![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 17, Issue 9, 3989-4001, September 2006
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
*Department of Biochemistry, The Norwegian Radium Hospital and the University of Oslo, Montebello, N-0310 Oslo, Norway; Department of Developmental Biology, Wenner-Gren Institute, Stockholm University, S-106 91 Stockholm, Sweden; and Department of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
Submitted March 27, 2006;
Revised June 13, 2006;
Accepted July 3, 2006
Monitoring Editor: Jean Gruenberg
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Gonzalez-Gaitan, 2003; Piddini and Vincent, 2003; Polo et al., 2004).
Receptor density at the cell surface is an important determinant of signaling responses, and there are both slow and fast mechanisms attenuating receptor levels. Transcriptional down-regulation is a slow and long-lasting mechanism, whereas posttranslational modification and/or internalization represent fast ways to reduce the amounts of functional receptors on the cell surface. Internalization of many receptors, including Notch and Wnt receptors, is followed by their transport from endosomes to lysosomes, where they become degraded, resulting in a transient reduction in the ability of cells to receive signals. Adding to the complexity of signaling regulation is the fact that ligand-bound receptors may also signal from endosomal membranes, and their signaling output from endosomes may differ from the output triggered from the plasma membrane (Ceresa and Schmid, 2000; Sorkin and Von Zastrow, 2002).
The key roles of the endocytic pathway in cell signaling are highlighted by the analyses of mutants interfering with endocytic trafficking. Such an example is provided by Hrs, a protein that sorts ubiquitinated receptors into intraluminal vesicles of multivesicular bodies (MVBs), destined for degradation in lysosomes. Drosophila hrs mutants show impaired sorting of receptors into MVBs, causing their accumulation in early endosomes (Lloyd et al., 2002; Jekely and Rorth, 2003). In hrs mutants, Dpp (a transforming growth factor-
homologue) and epidermal growth factor receptor signaling is enhanced, presumably because the activated receptors have a prolonged residence time in the limiting membrane of endosomes. Likewise, mutations of two subunits of the endosomal sorting complex required for transport (ESCRT)-I and -II, Tsg101 and Vps25, which are thought to function immediately downstream of Hrs, cause endosomal accumulation of receptors and tumor-like overproliferation in a cell nonautonomous manner due to increased Notch signaling (Moberg et al., 2005; Thompson et al., 2005; Vaccari and Bilder, 2005). This supports the view that proper endocytic traffic has an important antitumorigenic function (Bache et al., 2004; Polo et al., 2004).
Hrs is recruited to endosome membranes by binding the phosphoinositide (PI) phosphatidylinositol (PtdIns) 3-phosphate [PtdIns(3)P], formed by phosphorylation of PtdIns by a class III PI 3-kinase (Raiborg et al., 2001). PtdIns(3)P is specifically localized to endosomal membranes (Gillooly et al., 2000) and not only recruits Hrs but also several other proteins containing FYVE or PX domains (Ellson et al., 2002; Stenmark et al., 2002). Class III PI 3-kinase and PtdIns(3)P are thus crucial regulators of endocytic trafficking, mediating endosome fusion as well as degradative sorting, recycling, and retrograde trafficking to the biosynthetic pathway (Lindmo and Stenmark, 2006). PtdIns(3)P is metabolized by dephosphorylation and by lysosomal lipases (Stenmark and Gillooly, 2001). In addition, this PI can be phosphorylated in the 5-position of the inositol headgroup, giving rise to phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] (Dove et al., 1997; Cooke, 2002; Efe et al., 2005). The kinase catalyzing this phosphorylation, Fab1, was first characterized in yeast. Saccharomyces cerevisiae fab1 mutants have abnormally enlarged vacuoles and show impaired trafficking of the ubiquinated cargo carboxypeptidase S to the vacuole lumen (Odorizzi et al., 1998). Fab1 is evolutionarily conserved, and overexpression of a kinase-dead mutant of the mammalian Fab1 homologue PIKfyve in cultured cells has been reported to inhibit fluid-phase transport of endocytic markers but not recycling/degradation of endocytosed receptors or sorting of procathepsin D (Ikonomov et al., 2001, 2003). Moreover, PIKfyve has been found to be phosphorylated by the PI 3-kinase–regulated protein kinase, PKB, after insulin stimulation, and PIKfyve colocalizes with a highly motile subpopulation of vesicles containing insulin-responsive aminopeptidase (Berwick et al., 2004).
