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The cell wall regulates dynamics and size of plasma-membrane nanodomains in Arabidopsis.

Joseph Franics McKenna, Daniel Rolfe, Stephen E D Webb, Andrea Frances Tolmie, Stanley W Botchway, Marisa Martin-Fernandez, Chris Hawes, John Runions

Preprint posted on November 06, 2018 https://www.biorxiv.org/content/early/2018/11/06/455782?versioned=true

Article now published in Proceedings of the National Academy of Sciences at http://dx.doi.org/10.1073/pnas.1819077116

Imaging plasma membrane nanodomains in planta – and how they connect the extracellular cell wall to the intracellular cytoskeleton

Selected by Marc Somssich

Background

The existence of lipid rafts within the plasma membrane (PM) has been a controversial topic over the past 20 years (Goñi, 2018). By now, their existence is widely accepted, even though the ‘burned’ name ‘lipid raft’ had to be replaced with ‘plasma membrane sub- or nano-domain’ (Goñi, 2018). One point central to this controversy has always been that these domains could not be visualized using conventional light/confocal microscopes (Goñi, 2018). Now, McKenna et al. report the sub-diffraction limit imaging of different PM-localized proteins in nanodomains of various sizes in Arabidopsis (McKenna et al., 2018).

Key findings

McKenna and colleagues use the Zeiss AiryScan detector unit to image the localization of several well-studied PM-localized proteins, such as PIN3, FLS2 and PIP2A. They find that these proteins are not homogenously distributed within the PM, but form nanodomains of varying sizes – all below the ~250 nm resolution limit of a standard confocal microscope. They then go on to study the mobility of these proteins in the PM using single particle tracking in TIRF imaging mode. They find that the nanodomain-localized proteins indeed diffuse at a slower speed than a freely diffusing control protein, and that their movement is confined to the area of the nanodomain. Finally, the authors perturb the cytoskeleton and cell wall, finding that the cytoskeleton is involved in regulating FLS2 diffusion in the PM, while perturbations of the cell wall affected diffusion rate and area size, as well as nanodomain size for both, FLS2 and PIN3, indicating that the wall is also involved in regulating protein-localization to PM-nanodomains.

Highlights of this Preprint

In contrast to mammalian systems, high-/super-resolution imaging of living plant cells is still challenging when using common methods, such as STED, dSTORM or PALM (Komis et al., 2018). The Zeiss AiryScan Unit offers a very interesting alternative to these super-resolution techniques (Huff, 2015). Using a 32-detector array, each operating with a minimum-sized pinhole, and providing an automated deconvolution tool embedded in their standard operating software, the AiryScan unit doesn’t allow full super-resolution imaging, but is able to go below the diffraction limit with minimal technical effort – and even in living plant cells. The successful implementation of this technique to image PM-nanodomains in vivo in Arabidopsis, is a great step forward for plant cell imaging, and in the challenge to finally characterize PM-nanodomains in closer detail in plant cells. Similarly, the use of TIRF-based single particle tracking in living Arabidopsis cells is another great technical accomplishment. Most reports on protein diffusion rates in plant PMs are based on FRAP-measurements, and therefore crude averages of protein populations within a large region (Martinière et al., 2012). Here, determining the mobility on a single-particle level is highly informative. The findings of the group, that the PM-nanodomains are linked to the extracellular matrix of the cell wall, and also the intracellular cytoskeleton, furthermore helps to understand how pathways can signal from one cell to the other – using protein signaling platforms arranged within distinct domains of the PM, which are connected to both, the outside and inside of the cell.

Future directions

The results presented in this paper tie the cell wall, the plasma membrane and the cytoskeleton into a continuum. This is very exciting work, which is especially interesting when working on non-cell-autonomous signaling pathways. It would now be interesting to identify the physical connections between these three domains. There must be cell wall binding proteins involved, as well as connection points between the cytoskeleton and the PM-nanodomains. From a signaling standpoint, it would then be great to see what happens when a pathway gets activated, e.g. by addition of the ligand for a nanodomain-localized receptor. The authors briefly touch on this in the case of the FLS2 protein, when they add its ligand flg22, and find that this leads to a reduction in FLS2-mobility. But a more detailed study of the effects of signaling activation certainly holds the potential for another publication in itself. This could also be a major step in characterizing the PM-nanodomains as ‘signaling platforms’, as they were hypothesized to be (Lingwood and Simons, 2010). Furthermore, it would be interesting to study the role of PM-nanodomains in separating or integrating different pathways (He et al., 2018). Again, remaining with the FLS2-dependent flagellin-pathway, it would be interesting to see if FLS2 signaling partners, such as BAK1 or BIK1, localize to the same PM-nanodomains as FLS2, and if BRI1, which competes with FLS2 for BAK1 interaction, is in a different domain.

References

Goñi, F. M. (2018). “Rafts”: a nickname for putative transient nanodomains. Chem. Phys. Lipids.

He, Y., Zhou, J., Shan, L. and Meng, X. (2018). Plant cell surface receptor-mediated signaling – a common theme amid diversity. J. Cell Sci. 131,.

Huff, J. (2015). The Airyscan detector from ZEISS: confocal imaging with improved signal-to-noise ratio and super-resolution. Nat. Methods 12, i–ii.

Komis, G., Novák, D., Ovečka, M., Šamajová, O. and Šamaj, J. (2018). Advances in Imaging Plant Cell Dynamics. Plant Physiol. 176, 80–93.

Lingwood, D. and Simons, K. (2010). Lipid rafts as a membrane-organizing principle. Science 327, 46–50.

Martinière, A., Lavagi, I., Nageswaran, G., Rolfe, D. J., Maneta-Peyret, L., Luu, D.-T., Botchway, S. W., Webb, S. E. D., Mongrand, S., Maurel, C., et al. (2012). Cell wall constrains lateral diffusion of plant plasma-membrane proteins. Proc. Natl. Acad. Sci. U. S. A. 109, 12805–10.

McKenna, J. F., Rolfe, D. J., Webb, S. E. D., Tolmie, A. F., Botchway, S. W., Martin-Fernandez, M. L., Hawes, C. and Runions, J. (2018). The cell wall regulates dynamics and size of plasma-membrane nanodomains in Arabidopsis. bioRxiv.

Tags: airyscan, arabidopsis, lipid raft, plasma membrane nanodomains, signalling

Posted on: 10th December 2018 , updated on: 11th December 2018

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