The N-terminal portion of dVAP is secreted
We previously generated guinea pig polyclonal antibodies against dVAP which specifically detect the protein on Western blots (Pennetta et al., 2002
). Although these antibodies (GP33 or GP36) detect overexpressed dVAP, the presence of some immunofluorescence signals in dVAP
null mutant tissue (dVAPΔ20
) suggests that they recognize a non-specific component (data not shown). We generated new antisera against dVAP (Rb92) and confirmed antibody specificity (). To assess the cellular dVAP distribution, we co-stained fly tissues with anti-dVAP and cellular markers. We find that dVAP is associated with the ER in wing imaginal disc cells (data not shown) and salivary gland cells (Figure S1A-C
). dVAP is also localized to the cell membrane or just beneath the cell membrane based on its partial colocalization with Spectrin (Pesacreta et al., 1989
) (). In addition, we observed numerous punctae (arrows in ) in wild type tissue which neither colocalize with Spectrin or Boca, an ER marker ( and data not shown). These punctae are most often found near cell boundaries, suggesting that they are in the extracellular space.
To determine if dVAP is present extracellularly, we used protocols for distinguishing between extracellular and intracellular antigens in wing discs (Seto and Bellen, 2006
; Strigini and Cohen, 2000
). Extracellular dVAP is found in wild type discs (), but is absent in dVAP
null mutant discs (). These data indicate that a portion of dVAP or the whole protein can be secreted.
There are at least two possible alternatives. The wing cells reverse dVAP polarity such that it remains membrane-anchored but the N-terminus is extracellular. Alternatively, dVAP is cleaved intracellularly and a portion is secreted. To determine which portion of dVAP is extracellular, we generated transgenic flies carrying the dVAP cDNA tagged with an N-terminal FLAG tag and a C-terminal HA tag. This UAS construct is able to rescue the lethality associated with dVAP loss, indicating that it is functional (data not shown). To examine the localization of the tagged protein, we used the dpp-GAL4 driver (). Anti-FLAG antibody staining without membrane permeabilization shows that the N-terminal portion of dVAP is extracellular (), whereas staining with anti-HA antibody () shows that the C-terminal portion is intracellular. We then incubated the live discs expressing FLAG-dVAP-HA protein with anti-FLAG antibody, fixed, permeabilized the cells, and incubated with anti-HA antibody. As shown in , the N-terminal part of dVAP is more broadly distributed than the C-terminal portion, typically extending from 1-3 cells beyond the dpp-GAL4 expressing cells, which indicates that the N-terminal portion is secreted and diffuses away. In summary, our data provide strong evidence that the dVAP protein is cleaved and that an N-terminal portion is secreted.
The MSP domain of dVAP is cleaved
Western blots should allow identification of the processed forms of the protein. We extracted proteins from larvae expressing FLAG-dVAP-HA and performed western blotting with anti-dVAP (GP33) and anti-FLAG. The size of the full length protein is about 33kDa. Extracts from wild type third instar larvae (, Canton S, CS, left) show a broad set of bands around 33 kDa as well as less abundant smaller sized bands. At least four different proteins (235aa, 238aa, and two different 269aa proteins, which all seem to differ in the coiled-coil domain) can be derived from the dVAP locus (FlyBase ID; FBgn0029687). These proteins migrate in the 28-33kDa range whereas the 17 kDa band is probably a cleavage product. None of these proteins is present in dVAP null mutants (data not shown).
To determine the cleavage profile of the overexpressed FLAG-dVAP-HA protein we probed the same blot for the N-terminal FLAG tag. We observe no staining in wild type controls (CS) and dVAP null mutant animals (not shown). However, we observe 22-37 kDa bands as well as multiple bands in the 13-18 kDa range (, bracket). Since we expressed a single isoform of FLAG-dVAP-HA (around 38kDa), many of the bands labeled with anti-FLAG must be cleavage products. Probing the blot with the HA tag confirms that the FLAG-containing products are uniquely N-terminal. The size of the N-terminal cleavage product indicates that it must contain most or all the MSP domain (about 125 amino acids). The bands revealed by anti-HA (10-15kD) correspond to multiple cleavage products. In summary, our data suggest that the MSP domain of the dVAP protein is cleaved from the transmembrane domain.