These findings indicate that Fab1/PIKfyve plays a role in controlling specific membrane trafficking processes, but its functions in signal termination and in the physiology of a multicellular organism are not known. To address this, we have generated Drosophila fab1 mutants and studied their phenotype with respect to survival, growth, membrane trafficking and cell signaling. We find that the activity of Drosophila Fab1 is essential for development and cell volume control and that its inactivation leads to endosomal accumulation of Wingless and Notch. Remarkably, this accumulation is not accompanied by increased signaling, indicating that Fab1, unlike Hrs and ESCRT-I and -II, is not involved in receptor silencing.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The P-element vector pCaSpeR4 carrying a ubiquitously expressed tubulin promoter was modified by addition of enhanced green fluorescent protein (EGFP) from pEGFP-C2 (Clontech, Mountain View, CA) into the NotI and XbaI sites (Basler and Struhl, 1994). Atg18 was amplified by PCR from pGEX-4T1-Atg18 (Dove et al., 2004) by using specific primers encoding 5' and 3' XbaI and StuI sites, respectively, and ligated into the XbaI/StuI-digested pCaSpeR4-EGFP to generate pCaSpeR4-EGFP-Atg18 (here called tGFP-Atg18). The plasmids were injected into w1118 embryos for transformation. A minimum of four independent lines were established and tested in each case. The mutations in the fab18, fab121, and fab131 alleles were identified by sequencing at least two overlapping PCR products amplified from homozygous mutant genomic DNA in each case. Primer sequences are available upon request. For antibody production, a construct comprising the N-terminal 400 residues of Fab1 as a C-terminal fusion with maltose binding protein (MBP) was prepared in pMAL-C2 (New England Biolabs, Beverly, MA).
Drosophila Stocks and Genetics
Drosophila stocks included UAS-GFP-Rab5,UAS-GFP-Rab7, UAS-myc-GFP-2xFYVE (Entchev et al., 2000), Df(2R)w30 (Mohr and Gelbart, 2002), nub-Gal4 (Calleja et al., 1996), hrs28, UAS-hrp-Lamp1 (Lloyd et al., 2002), and UAS-hrp-wg (Dubois et al., 2001). For description of other alleles and balancer chromosomes, see FlyBase at http://flybase.bio.indiana.edu/ Crosses were performed at 25°C (unless otherwise specified), and imaginal disk and ovarian clones were generated by heat shocking first- (L1) and second-stage (L2) larvae for 1 h at 37°C when hs-flp was used as a source of Flip recombinase.
Antibody Generation
Polyclonal antiserum against Drosophila Lamp1 (CG3305) was generated by injection of rabbits with a synthetic peptide of 22 amino acids corresponding to the C-terminal cytoplasmic tail SYLCARRRSTSRGYMSF. Polyclonal antiserum toward the N-terminal 400 amino acids of Fab1 was generated by injection of a purified bacterially expressed MBP-Fab1 fusion protein into rabbits.
Immunohistochemistry, Dextran Uptake, and Microscopy
Dextran and bovine serum albumin (BSA) uptake experiments in imaginal disks were performed as described previously (Entchev et al., 2000). Imaginal disks and ovaries were fixed and stained by standard procedures using the following antibodies: mouse anti-Notch (1:40), Dl (1:20), Cut (1:50), Hindsight (1:40), Disk Large (1:40), and Wg (1:1000) (all from the Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), rabbit anti-Drosophila Lamp1 (1:1000), rabbit anti-horseradish peroxidase (HRP) (1:400) (Sigma-Aldrich, Steinheim, Germany), affinity-purified rabbit anti-Fab1 (1:250), rat anti-Spalt (Sal; 1:500) (De Celis and Barrio, 2000), guinea pig anti-Senseless (Sens; 1:1000) (Nolo et al., 2000), rabbit anti-phosphorylated Mothers Against Dpp (Mad; 1:1000) (Tanimoto et al., 2000), guinea pig anti-Hrs (Hrs; 1:1000) (Lloyd et al., 2002) guinea pig anti-Eyegone (Eyg; 1:1000) (Aldaz et al., 2003), rat anti-Distalless (Dll; 1:500) (Vachon et al., 1992), and mouse anti-poly- and monoubiquitin (Affiniti Research Products, Exeter, United Kingdom). Cy2-, Cy3-, and Cy5-conjugated secondary antibodies (1:1000) were from Jackson ImmunoResearch Laboratories (West Grove, PA). Phalloidin (1:50) was used to detect actin, Toto-3 (1:100) to stain DNA, and LysoTracker Red DND-99 (1:200) to detect acidic compartments (all from Invitrogen, Carlsbad, CA). Confocal images were recorded using a Zeiss LSM510 Meta microscope, and images of adult structures were obtained on a Leica MZ FLIII Leica microscope equipped with a Leica DC480 camera (Leica, Wetzlar, Germany), and processed using Adobe Photoshop, version 7.0 (Adobe Systems, Mountain View, CA). The area of ommatidia from eight eyes (4 animals) of each genotype was measured from images obtained at identical conditions using Adobe Photoshop, version 7.0.
