Confocal ̄uorescence microscopy of cells expressing 2 3 FYVE. BHK cells were transfected with 2 3 FYVE constructs, then permeabilized with saponin and ®xed for confocal microscopy. ( A ) myc-2 3 FYVE (red) and GFP-PLC d 1 (green). (B and C) myc-2 3 FYVE (red) in the absence ( B ) and presence ( C ) of 100 nm wortmannin for 30 min. (D and E) myc-2 3 FYVE (red) with a single ( D ) or double ( E ) C215S mutation. ( F ) myc-2 3 FYVE (red) and endogenous mannose 6-phosphate receptor (green). ( G and H ) myc-2 3 FYVE (red) and endogenous EEA1 (green). ( J ) GFP-2 3 FYVE (green) and endogenous LBPA (red). ( K and L ) myc-2 3 FYVE (red) and endogenous EEA1 (green). In the merged images (A, F, I and J), yellow colour indicates co-localization between 2 3 FYVE and the organelle marker molecule. The arrow in (K) and (L) points to an expanded vacuolar structure that contains a high level of 2 3 FYVE and a low level of EEA1. Bars, 5 m m. Nuclei are indicated with `n'. 

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... importance in signal transduction, membrane traf®cking, cytoskeletal regulation and apoptosis (Leevers et al ., 1999; Rameh and Cantley, 1999). PI(3,4,5)P 3 is produced upon agonist stimulation of mammalian cells and interacts with a number of proteins containing PH domains (Rameh and Cantley, 1999). In contrast, PI(3)P is constitutively produced in both yeast and mammalian cells, and binds to proteins containing FYVE ®nger domains (Stenmark and Aasland, 1999). In order to understand the functions of FYVE ®nger proteins, several of which have been shown to play crucial roles in physiology and cell biology (Stenmark and Aasland, 1999), it is essential to localize PI(3)P within the cell. Fluorescence microscopy has been used to show that the C-terminal part of the early endosomal autoantigen EEA1 (Mu et al ., 1995), which contains a FYVE ®nger domain, is targeted to early endosomes (Stenmark et al ., 1996; Burd and Emr, 1998; Gaullier et al ., 1998; Patki et al ., 1998). However, since this targeting also requires an adjacent domain that binds to the early endosomal GTPase Rab5 (Stenmark et al ., 1996; Simonsen et al ., 1998b; Lawe et al ., 2000), this provides only circumstantial evidence for the presence of PI(3)P on early endosomes and no information about the absence or presence of PI(3)P on other membranes. Here we have used biochemical and morphological assays to characterize a new probe with unique speci®city for PI(3)P. We have used this probe to determine the localization of PI(3)P in both mammalian and yeast cells. We now show that PI(3)P is found on the limiting membranes of early endosomes and, in addition, on the internal vesicles of multivesicular endosomes and the yeast vacuole. We considered the possibility of using a FYVE ®nger- derived construct as a probe for PI(3)P. The isolated FYVE ®nger of EEA1 apparently is unsuitable as a probe for PI(3)P (Stenmark et al ., 1996; Lawe et al ., 2000), presumably because it binds PI(3)P with too low af®nity. We therefore investigated whether the FYVE ®nger of the receptor tyrosine kinase substrate Hrs (Komada and Kitamura, 1995) could be used for this purpose. However, like the EEA1 FYVE ®nger, the FYVE ®nger of Hrs was mainly cytosolic when expressed in cells (data not shown). We therefore constructed a double FYVE ®nger of Hrs (2 3 FYVE), with the rationale that this should have a greater af®nity for PI(3)P. Indeed, in an in vitro assay, a fusion protein consisting of GST and 2 3 FYVE bound strongly to PI(3)P-containing liposomes, with maximal binding at ~0.5% (Figure 1A). Even at a very low PI(3)P concentration (0.05%), a signi®cant binding of 2 3 FYVE could be detected, in contrast to the single FYVE ®nger. We also found that 2 3 FYVE did not bind to liposomes containing any other phosphoinositides (Figure 1B), and a point mutation (C215S) (Gaullier et al ., 1998) introduced into both of its constituent FYVE ®ngers completely abolished PI(3)P binding (Figure 1C). In order to compare the binding kinetics of GST±FYVE and GST±2 3 FYVE, we studied their binding to PI(3)P using surface plasmon resonance. A sensor surface was coated with mixed phospholipids containing 2% PI(3)P, and sensorgrams were recorded upon addition of the GST fusion proteins. Although the FYVE ®nger of Hrs has been proposed to interact with PI(3)P as a dimer (Mao et al ., 2000), the interaction of GST±FYVE with PI(3)P showed Langmuir association and dissociation kinetics characteristic of a 1:1 binding, with a K D of 38 6 19 nM (Figure 1D). In contrast, GST±2 3 FYVE showed more complex associ- ation/dissociation kinetics that could be ®t into a bivalent binding model (Figure 1E). Because of the complex kinetics of GST±2 3 FYVE, it is dif®cult to draw direct comparisons between the two probes, but it is evident that GST±2 3 FYVE has slower dissociation kinetics than GST±FYVE. The ability of one GST±2 3 FYVE molecule to interact with two molecules of PI(3)P is thus likely to explain its superior PI(3)P binding compared with GST± FYVE. In order to ascertain whether 2 3 FYVE could be used as a probe for PI(3)P, we examined its distribution in transiently transfected BHK cells (Simonsen et al ., 1998a) using confocal immuno ̄uoresence microscopy. When a myc epitope-tagged 2 3 FYVE was co-expressed with a GFP-tagged PH domain from PLC d 1, 2 3 FYVE was localized on intracellular vesicular structures, in contrast to GFP±PLC- d 1±PH, which is known to localize mainly to the plasma