Impaired α-tubulin re-tyrosination leads to synaptic dysfunction and is a feature of Alzheimer's disease

In neurons, dynamic microtubules play regulatory roles in neurotransmission and synaptic plasticity. While stable microtubules contain detyrosinated tubulin, dynamic microtubules are composed of tyrosinated tubulin, suggesting that the tubulin tyrosination/detyrosination (Tyr/deTyr) cycle modulates microtubule dynamics and synaptic function. In the Tyr/deTyr cycle, the C-terminal tyrosine of alpha-tubulin is re-added by tubulin-tyrosine-ligase (TTL). Here we show that TTL+/- mice exhibit decreased tyrosinated microtubules, synaptic plasticity and memory deficits, and that reduced TTL expression is a feature of sporadic and familial Alzheimer's disease (AD), with human APPV717I neurons having less dynamic microtubules. We find that spines visited by dynamic microtubules are more resistant to Abeta1-42 and that TTL, by promoting microtubule entry into spines, prevents Abeta1-42-induced spine pruning. Our results demonstrate that the Tyr/deTyr cycle regulates synaptic plasticity, is protective against spine injury, and that tubulin re-tyrosination is lost in AD, providing evidence that a defective Tyr/deTyr cycle may contribute to neurodegeneration.

TTL-Expression Modulates Epithelial Morphogenesis

Epithelial monolayer formation depends on the architecture and composition of the microtubule cytoskeleton. Microtubules control bidirectional trafficking and determine the positioning of structural cellular proteins. We studied the role of tubulin tyrosination in epithelial cell shape and motility. Tubulin tyrosine ligase (TTL), the enzyme that adds tyrosine to the carboxy terminus of detyrosinated α-tubulin, was depleted or overexpressed in 2D epithelial monolayers as well as in 3D intestinal organoids. We demonstrate qualitatively and quantitatively that in the absence of TTL the cells comprise high levels of detyrosinated tubulin, change their shape into an initial flat morphology and retardedly acquire a differentiated columnar epithelial cell shape. Enhanced adhesion and accelerated migration patterns of TTL-knockout cells combined with reverse effects in TTL-overexpressing cells indicate that the loss of TTL affects the organization of cell adhesion foci. Precipitation of detyrosinated tubulin with focal adhesion scaffold components coincides with increased quantities and persistence of focal adhesion plaques. Our results indicate that the equilibrium between microtubules enriched in detyrosinated or tyrosinated tubulin modulates epithelial tissue formation, cell morphology, and adhesion.

Defective tubulin detyrosination causes structural brain abnormalities with cognitive deficiency in humans and mice

Reversible detyrosination of tubulin, the building block of microtubules, is crucial for neuronal physiology. Enzymes responsible for detyrosination were recently identified as complexes of vasohibins (VASHs) one or two with small VASH-binding protein (SVBP). Here we report three consanguineous families, each containing multiple individuals with biallelic inactivation of SVBP caused by truncating variants (p.Q28* and p.K13Nfs*18). Affected individuals show brain abnormalities with microcephaly, intellectual disability and delayed gross motor and speech development. Immunoblot testing in cells with pathogenic SVBP variants demonstrated that the encoded proteins were unstable and non-functional, resulting in a complete loss of VASH detyrosination activity. Svbp knockout mice exhibit drastic accumulation of tyrosinated tubulin and a reduction of detyrosinated tubulin in brain tissue. Similar alterations in tubulin tyrosination levels were observed in cultured neurons and associated with defects in axonal differentiation and architecture. Morphological analysis of the Svbp knockout mouse brains by anatomical magnetic resonance imaging showed a broad impact of SVBP loss, with a 7% brain volume decrease, numerous structural defects and a 30% reduction of some white matter tracts. Svbp knockout mice display behavioural defects, including mild hyperactivity, lower anxiety and impaired social behaviour. They do not, however, show prominent memory defects. Thus, SVBP-deficient mice recapitulate several features observed in human patients. Altogether, our data demonstrate that deleterious variants in SVBP cause this neurodevelopmental pathology, by leading to a major change in brain tubulin tyrosination and alteration of microtubule dynamics and neuron physiology.

