MAP9/MAPH-9 supports axonemal microtubule doublets and modulates motor movement.

Microtubule doublets (MTDs) comprise an incomplete microtubule (B-tubule) attached to the side of a complete cylindrical microtubule. These compound microtubules are conserved in cilia across the tree of life; however, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we identify microtubule-associated protein 9 (MAP9) as an MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. We find that loss of MAPH-9 causes ultrastructural MTD defects, including shortened and/or squashed B-tubules with reduced numbers of protofilaments, dysregulated axonemal motor velocity, and perturbed cilia function. Because we find that the mammalian ortholog MAP9 localizes to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in regulating ciliary motors and supporting the structure of axonemal MTDs.

Choroid plexuses carry nodal-like cilia that undergo axoneme regression from early adult stage.

Choroid plexuses (ChPs) produce cerebrospinal fluid and sense non-cell-autonomous stimuli to control the homeostasis of the central nervous system. They are mainly composed of epithelial multiciliated cells, whose development and function are still controversial. We have thus characterized the stepwise order of mammalian ChP epithelia cilia formation using a combination of super-resolution-microscopy approaches and mouse genetics. We show that ChP ciliated cells are built embryonically on a treadmill of spatiotemporally regulated events, starting with atypical centriole amplification and ending with the construction of nodal-like 9+0 cilia, characterized by both primary and motile features. ChP cilia undergo axoneme resorption at early postnatal stages through a microtubule destabilization process controlled by the microtubule-severing enzyme spastin and mitigated by polyglutamylation levels. Notably, this phenotype is preserved in humans, suggesting a conserved ciliary resorption mechanism in mammals.

MAP9/MAPH-9 supports axonemal microtubule doublets and modulates motor movement.

Microtubule doublets (MTDs) are a well conserved compound microtubule structure found primarily in cilia. However, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we characterize microtubule-associated protein 9 (MAP9) as a novel MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. Loss of MAPH-9 caused ultrastructural MTD defects, dysregulated axonemal motor velocity, and perturbed cilia function. As we found that the mammalian ortholog MAP9 localized to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in supporting the structure of axonemal MTDs and regulating ciliary motors.

Tubulin polyglutamylation differentially regulates microtubule-interacting proteins.

Tubulin posttranslational modifications have been predicted to control cytoskeletal functions by coordinating the molecular interactions between microtubules and their associating proteins. A prominent tubulin modification in neurons is polyglutamylation, the deregulation of which causes neurodegeneration. Yet, the underlying molecular mechanisms have remained elusive. Here, using in-vitro reconstitution, we determine how polyglutamylation generated by the two predominant neuronal polyglutamylases, TTLL1 and TTLL7, specifically modulates the activities of three major microtubule interactors: the microtubule-associated protein Tau, the microtubule-severing enzyme katanin and the molecular motor kinesin-1. We demonstrate that the unique modification patterns generated by TTLL1 and TTLL7 differentially impact those three effector proteins, thus allowing for their selective regulation. Given that our experiments were performed with brain tubulin from mouse models in which physiological levels and patterns of polyglutamylation were altered by the genetic knockout of the main modifying enzymes, our quantitative measurements provide direct mechanistic insight into how polyglutamylation could selectively control microtubule interactions in neurons.

VASH1-SVBP and VASH2-SVBP generate different detyrosination profiles on microtubules.