membrane (Stauffer et al ., 1998) (Figure 2A). A similar result was obtained with a 2 3 FYVE construct derived from EEA1, although the vesicular staining was somewhat fainter (data not shown). By two-hybrid analysis and co-expression experiments, we detected no binding of the 2 3 FYVE constructs to their native proteins (i.e. EEA1 and Hrs) (data not shown). Incubation of cells with wortmannin, an established inhibitor of PI3K, abolished the localization of 2 3 FYVE on the intracellular vesicular structures (Figure 2, compare C with B). Likewise, a point mutation that interferes with PI(3)P binding (Gaullier et al ., 1998) abolished the membrane localization of 2 3 FYVE when introduced into one (Figure 2D) or both (Figure 2E) of its constituent FYVE ®ngers. These results indicate that the 2 3 FYVE constructs were associated with vesicular membranes as a consequence of binding to PI(3)P. The localization of 2 3 FYVE was then compared with that of various organelle markers. myc- or GFP-tagged 2 3 FYVE co-localized extensively with endogenous EEA1 (Mu et al ., 1995), a well-characterized marker of early endosomes (Figure 2G±I). 2 3 FYVE did not, however, co-localize signi®cantly with any other markers of membranous compartments that were tested, including the Golgi marker, mannosidase II (not shown), the trans -Golgi network/late endosome marker, mannose-6-phosphate receptor (Figure 2F), the late endosome/lysosome marker, LAMP-1 (not shown), and the late endosome marker, lyso- bisphosphatidic acid (LBPA) (Kobayashi et al ., 1998) (Figure 2J). To investigate whether 2 3 FYVE could compete with endogenous FYVE ®nger proteins for PI(3)P binding, we examined the localization of EEA1 in cells that expressed a high level of 2 3 FYVE. While endogenous EEA1 co-localized with 2 3 FYVE at low expression (Figure 2K and L, right cell), EEA1 appeared to be partially displaced from membranes in the cells expressing 2 3 FYVE at a high level (Figure 2K and L, left cells). This was supported by quantitation of confocal images, which indicated that an ~2.5-fold relative increase in 2 3 FYVE signal was associated with an ~3-fold decrease in EEA1 signal (not shown). Moreover, the structures that were strongly positive for 2 3 FYVE were much larger than normal endosomes and had a vacuolar appearance (Figure 2L, inset). They could be labelled with internalized Alexa±transferrin (not shown), indicating that they were of endocytic origin. This effect is reminiscent of wortmannin treatment, which releases FYVE ®nger proteins from membranes and causes the vacuolation of endosomes (Patki et al ., 1997; Fernandez- Borja et al ., 1999; Komada and Soriano, 1999). While the size of the 2 3 FYVE-positive endosomes increased and the EEA1 labelling decreased as a function of the transfection time (not shown), we could also detect a nuclear labelling in the highly expressing cells (left cells in Figure 2L). The possible relevance of this labelling is discussed below. The ®nding that high level 2 3 FYVE expression led to alterations in endosome morphology underscores the potential caveats associated with the use of transfected phosphoinositide probes. We therefore investigated whether the 2 3 FYVE probe could be used to detect PI(3)P in untransfected cells. For this purpose, we permeabilized BHK cells by freeze±thawing prior to ®xation, and we stained the cells with biotinylated GST± 2 3 FYVE followed by Cy3±streptavidin. As shown in Figure 3A, a strong labelling of vesicular structures was detected, whereas unpermeabilized cells were not labelled (Figure 3D). The GST±2 3 FYVE labelling overlapped extensively with that of EEA1 (Figure 3B and C), indicating that this probe can be used for the direct detection of PI(3)P on endosomes. In agreement with this, the labelling was strongly reduced upon incubation of the cells with 100 nM wortmannin for 30 min prior to ®xation (not shown). However, we could not rule out the possibility that 2 3 FYVE might only recognize PI(3)P in a certain cellular context, for instance in association with an endosomal protein. In order to investigate this, we studied the labelling of unpermeabilized cells that had been pre- incubated brie ̄y with PI(3)P-containing liposomes, in order to incorporate PI(3)P into the outer lea ̄et of the plasma membrane. When these cells were stained with the 2 3 FYVE probe, a strong plasma membrane labelling was observed (Figure 3E). In contrast, no labelling was detected in cells incubated with buffer alone (Figure 3D), or with liposomes containing PI(3,4)P 2 (Figure 3F) or PI(3,4,5)P 3 (Figure 3G). This indicates that the 2 3 FYVE probe detects cell-associated PI(3)P as such, and not in conjunction with endosome-speci®c molecules. These data also con®rm the high speci®city of the 2 3 FYVE probe for PI(3)P. To investigate the distribution of PI(3)P at the ultrastructural level, we sought to develop a post-embedding, in situ labelling approach to localize this lipid. This approach avoids cell permeabilization and other possible artefacts associated with pre-embedding labelling and is the method of choice to localize both proteins and lipids. Importantly, it also allows quantitative estimation of the distribution of a protein or lipid assuming similar labelling ef®ciencies in different compartments. However, to date, lipid localization has been limited by the availability of speci®c lipid probes and the dif®culties in lipid retention. Ultrathin frozen sections (~60 nm) were compared with ...