Fidgetin regulates cultured astrocyte migration by severing tyrosinated microtubules at the leading edge

Microtubule (MT) organization is essential for many cellular events, including mitosis, migration, and cell polarity. Fidgetin (Fign), an ATP-dependent, MT-severing protein, contributes to the regulation of MT configuration by cutting and trimming MT polymers. Functions of Fign have been indicated in neurite outgrowth, mitosis, meiosis, and cellular migration. Here we focus on migration of astrocytes. We find that Fign plays an essential role in cultured astrocyte migration by preferentially targeting MTs (or regions of MTs) that are rich in tyrosinated tubulin, a marker for especially dynamic MTs or especially dynamic regions of MTs. Inhibition of cellular migration induced by Fign knockdown can be rescued with concomitant knockdown of kinesin-12, a motor protein best known for its role in mitosis. We propose a novel working model for MT reconfiguration underlying cellular migration elicited by the functional cooperation of two distinct MT-related proteins.

Evidence for new C-terminally truncated variants of α- and β-tubulins

Cellular α-tubulin can bear various carboxy-terminal sequences: full-length tubulin arising from gene neosynthesis is tyrosinated, and two truncated variants, corresponding to detyrosinated and Δ2 α‑tubulin, result from the sequential cleavage of one or two C-terminal residues, respectively. Here, by using a novel antibody named 3EG that is highly specific to the –EEEG C-terminal sequence, we demonstrate the occurrence in neuronal tissues of a new αΔ3‑tubulin variant corresponding to α1A/B‑tubulin deleted of its last three residues (EEY). αΔ3‑tubulin has a specific distribution pattern: its quantity in the brain is similar to that of αΔ2-tubulin around birth but is much lower in adult tissue. This truncated α1A/B-tubulin variant can be generated from αΔ2-tubulin by the deglutamylases CCP1, CCP4, CCP5, and CCP6 but not by CCP2 and CCP3. Moreover, using 3EG antibody, we identify a C‑terminally truncated β-tubulin form with the same –EEEG C-terminal sequence. Using mass spectrometry, we demonstrate that β2A/B-tubulin is modified by truncation of the four C-terminal residues (EDEA). We show that this newly identified βΔ4-tubulin is ubiquitously present in cells and tissues and that its level is constant throughout the cell cycle. These new C-terminally truncated α- and β-tubulin variants, both ending with –EEEG sequence, are expected to regulate microtubule physiology. Of interest, the αΔ3-tubulin seems to be related to dynamic microtubules, resembling tyrosinated-tubulin rather than the other truncated variants, and may have critical function(s) in neuronal development.

The larval nervous system of the penis worm Priapulus caudatus (Ecdysozoa)

The origin and extreme diversification of the animal nervous system is a central question in biology. While most of the attention has traditionally been paid to those lineages with highly elaborated nervous systems (e.g. arthropods, vertebrates, annelids), only the study of the vast animal diversity can deliver a comprehensive view of the evolutionary history of this organ system. In this regard, the phylogenetic position and apparently conservative molecular, morphological and embryological features of priapulid worms (Priapulida) place this animal lineage as a key to understanding the evolution of the Ecdysozoa (i.e. arthropods and nematodes). In this study, we characterize the nervous system of the hatching larva and first lorica larva of the priapulid worm Priapulus caudatus by immunolabelling against acetylated and tyrosinated tubulin, pCaMKII, serotonin and FMRFamide. Our results show that a circumoral brain and an unpaired ventral nerve with a caudal ganglion characterize the central nervous system of hatching embryos. After the first moult, the larva attains some adult features: a neck ganglion, an introvert plexus, and conspicuous secondary longitudinal neurites. Our study delivers a neuroanatomical framework for future embryological studies in priapulid worms, and helps illuminate the course of nervous system evolution in the Ecdysozoa.

Tyrosination of α-Tubulin Controls The Initiation of Processive Dynein-Dynactin Motility

Post-translational modifications (PTMs) of αβtubulin are believed to regulate interactions with microtubule binding proteins. A well-characterized PTM involves the removal and re-ligation of the C-terminal tyrosine on α-tubulin, but the purpose of this tyrosination-detyrosination cycle remains elusive. Here, we examined the processive motility of mammalian dynein complexed with dynactin and BicD2 (DDB) on tyrosinated versus detyrosinated microtubules. Motility was decreased ~4-fold on detyrosinated microtubules, constituting the largest effect of a tubulin PTM on motor function observed to date. This preference is mediated by dynactin's microtubule binding p150 subunit rather than dynein itself. Interestingly, on chimeric microtubules, DDB molecules that initiated movement on tyrosinated tubulin continued moving into a region of detyrosinated tubulin. This result indicates that the α-tubulin tyrosine facilitates initial motor-tubulin encounters, but is not needed for subsequent motility. Our results reveal a strong effect of the C-terminal α-tubulin tyrosine on dynein-dynactin motility and suggest that the tubulin tyrosination cycle could modulate the initiation of dynein-driven motility in cells.

