Close

Genetically encoded multimeric tags for intracellular protein localisation in cryo-EM

Herman KH Fung, Yuki Hayashi, Veijo T Salo, Anastasiia Babenko, Ievgeniia Zagoriy, Andreas Brunner, Jan Ellenberg, Christoph W Müller, Sara Cuylen-Haering, Julia Mahamid

Preprint posted on 10 December 2022 https://www.biorxiv.org/content/10.1101/2022.12.10.519870v1

A new development of a small molecule-inducible approach to label proteins, enables nanometer-scale localization of molecules via cryo-ET combined with fluorescence microscopy.

Selected by Martyna Kosno-Vega

Background:

To understand how cells function in a given process, we need to know the precise sub-cellular localization of all relevant molecules. However, the crowded cellular environment makes this a difficult task. Cryo-electron tomography (cryo-ET) and cryo-correlative light and electron microscopy (CLEM) are some of the techniques that can help address this problem [1, 2]. While much progress has been made in recent years, uncovering fine details using cellular imaging remains very difficult due to the fluorescence imaging resolution limit and potential distortion of samples during the preparation process. Thus, further developments of these imaging methods are needed.

In this collaborative study, the Mahamid and Culyen-Haering research groups from EMBL developed a novel strategy to tag proteins in cells in order to precisely identify their location. To do so, they designed a genetically encoded multimeric (GEM) protein tag, which binds to GFP upon small molecule treatment, through an FRB-FKBP interaction. They wanted the tag to not only provide fluorescent signal, but also be visible when using cryo-ET. Thus, the scientists based their labeling system on encapsulins – 25-42 nm proteins from archaea and bacteria with an unusual, icosahedral shape, which can be easily seen via electron microscopy [3].

Preprint Figure 1a: Schematic of the system designed by the authors.

Key Findings

GEM2 is the best candidate to tag proteins of interest

The group performed a library screen of multiple GEMs, including 10 natural encapsulins and 3 engineered proteins in HeLa cells. They found that one of the candidates exhibited uniform distribution both in the cytoplasm and the nucleus of mammalian cells (GEM2). Next, the authors used the GEM2 tag with several known targets, to assess its accuracy of labeling, its applicability to natural cellular systems and visibility in cryo-ET.

Functional assays confirm GEM2’s utility in structural and cellular studies

The scientists chose three known and diverse targets: the mitotic chromosome surface, nuclear pore, and endoplasmic reticulum-lipid droplet junction. In all cases they observed small molecule-inducible colocalization of the GEM tag with the chosen target protein. However, the dynamics of recruitment were dependent on the target protein’s abundance (the more abundant the protein, the faster the dynamics of recruitment). The icosahedral particles of the GEM tag were visible in cryo-ET, a majority within ~50nm of the expected location, making this system very accurate. Due to the multimeric nature of the GEMs, each particle comes with 60 Halo tags to provide strong fluorescence and thus help guide cryo-ET data collection.

Preprint Figure 2e: From left to right: cryo-TEM image overlaid with a fluorescent image. Center and right images show tomographic slice and itssegmentation with insets showing details of the ER-LD contact site with the GEM tags in close proximity.

In summary, this study provides a novel, small molecule-inducible approach to label proteins, allowing nanometer-scale localization of molecules. The method seems promising for many types of experiments, with some additional optimization required depending on the application. The authors claim that it is easy to implement and optimize for any system and that it should help expand our structural knowledge of diverse subcellular compartments.

Why I chose this preprint

I have been working towards my PhD in the Department of Biophysics at UT Southwestern, where a large focus of many labs is on structural biology. Nonetheless, I am a cell biologist at heart. This preprint provides a very interesting method to combine the two: structural biology and cell biology, promising to unravel fine details of subcellular organization.

I am also incredibly impressed when scientists decide to utilize a system that nature already came up with and optimize it for scientific purposes. We have seen a similar thing with CRISPR, back in 2012 [4]. Here, the two labs “borrowed” encapsulin proteins, which – in bacteria and archaea – serve as structural blocks to build protein cages. These cages provide a means of compartmentalization for an otherwise organelle-less organism. The scientists decided to utilize the large size and unique shape of these proteins to help guide cryo-ET in human cells.

Future directions and questions for the authors

It will be fascinating to see where this technology could lead structural studies in the coming years and how much it can be optimized. It would be great – for example – if the technology could be less dependent on protein abundance and potential protein shape. It will also be important to determine if in fact the applicability of this system is as wide as the authors claim.

  • Could GEM tags affect the function of the target protein in systems other than the three tested by the authors?
  • Due to their size, could GEMs sterically hinder the proper protein localization?

References

  1. Dahlberg, P.D., et al., Cryogenic single-molecule fluorescence annotations for electron tomography reveal in situ organization of key proteins in Caulobacter. Proc Natl Acad Sci U S A, 2020. 117(25): p. 13937-13944.
  2. Rigort, A., et al., Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography. Proc Natl Acad Sci U S A, 2012. 109(12): p. 4449-54.
  3. Andreas, M.P. and T.W. Giessen, Large-scale computational discovery and analysis of virus-derived microbial nanocompartments. Nat Commun, 2021. 12(1): p. 4748.
  4. Jinek, M., et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012. 337(6096): p. 816-21.

 

Posted on: 16 January 2023

doi: https://doi.org/10.1242/prelights.33493

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

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

ASCB/EMBO Annual Meeting 2018

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

 



List by Dey Lab, Amanda Haage

Also in the molecular 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

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23

 



List by Alex Eve

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.

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

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.

Lung Disease and Regeneration

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

 



List by Rob Hynds

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
Close