Close

Pick-ya actin: A method to purify actin isoforms with bespoke key post-translational modifications

Tomoyuki Hatano, Lavanya Sivashanmugam, Andrejus Suchenko, Hamdi Hussain, Mohan K. Balasubramanian

Preprint posted on 6 November 2019 https://www.biorxiv.org/content/10.1101/833152v1

Article now published in Journal of Cell Science at http://dx.doi.org/10.1242/jcs.241406

Biochemically pure actin, now available with customised PTMs - a whole new playground for in vitro characterisation of actin behaviour.

Selected by Angika Basant

Categories: biochemistry, cell biology

Background:

Actin and tubulin are major components of the cytoskeleton that can polymerise into microfilaments to perform a plethora of functions in the life of a cell. Both are well-understood to be regulated by their inherent assembly and disassembly dynamics and by interactor proteins. Fundamental properties of these cytoskeletal elements have been revealed through in vitro assays where the source of pure actin and tubulin is generally animal tissue such as muscle or brain.

An important factor contributing to cellular properties of tubulin and actin proteins, and often unaccounted for in vitro, is their post-translational modification (PTMs). In recent years we have made progress in elucidating how both actin and tubulin can be post-translationally modified. For example, the microtubule detyrosinylation enzyme was only recently identified (1, 2). Here we focus on actin modifications.

All known actin PTMs are highly species and cell type-specific. Therefore, actin used from animal tissues for in vitro assays either contain monomers with mixed or highly limited PTMs. This greatly restricts our understanding of actin behaviour in different organisms and of the most commonly found cytosolic actin.

Six isoforms of actin are known and they can be post-translationally modified in many ways (3). Though these isoforms are highly similar they have unique N-termini that are subject to modification. Human cytosolic b and g actin isoforms undergo a multistep maturation process. The initiator methionine is removed, and the following aspartate or glutamate residue is frequently acetylated or arginylated. As the actin N-terminus is highly acidic, these PTMs dramatically affect the charge of this region. Moreover, the N-terminus of both monomeric and filamentous actin is exposed on the surface, which suggests that these modifications very likely impact function.

In 2018, Hatano et al developed a method to cost-effectively purify heterologous actin and arginylated actin in large scales by expression in the methylotropic yeast P. pastoris; another feather in the cap of this powerful protein production host system (4, 5). However, the full suite of isoform-specific actin PTMs was still unavailable in biochemically pure form.

Solving long-standing mysteries, key enzymes involved in post-translational processing of actin have been recently identified. NAA80 was revealed to be the N-terminal acetyltransferase that completes N-terminal maturation of actin (6, 7). Meanwhile SETD3 is the methyltransferase that methylates actin at histidine 73 (8, 9). Taking advantage of these findings, in this preprint Hatano et al expand the scope of their protein expression method to purify actin tailored with specific and combinatorial PTMs.

Key findings:

The central approach in their methodology is to express in P. pastoris a quadruple fusion construct of ubiquitin followed by the desired actin isoform (without the initiator methionine) followed by the human thymosinb4 gene with 8 His residues at the C-terminus. The His-tag permits purification of the protein. The thymosinb4 forms an intramolecular interaction with actin preventing polymerisation of this monomer. A natural, conserved phenylalanine residue at the C-terminus of actin allows cleavage of thymosinb4 by addition of chymotrypsin at a desired time. The ubiquitin moiety is cleaved by an endogenous protease leaving the N-terminal residue of actin accessible for modification.

To acetylate the N-terminus of the expressed actin, NAA80 enzyme was co-expressed. Alternately or in combination, by co-expressing the SETD3 enzyme, the authors could also successfully purify actin methylated at position 73. The presence of the resulting PTMs was confirmed by mass spectrometry and purified actin monomers were demonstrated to polymerise normally in in vitro assays.

Using these approaches human b and g actin of the following forms were produced with good yields (a) unmodified (b) N- Asp/Glu acetylated (c) N-arginylated (d) His 73-methylated and (e) N- Asp/Glu acetylated; His 73-methylated. Additionally, this method was expanded to plant actin. An Arabidopsis isoform was expressed that was N-Ala acetylated (by an endogenous P. pastoris enzyme) and/or methylated at His 74 by exogenous SETD3.

What I like about this preprint:

This study resolves a major obstacle in the field of actin biology with an easy methodology that other labs could readily adapt and use, provided the modifying enzymes are known and readily expressed. I look forward to the first in vitro assays that characterise these specific, modified actin isoforms in detail.

Questions for the authors:

  1. How do the time investment, yield and costs of this method compare to conventional methods of actin purification from tissue?
  2. An endogenous pastoris acetylase can acetylate Arabidopsis actin. What other PTMs can this yeast confer, and do you envisage a scenario where endogenous enzymes would need to be deleted to generate the desired actin monomer?
  3. What challenges remain in expressing recombinant, tailor-made actin?

References:

  1. Aillaud et al., Science (2017)
  2. Nieuwenhuis et al., Science (2017)
  3. Varland et al., Trends in Biochem Sciences (2019)
  4. Hatano et al., Journal of Cell Science (2018)
  5. Ahmad et al., Applied Microbiol Biotechnol (2014)
  6. Drazic et al., PNAS (2018)
  7. Goris et al., PNAS (2018)
  8. Kwiatkowski et al., eLife (2018)
  9. Wilkinson et al., Nature (2018)

 

Posted on: 19 December 2019

doi: https://doi.org/10.1242/prelights.15949

Read preprint (No Ratings Yet)

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the biochemistry category:

Preprint Peer Review – Biochemistry Course at UFRJ, Brazil

Communication of scientific knowledge has changed dramatically in recent decades and the public perception of scientific discoveries depends on the peer review process of articles published in scientific journals. Preprints are key vehicles for the dissemination of scientific discoveries, but they are still not properly recognized by the scientific community since peer review is very limited. On the other hand, peer review is very heterogeneous and a fundamental aspect to improve it is to train young scientists on how to think critically and how to evaluate scientific knowledge in a professional way. Thus, this course aims to: i) train students on how to perform peer review of scientific manuscripts in a professional manner; ii) develop students' critical thinking; iii) contribute to the appreciation of preprints as important vehicles for the dissemination of scientific knowledge without restrictions; iv) contribute to the development of students' curricula, as their opinions will be published and indexed on the preLights platform. The evaluations will be based on qualitative analyses of the oral presentations of preprints in the field of biochemistry deposited in the bioRxiv server, of the critical reports written by the students, as well as of the participation of the students during the preprints discussions.

 



List by Marcus Oliveira

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

20th “Genetics Workshops in Hungary”, Szeged (25th, September)

In this annual conference, Hungarian geneticists, biochemists and biotechnologists presented their works. Link: http://group.szbk.u-szeged.hu/minikonf/archive/prg2021.pdf

 



List by Nándor Lipták

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra

Also in the cell biology category:

‘In preprints’ from Development 2022-2023

A list of the preprints featured in Development's 'In preprints' articles between 2022-2023

 



List by Alex Eve, Katherine Brown

preLights peer support – preprints of interest

This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.

 



List by preLights peer support

The Society for Developmental Biology 82nd Annual Meeting

This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.

 



List by Joyce Yu, Katherine Brown

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23

 



List by Alex Eve

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.

 



List by Helen Zenner

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.

 



List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.

 



List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria

 



List by Dey Lab, Samantha Seah

1

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA

 



List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.

 



List by Dey Lab, Amanda Haage
Close