To determine if secretion of the MSP domain is observed in humans, we examined the expression of hVAP in flies, human leukocytes, and human serum. The anti-hVAP antibody was raised against the full length (30 kDa) human protein (Amarilio et al., 2005
). This antibody does not crossreact with the Drosophila
protein (, control, lane C155/+). When hVAP is expressed in flies, we detect full length protein and cleavage products. In human leukocytes, we detect the full length protein as well as additional 25 kDa and 18 kDa bands (, lane WBC). In contrast, we only detect the 18 kDa band in human serum (, lane Serum and Figure S1 D
). Taken together, our data indicate that VAP MSP domains are secreted and suggest that the hVAP MSP domain is found in human serum.
P58S dVAP fails to be secreted, aggregates in the cytoplasm, and is ubiquitinated
In humans, the P56S mutation in hVAP
causes ALS8 (Nishimura et al., 2004
). To better understand the nature of the mutation, we generated a UAS-P58S dVAP construct which corresponds to the P56S mutation in hVAP. We examined the subcellular localization of P58S dVAP and wild type (WT) dVAP proteins expressed ectopically in the wing disc under the control of the dpp-GAL4 driver. As shown in , when the cells are not permeabilized we observe extracellular staining of WT dVAP. However, little or no extracellular staining is observed when P58S dVAP is overexpressed (). To further examine the effect of the mutation on secretion, we overexpressed the WT and P58S dVAP MSP domains in S2 cells. As shown in Figure S2A
, WT dVAP MSP, but not mutant dVAP MSP, is present in the conditioned medium. These data indicate that the P58S mutation prevents secretion of the MSP domain.
The P58S mutation leads to a failure to secrete dVAP and forms ubiquitinated inclusions
The staining of permeabilized wing disc cells with anti-dVAP antibodies shows that the P58S mutation causes the dVAP protein to localize to intracellular aggregates (). These aggregates resemble cytoplasmic protein inclusions found in ALS patients (Boillee et al., 2006
; Bruijn et al., 2004
). To compare the localization of WT and P58S in motor neurons, we expressed the transgenes under control of C155- and C164-GAL4 in a dVAP
null mutant background. WT dVAP is present in the cytoplasm and axons (; Figure S2B-C and F-G
), whereas P58S dVAP exhibits a very different profile with numerous cytoplasmic punctae (; Figure S2D-E
) and axonal aggregates (Figure S2H-I
To test whether P58S dVAP is ubiquitinated, we co-stained flies expressing WT or P58S dVAP with anti-dVAP and anti-Ubiquitin antibodies. Expression of wild type protein does not cause ubiquitination (), whereas aggregates induced by P58S dVAP expression stain positively with anti-Ubiquitin antibodies (). As shown in , these aggregates are likely to contain insoluble P58S dVAP. The high molecular weight ladder seen in lane 8 (bracket) is consistent with ubiquitination. To confirm that P58S dVAP is ubiquitinated, we performed immunoprecipitation (IP) assays to isolate dVAP protein from flies expressing WT or P58S dVAP. We immunoblotted the IP products with anti-Ubiquitin antibodies. As shown in (bracket), the IP product from P58S dVAP, but not WT dVAP, is positive for Ubiquitin. In summary, P58S dVAP fails to be secreted, is ubiquitinated, and accumulates in cytoplasmic inclusions.
P58S dVAP accumulates in and disrupts the ER, and induces an unfolded protein response
VAPs are ER associated proteins that can form homodimers and heterodimers (Kaiser et al., 2005
). To test whether P58S dVAP can recruit wild type dVAP to mutant inclusions, we first expressed WT FLAG-dVAP-HA and WT dVAP together in motor neurons. As shown in , we find that WT FLAG-dVAP-HA is localized diffusely in the cytoplasm when it is co-expressed with WT dVAP.
P58S dVAP protein accumulates in the ER, causes morphological changes in the ER, and induces an UPR
When we overexpress WT FLAG-dVAP-HA and P58S dVAP together, we find that the tags are localized to inclusions (), indicating that WT dVAP is recruited into the inclusions. To determine if other ER associated proteins are present in these inclusions, we co-stained flies expressing P58S dVAP with anti-dVAP and anti-Boca, an ER marker (Culi and Mann, 2003
). As shown in , Boca is present in the inclusions. Similarly, Protein disulfide isomerase (PDI), another ER marker (Herpers and Rabouille, 2004
), is also present in the inclusions (). Transmission electron microscopy (TEM) of wing disc cells overexpressing WT or P58S dVAP shows that mutant dVAP causes abnormalities in the ER (). Flies expressing P58S dVAP exhibit clusters of aberrant electron-dense material (bracket in ) that is continuous with the rough ER (arrow) in numerous cells. In summary, P58S dVAP recruits WT dVAP to cytosolic inclusions, alters the distribution of ER markers, and affects ER structure.