The detyrosination/tyrosination cycle of α-tubulin is critical for proper cell functioning. VASH1-SVBP and VASH2-SVBP are ubiquitous enzymes involved in microtubule detyrosination, whose mode of action is little known. Here, we show in reconstituted systems and cells that VASH1-SVBP and VASH2-SVBP drive the global and local detyrosination of microtubules, respectively. We solved the cryo-electron microscopy structure of VASH2-SVBP bound to microtubules, revealing a different microtubule-binding configuration of its central catalytic region compared to VASH1-SVBP. We show that the divergent mode of detyrosination between the two enzymes is correlated with the microtubule-binding properties of their disordered N- and C-terminal regions. Specifically, the N-terminal region is responsible for a significantly longer residence time of VASH2-SVBP on microtubules compared to VASH1-SVBP. We suggest that this VASH region is critical for microtubule detachment and diffusion of VASH-SVBP enzymes on lattices. Our results suggest a mechanism by which VASH1-SVBP and VASH2-SVBP could generate distinct microtubule subpopulations and confined areas of detyrosinated lattices to drive various microtubule-based cellular functions.

Actin-microtubule cytoskeletal interplay mediated by MRTF-A/SRF signaling promotes dilated cardiomyopathy caused by LMNA mutations.

Mutations in the lamin A/C gene (LMNA) cause dilated cardiomyopathy associated with increased activity of ERK1/2 in the heart. We recently showed that ERK1/2 phosphorylates cofilin-1 on threonine 25 (phospho(T25)-cofilin-1) that in turn disassembles the actin cytoskeleton. Here, we show that in muscle cells carrying a cardiomyopathy-causing LMNA mutation, phospho(T25)-cofilin-1 binds to myocardin-related transcription factor A (MRTF-A) in the cytoplasm, thus preventing the stimulation of serum response factor (SRF) in the nucleus. Inhibiting the MRTF-A/SRF axis leads to decreased α-tubulin acetylation by reducing the expression of ATAT1 gene encoding α-tubulin acetyltransferase 1. Hence, tubulin acetylation is decreased in cardiomyocytes derived from male patients with LMNA mutations and in heart and isolated cardiomyocytes from Lmnap.H222P/H222P male mice. In Atat1 knockout mice, deficient for acetylated α-tubulin, we observe left ventricular dilation and mislocalization of Connexin 43 (Cx43) in heart. Increasing α-tubulin acetylation levels in Lmnap.H222P/H222P mice with tubastatin A treatment restores the proper localization of Cx43 and improves cardiac function. In summary, we show for the first time an actin-microtubule cytoskeletal interplay mediated by cofilin-1 and MRTF-A/SRF, promoting the dilated cardiomyopathy caused by LMNA mutations. Our findings suggest that modulating α-tubulin acetylation levels is a feasible strategy for improving cardiac function.

Lysate-based pipeline to characterize microtubule-associated proteins uncovers unique microtubule behaviours.

The microtubule cytoskeleton forms complex macromolecular assemblies with a range of microtubule-associated proteins (MAPs) that have fundamental roles in cell architecture, division and motility. Determining how an individual MAP modulates microtubule behaviour is an important step in understanding the physiological roles of various microtubule assemblies. To characterize how MAPs control microtubule properties and functions, we developed an approach allowing for medium-throughput analyses of MAPs in cell-free conditions using lysates of mammalian cells. Our pipeline allows for quantitative as well as ultrastructural analyses of microtubule-MAP assemblies. Analysing 45 bona fide and potential mammalian MAPs, we uncovered previously unknown activities that lead to distinct and unique microtubule behaviours such as microtubule coiling or hook formation, or liquid-liquid phase separation along the microtubule lattice that initiates microtubule branching. We have thus established a powerful tool for a thorough characterization of a wide range of MAPs and MAP variants, thus opening avenues for the determination of mechanisms underlying their physiological roles and pathological implications.

Distinct roles of α- and ß-tubulin polyglutamylation in controlling axonal transport and in neurodegeneration.