Effects of kinesin-5 inhibition on dendritic architecture and microtubule organization

Kinesin-5 is a slow homotetrameric motor protein best known for its essential role in the mitotic spindle, where it limits the rate at which faster motors can move microtubules. In neurons, experimental suppression of kinesin-5 causes the axon to grow faster by increasing the mobility of microtubules in the axonal shaft and the invasion of microtubules into the growth cone. Does kinesin-5 act differently in dendrites, given that they have a population of minus end–distal microtubules not present in axons? Using rodent primary neurons in culture, we found that inhibition of kinesin-5 during various windows of time produces changes in dendritic morphology and microtubule organization. Specifically, dendrites became shorter and thinner and contained a greater proportion of minus end–distal microtubules, suggesting that kinesin-5 acting normally restrains the number of minus end–distal microtubules that are transported into dendrites. Additional data indicate that, in neurons, CDK5 is the kinase responsible for phosphorylating kinesin-5 at Thr-926, which is important for kinesin-5 to associate with microtubules. We also found that kinesin-5 associates preferentially with microtubules rich in tyrosinated tubulin. This is consistent with an observed accumulation of kinesin-5 on dendritic microtubules, as they are known to be less detyrosinated than axonal microtubules.

CLUH regulates mitochondrial biogenesis by binding mRNAs of nuclear-encoded mitochondrial proteins

Mitochondrial function requires coordination of two genomes for protein biogenesis, efficient quality control mechanisms, and appropriate distribution of the organelles within the cell. How these mechanisms are integrated is currently not understood. Loss of the Clu1/CluA homologue (CLUH) gene led to clustering of the mitochondrial network by an unknown mechanism. We find that CLUH is coregulated both with genes encoding mitochondrial proteins and with genes involved in ribosomal biogenesis and translation. Our functional analysis identifies CLUH as a cytosolic messenger ribonucleic acid (RNA; mRNA)–binding protein. RNA immunoprecipitation experiments followed by next-generation sequencing demonstrated that CLUH specifically binds a subset of mRNAs encoding mitochondrial proteins. CLUH depletion decreased the levels of proteins translated by target transcripts and caused mitochondrial clustering. A fraction of CLUH colocalizes with tyrosinated tubulin and can be detected close to mitochondria, suggesting a role in regulating transport or translation of target transcripts close to mitochondria. Our data unravel a novel mechanism linking mitochondrial biogenesis and distribution.

Visualization of microtubule growth in living platelets reveals a dynamic marginal band with multiple microtubules

The marginal band of microtubules maintains the discoid shape of resting blood platelets. Although studies of platelet microtubule coil structure conclude that it is composed of a single microtubule, no investigations of its dynamics exist. In contrast to previous studies, permeabilized platelets incubated with GTP-rhodamine-tubulin revealed tubulin incorporation at 7.9 (± 1.9) points throughout the coil, and anti-EB1 antibodies stained 8.7 (± 2.0) sites, indicative of multiple free microtubules. To pursue this result, we expressed the microtubule plus-end marker EB3-GFP in megakaryocytes and examined its behavior in living platelets released from these cells. Time-lapse microscopy of EB3-GFP in resting platelets revealed multiple assembly sites within the coil and a bidirectional pattern of assembly. Consistent with these findings, tyrosinated tubulin, a marker of newly assembled microtubules, localized to resting platelet microtubule coils. These results suggest that the resting platelet marginal band contains multiple highly dynamic microtubules of mixed polarity. Analysis of microtubule coil diameters in newly formed resting platelets indicates that microtubule coil shrinkage occurs with aging. In addition, activated EB3-GFP–expressing platelets exhibited a dramatic increase in polymerizing microtubules, which travel outward and into filopodia. Thus, the dynamic microtubules associated with the marginal band likely function during both resting and activated platelet states.