Designer organelles bring new functionalities into cells

Authored by embl.de and submitted by mvea
image for Designer organelles bring new functionalities into cells

Skip to German: 'Designer-Organellen in Zellen stellen künstliche Proteine her'

Designer organelles bring new functionalities into cells

EMBL scientists create membraneless organelle to build proteins in living cell

For the first time, scientists have engineered the complex biological process of translation into a designer organelle in a living mammalian cell. Research by the Lemke group at the European Molecular Biology Laboratory (EMBL) – in collaboration with JGU Mainz and IMB Mainz – used this technique to create a membraneless organelle that can build proteins from natural and synthetic amino acids carrying new functionality. Their results – published in Science on 29 March – allow scientists to study, tailor, and control cellular function in more detail.

The authors see the newly developed synthetic host as a city. On the one hand, typical cellular processes – seen as encapsulated, isolated, and made up of non-interchangeable elements – are represented as repetitive structures: squared, isolated blocks which are always fenced, just like membranous organelles. On the other hand, the image highlights the making of a new organelle – a new building that is not fenced – which is accessible to the rest of the city while having its own identity, a building which is more dynamic and flexible. IMAGE: Gemma Estrada Girona

During evolution, the development of new organelles allows cells and organisms to become more complex, due to the ability to sort cellular processes into specific hotspots. “Our tool can be used to engineer translation, but potentially also other cellular processes like transcription and post-translational modifications. This might even allow us to engineer new types of organelles that extend the functional repertoire of natural complex living systems,” explains Christopher Reinkemeier, PhD student at EMBL and JGU Mainz and co-first author of the paper. “We could for example incorporate fluorescent building blocks that allow a glimpse inside the cell using imaging methods.”

“The organelle can make proteins by using synthetic non-canonical amino acids. Currently we know of more than 300 different non-canonical amino acids – compared to 20 which are naturally occurring. We are no longer restricted to the latter ones,” says co-first author Gemma Estrada Girona. “The novelty we introduce is the ability to use these in a confined space, the organelle, which minimises the effects on the host.”

Translation is such a complex process that it cannot be contained in one single organelle surrounded by a membrane. Therefore, inspiration was drawn from phase separation: the process responsible for the formation of membraneless organelles in vivo, such as nucleoli or stress granules. Phase separation is used by cells to locally concentrate specific proteins and RNAs. Even though these wall-less organelles have wobbly boundaries as they dynamically interact with the surrounding cytoplasm, they can still do very precise tasks. The team combines phase separation with cellular targeting to create their membraneless organelle and to make sure that only one organelle per cell is formed.

The genetic code is made up of three-letter sequences called codons. Each one codes for an amino acid, except for three 'stop' codons, which signal that an amino acid chain is complete. The Lemke group were able to develop a cell organelle that uses a reprogrammed stop codon, so that it codes for a new amino acid - not one of the 20 that occur naturally in living organisms.

In the end, only five new components have to be engineered into a cell to build it. The assembly of these components generates a large structure, which might create some burden on the cell. In the future, the group aims to engineer minimal designer organelles, to minimise any impact on the physiology of the healthy organism.

Edward Lemke – visiting group Leader at EMBL, Professor at JGU Mainz and Adjunct Director at the IMB – led the project. He concludes: “In the end, we aim to develop a technique to engineer synthetic cellular organelles and proteins that do not affect the host machinery at all. We want to create a tool that does not have any uncharacterised effects. The organelle should be a simple add-on that allows organisms to do custom-designed novel things in a controlled fashion.”

Reinkemeier, C.D., Girona, G.E., Lemke, E.A. ‘Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes’. Science, published online 29 March 2019.

This post was originally published on EMBL news.

