Posttranslational glutamylation of tubulin is present on selected subsets of microtubules in cells. Although the modification is expected to contribute to the spatial and temporal organization of the cytoskeleton, hardly anything is known about its functional relevance. Here we demonstrate that glutamylation, and in particular the generation of long glutamate side chains, promotes the severing of microtubules. In human cells, the generation of long side chains induces spastin-dependent microtubule disassembly and, consistently, only microtubules modified by long glutamate side chains are efficiently severed by spastin in vitro. Our study reveals a novel control mechanism for microtubule mass and stability, which is of fundamental importance to cellular physiology and might have implications for diseases related to microtubule severing.

Mature megakaryocytes extend long processes called proplatelets from which platelets are released in the blood stream. The Rho GTPases Cdc42 and Rac as well as their downstream target, p21-activated kinase 2 (PAK2), have been demonstrated to be important for platelet formation. Here we address the role, during platelet formation, of PAK1, another target of the Rho GTPases. PAK1 decorates the bundled microtubules (MTs) of megakaryocyte proplatelets. Using a validated cell model which recapitulates proplatelet formation, elongation and platelet release, we show that lack of PAK1 activity increases the number of proplatelets but restrains their elongation. Moreover, in the absence of PAK1 activity, cells have hyperacetylated MTs and lose their MT network integrity. Using inhibitors of the tubulin deacetylase HDAC6, we demonstrate that abnormally high levels of MT acetylation are not sufficient to increase the number of proplatelets but cause loss of MT integrity. Taken together with our previous demonstration that MT acetylation is required for proplatelet formation, our data reveal that MT acetylation levels need to be tightly regulated during proplatelet formation. We identify PAK1 as a direct regulator of the MT acetylation levels during this process as we found that PAK1 phosphorylates the MT acetyltransferase MEC-17 and inhibits its activity.

Microtubules (MTs) play crucial roles during neuronal life. They are formed by heterodimers of alpha and beta-tubulins, which are subjected to several post-translational modifications (PTMs). Amongst them, glutamylation consists in the reversible addition of a variable number of glutamate residues to the C-terminal tails of tubulins. Glutamylation is the most abundant MT PTM in the mammalian adult brain, suggesting that it plays an important role in the nervous system (NS). Here, we show that the previously uncharacterized CG31108 gene encodes an alpha-tubulin glutamylase acting in the Drosophila NS. We show that this glutamylase, which we named DmTTLL5, initiates MT glutamylation specifically on alpha-tubulin, which are the only glutamylated tubulin in the Drosophila brain. In DmTTLL5 mutants, MT glutamylation was not detected in the NS, allowing for determining its potential function. DmTTLL5 mutants are viable and we did not find any defect in vesicular axonal transport, synapse morphology and larval locomotion. Moreover, DmTTLL5 mutant flies display normal negative geotaxis behavior and their lifespan is not altered. Thus, our work identifies DmTTLL5 as the major enzyme responsible for initiating neuronal MT glutamylation specifically on alpha-tubulin and we show that the absence of MT glutamylation is not detrimental for Drosophila NS function.

Polyglutamylases are enzymes that form polyglutamate side chains of variable lengths on proteins. Polyglutamylation of tubulin is believed to regulate interactions of microtubules (MTs) with MT-associated proteins and molecular motors. Subpopulations of MTs are differentially polyglutamylated, yet only one modifying enzyme has been discovered in mammals. In an attempt to better understand the heterogeneous appearance of tubulin polyglutamylation, we searched for additional enzymes and report here the identification of six mammalian polyglutamylases. Each of them has a characteristic mode of catalysis and generates distinct patterns of modification on MTs, which can be further diversified by cooperation of multiple enzymes. Polyglutamylases are restricted to confined tissues and subtypes of MTs by differential expression and localization. In conclusion, we propose a multienzyme mechanism of polyglutamylation that can explain how the diversity of polyglutamylation on selected types of MTs is controlled at the molecular level.

