from chance to function: the story of one gene (part III)

This is the third and final post on the story of FAPP2. You might want to read part I and part II before diving into this one, unless you are already familiar with secretory transport and the various organelles involved in it.

In this post, I am going to go over a 2006 paper written by Kai Simons and some of his grad students, in collaboration with other researchers from the University of Coimbra (Portugal). In this paper, the authors finally link FAPP2 to membrane polarization and cilium formation in the apical membrane of MDCK cells.

In case you completely forgot how polarized epithelial cells - such as MDCK cells - look like, here is a pictorial reminder from the previous post...


These cells are ciliated - and their cilia help them sense the flow of fluid within the kidney. The cilia themselves, however, are immotile - which means they are not flagella. Here is a schematic explanation of how the cilium can sense flow. For more information on the primary cilium and its many functions in signalling, you can turn to the review this figure belongs to (simply follow the citation link at the end of the post, or click on the figure).


You can imagine that defects in cilia structure would impact the ability of these cells to sense flow, and that they would therefore lead to a serious impairment in kidney function. And this is where the study of FAPP2, that until now might have looked like it was of remote importance for biomedical applications, gives us an insight on the maintenance of a very important cellular organelle.

More on FAPP2 and cilium formation below the fold.

First of all, the authors investigated whether cilium structure was affected in FAPP2 knockdown cells. They used fluorescence microscopy to visualize the cilia (staining acetylated tubulin) and the cell nuclei (using DAPI staining). They looked at cells that were grown in a layer and in a cyst. These cells will normally form cyst-like structures in the kidneys, but growing them in layers can be useful to run experiments in the lab; however, one would always want to check that the results obtained by the two different methods are comparable and consistent with each other.


As you can see, the cells suffering a FAPP2 knockdown (B, E and white bars in graphs) suffer from decreased or abolished ciliogenesis. But it must be noted that these cells' cilia end up growing back to normal over time - which might be due to the fact that the knockdown is 'leaky', or that FAPP2 knockdown alone is not sufficient to stop ciliogenesis. As we already know from previous studies that the knockdown method still leaves about 10% FAPP2 protein expression as compared to untreated cells, probably that 10% was enough, over time, to promote ciliary growth.

These figures alone, however, are already able to show that FAPP2 is somehow involved in the maintenance of the primary cilium.

The authors then check whether the lipid composition of the apical and basolateral membranes of the MDCK cells has been affected by the knockdown. To do this, they use a technique known as Laurdan staining. Laurdan is a dye that intercalates between lipids, and membrane fluidity affects the emission spectrum of Laurdan. This shift can then be normalized and quantified, giving us a measure of whether the membrane in FAPP2-depleted cells is moving toward a more disordered (fluid) or more ordered (lipid rafts) state.

We have already seen in previous papers how FAPP2 was shown to have some involvement with lipid rafts, so this experiment is the next logical step. Through Laurdan staining (I am not going to show the figure here), Simons and colleagues discovered that the lipid composition, and the state of the two different membranes in MDCK cells is affected by the lack of FAPP2 protein. In fact, while the usually raft-rich apical membrane shifts toward a more disordered state, the basolateral membrane seems to be gaining lipid rafts.

From this, one can deduce that the actual chemical composition of the two membranes has been affected, and that FAPP2 might be affecting the targeting of different lipids to the different membranes: this would explain why its lack causes mistargeting of lipids, usually destined to the apical surface, to basolateral surfaces.

Finally, the authors look at the membrane structure at the base of the cilium, using both Laurdan staining and standard fluorescence microscopy, and show that, in wild-type MDCK cells, there is a domain of condensed lipids (rafts?) around the base of the cilium. What is notably lacking in this paper is the corresponding experiment with the knockdown cells - which makes me wonder whether they did try and saw no difference with the wild-type at all.


But in absence of such evidence...one can argue that FAPP2 depletion is affecting lipid targeting, and probably this also has an impact on the state of the membrane at the base of the cilium - and maybe both these issues will then lead to delayed/impaired ciliogenesis in FAPP2 knockdowns.

In conclusion, we have seen, throughout the three posts, how a gene discovered through a random screen for proteins binding to a lipid mediator was then characterized, and how a putative function for this gene emerged over the years, thanks to the work of multiple groups around the world. From FAPP2 thought of as a gene with unknown function, we arrived to the point where we know that FAPP2 is most probably important for the maintenance of proper kidney function.

Singla, V. (2006). The Primary Cilium as the Cell's Antenna: Signaling at a Sensory Organelle. Science, 313(5787), 629-633. DOI: 10.1126/science.1124534

Vieira, O.V., Gaus, K., Verkade, P., Fullekrug, J., Vaz, W.L., Simons, K. (2006). From the Cover: FAPP2, cilium formation, and compartmentalization of the apical membrane in polarized Madin-Darby canine kidney (MDCK) cells. Proceedings of the National Academy of Sciences, 103(49), 18556-18561. DOI: 10.1073/pnas.0608291103


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