Notch signaling is disrupted in FK39 and FV10 mutant follicle cells and imaginal disc cells
In a genetic mosaic screen aimed at identifying genes regulating the organization and morphogenesis of the Drosophila follicular epithelium (Denef et al., 2008
), we isolated two complementation groups which caused defects reminiscent of those observed in follicle cells mutant for Notch
. The two complementation groups were initially named after the representative alleles FK39
and contained 13 and 17 alleles respectively.
Wild-type follicle cells divide mitotically from stage 2 to stage 6 of oogenesis and then switch from a mitotic cycle to an endocycle. From stage 7 to 10A follicle cells go through three rounds of DNA duplication without cell division (Horne-Badovinac and Bilder, 2005
). This mitotic cycle to endocycle switch is regulated by the Notch pathway. Follicle cells in which Notch signaling is compromised keep dividing after stage 6 and fail to undergo endoreplication. As a result, follicle cell clones mutant for Notch
form patches of cells with smaller nuclei than neighboring wild-type cells (Deng et al., 2001
; Lopez-Schier and St Johnston, 2001
). Follicle cell clones mutant for FK39
show a similar phenotype ( and data not shown). To confirm that the mutant cells continue dividing after stage 6 we stained for the mitotic marker phosphorylated Histone H3 (PH3) and found that, in contrast to wild-type egg chambers where PH3 positive cells are only found up to stage 6, PH3 staining was frequently present after stage 6 in these mutant cells ( and data not shown).
Loss of Notch signaling in FK39 and FV10 mutant cells
The failure of FK39
mutant follicle cells to switch from a mitotic cycle to an endocycle suggests defective Notch signaling. To test this more directly, we examined the expression of several known Notch targets in the follicular epithelium. The transcription factor Hindsight (Hnt) is upregulated by Notch signaling at stage 6/7 and this upregulation is required for the mitotic-cycle-to-endocycle switch in follicle cells (Sun and Deng, 2007
). In contrast to wild-type cells Hnt upregulation was not detected in FK39
mutant follicle cells in egg chambers beyond stage 6 ( and data not shown). The homeodomain protein Cut is downregulated in wild-type follicle cells upon Notch activation at stage 6 and this downregulation is critical for follicle cells to enter the endocycle (Sun and Deng, 2005
). In FK39
mutant follicle cells Cut expression fails to be repressed after stage 6 ( and data not shown). Finally, we examined the expression of a transcriptional reporter for Notch signaling, Gbe+Su(H)-lacZ (Furriols and Bray, 2001
). In 77% of FK39
mutant clones (n=84), β-galactosidase staining was either absent or strongly reduced (). Taken together, these results indicate that Notch signaling is lost in FK39
mutant follicle cells.
To ask whether the gene products disrupted in these mutants were required specifically in the follicular epithelium for Notch signaling or whether they also affected Notch signaling in other tissues, we examined the expression of the Gbe+Su(H)-lacZ reporter in FK39 mutant eye disc cells. Similar to our results in the follicle cells, we detected much lower levels of β-galactosidase in FK39 mutant eye disc cells compared to neighboring wild-type cells (, n>30), demonstrating that Notch signaling is compromised in FK39 mutant eye disc cells.
The observed loss of Notch signaling in FK39
mutant follicle cells indicates that the gene products disrupted in FK39
are required for Notch signaling in the signal receiving cells. Indeed, it was shown that the Notch ligand Delta is required in the germ line but not in the follicle cells to induce the mitotic to endocycle switch (Deng et al., 2001
; Lopez-Schier and St Johnston, 2001
). Upon ligand binding, Notch undergoes an extracellular cleavage (S2), generating the membrane bound NEXT, followed by an intramembranous cleavage (S3) producing NICD (Lai, 2004
; Schweisguth, 2004
). To further define the step where Notch signaling is disrupted in the mutants, we overexpressed full length Notch, NEXT or NICD constructs in FK39
mutant clones, using the MARCM system (Lee and Luo, 1999
). With this system of clone generation, Hindsight failed to be upregulated in 83% of FK39
follicle cell clones (n=180) in stage 6/7 egg chambers, again indicating defective Notch signaling. Disrupted Notch signaling was similarly observed in 83% of FK39
clones with coexpression of full length Notch (n=121), and in 71% of FK39
clones which expressed NEXT (n=139), but only in 10% of FK39
clones which expressed NICD (n=122). () Therefore, only the expression of NICD but not full length Notch or NEXT, can restore the loss of Notch signaling in FK39
mutant follicle cells.