Soft tissue sarcomas with complex genomics are very heterogeneous tumors lacking simple prognosis markers or targeted therapies. Overexpression of a subset of mitotic genes from a signature called CINSARC is of bad prognosis, but the significance of this signature remains elusive. Here we precisely measure the cell cycle and mitosis duration of sarcoma cell lines and we found that the mitotic gene products overexpression does not reflect variation in the time spent during mitosis or G2/M. We also found that the CINSARC cell lines, we studied, are composed of a mixture of aneuploid, diploid, and tetraploid cells that are highly motile in vitro. After sorting diploid and tetraploid cells, we showed that the tetraploid cell clones do not possess a proliferative advantage, but are strikingly more motile and invasive than their diploid counterparts. This is correlated with higher levels of mitotic proteins overexpression. Owing that mitotic proteins are almost systematically degraded at the end of mitosis, we propose that it is the abnormal activity of the mitotic proteins during interphase that boosts the sarcoma cells migratory properties by affecting their cytoskeleton. To test this hypothesis, we designed a screen for mitotic or cytoskeleton protein inhibitors affecting the sarcoma cell migration potential independently of cytotoxic activities. We found that inhibition of several mitotic kinases drastically impairs the CINSARC cell invasive and migratory properties. This finding could provide a handle by which to selectively inhibit the most invasive cells.

Polyglutamylation is a posttranslational modification that generates glutamate side chains on tubulins and other proteins. Although this modification has been shown to be reversible, little is known about the enzymes catalyzing deglutamylation. Here we describe the enzymatic mechanism of protein deglutamylation by members of the cytosolic carboxypeptidase (CCP) family. Three enzymes (CCP1, CCP4, and CCP6) catalyze the shortening of polyglutamate chains and a fourth (CCP5) specifically removes the branching point glutamates. In addition, CCP1, CCP4, and CCP6 also remove gene-encoded glutamates from the carboxyl termini of proteins. Accordingly, we show that these enzymes convert detyrosinated tubulin into Δ2-tubulin and also modify other substrates, including myosin light chain kinase 1. We further analyze Purkinje cell degeneration (pcd) mice that lack functional CCP1 and show that microtubule hyperglutamylation is directly linked to neurodegeneration. Taken together, our results reveal that controlling the length of the polyglutamate side chains on tubulin is critical for neuronal survival.

Upon maturation in the bone marrow, polyploid megakaryocytes elongate very long and thin cytoplasmic branches called proplatelets. Proplatelets enter the sinusoids blood vessels in which platelets are ultimately released. Microtubule dynamics, bundling, sliding, and coiling, drive these dramatic morphological changes whose regulation remains poorly understood. Microtubule properties are defined by tubulin isotype composition and post-translational modification patterns. It remains unknown whether microtubule post-translational modifications occur in proplatelets and if so, whether they contribute to platelet formation.

Polyglycylation is a posttranslational modification that generates glycine side chains on proteins. Here we identify a family of evolutionarily conserved glycine ligases that modify tubulin using different enzymatic mechanisms. In mammals, two distinct enzyme types catalyze the initiation and elongation steps of polyglycylation, whereas Drosophila glycylases are bifunctional. We further show that the human elongating glycylase has lost enzymatic activity due to two amino acid changes, suggesting that the functions of protein glycylation could be sufficiently fulfilled by monoglycylation. Depletion of a glycylase in Drosophila using RNA interference results in adult flies with strongly decreased total glycylation levels and male sterility associated with defects in sperm individualization and axonemal maintenance. A more severe RNAi depletion is lethal at early developmental stages, indicating that protein glycylation is essential. Together with the observation that multiple proteins are glycylated, our functional data point towards a general role of glycylation in protein functions.

Tubulin post-translational modifications regulate microtubule properties and functions. Mitotic spindle microtubules are highly modified. While tubulin detyrosination promotes proper mitotic progression by recruiting specific microtubule-associated proteins motors, tubulin acetylation that occurs on specific microtubule subsets during mitosis is less well understood. Here, we show that siRNA-mediated depletion of the tubulin acetyltransferase ATAT1 in epithelial cells leads to a prolonged prometaphase arrest and the formation of monopolar spindles. This results from collapse of bipolar spindles, as previously described in cells deficient for the mitotic kinase PLK1. ATAT1-depleted mitotic cells have defective recruitment of PLK1 to centrosomes, defects in centrosome maturation and thus microtubule nucleation, as well as labile microtubule-kinetochore attachments. Spindle bipolarity could be restored, in the absence of ATAT1, by stabilizing microtubule plus-ends or by increasing PLK1 activity at centrosomes, demonstrating that the phenotype is not just a consequence of lack of K-fiber stability. We propose that microtubule acetylation of K-fibers is required for a recently evidenced cross talk between centrosomes and kinetochores.