Designer-Organellen in Zellen stellen künstliche Proteine her

Forscherteam erzeugt membranlose Organellen in lebender Zelle für die Proteinsynthese – Einbau von synthetischen Aminosäuren ermöglicht komplett neue chemische Funktionalität

Einem Forscherteam um den biophysikalischen Chemiker Prof. Dr. Edward Lemke ist es gelungen, eine membranlose Organelle in einer lebenden Zelle zu erzeugen und damit selektiv Proteine herzustellen, in die synthetische Aminosäuren eingebaut sind. Über diese chemisch erzeugten Aminosäuren ist es möglich, die Zellen mit völlig neuen Funktionen auszustatten. Beispielsweise könnten fluoreszierende Bausteine eingebaut werden, die mit bildgebenden Verfahren einen Blick ins Innere der Zelle erlauben. Die Forschungsarbeit der Gruppe ist in Zusammenarbeit der Johannes Gutenberg-Universität Mainz (JGU), dem Institut für Molekulare Biologie (IMB) und dem European Molecular Biology Laboratory (EMBL) erfolgt und wurde in dem renommierten Wissenschaftsmagazin Science veröffentlicht.

Organellen sind Kompartimente in Zellen, die wie der Kern oder die Mitochondrien bestimmte Funktionen erfüllen. Die Gruppe um Lemke hat nun ein neues Kompartiment erzeugt, in dem spezielle Proteine synthetisiert werden können. „Bildlich gesprochen suchen wir uns eine Ecke in der Zelle aus, wo wir unser Haus bauen und holen dann einen Teil der Ribosomen, die in der Zelle vorhanden sind, herein“, beschreibt Edward Lemke das Vorgehen. An den Ribosomen erfolgt die Biosynthese von Proteinen. Über den genetischen Code wird dabei die Boten-RNA (mRNA) in die Abfolge der Aminosäuren für das neu zu bildende Protein übersetzt.

Beim Bau der Designer-Organelle hat sich das Team um Lemke vom Prinzip der Phasenseparation inspirieren lassen: Phasenseparation wird von der Zelle verwendet, um spezielle Proteine und RNA lokal zu konzentrieren und neue, membranlose Kompartimente zu bauen. „Unsere membranlose Organelle ist quasi ein offenes Reaktionszentrum“, so Lemke.

Damit kann die Proteinbiosynthese an einem genau definierten Ort ablaufen, was für die Arbeit mit künstlichen Aminosäuren wichtig ist. Denn die Technik, mit Hilfe einer nicht natürlichen Aminosäure ein neues Protein zu schaffen, ist bereits bekannt. Wenn dieser Einbau aber unspezifisch in der ganzen Zelle erfolgt, ist die Belastung groß und die Zelle wird unter Umständen stark beeinträchtigt. Mit ihrer Methode der sogenannten orthogonalen Translation vermeiden die Wissenschaftler dieses Problem.

Großer Fundus an natürlichen und synthetischen Aminosäuren für Proteinsynthese an Designer-Organellen

„Unsere Organelle kann Proteine erzeugen, indem sie synthetisch hergestellte nicht-kanonische Aminosäuren verwendet. Davon gibt es zurzeit über 300. Das heißt es gibt nun keine Beschränkungen mehr, nur die 20 kanonischen Aminosäuren zu nutzen“, erklärt Gemma Estrada Girona, zusammen mit Christopher Reinkemeier Erstautorin der Science-Veröffentlichung. Beim Menschen bestehen die Proteine aus 20 natürlich vorkommende Aminosäuren, auch kanonische Aminosäuren genannt. Darüber hinaus gibt es nicht-kanonische Aminosäuren, die nicht in normalen menschlichen Proteinen vorkommen. Die Erweiterung des genetischen Codes ermöglicht es, dass auch nicht-kanonische Aminosäuren eingebaut werden. Die neue Designer-Organelle ist in der Lage, den genetischen Code selektiv zu erweitern. Dadurch wird innerhalb der Organelle die RNA anders übersetzt als im Rest der Zelle. „Wir haben uns die Natur zum Vorbild genommen, speziell den membranlosen Nukleolus, der im Zellkern an der Synthese von RNA beteiligt ist“, erklärt Lemke. „Wir waren dann aber doch überrascht, dass wir eine so komplizierte Struktur und Funktion tatsächlich mit wenigen Schritten selber bauen können.“

