scholarly journals ATAT1-enriched vesicles promote microtubule acetylation via axonal transport

2019 ◽  
Vol 5 (12) ◽  
pp. eaax2705 ◽  
Author(s):  
Aviel Even ◽  
Giovanni Morelli ◽  
Loïc Broix ◽  
Chiara Scaramuzzino ◽  
Silvia Turchetto ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Vesicular Transport ◽  
Microtubule Dynamics ◽  
Tubulin Acetylation ◽  
Cytosolic Side ◽  
Neuronal Vesicles ◽  
Β Tubulin

Microtubules are polymerized dimers of α- and β-tubulin that underlie a broad range of cellular activities. Acetylation of α-tubulin by the acetyltransferase ATAT1 modulates microtubule dynamics and functions in neurons. However, it remains unclear how this enzyme acetylates microtubules over long distances in axons. Here, we show that loss of ATAT1 impairs axonal transport in neurons in vivo, and cell-free motility assays confirm a requirement of α-tubulin acetylation for proper bidirectional vesicular transport. Moreover, we demonstrate that the main cellular pool of ATAT1 is transported at the cytosolic side of neuronal vesicles that are moving along axons. Together, our data suggest that axonal transport of ATAT1-enriched vesicles is the predominant driver of α-tubulin acetylation in axons.

scholarly journals ATAT1-enriched vesicles promote microtubule acetylation via axonal transport

2019 ◽  
Author(s):  
Aviel Even ◽  
Giovanni Morelli ◽  
Chiara Scaramuzzino ◽  
Ivan Gladwyn-Ng ◽  
Romain Le Bail ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Vesicular Transport ◽  
Microtubule Dynamics ◽  
Tubulin Acetylation ◽  
Cytosolic Side ◽  
Neuronal Vesicles ◽  
Β Tubulin

Microtubules are polymerized dimers of α- and β-tubulin that underlie a broad range of cellular activities. Acetylation of α-tubulin by the acetyl-transferase ATAT1 modulates microtubule dynamics and functions in neurons. However, it remains unclear how and why this enzyme acetylates microtubules over long distances in axons. Here, we show that loss of ATAT1 impairs axonal transport in neurons and cell free motility assays confirm a requirement of tubulin acetylation for proper bidirectional vesicular transport. Moreover, we demonstrate that the main cellular pool of ATAT1 is transported at the cytosolic side of neuronal vesicles that are moving along axons. Altogether, our data suggest that axonal transport of ATAT1-enriched vesicles is the predominant driver of α-tubulin acetylation in axons.

scholarly journals β-Tubulin C354 Mutations that Severely Decrease Microtubule Dynamics Do Not Prevent Nuclear Migration in Yeast

2002 ◽  
Vol 13 (8) ◽  
pp. 2919-2932 ◽  
Author(s):  
Mohan L. Gupta ◽  
Claudia J. Bode ◽  
Douglas A. Thrower ◽  
Chad G. Pearson ◽  
Kathy A. Suprenant ◽  
...  
Keyword(s):  
Essential Gene ◽  
Dynamic Properties ◽  
Microtubule Dynamics ◽  
Cytoplasmic Microtubule ◽  
Bud Emergence ◽  
Spindle Elongation ◽  
Mutant Cells ◽  
Β Tubulin

Microtubule dynamics are influenced by interactions of microtubules with cellular factors and by changes in the primary sequence of the tubulin molecule. Mutations of yeast β-tubulin C354, which is located near the binding site of some antimitotic compounds, reduce microtubule dynamicity greater than 90% in vivo and in vitro. The resulting intrinsically stable microtubules allowed us to determine which, if any, cellular processes are dependent on dynamic microtubules. The average number of cytoplasmic microtubules decreased from 3 in wild-type to 1 in mutant cells. The single microtubule effectively located the bud site before bud emergence. Although spindles were positioned near the bud neck at the onset of anaphase, the mutant cells were deficient in preanaphase spindle alignment along the mother-bud axis. Spindle microtubule dynamics and spindle elongation rates were also severely depressed in the mutants. The pattern and extent of cytoplasmic microtubule dynamics modulation through the cell cycle may reveal the minimum dynamic properties required to support growth. The ability to alter intrinsic microtubule dynamics and determine the in vivo phenotype of cells expressing the mutant tubulin provides a critical advance in assessing the dynamic requirements of an essential gene function.

scholarly journals Tubulin cofactors and Arl2 are cage-like chaperones that regulate the soluble αβ-tubulin pool for microtubule dynamics