The FK39 gene product is required for Notch signaling after the S2 cleavage of Notch
In wing imaginal discs, overexpression of NEXT results in ectopic activation of the Notch target gene Cut (Rebay et al., 1993
; Struhl and Greenwald, 1999
). We therefore compared wing discs expressing NEXT in FK39
mutant clones with discs expressing NEXT in control clones. Cut was ectopically expressed in all cells of 59% of control clones (n=172) but only in 33% of FK39
mutant clones (n=152) (p<10-3
, binomial probability) (). It is possible that perdurance of the wild-type gene product between the time of clone induction and dissection of the discs prevented a completely penetrant loss of signaling activity. Nevertheless, this result indicates that NEXT signals less efficiently in FK39
mutant cells than in control cells. We also attempted to assess whether ectopic Notch signaling induced by overexpession of NICD in wing discs could be suppressed by the loss of the FK39
gene product. However, we were unable to obtain a significant number of surviving FK39
clones expressing NICD in this experiment.
Taking together the evidence in both follicle cells and wing disc cells, we conclude that the gene product disrupted in FK39 mutant cells is required for signaling after the S2 cleavage of Notch but does not significantly affect Notch signaling after the release of the intracellular domain into the cytoplasm.
The genes disrupted in FV10 and FK39 encode the Drosophila homologues of mammalian Rabconnectin-3 alpha and beta respectively
Using meiotic recombination with visible recessive markers and P element insertions we mapped the lethal phenotype of FK39
to the chromosomal regions 2B15 and 5E respectively. The fact that these two complementation groups were mapped to distinct loci, yet shared identical phenotypes suggested that the two gene products might be functionally related. Two ORFs in these regions therefore stood out; CG3585 in region 5E and CG17766 in region 2B15 encode the fly homologues of the mammalian WD-40 proteins Rabconnectin-3 alpha and beta (Rbcn-3A and B) respectively. Rbcn-3A and B were identified as binding partners of Rab3-GEP and Rab3-GAP in synaptic vesicle extracts of rat brain (Kawabe et al., 2003
; Nagano et al., 2002
; Sakisaka and Takai, 2005
). The two proteins appear to form a stable complex but the biochemical function of this complex remains unknown. The Drosophila Rbcn-3A
gene was originally named DmX
(Kraemer et al., 1998
) and its mammalian homologues Dmx-like1
, with Rbcn-3A
(Kraemer et al., 2000
; Nagano et al., 2002
). Because of the functional connection between Rbcn-3A (DmX) and Rbcn-3B we will use the name Rbcn-3A in this paper.
To ask whether the loss of Rbcn-3A and B was indeed responsible for the phenotypes observed in FK39 and FV10 mutants, we sequenced the Rbcn-3B gene in seven alleles of the FK39 group and found that all of them contained mutations in Rbcn-3B (). Moreover, expression of a HA-tagged Rbcn-3B cDNA in FK39 mutant follicle cell clones rescued the Notch-like phenotype (, n>50). Similarly, a genomic rescue construct containing the Rbcn-3A gene and ~1.5 kb upstream and downstream sequences rescued the lethality and ovarian phenotypes of FV10 mutants ().
The defects in FV10 and FK39 mutant follicle cells are caused by mutations in the fly homologues of Rabconnectin-3alpha and beta
Taken together, our results demonstrate that the loss of Notch signaling observed in FV10 and FK39 mutants is caused by the loss of function of the Drosophila homologues of Rbcn-3A and Rbcn-3B respectively. Since the two genes share the same mutant phenotypes in all aspects of our analysis we will refer to them collectively as Rbcn-3, where appropriate.