Tubulin polyglutamylation is a post-translational modification of the microtubule cytoskeleton, which is generated by a variety of enzymes with different specificities. The "tubulin code" hypothesis predicts that modifications generated by specific enzymes selectively control microtubule functions. Our recent finding that excessive accumulation of polyglutamylation in neurons causes their degeneration and perturbs axonal transport provides an opportunity for testing this hypothesis. By developing novel mouse models and a new glutamylation-specific antibody, we demonstrate here that the glutamylases TTLL1 and TTLL7 generate unique and distinct glutamylation patterns on neuronal microtubules. We find that under physiological conditions, TTLL1 polyglutamylates α-tubulin, while TTLL7 modifies ß-tubulin. TTLL1, but not TTLL7, catalyses the excessive hyperglutamylation found in mice lacking the deglutamylase CCP1. Consequently, deletion of TTLL1, but not of TTLL7, prevents degeneration of Purkinje cells and of myelinated axons in peripheral nerves in these mice. Moreover, loss of TTLL1 leads to increased mitochondria motility in neurons, while loss of TTLL7 has no such effect. By revealing how specific patterns of tubulin glutamylation, generated by distinct enzymes, translate into specific physiological and pathological readouts, we demonstrate the relevance of the tubulin code for homeostasis.

Tubulin polyglutamylation, a regulator of microtubule functions, can cause neurodegeneration.

Neurodegenerative diseases lead to a progressive demise of neuronal functions that ultimately results in neuronal death. Besides a large variety of molecular pathways that have been linked to the degeneration of neurons, dysfunctions of the microtubule cytoskeleton are common features of many human neurodegenerative disorders. Yet, it is unclear whether microtubule dysfunctions are causative, or mere bystanders in the disease progression. A so-far little explored regulatory mechanism of the microtubule cytoskeleton, the posttranslational modifications of tubulin, emerge as candidate mechanisms involved in neuronal dysfunction, and thus, degeneration. Here we review the role of tubulin polyglutamylation, a prominent modification of neuronal microtubules. We discuss the current understanding of how polyglutamylation controls microtubule functions in healthy neurons, and how deregulation of this modification leads to neurodegeneration in mice and humans.

Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles.

One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They allow the analysis of the intrinsic properties of microtubules, such as dynamics, and their interactions with microtubule-associated proteins (MAPs). The "tubulin code" is an emerging concept that points to different tubulin isotypes and various posttranslational modifications (PTMs) as regulators of microtubule properties and functions. To explore the molecular mechanisms of the tubulin code, it is crucial to perform in vitro reconstitution experiments using purified tubulin with specific isotypes and PTMs. To date, this was technically challenging as brain tubulin, which is widely used in in vitro experiments, harbors many PTMs and has a defined isotype composition. Hence, we developed this protocol to purify tubulin from different sources and with different isotype compositions and controlled PTMs, using the classical approach of polymerization and depolymerization cycles. Compared to existing methods based on affinity purification, this approach yields pure, polymerization-competent tubulin, as tubulin resistant to polymerization or depolymerization is discarded during the successive purification steps. We describe the purification of tubulin from cell lines, grown either in suspension or as adherent cultures, and from single mouse brains. The method first describes the generation of cell mass in both suspension and adherent settings, the lysis step, followed by the successive stages of tubulin purification by polymerization-depolymerization cycles. Our method yields tubulin that can be used in experiments addressing the impact of the tubulin code on the intrinsic properties of microtubules and microtubule interactions with associated proteins.

The tubulin code and its role in controlling microtubule properties and functions.

Microtubules are core components of the eukaryotic cytoskeleton with essential roles in cell division, shaping, motility and intracellular transport. Despite their functional heterogeneity, microtubules have a highly conserved structure made from almost identical molecular building blocks: the tubulin proteins. Alternative tubulin isotypes and a variety of post-translational modifications control the properties and functions of the microtubule cytoskeleton, a concept known as the 'tubulin code'. Here we review the current understanding of the molecular components of the tubulin code and how they impact microtubule properties and functions. We discuss how tubulin isotypes and post-translational modifications control microtubule behaviour at the molecular level and how this translates into physiological functions at the cellular and organism levels. We then go on to show how fine-tuning of microtubule function by some tubulin modifications can affect homeostasis and how perturbation of this fine-tuning can lead to a range of dysfunctions, many of which are linked to human disease.

