Close

PUFFFIN: A novel, ultra-bright, customisable, single-plasmid system for labelling cell neighbourhoods

Tamina Lebek, Mattias Malaguti, Alistair Elfick, Sally Lowell

Posted on: 9 October 2023

Preprint posted on 8 September 2023

PUFFFIN: Shining light on cell chat

Selected by Jennifer Ann Black

Categories: developmental biology

Background:

Understanding how cells communicate with each other is important to understand how biological processes in our bodies are coordinated (1). One way in which scientists can study how cells talk to their neighbours is to design an artificial pathway that allows some cells to transfer (or secrete) fluorescent proteins for neighbouring cells to potentially take up. The ‘labelled neighbours’ can then be selected specifically based on this fluorescent signal and investigated further. But how can a fluorescent protein such as green fluorescent protein (GFP) be engineered such that it is efficiently taken up any neighbouring cell type? And how can the relatively modest GFP fluorescence be amplified to make it bright enough for rapid, reliable, and unambiguous detection of labelled neighbours?

In this study, the authors propose the use of ‘Supercharged GFP (or +36GFP)’ fusion proteins for this purpose. +36GFP has been engineered to be better suited to interact with cell membranes meaning +36GFP does not need the help of a cell penetrating peptide to deliver proteins into a cell (2). Addition of the human serum album signal peptide to supercharged GFP (generating ‘s36GFP’) facilitates secretion by the expressing cells and the signal is amplified by fusion to a second ultra-bright protein, or by fusion to a HaloTag that activates ultra-bright Halo-compatible dyes. Herein, the authors then describe the development and use of a new modular fluorescent labelling system for investigating cell-cell interactions called Positive Ultra-Bright Fluorescent Fusion For Identifying Neighbours, or, PUFFFIN.

 

Key Findings

1) PUFFFIN rapidly labels neighbouring cells

The PUFFFIN system is a plasmid-based system for use in mammalian cells to investigate cell-cell communication. To use the system, a plasmid is transfected into a target set of cells which can be left to interact with their neighbours. Within as little as 15 minutes of contact, these ‘secretor’ cells have the capacity to transfer a fluorescent protein to neighbouring cells. These neighbour cells can then be isolated based on their fluorescence.

The plasmid was structured to contain initially +36GFP fused to a secretion signal (human serum albumin) and a super-bright fluorescent protein called mNeonGreen (mNG) to enhance the fluorescent signal, and another separate fluorescent protein called mCherry. mCherry is fused to a Nuclear Localisation Signal (NLS) which allows the user to locate which cells were the secretor cells as the mCherry will become expressed in the nuclei of these cells. Both fluorescent proteins are expressed using a strong promoter to ensure strong expression. This system does not require any modifications to be made in the neighbouring cells.

When transfected, the authors show that PUFFFIN is, 1) capable of labelling neighbouring cells, 2) the cells labelled are nearby the secretor cell and, 3) they could detect signal as early as 15 mins in neighbour cells nearby PUFFFIN secretors and that the signal can be long lasting – in this study, they tested for signal up to 22hr via live imaging and 48 hrs by flow cytometry after secretor cells were mixed into the population.

Figure shows select data from Lebek et al. Figure 1A illustrates how PUFFFIN can be used to examine cell-cell communication and the modularity of the PUFFFIN system. The construct for PUFFFIN is shown in full colour and below shows how modifications can be made to the plasmid to suit the user. Figure 1 D&E show flow cytometry results at 0 and 48 hrs after unmodified cells and secretor cells are mixed. At 48 hrs, most cells that were previously unlabelled have become GFP positive. Figure 2A shows a similar experiment performed using live cell imaging.

2) PUFFFIN is modular

To ensure PUFFFIN is fully customisable, the authors used the EMMA modular assembly system to generate one single plasmid with all the necessary PUFFFIN components that could be modified easily i.e, swapping fluorescent proteins, resistance genes and promoter sequences. EMMA, or Extensible Mammalian Modular Assembly Toolkit, is a vector-based system used for the rapid assembly of custom mammalian expression vectors. This highly modular system allows researchers to design their desired expression vector, select the required ‘parts’ for their vector from a DNA library, then assemble up to 25 of these DNA parts using a single step reaction within a single tube to create their final vector (3).

Additionally, they created a HaloTag compatible vector known as PUFFHalo. HaloTags are self-labelling protein tags originating from a bacterial enzyme. Once your protein of interest is tagged, you incubate your cells with a HaloTag compatible ligand, for instance a fluorescent ligand, then visualise your protein of interest (4). This allows labelling to be performed in any colour of choice from the same PUFFFIN-transfected cells.

 

What I liked about this preprint

I think the PUFFFIN system will provide a simple and effective way of studying cell-cell communication that keeps costs down. I like that PUFFFIN is so modular giving the user greater flexibility to adapt this approach to their own laboratory.

 

Questions for the Authors

Q1: Do you find any specific combinations of fluorescent proteins work best for cell-cell signalling studies?

Q2: Does PUFFFIN work outside of mammalian systems to look at how single cell organisms communicate with each other?

Q3: What was the most challenging aspect of developing PUFFFIN?

 

References

1.https://www.nature.com/scitable/topicpage/cell-adhesion-and-cell-communication-14050486/

2. McNaughton BR, Cronican JJ, Thompson DB, Liu DR. Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins. PNAS. 2009 14;106(15):6111-6. doi: 10.1073/pnas.0807883106.