Der genetische Code besteht aus einer Sequenz von drei aufeinanderfolgenden Nukleobasen (Adenin, Guanin, Cytosin, Thymin) der Nukleinsäuren, den sogenannten Codons. Jedes Triplett codiert für eine Aminosäure, mit Ausnahme von drei "Stop"-Codons, die signalisieren, dass eine Aminosäurekette vollständig ist. Prof. Dr. Edward Lemke und sein Team konnten eines dieser Stop-Codons so umprogrammieren, dass es eine neue Aminosäure codiert, die nicht zu den 20 Aminosäuren gehört, die in lebenden Organismen natürlich vorkommen. IMAGE: Aleks Krolik/EMBL

Das Konzept kann möglicherweise als Plattform für das Design weiterer Organellen dienen und einen Weg aufzeigen, um semisynthetische Zellen und semisynthetische Organismen zu schaffen. „Unser Werkzeug ist in der Lage, Translation in Zellen durchzuführen, potenziell aber auch andere Zellprozesse wie die Transkription. Dies könnte es uns ermöglichen, neue Typen von Organellen zu erzeugen, die das funktionelle Repertoire komplexer lebender Systeme erweitern“, erläutert Christopher Reinkemeier.

Die Designer-Organellen verbinden also Biologie und Chemie, um eine komplett neue chemische Funktionalität zu erreichen. Anwendungen ergeben sich außer der erwähnten Fluoreszenz-Methode für die Bildgebung etwa auch bei der Herstellung von Antikörpern für therapeutische Zwecke. Zunächst wollen Lemke und sein Team jedoch die Designer-Organellen weiter verkleinern, um jeden Einfluss auf die Physiologie eines gesunden Organismus zu minimieren.

Edward Lemke ist Visiting Group Leader am European Molecular Biology Laboratory, Professor für synthetische Biophysik an der Johannes Gutenberg-Universität Mainz und Adjunct Director am Institut für Molekulare Biologie. Er koordiniert auch das neue DFG-Schwerpunktprogramm „Molekulare Mechanismen funktioneller Phasenseparation".

Morphie12121 on March 31st, 2019 at 14:35 UTC »

Title sounds super impressive, but could someone break what is going on in the study down to a layperson level?

stars9r9in9the9past on March 31st, 2019 at 13:49 UTC »

“The organelle can make proteins by using synthetic non-canonical amino acids. Currently we know of more than 300 different non-canonical amino acids – compared to 20 which are naturally occurring. We are no longer restricted to the latter ones,” says co-first author Gemma Estrada Girona. “The novelty we introduce is the ability to use these in a confined space, the organelle, which minimises the effects on the host.”

So is the major takeaway more that the researchers were able to make microscopic machinery that can use synthetic AAs to build things in a way that is normally only seen with ribosomes (and the natural 20 AAs they build with), or is the takeaway that they made a process that can build proteins outside of a membraneous space?

mvea on March 31st, 2019 at 11:18 UTC »

The title of the post is a copy and paste from the first paragraph of the linked academic press release here:

For the first time, scientists have engineered the complex biological process of translation into a designer organelle in a living mammalian cell. Research by the Lemke group at the European Molecular Biology Laboratory (EMBL) – in collaboration with JGU Mainz and IMB Mainz – used this technique to create a membraneless organelle that can build proteins from natural and synthetic amino acids carrying new functionality. Their results – published in Science on 29 March – allow scientists to study, tailor, and control cellular function in more detail.

Journal Reference:

Journal Reference:

Christopher D. Reinkemeier, Gemma Estrada Girona, Edward A. Lemke.

Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes.

Science, 2019

DOI: 10.1126/science.aaw2644

Link: http://science.sciencemag.org/content/363/6434/eaaw2644

How to make an organelle in eukaryotes

A key step in the evolution of complex organisms like eukaryotes was the organization of specific tasks into organelles. Reinkemeier et al. designed an artificial, membraneless organelle into mammalian cells to perform orthogonal translation. In response to a specific codon in a selected messenger RNA, ribosomes confined to this organelle were able to introduce chemical functionalities site-specifically, expanding the canonical set of amino acids. This approach opens possibilities in synthetic cell engineering and biomedical research.