Electron Microscopy
Garland cells and wing disks were prepared for conventional plastic embedding according to the following protocol. Cells and tissue were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 1 h at room temperature. Postfixation was performed in 1% OsO4 and 1.5% KFeCN (1 h), followed by en bloc staining in 4% uranyl acetate (30 min), dehydration in graded alcohol concentrations, and embedding with Epon 812. Ultrathin sections were cut on a Leica Ultracut, counterstained with 1% lead citrate and observed in a Philips CM10 at 60–80 kV. Substrate reactions in eye disks expressing HRP-Lamp or HRP-wg were done as follows. After initial glutaraldehyde fixation, the disks were washed in phosphate buffer, incubated for 5 min with 5 mg/ml diaminobenzidine (DAB), followed by 45 min incubation with DAB added to H2O2 to a final concentration of 0.02%. After washing, the samples were further processed for postfixation as described above. Wing disks were also prepared for cryosectioning after fixation in 4% formaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer. The eye disks were embedded in 10% gelatin followed by infusion with 2.3 M sucrose (1 h), mounted on silver pins, and frozen in liquid nitrogen. Ultrathin sections were obtained at –110°C, picked up with a 1:1 mixture of 2.3 M sucrose and 2% methyl cellulose. Sections were then embedded with 2% methyl cellulose and 0.4% uranyl acetate and observed in the microscope as described above.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
). Genomic sequencing of three of the alleles l(2)SJB8, l(2)SJB21, and l(2)SJB31 revealed nonsense mutations in the fab1 coding region, resulting in predicted truncated proteins lacking a functional kinase domain (Figure 1A). Heterozygotes of either of the three alleles over either Df(2R)w30 or Df(2R)Pcl7b, which both delete the fab1 locus, died as late pupae or rare pharate adults in the process of hatching (Figure 1, A and B). Moreover, the lethal phase of l(2)SJB8/l(2)SJB21, l(2)SJB8/l(2)SJB31, or l(2)SJB31/l(2)SJB21 was identical to that of l(2)SJB8, l(2)SJB21, or l(2)SJB31 over Df(2R)w30, suggesting they all represent null, or close to null alleles (Figure 1A). We hereafter refer to the fab1 alleles as fab18, fab121, and fab131, respectively.
We noticed that fab1 pupae were larger than control animals, even though fab1 larvae did not display an extended larval phase (Figure 1B). This size difference was also evident when comparing the sizes of the appendages of fab1 homozygotes dissected from the pupal cases to those of wild-type pupae. The legs were of correct length but considerably thicker than in control animals. Loss of fab1 can autonomously cause tissue-specific overgrowth, because mosaic adult flies (Newsome et al., 2000) with heads consisting primarily of fab1 mutant cells were viable and showed a clear enlargement of the head and eyes (Figure 1, C and D). The overgrowth phenotypes were also distinct in the larval tissues of fab1 mutants. Salivary glands, gut tissue, brain lobes, and imaginal disks looked larger and swollen compared with wild type (our unpublished data).
To address whether Fab1 is the responsible kinase for producing PtdIns(3,5)P2 on endosomal structures, we took advantage of the fact that the yeast green fluorescent protein (GFP)-Atg18/Svp1 fusion protein can serve as an in vivo probe for the detection of PtdIns(3,5)P2 lipids on endosomes (Dove et al., 2004). In transgenic flies expressing GFP-Atg18 under the control of the ubiquitously expressed tubulin promoter, punctate structures were preferentially observed in the apical cytoplasm of eye imaginal disk cells (Figure 1, E–G). A partial colocalization was observed with LysoTracker (Figure 1E), which labels late acidic endosomes and lysosomes, and with endosomes labeled with internalized Texas Red dextran (TRD) (Figure 1, F and G), demonstrating that GFP-Atg18 can be found on late acidic endosomal structures. Like previously reported in fab1 yeast mutants, the punctate localization of GFP-Atg18 was lost in fab1 mutant cells and instead occurred diffusely in the cytoplasm (Figure 1H). Thus, Fab1 seems to be the sole kinase producing PtdIns(3,5)P2 on endosomal membranes (Figure 1I).
To detect endogenous Fab1, we generated polyclonal antibodies against the N-terminal part of recombinant Fab1. Distinct punctate structures were labeled in Garland cells isolated from wandering L3 control animals, whereas no staining was observed in Df(2R)w30/Df(2R)Pcl7b larvae, demonstrating the specificity of the antibody (Figure 2, A and B). We used the antibody to determine the subcellular localization of Fab1 relatively to markers of the endocytic machinery in wing imaginal disks and in Garland cells. Fab1 showed little colocalization with the fluid-phase marker Oregon green dextran (OGD) after a 5-min internalization in Garland cells (Figure 2C), whereas there was significant colocalization after a 40-min chase (Figure 2D). Moreover, Fab1 colocalized partially with markers of early (GFP-Rab5; Figure 2E) and late endosomes (GFP-Rab7; Figure 2H). Interestingly, only limited colocalization, but still proximity, was observed between Fab1 and Hrs (Figure 1F) and a probe specific for PtdIns(3)P (GFP-2xFYVE; Figure 1G), perhaps reflecting the rapid turnover of PtdIns(3)P on a subset of endosomal membranes (Gillooly et al., 2000; Lloyd et al., 2002; Wucherpfennig et al., 2003). This finding is in agreement with the distribution of Fab1 in mammalian cells, where Fab1, Hrs, and PtdIns(3)P are found on different microdomains of endosomal membranes (Cabezas et al., 2006). Close examination of unfixed fab1 mutant Garland cells showed that these had up to 3 times their normal diameter (Figure 2I). Investigation of the imaginal wing disk epithelium from fab1 mutant larvae revealed that the overall cell polarity was intact, whereas the cells were larger and showed a disorganized distribution of nuclei relative to wild-type wing disks (Figure 2, J and K). Together with the increased organ size in fab1 mutants (Figure 1, B–D), this indicates that Fab1 exerts a negative cell size control but is not required for epithelial polarization.