3. Martella A, Matjusaitis M, Auxillos J, Pollard, S.M. and Cai, Y. EMMA: An Extensible Mammalian Modular Assembly Toolkit for the Rapid Design and Production of Diverse Expression Vectors. ACS Synthetic Biology. 2017. doi.org/10.1021/acssynbio.7b00016

4. https://www.promega.com.br/resources/technologies/halotag/

Tags: cell communication, emma, expression vector, gfp, mammalian expression

doi: https://doi.org/10.1242/prelights.35693

Read preprint (1 votes)

Author's response

Tamina Lebek shared

Q1: Do you find any specific combinations of fluorescent proteins work best for cell-cell signalling studies?

We think the answer will be different for everyone, which is why we’ve used HaloTag technology so that anyone can label in any colour of choice by simply switching the Halo-compatible dye. For example, Red-Green are a naturally popular colour combination because their spectra are well separated, but that means that many researchers will be using cell lines or animal models that already contain green or red fluorescent molecules, and will need a different colour for their “neighbour labelling”. We can also use our ‘Lego-like’ modular plasmid to easily switch the colour of the nuclear fluorescent molecule that marks ‘secretor’ cells.

 

Q2: Does PUFFFIN work outside of mammalian systems to look at how single cell organisms communicate with each other?

We would be excited to see PUFFFIN in different model organisms. The supercharged fluorescent label attaches to neighbouring cells based on charge interaction with negatively charged cell membranes. All membranes are negatively changed so this should be a universal receptor-independent uptake mechanism (see PMID: 19307578). Mammalian cells subsequently internalise the label by endocytosis, but of course internalisation is not essential for effective labelling. There is also the question of how the label gets out of the secretor cell in the first place: we use a signal peptide as a secretion signal, and these tend to be well conserved, but if necessary it should be straightforward to re-engineer the secretion signal of the PUFFFIN label to suit the organism of interest.

We hope that this simple universal mechanism for labelling will allow broad applications even beyond mammalian cells.

 

Q3: What was the most challenging aspect of developing PUFFFIN?

I think the moment I will never forget was when we first came up with a really effective signal-amplification strategy: the PUFFFIN sparkles were so bright that we could see single vesicles moving around in the cells on the TC microscope and everyone from our lab ended up in that tiny space excited about the sparkly cells – even people from other labs started looking. This was such a powerful moment because we knew that neighbour-labelling would only be really effective if the signal is very, very bright, making it possible to identify neighbours by live imaging and separate them by flow cytometry even after relatively brief interactions.

We also set ourselves the major challenge of compiling all PUFFFIN components onto a single plasmid with modular exchangeable parts, because we knew this would make the system so much more useful and adaptable for different users and different experiments. We have the amazing EMMA modular assembly toolkit (plus a LOT of hard work) to thank for overcoming that particular challenge (see PMID: 28418644).

 

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

Also in the developmental biology category:

Notch3 is a genetic modifier of NODAL signalling for patterning asymmetry during mouse heart looping

Tobias Holm Bønnelykke, Marie-Amandine Chabry, Emeline Perthame, et al.

Selected by 06 June 2024

Bhaval Parmar

Developmental Biology

Lipid-Based Transfection of Zebrafish Embryos: A Robust Protocol for Nucleic Acid Delivery

Aslihan Terzi, Tiger Liao, Adrian Jacobo

Selected by 09 May 2024

Roberto Rodríguez-Morales

Genetics

Topology changes of the regenerating Hydra define actin nematic defects as mechanical organizers of morphogenesis

Yamini Ravichandran, Matthias Vogg, Karsten Kruse, et al.

Selected by 08 May 2024

Rachel Mckeown

Developmental Biology

preLists in the developmental biology category:

BSDB/GenSoc Spring Meeting 2024

A list of preprints highlighted at the British Society for Developmental Biology and Genetics Society joint Spring meeting 2024 at Warwick, UK.

 



List by Joyce Yu, Katherine Brown

GfE/ DSDB meeting 2024

This preList highlights the preprints discussed at the 2024 joint German and Dutch developmental biology societies meeting that took place in March 2024 in Osnabrück, Germany.

 



List by Joyce Yu

‘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.

2nd Conference of the Visegrád Group Society for Developmental Biology

Preprints from the 2nd Conference of the Visegrád Group Society for Developmental Biology (2-5 September, 2021, Szeged, Hungary)

 



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

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

EMBL Conference: From functional genomics to systems biology

Preprints presented at the virtual EMBL conference "from functional genomics and systems biology", 16-19 November 2020

 



List by Jesus Victorino

Single Cell Biology 2020

A list of preprints mentioned at the Wellcome Genome Campus Single Cell Biology 2020 meeting.

 



List by Alex Eve

Society for Developmental Biology 79th Annual Meeting

Preprints at SDB 2020

 



List by Irepan Salvador-Martinez, Martin Estermann

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

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

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.

EDBC Alicante 2019

Preprints presented at the European Developmental Biology Congress (EDBC) in Alicante, October 23-26 2019.

 



List by Sergio Menchero 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

SDB 78th Annual Meeting 2019

A curation of the preprints presented at the SDB meeting in Boston, July 26-30 2019. The preList will be updated throughout the duration of the meeting.

 



List by Alex Eve

Lung Disease and Regeneration

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

 



List by Rob Hynds

Young Embryologist Network Conference 2019

Preprints presented at the Young Embryologist Network 2019 conference, 13 May, The Francis Crick Institute, London

 



List by Alex Eve

Pattern formation during development

The aim of this preList is to integrate results about the mechanisms that govern patterning during development, from genes implicated in the processes to theoritical models of pattern formation in nature.

 



List by Alexa Sadier

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

Zebrafish immunology

A compilation of cutting-edge research that uses the zebrafish as a model system to elucidate novel immunological mechanisms in health and disease.

 



List by Shikha Nayar
Close