Tubulin polyglutamylation is a general traffic-control mechanism in hippocampal neurons.

Neurons are highly complex cells that heavily rely on intracellular transport to distribute a range of functionally essential cargoes within the cell. Post-translational modifications of tubulin are emerging as mechanisms for regulating microtubule functions, but their impact on neuronal transport is only marginally understood. Here, we have systematically studied the impact of post-translational polyglutamylation on axonal transport. In cultured hippocampal neurons, deletion of a single deglutamylase, CCP1 (also known as AGTPBP1), is sufficient to induce abnormal accumulation of polyglutamylation, i.e. hyperglutamylation. We next investigated how hyperglutamylation affects axonal transport of a range of functionally different neuronal cargoes: mitochondria, lysosomes, LAMP1 endosomes and BDNF vesicles. Strikingly, we found a reduced motility for all these cargoes, suggesting that polyglutamylation could act as a regulator of cargo transport in neurons. This, together with the recent discovery that hyperglutamylation induces neurodegeneration, makes it likely that perturbed neuronal trafficking could be one of the central molecular causes underlying this novel type of degeneration.This article has an associated First Person interview with the first author of the paper.

Knocking Out Multiple Genes in Cultured Primary Neurons to Study Tubulin Posttranslational Modifications.

Microtubules, as integral part of the eukaryotic cytoskeleton, exert numerous essential functions in cells. A mechanism to control these diverse functions are the posttranslational modifications of tubulin. Despite being known for decades, relatively little insight into the cellular functions of these modifications has been gained so far. The discovery of tubulin-modifying enzymes and a growing number of available knockout mice now allow working with primary cells from those mouse models to address biological functions and molecular mechanisms behind those modifications. However, a number of those mouse models show either lethality or sterility, making it difficult to impossible to obtain a sufficient number of animals for a systematic study with primary cells. Moreover, many of those modifications are controlled by several redundant enzymes, and it is often necessary to knock out several enzymes in parallel to obtain a significant change in a given tubulin modification. Here we describe a method to generate primary cells with combinatorial knockout genotypes using conditional knockout mice. The conditional alleles are converted into knockout in the cultured primary cells by transduction with a lentivirus encoding cre-recombinase. This approach has allowed us to knock out the two main brain deglutamylases in mouse primary neurons, which leads to strongly increased polyglutamylation in these cells. Our method can be applied to measure different cellular processes, such as axonal transport, for which it can be combined with the expression of different fluorescent reporters to label intracellular proteins. Using a panel of conditional knockout mice, our method can further be applied to study the functions of a variety of tubulin modifications that require simultaneous knockout of multiple genes.

Measuring the Impact of Tubulin Posttranslational Modifications on Axonal Transport.

Axonal transport is a process essential for neuronal function and survival that takes place on the cellular highways-the microtubules. It requires three major components: the microtubules that serve as tracks for the transport, the motor proteins that drive the movement, and the transported cargoes with their adaptor proteins. Axonal transport could be controlled by tubulin posttranslational modifications, which by decorating specific microtubule tracks could determine the specificity of cargo delivery inside neurons. However, it appears that the effects of tubulin modifications on transport can be rather subtle, and might thus be easily overlooked depending on which parameter of the transport process is analyzed. Here we propose an analysis paradigm that allows detecting rather subtle alterations in neuronal transport, as induced for instance by accumulation of posttranslational polyglutamylation. Analyzing mitochondria movements in axons, we found that neither the average speed nor the distance traveled were affected by hyperglutamylation, but we detected an about 50% reduction of the overall motility, suggesting that polyglutamylation controls the efficiency of mitochondria transport in axons. Our protocol can readily be expanded to the analysis of the impact of other tubulin modifications on the transport of a range of different neuronal cargoes.

