Visualization of cellular phosphoinositide pools with GFP-fused protein-domains

Abstract

This unit describes the method of following phosphoinositide dynamics in live cells. Inositol phospholipids have emerged as universal signaling molecules present in virtually every membrane of eukaryotic cells. Phosphoinositides are present in only tiny amounts as compared to structural lipids, but they are metabolically very active as they are produced and degraded by the numerous inositide kinase and phosphatase enzymes. Phosphoinositides control the membrane recruitment and activity of many membrane protein signaling complexes in specific membrane compartments, and they have been implicated in the regulation of a variety of signaling and trafficking pathways. It has been a challenge to develop methods that allow detection of phosphoinositides at the single-cell level. The only available technique in live cell applications is based on the use of the same protein domains selected by evolution to recognize cellular phosphoinositides. Some of these isolated protein modules, when fused to fluorescent proteins, can follow dynamic changes in phosphoinositides. While this technique can provide information on phosphoinositide dynamics in live cells with subcellular localization, and it has rapidly gained popularity, it also has several limitations that must be taken into account when interpreting the data. This unit summarizes the design and practical use of these constructs and also reviews important considerations for interpretation of the data obtained by this technique.

Figure 1

Cellular localization of the PLCδ1PH-GFP. (A) Clusters of HEK293 cells transfected for 24h, and expressing the domain at various levels. Note the vesicular structures in the cell pointed to by the arrow that expresses high level of the fusion protein. This is an example of the toxic effects of the protein. Also note that with these illumination settings (panel a), cells 1 and 2 are not even visible, yet these are the cells that one should chose for analysis as shown by the higher illumination that already saturates the signal from the other cells (b and c). (B) Confocal imagaes of a COS-7 cell trasnfected with PLCδ1PH-GFP for 24 h and analyzed by z-sectioning. Panel a shows an image taken close to the bottom of the cell where it attaches to the coverslip. Note that there is no sharp outline of the cell and the signal covers the whole area of the cell. In panel b, the picture is taken at a z-plane higher up in the cell and again, there is no clear outline of the plasma membrane. Compare it with HEK293 cells that are not as flat and show a clear image of the plasma membrane (A). The side views of this COS-7 cell at the cross-sections (top and right side) show better the plasma membrane localization and the shape of the cell.

Figure 2

Localization of the various domains used for imaging PtdIns4P in COS-7 and HEK293 cells. (A) COS-7 cells transfected with the indicated domains for 24 h. Note the sharp contrast and prominent recruitment of the FAPP1-PH domain and the higher nuclear staining of the OSBP-PH domain. The yeast OSH1-PH-GFP also shows the Golgi but is also localized to a small extent to the plasma membrane (better seen on panel B). The OSH2-2xPH-GFP prominently labels the plasma membrane but does not show Golgi localization. For this picture cells were selected that are not so flat, do demonstrate better the plasma membrane localization. (B) Localization of the OSH1-PH-GFP and OSH2-PH-GFP constructs in HEK293 cells. Note the Golgi and moderate plasma membrane localization of the OSH1-PH and the lack of Golgi localization and high nuclear signal with the OSH2-PH domain. The nuclear localization is less prominent with the OSH2-2xPH-GFP construct. (C) Examples for interference of the domains with cellular functions. Both the OSBP- and FAPP1-PH domains cause tubulation of the Golgi. Remarkably, this always occurs at moderate level of expression and never at high expression levels and only in a fraction of cells. This indicates that this effect is conditional and requires a certain functional state of the Golgi. At high expression levels, the two constructs have very different effects: the OSBP-PH brakes the Golgi to small vesicles that eventually cover the whole cytoplasm. These are completely resistant to brefeldin A, a treatment that rapidly eliminates the Golgi localization of the construct indicating the Arf1-GTP requirement of the localization (not shown). In contrast, the FAPP1-PH domain shows no Golgi localization at high expression levels and instead produces large vacuoles in the cell with FAPP1-PH domain attached to their limiting membranes. (C)

Figure 3

Quantification of the plasma membrane localization of an inositide binding domain. HEK293 cells are shown expressing the PLCδ1PH-GFP. The pixel intensity histograms are calculated for the three lines placed on this recording. Note that the scale shows that this is a 12 bit image. An 8 bit image would only have only 256 levels of intensities. Also note that the peak intensities are not at saturation. The lower panel shows how the intensity values from the membrane and the cytosol are calculated. Their ratio is than a good measure of localization. Also note that no lines are placed over areas where two cells are joined. These calculations have to be made for each picture from a sequence to obtain a full time-course of change. It is advised that more than one line is placed on a cell to get a more accurate value as the intensities even vary along the perimeter.