Science, this issue p. eaaw2644

Structured Abstract

INTRODUCTION

The ability to engineer translation of noncanonical (unnatural) amino acids (ncAAs) site-specifically into proteins in living cells greatly expands the chemical space that can be used to control, tailor, and study cellular function. However, translation is a complex multistep process in which at least 20 different aminoacylated tRNAs, their cognate tRNA synthetases, ribosomes, and other factors need to act in concert to synthesize a polypeptide chain encoded by an mRNA transcript. To minimize interference with the host machinery, we aimed to engineer fully orthogonal translation into eukaryotes: to encode a new functionality in response to a specific codon in only one targeted mRNA, leading to site-specific ncAA incorporation only into the selected protein of choice. Although codon specificity can be achieved with genetic code expansion (GCE), this technology relies on using an orthogonal tRNA/tRNA synthetase pair (one that does not cross-react with any of the endogenous pairs) to reprogram a stop codon. Most commonly, the Amber (UAG) stop codon is used (20% abundance in human cells), and in principle, stop codon suppression can happen for every cytoplasmic mRNA that terminates naturally on this codon. Here, we present a strategy to generate a distinctly expanded genetic code for only selected mRNAs.

RATIONALE

We hypothesized that it should be possible to create an orthogonal translation system by spatially enriching the key components of the GCE machinery in an orthogonally translating (OT) synthetic designer organelle and by targeting a specific mRNA to it. In order to perform protein translation, such an OT organelle would need to be readily accessible to the entire translational machinery of the host, thus precluding membrane encapsulation. Inspired by the concept of phase separation, which is used by cells to concentrate specific proteins and RNA locally, we hypothesized that it might be possible to use this principle to create such membraneless OT organelles. In our design, only a spatially distinct set of ribosomes associated with OT organelles can use the aminoacylated suppressor tRNA and thus will decode Amber codons only in the selected mRNA translated by the OT organelle, leading to a protein containing the ncAA.

RESULTS

To bring the modified suppressor tRNA and the translated mRNA of choice in close proximity to each other, we used different strategies to generate highly concentrated assemblies and spatial separation inside cells: (i) proteins undergoing phase separation in cells [fused-in sarcoma (FUS), Ewing sarcoma breakpoint region 1 (EWSR1), and spindle-defective protein 5 (SPD5), which contain long intrinsically disordered domains] and (ii) kinesin motor proteins, which spatially enrich at microtubule plus ends (KIF13A and KIF16B). We fused each of these to the suppressor tRNA synthetase as well as an RNA-binding domain major capsid protein (MCP) that binds to a specific RNA motif (ms2 loops) engineered into the untranslated region of the mRNA of choice, forming an ms2-MCP complex. Each of these approaches yielded the desired local enrichment and preferential stop codon suppression of the mRNA tagged with ms2 loops. However, by far the best performing system was a combination of phase and spatial separation, which typically formed a micrometer-sized organelle-like structure per cell. Cells that contained this organelle efficiently and selectively performed Amber suppression of only the targeted mRNA. We were able to demonstrate the utility and robustness of these OT organelles by selectively decoding any of the three stop codons in a variety of proteins with different ncAA functionalities in two different mammalian cell lines.

CONCLUSION

Our results show how to combine phase and spatial separation inside cells to allow the concentration of a custom designed task into a distinct specially designed membraneless organelle. We successfully demonstrated that specific and selective protein translation could be achieved within these OT organelles, which allowed the introduction of noncanonical functionalities into proteins in a codon-specific and mRNA-selective manner. The system only requires engineering five components into the cell and can be reprogrammed to other stop codons in a single step. We expect this concept to be a scalable platform for further organelle engineering and to provide a route toward generation of semisynthetic eukaryotic cells and organisms.