ATAT1-enriched vesicles promote microtubule acetylation via axonal transport

Even, Aviel; Morelli, Gabriele; Broix, Loïc; Scaramuzzino, Chiara; Turchetto, Silvia; Gladwyn-Ng, Ivan; Bail, Romain Le; Shilian, Michal; Freeman, Stephen; Magiera, Maria M.; Jijumon, A S; Krusy, Nathalie; Malgrange, Brigitte; Brône, Bert; Dietrich, Paula; Dragatsis, Ioannis; Janke, Carsten; Saudou, Frédéric; Weil, Miguel; Nguyen, Laurent

doi:10.1126/sciadv.aax2705

Cited by 59 publications

(50 citation statements)

References 44 publications

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“…Whether SETD2 methylates α−tubulin prior to or after assembly of α/β tubulin dimers into microtubules is not known. However, Even et al, reported that ATAT-1 is transported by vesicles along the length of microtubules, accessing K40 to acetylate this residue via pores in the microtubule shaft 15 . These and other studies have led to the growing appreciation that lattice dynamics of even stable microtubules can provide access to luminal residues along the shaft of microtubule polymers.…”

Section: Discussionmentioning

confidence: 99%

“…The pellet was removed and supernatants collected as whole cell extract for immunoblotting and immunoprecipitation analysis for soluble proteins. For subcellular fractionation, the pellet was collected after centrifugation as above, and resuspended in a hypotonic buffer (10 mM HEPES, pH 7.2, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA, 20 mM NaF, and 100 μM Na3VO4), and disrupted using a manual homogenizer for [15][16][17][18][19][20] times. Disruption of the cells was confirmed using a hemacytometer.…”

Section: Whole Cell Lysis and Sub-cellular Fractionationmentioning

confidence: 99%

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A Cytoskeletal Function for PBRM1 Reading Methylated Microtubules

Karki

Jangid

Seervai

et al. 2020

Preprint

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The chromatin modifier SETD2 was recently shown to be a dual-function methyltransferase that "writes" methyl marks on both chromatin and the mitotic spindle, revealing α-tubulin methylation as a new posttranslational modification of microtubules.Here, we report the first cytoskeletal "reader" for this SETD2 methyl mark: the polybromo protein PBRM1. We found PBRM1 directly binds the α-Tub-K40me3 mark on tubulin, and localizes to the mitotic spindle and spindle pole during cell division. PBRM1 can assemble a PBAF complex in the absence of chromatin as revealed by mass spectrometry, and can recruit other PBAF complex components including SMARCA4 and ARID2 to α-tubulin.In addition to PBRM1, other PBAF components were also localized to the mitotic spindle and spindle pole. This PBAF localization was dependent on recruitment to microtubules by PBRM1, and loss of spindle-associated PBRM1/PBAF led to genomic instability as assessed by increased formation of micronuclei. These data reveal a previously unknown function for PBRM1 beyond its role remodeling chromatin, and expand the repertoire of chromatin remodelers involved in writing and reading methyl marks on the cytoskeleton.The results of this study lay the foundation for a new paradigm for the epigenetic machinery as chromatocytoskeletal modifiers, with coordinated nuclear and cytoskeletal functions.3 Introduction:

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Section: Discussionmentioning

confidence: 99%

Section: Whole Cell Lysis and Sub-cellular Fractionationmentioning

confidence: 99%

A Cytoskeletal Function for PBRM1 Reading Methylated Microtubules

Karki

Jangid

Seervai

et al. 2020

Preprint

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“…Among these, BCL9L was the most highly enriched and most regulated interactor ( Fig. 4C-E); RNF20 forms a complex with RNF40 and participates in mitotic spindle assembly by monoubiquitinating the microtubule motor protein EG5 [28]; IFITM3 shares the same membrane topology and CD225/IFITM domain with TRARG1 [29,30]; ATAT1 and SPECC1L are involved in microtubule stabilization [31,32]; STEAP3 is a NADPH oxidoreductase involved in transferrin-dependent iron uptake [33,34]. Genes were knocked down by siRNA and GLUT4 translocation to the PM was measured in adipocytes expressing a GLUT4 reporter construct with an HA-tag in the first exofacial loop and mRuby3 at the cytosolic C-terminal tail (HA-GLUT4-mRuby3).…”

Section: Functional Screening Of Selected Trarg1 Interactorsmentioning

confidence: 99%

Trafficking regulator of GLUT4-1 (TRARG1) is a GSK3 substrate

Duan

Norris

Humphrey

et al. 2020

Preprint

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Trafficking regulator of GLUT4-1, TRARG1, positively regulates insulin-stimulated GLUT4 trafficking and insulin sensitivity. However, the mechanism(s) by which this occurs remain(s) unclear. Using biochemical and mass spectrometry analyses we found that TRARG1 is dephosphorylated in response to insulin in a PI3K/Akt-dependent manner and is a novel substrate for GSK3. Priming phosphorylation of mouse TRARG1 at serine 84 allows for GSK3-directed phosphorylation at serines 72, 76 and 80. A similar pattern of phosphorylation was observed in human TRARG1, suggesting that our findings are translatable to human TRARG1. Pharmacological inhibition of GSK3 increased cell surface GLUT4 in cells stimulated with a submaximal insulin dose, and this was impaired following Trarg1 knockdown, suggesting that TRARG1 acts as a GSK3-mediated regulator in GLUT4 trafficking. TRARG1 dephosphorylation in response to insulin or GSK3 inhibition regulated its protein-protein interactions, decreasing the interaction between TRARG1 and BCL9L, JAGN1, PYGO2 and HSD17B12. Knock-down of Bcl9l promoted GLUT4 translocation to the plasma membrane. These data place TRARG1 within the insulin signaling network and provide insights into how TRARG1 regulates GLUT4 trafficking in adipocytes.

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“…This ex vivo protocol has been developed to investigate axonal transport in a physiological setting closely reproducing the in vivo environment. For complete details on the use and execution of this protocol, please refer to Even et al. (2019) .…”

mentioning

confidence: 99%

“…For complete details on the use and execution of this protocol, please refer to Even et al. (2019) .…”

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confidence: 99%

Ex Vivo Recording of Axonal Transport Dynamics on Postnatal Organotypic Cortical Slices

2020

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Summary Axonal transport is a physiological process adopted by neurons to transport organelles, proteins, and other molecules along their axonal projections. Here, we describe a step-by-step protocol to record the dynamics of axonal transport along the projections of callosal neurons by combining the in utero electroporation technique with the preparation of postnatal organotypic cortical slices. This ex vivo protocol has been developed to investigate axonal transport in a physiological setting closely reproducing the in vivo environment. For complete details on the use and execution of this protocol, please refer to Even et al. (2019) .

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ATAT1-enriched vesicles promote microtubule acetylation via axonal transport

Abstract: The axonal transport of vesicles promotes microtubule acetylation across species.

Cited by 59 publications

References 44 publications

A Cytoskeletal Function for PBRM1 Reading Methylated Microtubules

A Cytoskeletal Function for PBRM1 Reading Methylated Microtubules

Trafficking regulator of GLUT4-1 (TRARG1) is a GSK3 substrate

Ex Vivo Recording of Axonal Transport Dynamics on Postnatal Organotypic Cortical Slices

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