraints were applied to the neurites trajectories we demonstrated that axonal specification was inhibited by curved lines. Altogether these results indicated that intrinsic mechanical tensions occur during neuritic growth and that maximal tension was developed by the axon and expressed on straight trajectories. The strong inhibitory effect of curved lines on axon specification was further demonstrated by their ability to prevent formation of multiple axons normally induced by cytochalasin or taxol treatments. Finally we provided evidence that microtubules were involved in the tension-mediated axonal polarization, acting as curvature sensors during neuronal differentiation. Thus, biomechanics coupled to physical constraints might be the first level of regulation during neuronal development, primary to biochemical and guidance regulations.

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.

Background The skeletal muscle fiber has a specific and precise intracellular organization which is at the basis of an efficient muscle contraction. Microtubules are long known to play a major role in the function and organization of many cells, but in skeletal muscle, the contribution of the microtubule cytoskeleton to the efficiency of contraction has only recently been studied. The microtubule network is dynamic and is regulated by many microtubule-associated proteins (MAPs). In the present study, the role of the MAP6 protein in skeletal muscle organization and function has been studied using the MAP6 knockout mouse line. Methods The presence of MAP6 transcripts and proteins was shown in mouse muscle homogenates and primary culture using RT-PCR and western blot. The in vivo evaluation of muscle force of MAP6 knockout (KO) mice was performed on anesthetized animals using electrostimulation coupled to mechanical measurement and multimodal magnetic resonance. The impact of MAP6 deletion on microtubule organization and intracellular structures was studied using immunofluorescent labeling and electron microscopy, and on calcium release for muscle contraction using Fluo-4 calcium imaging on cultured myotubes. Statistical analysis was performed using Student’s t test or the Mann-Whitney test. Results We demonstrate the presence of MAP6 transcripts and proteins in skeletal muscle. Deletion of MAP6 results in a large number of muscle modifications: muscle weakness associated with slight muscle atrophy, alterations of microtubule network and sarcoplasmic reticulum organization, and reduction in calcium release. Conclusion Altogether, our results demonstrate that MAP6 is involved in skeletal muscle function. Its deletion results in alterations in skeletal muscle contraction which contribute to the global deleterious phenotype of the MAP6 KO mice. As MAP6 KO mouse line is a model for schizophrenia, our work points to a possible muscle weakness associated to some forms of schizophrenia.

In the central nervous system, microtubule-associated protein 6 (MAP6) is expressed at high levels and is crucial for cognitive abilities. The large spectrum of social and cognitive impairments observed in MAP6-KO mice are reminiscent of the symptoms observed in psychiatric diseases, such as schizophrenia, and respond positively to long-term treatment with antipsychotics. MAP6-KO mice have therefore been proposed to be a useful animal model for these diseases. Here, we explored the brain anatomy in MAP6-KO mice using high spatial resolution 3D MRI, including a volumetric T 1w method to image brain structures, and Diffusion Tensor Imaging (DTI) for white matter fiber tractography. 3D DTI imaging of neuronal tracts was validated by comparing results to optical images of cleared brains. Changes to brain architecture included reduced volume of the cerebellum and the thalamus and altered size, integrity and spatial orientation of some neuronal tracks such as the anterior commissure, the mammillary tract, the corpus callosum, the corticospinal tract, the fasciculus retroflexus and the fornix. Our results provide information on the neuroanatomical defects behind the neurological phenotype displayed in the MAP6-KO mice model and especially highlight a severe damage of the corticospinal tract with defasciculation at the location of the pontine nuclei.