We next tested whether P58S dVAP induces an unfolded protein response (UPR), which has been documented in sporadic ALS as well as mutant SOD1
transgenic animals (Atkin et al., 2006
; Kikuchi et al., 2006
). To evaluate the UPR, we assessed the expression of BiP/Hsc3, a member of the Hsp70 chaperone family and a primary sensor in the UPR (Elefant and Palter, 1999
; Morris et al., 1997
; Ryoo et al., 2007
). As shown in , adult brains overexpressing P58S dVAP exhibit significantly higher levels of Hsc3 (see Figure S3
for detail and western blot). These data suggest that P58S dVAP causes an UPR in vivo
The phenotypic consequences of P58S dVAP overexpression
To assess the phenotypic consequences of P58S we compared overexpression of wild type and mutant protein in three different assays. First, presynaptic overexpression of the WT dVAP protein using the C155-GAL4 driver causes a significant increase in bouton number (Pennetta et al., 2002
), whereas overexpression of the P58S mutant dVAP (Figure S4A
) does not cause a severe overgrowth phenotype. Second, we compared the ability of flies overexpressing mutant and wild type protein in a flight assay. Overexpression of WT dVAP significantly reduces flight ability, whereas overexpression of P58S dVAP causes only a mild phenotype (Figure S4B
). These phenotypic differences are not due to differences in transgene expression (Figure S4C
Finally, we performed ultrastructural studies of the indirect flight muscles. Wild type control animals () display regular organization and uniform size of the myofibrils, and each myofibril (arrow) is smooth and surrounded by mitochondria (arrowhead). When the WT dVAP protein is expressed under the control of a neuronal driver (C155-GAL4) (), we find severe defects in 7% of the myofibrils. We do not observe these defects in wild type controls and flies expressing P58S dVAP (). In addition, the myofibrils are much more heterogeneous (mean of SD = 0.61 +/- 0.90, P<0.01 when compared to the control animals) in size when WT dVAP is overexpressed compared to when P58S is overexpressed (mean of SD = 0.33 +/- 0.70, P<0.05 when compared to the control animals; ). These results indicate that overexpression of WT dVAP in neurons can cause muscle defects and that P58S dVAP does not replicate the phenotypes of overexpressed WT protein.
Overexpression of P58S dVAP does not phenocopy WT dVAP overexpression
To determine the consequences of loss and overexpression of dVAP in synaptic transmission, we compared the electrophysiological properties of various animals. As shown in Figure S5 A and B
, loss of function causes a moderate but significant increase in Excitatory Junctional Amplitude (EJP) amplitude and MiniEJP (mEJP) whereas overexpression of wild type or P58S induces a subtle reduction in EJP which is however not significant (Figure S5 C
In summary, we have shown that the dVAP MSP domain is secreted and that WT dVAP overexpression can cause a cell non-autonomous defect. The P58S mutation prevents secretion, and potentially affects other functions such that overexpression of the mutant protein does not phenocopy that of wild type dVAP. P58S dVAP must retain some activity because overexpression of P58S dVAP in the nervous system (C155-GAL4) is able to rescue the lethality associated with the null mutations [data not shown and (Chai et al., 2008
)]. Thus, P58S dVAP may be secreted at very low levels or secretion may not be required for all activities.
