Tetraspanins TSP-12 and TSP-14 function redundantly to regulate the trafficking of the type II BMP receptor in Caenorhabditis elegans

Significance

Tetraspanins are a unique family of eukaryotic 4-pass transmembrane proteins. However, their functions in vivo are poorly understood. We find that 2 tetraspanins that share redundant functions are primarily localized to endosomes, which are major sorting centers for membrane proteins and lipids within the cell. We show that these 2 tetraspanins regulate the recycling of the type II receptor of the bone morphogenetic protein (BMP) signaling pathway. In their absence, the BMP type II receptor is mistargeted to lysosomes, leading to reduced cell-surface levels available to receive extracellular signals. Our work highlights the importance of tetraspanin-mediated intracellular trafficking in regulating BMP signaling in vivo, and suggests a similar mode of action for the mammalian counterparts of these 2 tetraspanins.

Abstract

Tetraspanins are a unique family of 4-pass transmembrane proteins that play important roles in a variety of cell biological processes. We have previously shown that 2 paralogous tetraspanins in Caenorhabditis elegans, TSP-12 and TSP-14, function redundantly to promote bone morphogenetic protein (BMP) signaling. The underlying molecular mechanisms, however, are not fully understood. In this study, we examined the expression and subcellular localization patterns of endogenously tagged TSP-12 and TSP-14 proteins. We found that TSP-12 and TSP-14 share overlapping expression patterns in multiple cell types, and that both proteins are localized on the cell surface and in various types of endosomes, including early, late, and recycling endosomes. Animals lacking both TSP-12 and TSP-14 exhibit reduced cell-surface levels of the BMP type II receptor DAF-4/BMPRII, along with impaired endosome morphology and mislocalization of DAF-4/BMPRII to late endosomes and lysosomes. These findings indicate that TSP-12 and TSP-14 are required for the recycling of DAF-4/BMPRII. Together with previous findings that the type I receptor SMA-6 is recycled via the retromer complex, our work demonstrates the involvement of distinct recycling pathways for the type I and type II BMP receptors and highlights the importance of tetraspanin-mediated intracellular trafficking in the regulation of BMP signaling in vivo. As TSP-12 and TSP-14 are conserved in mammals, our findings suggest that the mammalian TSP-12 and TSP-14 homologs may also function in regulating transmembrane protein recycling and BMP signaling.

The evolutionarily conserved bone morphogenetic proteins (BMPs) compose the largest subgroup of the transforming growth factor β (TGFβ) superfamily, and are key signaling molecules in embryonic development and tissue homeostasis. The BMP signal is transduced through a heteromeric complex of 2 single-pass transmembrane serine-threonine kinase receptors, known as BMP type I and BMP type II receptors (BMPRI and BMPRII). Ligand binding triggers the intracellular phosphorylation and subsequent activation of the type I receptor kinase by the constitutively active type II receptor kinase, which then activates downstream signaling either through the canonical Smad signaling cascade or the noncanonical MAP kinase pathway. Misregulation of the BMP pathway can cause a variety of diseases in humans (for reviews, see refs. 1–3).

Multiple levels of regulation exist to ensure proper spatiotemporal activation and specific downstream responses of BMP signaling (for recent reviews, see refs. 4 and 5). One of these regulatory mechanisms involves endocytosis and postendocytic trafficking of BMP receptors, which serve to fine-tune the signaling responses in specific cellular contexts (6–8). In particular, receptors internalized via clathrin-dependent or -independent mechanisms can be delivered to signaling endosomes, facilitating the amplification of BMP signaling, to lysosomes, leading to attenuation of signaling, or can recycle back to the cell surface for signal renewal (9). The detailed mechanisms involved in directing the receptors to each of these different routes are not fully understood. Caenorhabditis elegans provides an in vivo model system for dissecting these regulatory mechanisms in an intact living animal.