|
; Ikonomov et al., 2001), we found a strongly reduced lysosomal acidity in fab1 mutant Garland cells (Figure 2I). To investigate the role of Fab1 in fluid-phase transport, trafficking of fluorescently labeled internalized dextran was investigated in wandering third instar Garland cells. After 5 min of uptake in control cells, OGD was observed in distinct punctate endocytic structures close to the plasma membrane (Figures 2C and 3A). The predominant portion of OGD reached the more centrally located lysosomal compartment labeled with the pH-sensitive vital dye LysoTracker, after 40 min of chase (Figure 3B). OGD remained mainly in the periphery of the cells and failed to be transported to the weakly acidic compartments in fab1 mutant cells, suggesting a failure of fluid-phase transport to the lysosomes (Figure 3, C and D). This is consistent with the finding that overexpression of a kinase-dead PIKfyve mutant inhibits lysosomal transport of endocytosed HRP in human embryonic kidney cells (Ikonomov et al., 2003).
|
), but the transport of fluid phase tracer to lysosomes is intact in such cells (our unpublished data). The failure of transport of endocytic fluid phase tracer into the electron-dense structures observed by EM and LysoTracker-positive structures observed by immunofluorescence suggests a failure of endocytosed material to reach the lysosome or a failure of maturation of the endosome into a functional degradative compartment.
fab1 Mutant Cells Show Endosomal Accumulation of Receptors but Not of Ubiquitin
Because of the proposed relationship between endocytic trafficking and signaling (Lloyd et al., 2002; Gonzalez-Gaitan, 2003; Piddini and Vincent, 2003), we asked whether endocytic trafficking and degradation of signaling proteins were affected in fab1 mutant polarized epithelial tissue. The receptors Notch (N) and PVR and the ligands Wingless (Wg), Delta (Dl), and Boss accumulated in punctate internal structures specifically in fab1 mutant cells generated by the FLP/FRT technique (Golic, 1991; Xu and Rubin, 1993) or in dissected tissues from fab1 mutants (Figure 4, A–F, K, and L; our unpublished data). Importantly, unlike hrs and vps25 mutant cells, which showed dramatic accumulation of ubiquitin positive structures (Jekely and Rorth, 2003; Jekely et al., 2005; Thompson et al., 2005) (Figure 4M), fab1 mutant cells did not accumulate any higher ubiquitin levels compared with neighboring control cells (Figure 4N). The strong accumulation of ligands and receptors but not of ubiquitin in fab1 mutants suggests a distinct compartmentalization of cargo accumulation compared with the tsg101, vps25, and hrs mutants, in which ubiquitinated cargoes are retained in early Rab5/PtdIns(3)P-positive endosomes (Jekely and Rorth, 2003; Moberg et al., 2005; Thompson et al., 2005; Vaccari and Bilder, 2005).
|
). Intracellular Wg in endocytic 0.5- to 1-µm-sized structures was recognized within receiving cells by immunolocalization up to 20 µm away from the source in both the wing pouch and the notum (Figure 4, A–C and I). Detection of Wg in isolated wing imaginal disks from fab1 mutants revealed a striking accumulation of Wg in internal vesicles up to 40 µm away from the source of producing cells in the wing pouch (Figure 4, D, E, and J). The aberrant Wg accumulation was caused by the lack of Fab1, because expression of full-length Fab1 specifically in the wing pouch of fab1 mutant animals showed a nearly complete rescue of the defect in the wing pouch (Figure 4G). The selective accumulation of Wg in receiving cells shows that the biosynthetic pathway is functional in fab1 mutant cells. Moreover, Wg was specifically detected in fab1 mutant cells and not in heterozygous cells or wild-type twin spot cells located at a similar distance from the signaling source, suggesting that the excess of Wg protein in fab1 cells derived from defective endocytic trafficking in the receiving cells (Figure 4F). Comparison of confocal Z-sections revealed that although small 0.5- to 1.5-µm vesicles could be observed in the apical and basal portion of the receiving cells from both fab1 mutants and control wing disks, the larger Wg-containing structures (up to 2–5 µm in diameter) were located further toward the perinuclear region (our unpublished data). Like in control cells (Figure 5, A, C, and E), Wg was observed in Hrs- and 2xFYVE (dbFYVE)-positive early endosomes as well as in Rab7-positive endosomes in fab1 mutant tissue (Figure 5, B, D, and F). These endosomes seemed 1.5 to 2 times larger in diameter than in control cells and some contained Wg in the lumen, strongly suggesting that MVBs can form in fab1 mutants and that Wg can be sorted to their inner vesicles. In control cells, Wg was rarely observed in GFP-Rab7– and GFP-2xFYVE–negative intracellular compartments, suggesting that it is rapidly degraded after transport to the later lysosomal structures (Figure 5, C and E). The larger 2- to 5-µm Wg-positive vesicles in fab1 mutant cells were labeled by an antibody against the late-endosomal/lysosomal marker Lamp1 and contained an HRP-Lamp1 transgenic fusion protein (Figure 5, H and I). This suggests that although Wg traffics to late endosomes in fab1 mutants, its degradation is inhibited.