Endocytic trafficking is disrupted in Rbcn-3 mutant follicle cells and eye disc cells
We have shown that Rbcn-3 is required for Notch signaling in the receiving cells. Since effective Notch signaling relies on intracellular trafficking of Notch in the signal receiving cells (Le Borgne, 2006
; Nichols et al., 2007
) we asked whether Notch trafficking was defective in Rbcn-3
mutant cells. We therefore compared the subcellular distribution of Notch in Rbcn-3
mutant follicle cells and eye disc cells with that in wild-type cells. We found that in Rbcn-3
mutant follicle cells as well as eye disc cells, Notch accumulated in enlarged intracellular compartments ( and data not shown). Notch accumulation in Rbcn-3
mutant cells was observed with antibodies against both the intracellular domain (NICD, ) and the extracellular domain (NECD, ), suggesting that full length Notch is present in these enlarged compartments.
Transmembrane proteins accumulate in enlarged intracellular compartments in Rbcn-3 mutant cells
We next asked whether the loss of Rbcn-3 specifically disrupts the distribution of Notch or whether other transmembrane proteins were also affected. We examined the subcellular localization of Delta (), EGF receptor (Supplemental Figures 1A, B
), Domeless (Supplemental Figure 1C
), Fas II (), FasIII and DE-Cadherin (data not shown) and found that they all accumulate in similar enlarged compartments in the absence of Rbcn-3. In contrast, membrane-associated proteins, such as aPKC, Crag and Dlg maintained a normal staining pattern in Rbcn-3
mutant follicle cells ( and data not shown). Interestingly, despite the intracellular accumulation of the EGF receptor and of Domeless, which activates the JAK/STAT pathway, signaling downstream of these two receptors occurs normally in Rbcn-3
mutant follicle cells (Supplemental Figure 1
The intracellular accumulation of Notch and other transmembrane proteins in Rbcn-3
mutant cells clearly demonstrates a role for Rbcn-3 in regulating protein trafficking. To ask whether the accumulation of membrane proteins in Rbcn-3
mutant cells is due to a defect in endocytic or exocytic trafficking, we performed a Notch trafficking assay in eye discs (Le Borgne and Schweisguth, 2003
; Lu and Bilder, 2005
mosaic eye discs were incubated in cold medium containing an antibody against NECD to specifically label Notch at the cell surface. Pulse-labeled discs were either fixed immediately to examine the amount of Notch at the cell surface or incubated for different times in fresh medium at 25°C prior to fixation to follow endocytosis of labeled Notch. We did not observe a significant difference in the amount of Notch at the cell surface between wild-type and Rbcn-3B
mutant cells, indicating that exocytosis of Notch is not noticeably affected in the absence of Rbcn-3B ( and data not shown). Significantly, after a 1.5 h chase, abnormal accumulation of internalized Notch with bound antibody was observed in Rbcn-3B
mutant cells compared to neighboring wild-type cells (). These results indicate that, in the absence of Rbcn-3, Notch reaches the surface of eye disc cells, is internalized, but then accumulates in an endocytic compartment.
Endocytic trafficking of Notch is defective and Notch accumulates in an enlarged late endosomal compartment in Rbcn-3 mutant cells
A similar trafficking experiment is technically not possible in the follicle cells. To address whether Notch accumulation in Rbcn-3
mutant follicle cells results from defects in endocytic trafficking, as it does in eye disc cells, we compared Notch localization in Rbcn-3B
mutant follicle cells with that in cells mutant for both Rbcn-3B
and either Clathrin heavy chain
) or shibire
), the fly orthologue of dynamin. Both Chc and Shi are required for the formation of endocytic vesicles at the plasma membrane (Ungewickell and Hinrichsen, 2007
). In Chc
mutant follicle cells, Notch accumulates at the cell surface (, n>50 and data not shown)(Lu and Bilder, 2005
; Vaccari et al., 2008
). As described earlier, in Rbcn-3
mutant follicle cells Notch accumulates in enlarged intracellular compartments (e.g. , n>100). By contrast, in Rbcn-3B Chc
and Rbcn-3B shi
double mutant follicle cells Notch accumulated around the cell periphery as in Chc
single mutant follicle cells (, n>50 and data not shown). These results indicate that Rbcn-3 is not required for Notch to reach the cell surface and that the intracellular accumulation of Notch in the absence of Rbcn-3 requires Chc and Shi-dependent internalization.
