Background

Plasma membranes are permeable barriers between cells and their environment, and they control communication and substance trafficking in and out of cells. Important physiological processes, such as receptor-ligand interactions, lipid flip-flops and lipid-raft formation, are accompanied with the motions and structural changes of biomolecules on plasma membranes. Despite various advanced fluorescence tools being available to study membranes in vivo, novel technologies are required to make it possible to measure conformational changes of biomolecules in real time in live cell plasma membranes. Fluorescence imaging and spectroscopic techniques are often the methods of choice for investigating membrane proteins in live cells, however they face various hindrances. In previous work, the authors developed an in vitro fluorescence method to measure positions of membrane proteins in lipid bilayers, however this in vitro approach cannot be directly applied to study the trafficking of biomolecules in plasma membranes of live cells. In the present work, Hou et al (1) developed a one donor-multiple quenchers Förster resonance energy transfer method by adding non-fluorescent quenchers in the extracellular environment during cell imaging. The key for this approach is the appropriate selection of quenchers, which should be neutral, water-soluble and biocompatible.

Key findings and developments

Method principle

The underlying mechanism of the method presented in this preprint is FRET between a single fluorophore and a multitude of quenchers in the extracellular environment (termed queenFRET).  The FRET between the fluorophore and the quenchers results in a very steep change in fluorescence as the fluorophore-labeled molecule moves from the outer to the inner surface of the membrane or vice versa. The positional changes of the fluorophore-labeled biomolecule in cell membranes can be reflected by its intensity or lifetime changes.

Environment-sensitive fluorophores should be excluded from the candidates of the donors if they displayed fluorescent fluctuations in the absence of the quenchers. Those that are fluorescently unstable should also be excluded. In the present work, the authors used blue dextran (BD). This dye has various favourable characteristics including neutrality, water solubility and biocompatibility.

Use and validation of queenFRET in various biological contexts

The authors began by measuring flip-flops of fluorophore-labeled lipids. Flippases often translocate aminophospholipids toward the cytosolic leaflet of plasma membranes to maintain their asymmetry. The authors used a vesicle-plasma membrane fusion method to incorporate fluorescent phosphatidylethanolamine into the membrane. Individual fluorophores were then tracked and the intensities of the fluorophores recorded. In the absence of BD, the fluorescence of the fluorophores was stable. Otherwise, the BD solution percolated into the gap to quench the fluorophores in the membrane. Given that the fluorophores in the opposite sides of the lipid bilayer have different distances to the quencher solution, they displayed two different relative fluorescent intensities. The centers of both fluorescence peaks are consistent with the thickness of the plasma membrane. Overall, this result indicates that queenFRET allows the precise measure of the location of single fluorophore in plasma membranes. The authors then propose that the positions of the fluorophores in the membrane can also be deduced from their fluorescence lifetimes (measured with FLIM by using a time-correlated single photon counting system). The lifetime images of the plasma membranes looked differently in the absence and presence of BD.

            The authors then went on to investigate whether queenFRET can monitor interactions of extracellular proteins with plasma membranes. For this, they labeled the human host peptide LL-37 with tetramethylrhodamine (TAMARA), and added the peptide to culture medium to monitor its membrane insertion dynamics in a lung epithelial carcinoma cell line. While the intensity of TAMARA-LL-37 was stable in the absence of BD, in the presence of BD, the intensity histogram of the dynamics TAMARA-LL-37 showed three major peaks (which can be converted into insertion depths). The results suggest that LL-37 forms oligomers and displays dynamic behaviour in live cell membranes- and demonstrates the validity of queenFRET.

The authors also investigated the insertion of proteins from the intracellular side of cell membranes, which is relevant when endogenous proteins are of interest. queenFRET enable distinguishing different membrane-inserting proteins, and also different insertion states of the same protein within a membrane. For this proof of principle, the authors used MLKL with TAMARA and delivered it into the cell line THP-1. The lifetime histogram of MLKL_2-154 indicated a shallow membrane insertion, while the one of MLKL_2-123 suggests a deeper insertion into the membrane at three different depths.

 

What I like about this preprint

I liked this preprint because while I find the topic of membrane dynamics and membrane contact sites extremely interesting, there is a gap in technological (and image analysis) tools that limits the type of work that can be currently performed in this field. I think the work here proposed by the authors is an interesting and exciting step to study membrane dynamics in live cells.

 

Open questions

1. Apart from BD, what other dyes do you recommend as useful alternatives, and possibly with other absorption spectra?
2. In your discussion you touch on the fact that your current work focuses on the transmembrane positions of fluorophores, disregarding their in-plane movements. How do you envisage the combination of queenFRET with other tools such as super-resolution imaging to better understand processes involving membranes?
3. Could you expand on how and if queenFRET could be used either solely or in combination with other tools, to study membrane-contact sites, and changes in membrane dynamics upon such events?

 

References

1. Hou et al, Imaging transmembrane dynamics of biomolecules at live cell plasma membranes using quenchers in extracellular environment, bioRxiv, 2020

 

doi: https://doi.org/10.1242/prelights.26099

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