|
For a more detailed analysis, we expressed Wg and Lamp1 fused to HRP orthogonally to the endogenous Wg expression pattern in the wing disk by using the dpp-Gal4 driver. The relatively high resistance of the HRP moiety of the fusion proteins to lysosomal degradation allows HRP signals to be detected in late endocytic compartments of receiving cells whose endogenous Wg is efficiently degraded, thus visualizing the entire endolysosomal system (Dubois et al., 2001). Indeed, in control wing disks, immunolocalization of HRP could be detected in numerous structures devoid of Wg (Figure 5L). In contrast, in fab1 mutant disks, large structures, up to 5 µm, filled with both HRP and Wg could be detected in receiving cells. This suggests that endolysosomal Wg degradation is inhibited in fab1 mutant tissue (Figure 5M).
The HRP moiety of fusion proteins of HRP-Lamp1 and HRP-Wg is enzymatically functional in fixed tissue, allowing its in situ detection by EM after conversion of the diaminobenzidine substrate into a dark precipitate (Sevrioukov et al., 1999; Dubois et al., 2001). Consistent with previous findings (Sevrioukov et al., 1999), we detected the HRP-Lamp1 fusion in MVBs by EM (Figure 6, A and B). Both normal-sized and enlarged MVBs containing HRP-Lamp1 in intraluminal vesicles could be observed in fab1 mutant tissue, showing that the Hrs-dependent formation of intraluminal vesicles (Lloyd et al., 2002) does not require Fab1 function (Figure 6, C–F). Although the MVBs in control cells were generally 0.5–1 µm, the HRP-Lamp1–positive structures in fab1 mutant cells were 1–5 µm. These larger MVBs likely correspond to Lamp1/Wg/Notch-containing structures observed by confocal microscopy (Figure 5, I–K). Investigation of cells expressing HRP-Wg revealed abundant concentration of HRP in normal-sized endosomes of wild-type larvae (Figure 6, G and H) as well as in the larger structures in fab1 mutants (Figure 6, I and J). In both cases, labeling was found both on limiting membranes and in intraluminal vesicles. Thus, both HRP-Wg and HRP-Lamp1 labeled similar MVBs and late endocytic compartments by EM. These results indicate that endocytosed Wg is transported into the lumen of MVBs and late endosomes in both wild-type and fab1 wing disks.
|
; Moberg et al., 2005; Thompson et al., 2005; Vaccari and Bilder, 2005), whereas Wg signaling is unaffected in hrs mutants (Piddini et al., 2005). Having observed that Wg and Notch trafficking is affected in fab1 mutants, we therefore asked whether the impaired endocytic trafficking was associated with enhanced receptor signaling. The expression of Wg in the wing pouch activates target genes at distinct thresholds of signaling in a nested pattern away from the source and provides a sensitive read-out of signaling activity (schematically illustrated in Figure 7I). Investigation of mosaic or whole fab1 mutant animals revealed no measurable defects on expression of the high-threshold target genes Senseless (Sens), Cut, and Hindsight (Hnt) or the low-threshold target gene Distalless (Dll) (Figure 7, A–K). This provides evidence that Wg signaling is unaffected in fab1 mutants.
|
). In contrast, we did not detect any increase of the expression of the low level Dpp target gene Spalt (Sal) in fab1 clones of the wing pouch, nor of phosphorylated Mothers against Dpp (p-Mad) in fab1 clones of ovarian follicle cells (Figure 8, A–F). This indicates that Dpp signaling is unaffected in fab1 mutants.
|
; Moberg et al., 2005; Thompson et al., 2005; Vaccari and Bilder, 2005). No ectopic expression of Eyg was observed in fab1 mutant clones in eye disks (Figure 8G), suggesting that Notch signaling ensues at the correct space and time despite dramatic accumulation of both Dl and Notch in endosomes.
In the wing disk, short-range Notch signaling is instructive in defining the dorsal-ventral boundary organizer. A direct downstream consequence is the expression of Wg in a stripe of cells along the boundary. Thus, expression of Wg also serves as a marker for Notch signaling activity in the wing pouch (Vincent and Dubois, 2002). Even though the Wg stripe seemed broader than normal in fab1 mutant disks, this was clearly due to accumulation of Wg in internal endocytic vesicles and not due to a broader area of cells producing Wg, which are easily recognized due to large amounts of Wg in the biosynthetic pathway (our unpublished data). Moreover, no defects in the sensitive Notch-controlled bristle patterns and sensory organ cell specification in the cuticles of fab1 mutants were observed (Figure 1, B and C). This supports the conclusion that Notch signaling is normal in fab1 mutants. Collectively, the above-mentioned results indicate that Wg, Dpp, and Notch signaling is normal in fab1 mutant cells. Thus, Fab1 does not share the ability of Hrs and ESCRT-I and -II to mediate receptor silencing.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Murthy and De Camilli, 2003). Nevertheless, the functions of PtdIns(3)P 5-kinase in multicellular organisms have not been investigated. In the present study, we have established Fab1 as an essential PtdIns(3)P 5-kinase in Drosophila, with profound functions in membrane traffic. Although the importance of Fab1 in endocytic membrane homeostasis has previously been highlighted by studies in yeast and mammalian cell culture (Odorizzi et al., 1998; Ikonomov et al., 2001, 2003; Bonangelino et al., 2002), its failure to mediate receptor silencing was unanticipated. Our data place Fab1 downstream of Hrs and ESCRT-I and -II, which are known to mediate receptor silencing in endosomes.