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Stanley Nithianantham ◽  
Sinh Le ◽  
Elbert Seto ◽  
Weitao Jia ◽  
Julie Leary ◽  
...  
Keyword(s):  
Ternary Complex ◽  
Microtubule Dynamics ◽  
Gtp Hydrolysis ◽  
Tubulin Dimer ◽  
Physical Mechanisms ◽  
Biochemical Assays ◽  
Sequential Binding ◽  
Β Tubulin

Microtubule dynamics and polarity stem from the polymerization of αβ-tubulin heterodimers. Five conserved tubulin cofactors/chaperones and the Arl2 GTPase regulate α- and β-tubulin assembly into heterodimers and maintain the soluble tubulin pool in the cytoplasm, but their physical mechanisms are unknown. Here, we reconstitute a core tubulin chaperone consisting of tubulin cofactors TBCD, TBCE, and Arl2, and reveal a cage-like structure for regulating αβ-tubulin. Biochemical assays and electron microscopy structures of multiple intermediates show the sequential binding of αβ-tubulin dimer followed by tubulin cofactor TBCC onto this chaperone, forming a ternary complex in which Arl2 GTP hydrolysis is activated to alter αβ-tubulin conformation. A GTP-state locked Arl2 mutant inhibits ternary complex dissociation in vitro and causes severe defects in microtubule dynamics in vivo. Our studies suggest a revised paradigm for tubulin cofactors and Arl2 functions as a catalytic chaperone that regulates soluble αβ-tubulin assembly and maintenance to support microtubule dynamics.

scholarly journals A novel acetylation of β-tubulin by San modulates microtubule polymerization via down-regulating tubulin incorporation

2011 ◽  
Vol 22 (4) ◽  
pp. 448-456 ◽  
Author(s):  
Chih-Wen Chu ◽  
Fajian Hou ◽  
Junmei Zhang ◽  
Lilian Phu ◽  
Alex V. Loktev ◽  
...  
Keyword(s):  
Hela Cells ◽  
Posttranslational Modifications ◽  
Positive Charge ◽  
Dynamic Instability ◽  
Tubulin Polymerization ◽  
Critical Property ◽  
Tubulin Acetylation ◽  
Microtubule Polymerization ◽  
Β Tubulin

Dynamic instability is a critical property of microtubules (MTs). By regulating the rate of tubulin polymerization and depolymerization, cells organize the MT cytoskeleton to accommodate their specific functions. Among many processes, posttranslational modifications of tubulin are implicated in regulating MT functions. Here we report a novel tubulin acetylation catalyzed by acetyltransferase San at lysine 252 (K252) of β-tubulin. This acetylation, which is also detected in vivo, is added to soluble tubulin heterodimers but not tubulins in MTs. The acetylation-mimicking K252A/Q mutants were incorporated into the MT cytoskeleton in HeLa cells without causing any obvious MT defect. However, after cold-induced catastrophe, MT regrowth is accelerated in San-siRNA cells while the incorporation of acetylation-mimicking mutant tubulins is severely impeded. K252 of β-tubulin localizes at the interface of α-/β-tubulins and interacts with the phosphate group of the α-tubulin-bound GTP. We propose that the acetylation slows down tubulin incorporation into MTs by neutralizing the positive charge on K252 and allowing tubulin heterodimers to adopt a conformation that disfavors tubulin incorporation.

scholarly journals SUMOylation of α-tubulin is a novel modification regulating microtubule dynamics

2021 ◽  
Author(s):  
Wenfeng Feng ◽  
Rong Liu ◽  
Xuan Xie ◽  
Lei Diao ◽  
Nannan Gao ◽  
...  
Keyword(s):  
Microtubule Dynamics ◽  
In Vitro Experiments ◽  
Post Translational Modification ◽  
Cellular Functions ◽  
Post Translational Modifications ◽  
Neurite Extension ◽  
Microtubule Polymerization ◽  
Β Tubulin

Abstract Microtubules are regulated by a number of known post-translational modifications on α/β-tubulin to fulfill diverse cellular functions. Here, we showed that SUMOylation is a novel post-translational modification on α-tubulin in vivo and in vitro. The SUMOylation on α-tubulin mainly occurred at Lys 96 (K96), K166, and K304 of soluble α-tubulin and could be removed by SUMO-specific peptidase 1. In vitro experiments showed that tubulin SUMOylation could reduce inter-protofilament interaction, promote microtubule catastrophe, and impede microtubule polymerization. In cells, mutation of the SUMOylation sites on α-tubulin reduced catastrophe frequency and increased the proportion of polymerized α-tubulin, while upregulation of SUMOylation with fusion of SUMO1 reduced α-tubulin assembly into microtubules. Additionally, overexpression of SUMOylation-deficient α-tubulin attenuated the neurite extension in Neuro-2a cells. Thus, SUMOylation on α-tubulin represents a new player in the regulation of microtubule properties.