The C. elegans BMP pathway shares all of the core pathway components with vertebrates, including the secreted ligand DBL-1/BMP, the type I receptor SMA-6/BMPRI, the type II receptor DAF-4/BMPRII, and the downstream Smad proteins SMA-2/R-Smad, SMA-3/R-Smad, and SMA-4/Co-Smad (10–13). Unlike in vertebrates and Drosophila, BMP signaling is not required for viability in C. elegans. However, BMP signaling is critical in regulating multiple processes, including body length, male tail patterning, postembryonic mesoderm development, and innate immunity (14, 15). In particular, reduced BMP signaling leads to a small body size phenotype, while increased BMP signaling makes C. elegans worms longer. Furthermore, altered BMP signaling can suppress the postembryonic mesoderm patterning defect caused by mutations in sma-9, which encodes a Zn finger-containing transcription factor (16, 17) (SI Appendix, Fig. S1B). Based on these different phenotypes caused by altered BMP signaling, multiple evolutionarily conserved regulators of BMP signaling have been identified in C. elegans (18–24). Importantly, endocytosis and postendocytic trafficking of BMP receptors have also been found to be critical in regulating BMP signaling in C. elegans (7, 25). In particular, Gleason and colleagues (7) showed that the type I receptor SMA-6 enters the cell through clathrin-dependent endocytosis, but the type II receptor DAF-4 likely enters through clathrin-independent mechanisms. The postendocytic trafficking pathways for the 2 receptors are also different: While trafficking of SMA-6/BMPRI is accomplished through a retromer-dependent recycling pathway, recycling of DAF-4/BMPRII is independent of the retromer and instead is mediated by an ARF-6 (ADP ribosylation factor 6)–dependent mechanism. These two different recycling pathways are hypothesized to separate the 2 receptors upon termination of signaling, preserving them for further rounds of activation (7). How the 2 receptors are sorted into different recycling pathways and what additional factors are involved in these processes are not well-understood.

In this study, we identify 2 redundant tetraspanin proteins, TSP-12 and TSP-14, to be critically important in the trafficking of the type II receptor DAF-4/BMPRII. Tetraspanins are a large family of unique and highly conserved 4-pass transmembrane proteins found in a wide range of organisms (26). There are 37 tetraspanins in Drosophila, 21 in C. elegans, and 33 in humans. Much work in mammalian cell-culture systems has shown that tetraspanins are localized to the plasma membrane, to various types of endocytic membranes, or to different extracellular vesicles (27, 28). Through a variety of homotypic and heterotypic interactions, tetraspanins can establish “tetraspanin webs” or tetraspanin-enriched microdomains, and regulate the lateral clustering or trafficking of various membrane or membrane-associated proteins (29, 30). Abnormal expression of certain tetraspanins has been found to be associated with different types of diseases, in particular cancer (31, 32). Therefore, understanding the in vivo functions of the different tetraspanin proteins is critical to our understanding of the link between tetraspanins and disease states, and to developing therapeutic strategies for treating these diseases.

We have previously shown that in C. elegans, 2 paralogous tetraspanins, TSP-12 and TSP-14 (SI Appendix, Fig. S1A), function redundantly to promote BMP signaling, and that TSP-12 is localized both on the plasma membrane and in intracellular vesicles (22, 23). In this study, we found that TSP-12 and TSP-14 are primarily localized to early, late, and recycling endosomes, and that TSP-12 and TSP-14 partially colocalize with each other. In animals devoid of TSP-12 and TSP-14, the BMP type II receptor DAF-4 is mislocalized to late endosomes and lysosomes, resulting in the reduction of both total and cell-surface DAF-4/BMPRII steady-state protein levels and reduced BMP signaling. Our work identifies a key role of TSP-12 and TSP-14 in the recycling of DAF-4/BMPRII and highlights the importance of intracellular trafficking in the regulation of BMP signaling.

Results

The BMP Type II Receptor DAF-4 Is Mislocalized in tsp-12(0); tsp-14(0) Double Mutants.

We have previously shown that TSP-12 and TSP-14 function redundantly to promote BMP signaling and that TSP-12 is required for the cell-surface localization of the ADAM protease SUP-17 (22, 23). Since both tetraspanins can also bind to the BMP receptors SMA-6/BMPRI and DAF-4/BMPRII in yeast (22), we asked whether TSP-12 and TSP-14 play a role in regulating the localization of these 2 receptors. Using CRISPR/Cas9-mediated homologous recombination, we generated C-terminally GFP::3xFLAG-tagged SMA-6 and DAF-4 (SI Appendix, Fig. S1C and Tables S1 and S2, and Materials and Methods). We focused our analysis on DAF-4::GFP::3xFLAG because it is fully functional and is detectable in live animals under the Airyscan confocal microscope (SI Appendix, Materials and Methods).