That endosomal sorting of ubiquitinated cargoes is of great physiological importance is illustrated by studies of Drosophila mutants of the two ESCRT subunits, Tsg101 and Vps25. Loss of these proteins yields endosomal accumulation of receptors and ubiquitin, similarly to hrs mutants. Importantly, loss of Tsg101 and Vps25 in clones of cells causes a tumor-like overproliferation of adjacent tissue due to increased Notch-mediated signaling (Moberg et al., 2005; Thompson et al., 2005; Vaccari and Bilder, 2005). We were unable to detect any such effects with fab1 mutant clones (our unpublished data) consistent with the finding that Notch signaling (as well as Wg and Dpp signaling) was unaffected in fab1 mutants. Thus, Fab1, unlike Hrs and ESCRT-I and -II proteins, does not seem to play any role in receptor silencing, even though it is important for receptor degradation. This is reminiscent of the ESCRT-III subunit hVps24, which mediates degradation but not silencing of the epidermal growth factor receptor (Bache et al., 2006). Moreover, it is interesting to note that impaired Hrs, Tsg101, or Vps25 function causes a strong accumulation of ubiquitinated proteins in endosomes (Bishop et al., 2002; Jekely and Rorth, 2003; Thompson et al., 2005), whereas this was not observed in fab1 mutant clones. These results, together with the fact that Fab1 mainly localizes to later endocytic structures than Hrs, suggest that Fab1 functions later than Hrs and ESCRT-I/-II in endocytic trafficking, at a point beyond receptor deubiquitination and signal termination.
Studies in yeast and mammalian cells have suggested a role for Fab1 in endocytic membrane homeostasis, although its exact functions are not known. Indeed, confocal and EM revealed the accumulation of larger late endosomes in fab1 mutant Drosophila cells, consistent with previous studies in fab1 yeast and overexpression of kinase-dead PIKfyve in mammalian cells. The findings that the enlarged vacuoles in fab1 yeast mutants and late endosomes in kinase-dead PIKfyve-overexpressing cells contain few internal vesicles have suggested the possibility that Fab1 could mediate formation of such vesicles (Odorizzi et al., 1998; Ikonomov et al., 2001). In agreement with this, in fab1 mutant Drosophila cells we frequently observed enlarged endosomes with few or no intraluminal vesicles. However, in the fab1 mutants we also observed highly enlarged MVBs that were filled with numerous normal-sized intraluminal vesicles. This indicates that the increased endosome size in the absence of Fab1 cannot be explained by an inhibited formation of intraluminal vesicles, in contrast to what has been reported for Hrs (Lloyd et al., 2002). A more likely explanation is that late endosomes expand in fab1 mutants because of inhibited retrograde membrane flux to the biosynthetic and early endocytic pathways (Dove et al., 2004; Efe et al., 2005).
Cell and organ size is controlled by genetic, hormonal, and environmental inputs (Hafen and Stocker, 2003). In particular, insulin signaling is important for growth, and the functions of the downstream class I PI 3-kinases in growth signaling are well characterized. The striking growth phenotypes observed in fab1 mutants indicate that PtdIns(3)P 5-kinase also regulates cell size. Interestingly, however, whereas PI 3-kinases promote growth, our findings indicate that Fab1 has an inhibitory effect on cell size. Garland cells were strongly enlarged in fab1 mutants, suggesting a function of Fab1 in negative cell size regulation. In addition, fab1 deficiency led to a thickening of legs and enlargement of wings and heads, demonstrating a role for Fab1 in attenuating organ size. Overexpression of Drosophila Fab1 did not cause any overt growth-inhibitory effects (our unpublished data), consistent with the finding that overexpression of Fab1 in yeast does not yield any increase in PtdIns(3,5)P2 levels, presumably because regulatory components are limiting (Odorizzi et al., 1998). We were unable to detect any strong genetic interactions between fab1 and mutants in components of the insulin signaling pathway (our unpublished data), suggesting that the increased cell size in fab1 mutants may not be due to up-regulation of this pathway. Instead, there was a striking correlation between cell size and endosome overgrowth in fab1 mutant larvae. Thus, the increased cell and organ size in fab1 mutants may be due to the volume expansion of endosomes. We therefore propose that Fab1, through its effects on endosome morphology, functions in negative regulation of cell volume. Further work will reveal whether Fab1 also regulates cell size by additional mechanisms.