Taken together, our results demonstrate that the major defect in Notch trafficking in Rbcn-3 mutant follicle cells and eye disc cells occurs in the endocytic pathway and that exocytosis occurs normally.
Notch and other membrane proteins accumulate in an enlarged late endosomal compartment in Rbcn-3 mutant cells
To determine the nature of the endocytic compartment in which Notch accumulates in the absence of Rbcn-3, we examined the distribution of known endocytic markers in Rbcn-3B
mutant follicle cells and found that the enlarged compartments in mutant cells were positively labeled with the late endosomal marker YFP-Rab7 () (Zhang et al, 2007
), but not with the early endosomal markers GFP-Rab5 (data not shown) or GFP-2xFYVE ()(Wucherpfennig et al., 2003
) or the recycling endosomal marker GFP-Rab11 (data not shown)(Emery et al., 2005
). These results indicate that Notch and other membrane proteins accumulate in an enlarged late endosomal compartment in the absence of Rbcn-3.
To further characterize the nature of this late endosomal compartment, we stained egg chambers with an antibody detecting ubiquitinated proteins. Ubiquitination of membrane proteins often serves as a signal for their internalization and endocytic sorting prior to lysosomal degradation (Raiborg et al., 2003
). In cells lacking components of the endosomal sorting complexes required for transport (ESCRTs), such as Hrs, Vps25 and Tsg101, which mediate protein sorting in the endocytic pathway, ubiquitinated proteins strongly accumulate in enlarged endosomes () (Jekely and Rorth, 2003
; Moberg et al., 2005
; Thompson et al., 2005
; Vaccari and Bilder, 2005
). In contrast, the enlarged endosomes containing Notch and other membrane proteins in Rbcn-3B
mutant cells do not contain increased levels of ubiquitin ().
Our data indicate that the loss of Rbcn-3 in Drosophila follicle cells causes membrane proteins to accumulate in enlarged late endosomes after deubiquitination has taken place. Intriguingly, other mutants that cause Notch accumulation in late endosomal compartments, such as the phosphatidylinositol-5-kinase Fab1
, and the HOPS complex components carnation
and deep orange
, have no effect on Notch signaling (Rusten et al., 2006
; Sevrioukov et al., 1999
; Sriram et al., 2003
and data not shown). The accumulation of Notch in these enlarged late endosomal compartments in Rbcn-3
mutant cells can therefore not account for the observed loss of Notch signaling.
Rbcn-3 regulates Notch signaling and endocytic trafficking by regulating V-ATPase activity
We have shown two major defects in Rbcn-3
mutant cells: a loss of Notch signaling and an accumulation of Notch and other membrane proteins in abnormal late endosomal compartments. Given that the late trafficking defect cannot explain the block in Notch signaling, we wanted to determine the underlying common cellular defect that could lead to these phenotypes. Upon closer inspection of the Rbcn-3A protein sequence we noticed that it shares homology with the yeast protein Rav1(Seol et al., 2001
; Sipos et al., 2004
), as well as the C. elegans Rav1
(wormbase). Rav1 is a component of the RAVE complex (regulator of the (H+
)-ATPase of the vacuolar and endosomal membranes), which regulates the activity of the vacuolar (H+) ATPase (V-ATPase) (Seol et al., 2001
; Smardon et al., 2002
). The V-ATPases are conserved ATP-driven proton pumps composed of two multi-component subcomplexes, the membrane bound V0, which mediates proton translocation, and the peripheral V1, responsible for ATP hydrolysis (Forgac, 2007
; Jefferies et al., 2008
). The yeast RAVE complex promotes the assembly of V1 and V0 to form the active holo-enzyme (Seol et al., 2001
; Smardon et al., 2002
). One of the major functions of V-ATPases is to acidify the lumens of various intracellular organelles, including endosomes, lysosomes and secretory vesicles (Forgac, 2007