We found that DAF-4::GFP::3xFLAG is expressed in multiple tissues, including the pharynx, hypodermis, intestine, and developing vulva (Fig. 1). Within each cell, DAF-4::GFP::3xFLAG is primarily localized to the cell surface. We also detected some GFP signal in intracellular vesicles in hypodermal cells (Fig. 1B). Notably, in both the intestine and the vulva, 2 types of polarized epithelial cells, DAF-4::GFP::3xFLAG is localized to the basolateral membrane but absent on the apical membrane (Fig. 1 D–F). The basolateral localization of DAF-4::GFP::3xFLAG in the intestinal cells has also been previously reported by Gleason and colleagues using a functional, intestinally expressed DAF-4::GFP (7) (see also Fig. 2 C and E).

Fig. 1.

Endogenously tagged DAF-4::GFP is expressed in multiple cell types and asymmetrically localized in polarized epithelial cells. Airyscan confocal images of DAF-4::GFP (A–F) and the corresponding differential interference contrast (DIC) images (A′–F′) showing DAF-4 expression and localization in the head (A), hypodermis (B and C), intestine (D, midsagittal plane; E, basolateral plane), and the developing vulva (F). DAF-4::GFP is primarily localized to the surface of hypodermal cells and seam cells (arrowheads in B and C), although intracellular DAF-4::GFP is also detectable in hypodermal cells (arrows in B). In polarized intestinal cells (D and E) and the developing vulva (F), DAF-4::GFP is localized to the basolateral membrane (arrows in D–F) but is not in the apical membrane (arrowheads in D and F). Asterisks in D–F indicate autofluorescence signals from intestinal granules. L2, L3, and L4 refer to larval stage 2, 3, and 4, respectively. (Scale bars, 10 µm.)

Fig. 2.

DAF-4/BMPRII is mislocalized in tsp-12(0); tsp-14(0) double mutants. (A and B) Airyscan confocal images of midsagittal planes of early L3-stage wild type (A) and tsp-12(0); tsp-14(0) double mutants (B) expressing the endogenously tagged DAF-4::GFP::3xFLAG. Corresponding DIC and merged images are shown in A′, A″, B′, and B″, respectively. (Scale bar, 10 µm.) (C–F) Airyscan confocal images of the intestinally expressed DAF-4::GFP in the wild type (C and E) and tsp-12(0); tsp-14(0) double mutants (D and F) at the early L3 stage. C and D are the basolateral focal plane, while E and F are the midsagittal focal plane. Arrows in D and F point to DAF-4::GFP–accumulating large vesicles. (Scale bar, 10 µm.) (G and H) Quantification of the relative DAF-4::GFP fluorescence intensity (wild type set as 1.0) at the basolateral membrane (G) and at intracellular structures (H), respectively. For quantification, 8 to 10 worms and 3 corresponding regions of each worm (representative regions are outlined with rectangles in C–F) were measured and averaged. (I) Quantification showing the percentage of intestinal DAF-4::GFP–expressing animals that accumulate GFP-positive large vesicles. About 90% of tsp-12(0); tsp-14(0) double mutants (n = 59) accumulated DAF-4::GFP–positive large vesicles. For G–I, error bars show standard error of the mean (SEM). Statistical analysis was done using a 2-tailed t test with 95% confidence intervals (CIs). Mut, tsp-12(ok239); tsp-14(jj95) double mutant; WT, wild type. **P < 0.01, ***P < 0.0001.

If TSP-12 and TSP-14 play a role in regulating the localization of DAF-4/BMPRII, we would expect the localization of DAF-4::GFP::3xFLAG to be altered in tsp-12(0); tsp-14(0) double mutants. We used the null alleles tsp-12(ok239) and tsp-14(jj95) for these studies, and will refer to them as tsp-12(0) and tsp-14(0), respectively. The cell-surface signal of DAF-4::GFP::3xFLAG in hypodermal cells was reduced in tsp-12(0); tsp-14(0) double mutants (SI Appendix, Fig. S2). Furthermore, the intestinal cells of double mutants accumulated abnormally large DAF-4::GFP–positive vesicles (Fig. 2 B, D, and F). We were interested in determining the nature of the vesicles that accumulate DAF-4::GFP in tsp-12(0); tsp-14(0) double mutants. However, the high background of intestinal granule autofluorescence combined with the relatively faint endogenously tagged DAF-4::GFP signal in the double mutants made these analyses difficult. We therefore switched to using a functional, intestine-specific DAF-4::GFP transgenic line reported in Gleason et al. (7) for these studies. The GFP signal in this strain is much brighter than the endogenously tagged DAF-4::GFP::3xFLAG. The tsp-12(0); tsp-14(0) double mutants exhibited a reduction of the basolateral cell-surface signal (Fig. 2 D and G) and a concomitant increase in the intracellular signal of DAF-4::GFP (Fig. 2 F and H). Furthermore, over 90% of the tsp-12(0); tsp-14(0) double mutants accumulated DAF-4::GFP–positive vesicles (Fig. 2I). These observations demonstrated that TSP-12 and TSP-14 are required for the proper trafficking of DAF-4/BMPRII.

