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

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

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