; Jefferies et al., 2008
). To test whether Rbcn-3A is required for this V-ATPase function in Drosophila as Rav1 is in yeast, we monitored the acidity of intracellular compartments with the pH-sensitive vital dye LysoTracker. In wild-type follicle cells acidic organelles were evident by intense LysoTracker staining (). By contrast, Rbcn-3A
mutant follicle cells showed strongly reduced or no LysoTracker staining (). This observation indicates a lack of acidic compartments in the absence of Rbcn-3 and is consistent with a role for Rbcn-3 in regulating V-ATPase-dependent acidification. In yeast, the RAVE complex was shown to bind the V1 subcomplex of the V-ATPase (Seol et al., 2001
; Smardon et al., 2002
). We therefore asked whether Rbcn-3 interacts with components of the V1 subcomplex in Drosophila. We immuno-precipitated two Drosophila V1 subunits, Vha55 (V1 B subunit) and VhaSFD (V1 H subunit) from ovaries expressing HA-tagged Rbcn-3B in follicle cells and found that HA-Rbcn-3B co-precipitated with both subunits ( and data not shown). This result further confirms that Rbcn-3 could serve to regulate V-ATPase activity in Drosophila.
Lack of acidic compartments in Rbcn-3A and Rbcn-3B mutant follicle cells
If Rbcn-3 indeed affects both Notch signaling and protein trafficking through regulating V-ATPase function, mutations in V-ATPase subunits should show similar defects as Rbcn-3
mutants. In the genetic screen that lead to the identification of Rbcn-3A and B, we found two additional mutants, FZ29
, that resulted in mosaic patches of cells with small nuclei. Upon closer inspection, we found that these mutants showed identical phenotypes to Rbcn-3
mutants. We mapped the lethality of these two mutants between two transposable elements at 3D4 and 4C2, and very close to the recessive marker echinus
at 3F2 (). Interestingly, a single V-ATPase subunit gene, VhaAC39
, maps exactly within this region (3F4) (Allan et al., 2005
). We therefore sequenced the VhaAC39
gene in FZ29
, and found that, in both alleles, part of the VhaAC39
ORF was deleted. FY38
contains a 385 bp deletion combined with a 2 bp insertion and FZ29
contains a 137 bp deletion leading to a frame shift and a premature stop codon ( and Supplemental Figure 2
encodes one of the two Drosophila V0 d subunits (Allan et al., 2005
; Nishi et al., 2003
; Smith et al., 2002
). As expected from the loss of VhaAC39
activity and thus V-ATPase function, LysoTracker staining was lost in VhaC39
mutant follicle cells (). In addition, Notch and other membrane proteins accumulated in enlarged intracellular compartments that were labeled strongly with the late endosomal marker GFP-Rab7 but not with the early endosomal markers GFP-Rab5, 2xFYVE-GFP, the recycling endosomal marker GFP-Rab11 or the Golgi marker Lava lamp ( and data not shown). Very significantly, we found that Notch signaling was lost in the absence of VhaAC39; VhaAC39
mutant follicle cells keep dividing after stage 6, fail to upregulate Hnt, fail to downregulate Cut and show strongly reduced levels of Gbe+Su(H)-lacZ (). Finally, VhaAC39
mutant eye discs cells behaved similarly to Rbcn
-3 mutant eye disc cells in the Notch trafficking assay; Notch reached the surface, was internalized and accumulated in intracellular compartments (data not shown).
Membrane proteins accumulate in enlarged late endosomal compartments in VhaAC39 mutant cells
Notch signaling is lost in VhaAC39 mutant follicle cells
In summary, our results demonstrate that mutations in the V0 subunit VhaAC39 phenocopy mutations in Rbcn-3 with respect to protein trafficking and Notch signaling. We can therefore conclude that Rbcn-3 acts primarily through its effect on V-ATPase activity to regulate Notch signaling and endocytic trafficking. Moreover, our results provide evidence for a role of the vacuolar proton pump in the regulation of Notch signaling.