Purification of tubulin with controlled post-translational modifications by polymerization-depolymerization cycles.

In vitro reconstitutions of microtubule assemblies have provided essential mechanistic insights into the molecular bases of microtubule dynamics and their interactions with associated proteins. The tubulin code has emerged as a regulatory mechanism for microtubule functions, which suggests that tubulin isotypes and post-translational modifications (PTMs) play important roles in controlling microtubule functions. To investigate the tubulin code mechanism, it is essential to analyze different tubulin variants in vitro. Until now, this has been difficult, as most reconstitution experiments have used heavily post-translationally modified tubulin purified from brain tissue. Therefore, we developed a protocol that allows purification of tubulin with controlled PTMs from limited sources through cycles of polymerization and depolymerization. Although alternative protocols using affinity purification of tubulin also yield very pure tubulin, our protocol has the unique advantage of selecting for fully functional tubulin, as non-polymerizable tubulin is excluded in the successive polymerization cycles. It thus provides a novel procedure for obtaining tubulin with controlled PTMs for in vitro reconstitution experiments. We describe specific procedures for tubulin purification from adherent cells, cells grown in suspension cultures and single mouse brains. The protocol can be combined with drug treatment, transfection of cells before tubulin purification or enzymatic treatment during the purification process. The amplification of cells and their growth in spinner bottles takes ~13 d; the tubulin purification takes 6-7 h. The tubulin can be used in total internal reflection fluorescence (TIRF)-microscopy-based experiments or pelleting assays for the investigation of intrinsic properties of microtubules and their interactions with associated proteins.

An essential role for α4A-tubulin in platelet biogenesis.

During platelet biogenesis, microtubules (MTs) are arranged into submembranous structures (the marginal band) that encircle the cell in a single plane. This unique MT array has no equivalent in any other mammalian cell, and the mechanisms responsible for this particular mode of assembly are not fully understood. One possibility is that platelet MTs are composed of a particular set of tubulin isotypes that carry specific posttranslational modifications. Although ß1-tubulin is known to be essential, no equivalent roles of α-tubulin isotypes in platelet formation or function have so far been reported. Here, we identify α4A-tubulin as a predominant α-tubulin isotype in platelets. Similar to ß1-tubulin, α4A-tubulin expression is up-regulated during the late stages of megakaryocyte differentiation. Missense mutations in the α4A-tubulin gene cause macrothrombocytopenia in mice and humans. Defects in α4A-tubulin lead to changes in tubulin tyrosination status of the platelet tubulin pool. Ultrastructural defects include reduced numbers and misarranged MT coils in the platelet marginal band. We further observed defects in megakaryocyte maturation and proplatelet formation in Tuba4a-mutant mice. We have, thus, discovered an α-tubulin isotype with specific and essential roles in platelet biogenesis.

Kinesin-3 Responds to Local Microtubule Dynamics to Target Synaptic Cargo Delivery to the Presynapse.

Neurons in the CNS establish thousands of en passant synapses along their axons. Robust neurotransmission depends on the replenishment of synaptic components in a spatially precise manner. Using live-cell microscopy and single-molecule reconstitution assays, we find that the delivery of synaptic vesicle precursors (SVPs) to en passant synapses in hippocampal neurons is specified by an interplay between the kinesin-3 KIF1A motor and presynaptic microtubules. Presynaptic sites are hotspots of dynamic microtubules rich in GTP-tubulin. KIF1A binds more weakly to GTP-tubulin than GDP-tubulin and competes with end-binding (EB) proteins for binding to the microtubule plus end. A disease-causing mutation within KIF1A that reduces preferential binding to GDP- versus GTP-rich microtubules disrupts SVP delivery and reduces presynaptic release upon neuronal stimulation. Thus, the localized enrichment of dynamic microtubules along the axon specifies a localized unloading zone that ensures the accurate delivery of SVPs, controlling presynaptic strength in hippocampal neurons.