VAP MSP domains signal in an extracellular environment
The C. elegans
genome contains a single VAP homolog, called F33D11.1 or VPR-1 (for VAP33-Related). However, 28 loci encode the sperm-specific MSPs, which are sperm-derived hormones that induce oocyte maturation and ovarian sheath contraction (Bottino et al., 2002
; Yamamoto et al., 2006
). When sperm and MSP are absent, oocyte maturation and sheath contraction rates are very slow, preventing oocyte loss (McCarter et al., 1999
). Microinjecting purified, sperm-derived or recombinant MSP into the reproductive tract () promotes oocyte maturation and sheath contraction (Miller et al., 2001
). These responses are identical to those induced by wild type sperm and are not induced by other sperm proteins, endogenous bacterial proteins or several commercially available hormones (Corrigan et al., 2005
; Miller et al., 2001
VAPs have extracellular signaling activity and act in common genetic pathways with Eph receptors
The primary sequences of VAP MSP domains and MSPs are about 25% identical. If VAP MSP domains are secreted ligands for conserved receptors, they should be able to mimic the signaling activities of MSPs. To test this prediction, we microinjected purified, recombinant VPR-1 MSP domain into gonads lacking MSP and sperm (). As shown in , VPR-1 MSP stimulates a strong increase in oocyte maturation and sheath contraction rates over the same concentration range as MSP. Microinjecting dVAP and hVAP MSP domains also induces both responses (), suggesting that all MSP domains can signal through a common mechanism(s). The hVAP P56S mutant functions similarly to the wild type protein in this assay, indicating that this mutation does not affect signaling activity in the worm gonad. We conclude that C. elegans MSP and VAP MSP domains have an evolutionarily conserved extracellular signaling activity.
VAPs and Eph receptors function in common genetic pathways
Previous studies indicate that sperm-derived MSPs binds to the VAB-1 Eph receptors and unidentified receptors expressed in oocytes and sheath cells (Govindan et al., 2006
; Miller et al., 2003
). We hypothesized that VAP MSP domains also regulate Eph receptors, which are widely expressed in fly, worm, and human nervous systems (George et al., 1998
; Palmer and Klein, 2003
). To test this hypothesis, we first sought to determine whether VAPs are required for the same in vivo
processes as Eph receptors. Mutations in Eph receptors cause defects in cell and axon guidance during development (Klein, 2004
). The Drosophila
Eph receptor and its ligand Ephrin are required for proper axon guidance of the mushroom body (MB) neurons. Eph receptor and Ephrin mutations cause loss of alpha-lobes (Boyle et al., 2006
). We found that dVAP
null mutants and double mutants (ΔVAP; ΔEph) lack alpha-lobes in late pupae and adult brains (Figure S6 B,D
; data not shown). The data indicate that both dVAP and Eph receptors are required for alpha lobe formation.
Overexpression of dVAP in neurons causes muscle defects in flies, consistent with a signaling function (). Eph receptor mutants suppress the muscle phenotypes (variable diameter of the myofibrils and myofibrillar degeneration) induced by overexpressing dVAP (Figure S6A-E
). Moreover, Ephrin is present on muscles (Figure S6E-H
). These data strongly argue that the non-autonomous and toxic activity of overexpressed dVAP is mediated by Eph receptors. Other requirements for Eph receptor and Ephrin in flies have yet to be determined, as the existing mutants are likely partial loss of function mutations (Dearborn et al., 2002
To examine the role of vpr-1
in C. elegans
development, we characterized a vpr-1(tm1411)
null mutant (). Phenotypes associated with vpr-1 (tm1411)
can be rescued by transgenic expression of a fosmid containing vpr-1
. Mutations in the VAB-1 Eph receptor or ephrin ligands cause incompletely penetrant and variably expressed phenotypes during development (Brenner, 1974
; Chin-Sang et al., 1999
; Chin-Sang et al., 2002
; George et al., 1998
; Wang et al., 1999
; Zallen et al., 1999
). We found that vpr-1(tm1411)
mutants, similar to vab-1
mutants, have incompletely penetrant and variably expressed defects. Loss of VPR-1, the ephrin EFN-2, or VAB-1 causes defects in distal tip cell (DTC) migration ( and Figure S7A-C
). The DTC is a gonadal leader cell whose migration path can be monitored using differential interference contrast (DIC) microscopy or the lag-2::GFP
transgenic reporter, which expresses GFP in the DTC (Blelloch et al., 1999