In conclusion, we have performed the first analysis of a PtdIns(3)P 5-kinase in a multicellular organism and found that Fab1 is essential for proper degradative receptor trafficking but dispensable for the termination of receptor signaling. This places the function of Fab1 at a late stage in endocytic receptor trafficking and shows that receptor degradation is not required for signal termination.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Address correspondence to: Harald Stenmark (stenmark{at}ulrik.uio.no)
Abbreviations used: BSA, bovine serum albumin; EM, electron microscopy; ESCRT, endosomal sorting complex required for transport; GFP, green fluorescent protein; HRP, horseradish peroxidase; MVB, multivesicular body; OGD, Oregon green dextran; PI, phosphoinositide; TRD, Texas Red dextran.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bache, K. G., Slagsvold, T., Stenmark, H. (2004). Defective downregulation of receptor tyrosine kinases in cancer. EMBO J 23, 2707–2712.[CrossRef][Medline]
Bache, K. G., Stuffers, S., Malerød, L., Slagsvold, T., Raiborg, C., Lechardeur, D., Wälchli, S., Lukacs, G., Brech, A., Stenmark, H. (2006). The ESCRT-III subunit hVps24 is required for degradation but not silencing of the epidermal growth factor receptor. Mol. Biol. Cell 17, 2513–2523.
Basler, K. and Struhl, G. (1994). Compartment boundaries and the control of Drosophila limb pattern by hedgehog protein. Nature 368, 208–214.[CrossRef][Medline]
Berwick, D. C., Dell, G. C., Welsh, G. I., Heesom, K. J., Hers, I., Fletcher, L. M., Cooke, F. T., Tavare, J. M. (2004). Protein kinase B phosphorylation of PIKfyve regulates the trafficking of GLUT4 vesicles. J. Cell Sci 117, 5985–5993.
Bishop, N., Horman, A., Woodman, P. (2002). Mammalian class E vps proteins recognize ubiquitin and act in the removal of endosomal protein-ubiquitin conjugates. J. Cell Biol 157, 91–101.
Bonangelino, C. J., Nau, J. J., Duex, J. E., Brinkman, M., Wurmser, A. E., Gary, J. D., Emr, S. D., Weisman, L. S. (2002). Osmotic stress-induced increase of phosphatidylinositol 3,5-bisphosphate requires Vac14p, an activator of the lipid kinase Fab1p. J. Cell Biol 156, 1015–1028.
Cabezas, A., Pattni, K., Stenmark, H. (2006). Cloning and subcellular localization of a human phosphatidylinositol 3-phosphate 5-kinase, PIKfyve/Fab1. Gene 371, 34–41.[CrossRef][Medline]
Calleja, M., Moreno, E., Pelaz, S., Morata, G. (1996). Visualization of gene expression in living adult Drosophila. Science 274, 252–255.
Ceresa, B. P. and Schmid, S. L. (2000). Regulation of signal transduction by endocytosis. Curr. Opin. Cell Biol 12, 204–210.[CrossRef][Medline]
Cooke, F. T. (2002). Phosphatidylinositol 3,5-bisphosphate: metabolism and function. Arch. Biochem. Biophys 407, 143–151.[CrossRef][Medline]
De Celis, J. F. and Barrio, R. (2000). Function of the spalt/spalt-related gene complex in positioning the veins in the Drosophila wing. Mech. Dev 91, 31–41.[CrossRef][Medline]
Dove, S. K., Cooke, F. T., Douglas, M. R., Sayers, L. G., Parker, P. J., Michell, R. H. (1997). Osmotic stress activates phoshatidylinositol-3,5-bisphosphate synthesis. Nature 390, 187–192.[CrossRef][Medline]
Dove, S. K. (2004). Svp1p defines a family of phosphatidylinositol 3,5-bisphosphate effectors. EMBO J 23, 1922–1933.[CrossRef][Medline]
Dubois, L., Lecourtois, M., Alexandre, C., Hirst, E., Vincent, J. P. (2001). Regulated endocytic routing modulates wingless signaling in Drosophila embryos. Cell 105, 613–624.[CrossRef][Medline]
Efe, J. A., Botelho, R. J., Emr, S. D. (2005). The Fab1 phosphatidylinositol kinase pathway in the regulation of vacuole morphology. Curr. Opin. Cell Biol 17, 402–408.[CrossRef][Medline]
Ellson, C. D., Andrews, S., Stephens, L. R., Hawkins, P. T. (2002). The PX domain: a new phosphoinositide-binding module. J. Cell Sci 115, 1099–1105.
Englund, C., Uv, A. E., Cantera, R., Mathies, L. D., Krasnow, M. A., Samakovlis, C. (1999). adrift, a novel bnl-induced Drosophila gene, required for tracheal pathfinding into the CNS. Development 126, 1505–1514.[Abstract]
Entchev, E. V., Schwabedissen, A., Gonzalez-Gaitan, M. (2000). Gradient formation of the TGF-beta homolog Dpp. Cell 103, 981–991.[CrossRef][Medline]
Gillooly, D. J., Morrow, I. C., Lindsay, M., Gould, R., Bryant, N. J., Gaullier, J.-M., Parton, R. G., Stenmark, H. (2000). Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells. EMBO J 19, 4577–4588.[CrossRef][Medline]
Golic, K. G. (1991). Site-specific recombination between homologous chromosomes in Drosophila. Science 252, 958–961.