scholarly journals Microtubule Regulation in Mitosis: Tubulin Phosphorylation by the Cyclin-dependent Kinase Cdk1

2006 ◽  
Vol 17 (3) ◽  
pp. 1041-1050 ◽  
Author(s):  
Anne Fourest-Lieuvin ◽  
Leticia Peris ◽  
Vincent Gache ◽  
Isabel Garcia-Saez ◽  
Céline Juillan-Binard ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Microtubule Dynamics ◽  
Cyclin Dependent Kinase ◽  
Gtp Binding ◽  
Tubulin Binding ◽  
A Site ◽  
Β Tubulin ◽  
In Cells

The activation of the cyclin-depdndent kinase Cdk1 at the transition from interphase to mitosis induces important changes in microtubule dynamics. Cdk1 phosphorylates a number of microtubule- or tubulin-binding proteins but, hitherto, tubulin itself has not been detected as a Cdk1 substrate. Here we show that Cdk1 phosphorylates β-tubulin both in vitro and in vivo. Phosphorylation occurs on Ser172 of β-tubulin, a site that is well conserved in evolution. Using a phosphopeptide antibody, we find that a fraction of the cell tubulin is phosphorylated during mitosis, and this tubulin phosphorylation is inhibited by the Cdk1 inhibitor roscovitine. In mitotic cells, phosphorylated tubulin is excluded from microtubules, being present in the soluble tubulin fraction. Consistent with this distribution in cells, the incorporation of Cdk1-phosphorylated tubulin into growing microtubules is impaired in vitro. Additionally, EGFP-β3-tubulinS172D/E mutants that mimic phosphorylated tubulin are unable to incorporate into microtubules when expressed in cells. Modeling shows that the presence of a phosphoserine at position 172 may impair both GTP binding to β-tubulin and interactions between tubulin dimers. These data indicate that phosphorylation of tubulin by Cdk1 could be involved in the regulation of microtubule dynamics during mitosis.

scholarly journals Stu2p binds tubulin and undergoes an open-to-closed conformational change

2006 ◽  
Vol 172 (7) ◽  
pp. 1009-1022 ◽  
Author(s):  
Jawdat Al-Bassam ◽  
Mark van Breugel ◽  
Stephen C. Harrison ◽  
Anthony Hyman
Keyword(s):  
Conformational Change ◽  
Conformational Transition ◽  
Budding Yeast ◽  
Microtubule Dynamics ◽  
Open Structure ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Common

Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat–containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.

scholarly journals C9orf72-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility

2021 ◽  
Vol 7 (15) ◽  
pp. eabg3013
Author(s):  
Laura Fumagalli ◽  
Florence L. Young ◽  
Steven Boeynaems ◽  
Mathias De Decker ◽  
Arpan R. Mehta ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Motor Neurons ◽  
Single Molecule ◽  
Repeat Expansion ◽  
Physical Interaction ◽  
Hexanucleotide Repeat ◽  
Hexanucleotide Repeat Expansion ◽  
Axonal Trafficking ◽  
Inhibitory Interactions

A hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this mutation leads to these neurodegenerative diseases remains unclear. Here, we show using patient stem cell–derived motor neurons that the repeat expansion impairs microtubule-based transport, a process critical for neuronal survival. Cargo transport defects are recapitulated by treating neurons from healthy individuals with proline-arginine and glycine-arginine dipeptide repeats (DPRs) produced from the repeat expansion. Both arginine-rich DPRs similarly inhibit axonal trafficking in adult Drosophila neurons in vivo. Physical interaction studies demonstrate that arginine-rich DPRs associate with motor complexes and the unstructured tubulin tails of microtubules. Single-molecule imaging reveals that microtubule-bound arginine-rich DPRs directly impede translocation of purified dynein and kinesin-1 motor complexes. Collectively, our study implicates inhibitory interactions of arginine-rich DPRs with axonal transport machinery in C9orf72-associated ALS/FTD and thereby points to potential therapeutic strategies.

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