). Analysis of vpr-1
double mutants is consistent with these two genes acting in a common pathway during DTC migration ( and Figure S7A
VPR-1 and VAB-1 also function together to regulate the embryonic migrations of ventral hypodermal cells undergoing enclosure (Figure S7D,E
) and amphid neurons (), whose final positions in adults can be visualized with dye DiI (Zallen et al., 1999
). The amphids, as well as other head neurons, often lie too far posterior in vpr-1(tm1411)
mutants compared to the wild type (). The amphids are sometimes positioned too far anterior in vab-1(dx31)
mutants (). Analysis of vpr-1
double mutants is consistent with vpr-1
acting in vab-1
-dependent and vab-1
-independent mechanisms during embryogenesis ( and S7D,E
). Taken together, the data support the hypothesis that VAPs regulate Eph receptor signaling in vivo
VAP MSP domains bind to Eph receptors
In the worm proximal gonad, the VAB-1 Eph receptor is expressed on oocyte and sheath cell surfaces (). VAB-1 and other MSP receptor sites can be visualized using fluorescein–labeled MSP (MSP-FITC)(Miller et al., 2003
). MSP-FITC is biologically active and binding can be out-competed with a 25-fold molar excess of unlabelled MSP (). To test whether VAP MSP domains can bind to cell surface receptors, we incubated ~200 nM FITC–labeled MSP domain conjugates with dissected gonads. VPR-1, hVAP, and dVAP MSP domains bind to oocyte and sheath cell plasma membranes in a pattern identical to MSP-FITC (, data not shown). Pre-incubation of worm gonads with a 25-fold molar excess of hVAP MSP domain out-competes hVAP-FITC binding, indicating that binding is specific. Moreover, MSP and hVAP likely bind to common receptors, as MSP-FITC binding can be out-competed with an excess of hVAP (). These results strongly support the hypothesis that VAP MSP domains bind to cell surface receptors.
VAP MSP domains bind to Eph receptor extracellular domains
To test whether VAP MSP domains can directly bind to the Eph receptor extracellular domain, we examined the interaction between VPR-1 MSP and VAB-1 Eph receptor. We produced a V5-tagged VAB-1 ectodomain (VAB-1 Ex V5) in HEK293T cells. We also expressed and purified a FLAG tagged VPR-1 MSP (VPR-1 MSP-FLAG) in bacterial cells and incubated the VPR-1 MSP-FLAG and VAB-1 Ex V5 proteins to assess binding by co-immunoprecipitation (co-IP). As shown in , anti-FLAG antibody co-immunoprecipitates the VAB-1 ectodomain (top panel) together with VPR-1 MSP (bottom panel). Furthermore, the anti-V5 antibody co-immunoprecipitates the VPR-1 MSP (, bottom panel) together with the VAB-1 ectodomain (, top panel). These data indicate that VPR-1 MSP is able to directly bind the VAB-1 ectodomain.
To examine the interaction between VAP MSP and Eph receptor in mammals, we produced a His and V5-tagged mouse EphA4 ectodomain (mEphA4Ex-V5His) and His tagged hVAP MSP (hVAPMSP-His). We incubated mEphA4Ex-V5His together with hVAPMSP-His proteins and performed co-IP assays with the V5 antibody. The V5 antibody is able to co-immunoprecipitate the hVAP MSP protein (, top panel) together with the EphA4 ectodomain (, bottom panel), suggesting that hVAP MSP binds to the EphA4 ectodomain directly.
Since VAP MSP can bind to the Eph receptor, VAP MSP might compete with Ephrins for Eph receptor binding. To perform competition experiments between hVAP MSP and Ephrin in mammalian cells, we used conditioned medium from HEK293T cells expressing secreted hVAP MSP or the EphA4 ligand, EphrinB2. We preincubated HEK293T cells expressing FLAG-tagged mouse EphA4 (FLAG-mEphA4) with conditioned medium containing hVAP MSP. Subsequently, we applied conditioned medium containing Fc-tagged mouse EphrinB2 (mEphrinB2-Fc) to the cells. After incubation, we retrieved mEphrinB2-Fc and EphA4 proteins complexes and immunoblotted with the anti-FLAG antibody. As shown in the top panel of and quantified in , hVAP MSP suppresses the binding of EphrinB2 to EphA4 in a dose dependent fashion. To confirm competition, we incubated mEphA4Ex-V5His and hVAPMSP-His proteins together with EphrinB2 protein. We again performed co-IP assays with V5 antibody (similar assay as in ). Less hVAP MSP is recovered when EphrinB2 protein is increased in a dose dependent manner (, top panel), showing that EphrinB2 is able to out-compete hVAPB MSP for binding to EphA4 (quantified in ). In summary, these data indicate that VAP MSP domains bind to the extracellular domain of Eph receptors and that this binding influences ephrin interactions.