Gonzalez-Gaitan, M. (2003). Signal dispersal and transduction through the endocytic pathway. Nat. Rev. Mol. Cell Biol 4, 213–224.[CrossRef][Medline]
Hafen, E. and Stocker, H. (2003). How are the sizes of cells, organs, and bodies controlled? PLoS Biol 1, 319–323.[CrossRef]
Ikonomov, O. C., Sbrissa, D., Foti, M., Carpentier, J. L., Shisheva, A. (2003). PIKfyve controls fluid phase endocytosis but not recycling/degradation of endocytosed receptors or sorting of procathepsin D by regulating multivesicular body morphogenesis. Mol. Biol. Cell 14, 4581–4591.
Ikonomov, O. C., Sbrissa, D., Shisheva, A. (2001). Mammalian cell morphology and endocytic membrane homeostasis require enzymatically active phosphoinositide 5-kinase PIKfyve. J. Biol. Chem 276, 26141–26147.
Jekely, G. and Rorth, P. (2003). Hrs mediates downregulation of multiple signalling receptors in Drosophila. EMBO Rep 4, 1163–1168.[CrossRef][Medline]
Jekely, G., Sung, H. H., Luque, C. M., Rorth, P. (2005). Regulators of endocytosis maintain localized receptor tyrosine kinase signaling in guided migration. Dev. Cell 9, 197–207.[CrossRef][Medline]
Lindmo, K. and Stenmark, H. (2006). Regulation of membrane traffic by phosphoinositide 3-kinases. J. Cell Sci 119, 605–614.
Lloyd, T. E., Atkinson, R., Wu, M. N., Zhou, Y., Pennetta, G., Bellen, H. J. (2002). Hrs regulates endosome invagination and receptor tyrosine kinase signaling in Drosophila. Cell 108, 261–269.[CrossRef][Medline]
Luo, J., Manning, B. D., Cantley, L. C. (2003). Targeting the PI3K-Akt pathway in human cancer: rationale and promise. Cancer Cell 4, 257–262.[CrossRef][Medline]
Moberg, K. H., Schelble, S., Burdick, S. K., Hariharan, I. K. (2005). Mutations in erupted, the Drosophila ortholog of mammalian tumor susceptibility gene 101, elicit non-cell-autonomous overgrowth. Dev. Cell 9, 699–710.[CrossRef][Medline]
Mohr, S. E. and Gelbart, W. M. (2002). Using the P[wHy] hybrid transposable element to disrupt genes in region 54D–55B in Drosophila melanogaster. Genetics 162, 165–176.
Murthy, V. N. and De Camilli, P. (2003). Cell biology of the presynaptic terminal. Annu. Rev. Neurosci 26, 701–728.[CrossRef][Medline]
Newsome, T. P., Asling, B., Dickson, B. J. (2000). Analysis of Drosophila photoreceptor axon guidance in eye-specific mosaics. Development 127, 851–860.[Abstract]
Nolo, R., Abbott, L. A., Bellen, H. J. (2000). Senseless, a Zn finger transcription factor, is necessary and sufficient for sensory organ development in Drosophila. Cell 102, 349–362.[CrossRef][Medline]
Odorizzi, G., Babst, M., Emr, S. D. (1998). Fab1p PtdIns(3)P 5-kinase function essential for protein sorting in the multivesicular body. Cell 95, 847–858.[CrossRef][Medline]
Piddini, E., Marshall, F., Dubois, L., Hirst, E., Vincent, J. P. (2005). Arrow (LRP6) and Frizzled2 cooperate to degrade Wingless in Drosophila imaginal discs. Development 132, 5479–5489.
Piddini, E. and Vincent, J. P. (2003). Modulation of developmental signals by endocytosis: different means and many ends. Curr. Opin. Cell Biol 15, 474–481.[CrossRef][Medline]
Polo, S., Pece, S., Di Fiore, P. P. (2004). Endocytosis and cancer. Curr. Opin. Cell Biol 16, 1–6.[CrossRef][Medline]
Raiborg, C., Bremnes, B., Mehlum, A., Gillooly, D. J., Stang, E., Stenmark, H. (2001). FYVE and coiled-coil domains determine the specific localisation of Hrs to early endosomes. J. Cell Sci 114, 2255–2263.[Medline]
Rorth, P. (1998). Systematic gain-of-function genetics in Drosophila. Development 125, 1049–1057.[Abstract]
Sevrioukov, E. A., He, J. P., Moghrabi, N., Sunio, A., Kramer, H. (1999). A role for the deep orange and carnation eye color genes in lysosomal delivery in Drosophila. Mol. Cell 4, 479–486.[CrossRef][Medline]
Sorkin, A. and Von Zastrow, M. (2002). Signal transduction and endocytosis: close encounters of many kinds. Nat. Rev. Mol. Cell Biol 3, 600–614.[CrossRef][Medline]
Stenmark, H., Aasland, R., Driscoll, P. C. (2002). The phosphatidylinositol 3-phosphate-binding FYVE finger. FEBS Lett 513, 77–84.[CrossRef][Medline]
Stenmark, H. and Gillooly, D. J. (2001). Intracellular trafficking and turnover of phosphatidylinositol 3-phosphate. Semin. Cell Dev. Biol 12, 193–199.[CrossRef][Medline]