DAF-4/BMPRII Abnormally Accumulates in Lysosomes and Lysosome-Related Organelles in tsp-12(0); tsp-14(0) Double Mutants.

To identify the vesicles that accumulate DAF-4::GFP in tsp-12(0); tsp-14(0) double mutants, we generated strains carrying both DAF-4::GFP and red fluorescent protein-labeled intracellular organelle markers in tsp-12(0); tsp-14(0) double mutants (SI Appendix, Table S1). The markers used include the early endosome marker RAB-5 (33) (Fig. 3 A–D′), recycling endosome marker RAB-11 (34) (Fig. 3 E–H′), late endosome/lysosome/phagosome marker RAB-7 (35) (Fig. 3 I–L′), and lysosome marker LMP-1 (36) (Fig. 3 M–P′). We also used LysoTracker Red dye to label the acidic compartments (Fig. 3 Q–T′). We found an increase in DAF-4::GFP localization to LMP-1–positive lysosomes on the apical side of intestinal cells in tsp-12(0); tsp-14(0) double mutants (arrowheads in Fig. 3S″). Additional DAF-4::GFP–positive large vesicles present in tsp-12(0); tsp-14(0) double mutants are RAB-5–negative (Fig. 3 D and D′), RAB-11–negative (Fig. 3 H and H′), and LMP-1–negative (Fig. 3 P and P′) but RAB-7–positive (Fig. 3 L and L′) and LysoTracker-positive (Fig. 3 T and T′). The acidic nature of these vesicles and the presence of the late endosome/lysosome/phagosome marker RAB-7 on them suggest that they are late endosomes. Each of these DAF-4–positive large vesicles is often associated with a smaller vesicle, which exhibits autofluorescence under the DAPI channel, a unique characteristic displayed by an intestine-specific lysosome-related organelle required for zinc storage often called “gut granules” in C. elegans (SI Appendix, Fig. S3) (37–39). Thus, there is an increased accumulation of DAF-4 in lysosomes and late endosomes in tsp-12(0); tsp-14(0) double mutants, suggesting a failure to recycle DAF-4 without TSP-12 and TSP-14.

Fig. 3.

DAF-4::GFP is mislocalized to lysosomes and lysosome-related organelles in tsp-12(0); tsp-14(0) double mutants. Airyscan confocal images of the wild type (A–B′, E–F′, I–J′, M–N′, and Q–R′) and tsp-12(0); tsp-14(0) mutant (C–D′, G–H′, K–L′, O–P′, and S–T′) intestinal cells showing DAF-4::GFP (A′, C′, E′, G′, I′, K′, M′, O′, Q′, and S′) and various endolysosomal markers: mCherry::RAB-5 (early endosome; A–D′), RFP::RAB-11 (recycling endosome; E–H′), RFP::RAB-7 (late endosome; I–L′), LMP-1::TagRFP (lysosome; M–P′), and LysoTracker (lysosome and lysosome-related organelles; Q–T′). A″, C″, E″, G″, I″, K″, M″, O″, Q″, and S″ are the corresponding merged images. Regions marked by white boxes are enlarged and shown (Right) in each row. Large circles marked with white dashed lines outline the DAF-4::GFP–positive lysosome-related organelles, which appear to exhibit a lower fluorescence intensity of LysoTracker Red compared with the lysosomes. Smaller circles marked with yellow dotted lines in R and T outline the lysosomes. Arrowheads point to DAF-4::GFP–positive vesicles that are either LMP-1::TagRFP–positive (O″) or LysoTracker-positive (S″). (Scale bars, 15 µm.)

Two additional lines of evidence are consistent with the notion that DAF-4/BMPRII is mislocalized to lysosomes and late endosomes in tsp-12(0); tsp-14(0) double mutants. First, we detected a moderate but reproducible reduction of steady-state DAF-4 protein level in tsp-12(0); tsp-14(0) double mutants, relative to those in the wild type or tsp-12(0) or tsp-14(0) single mutants (Fig. 4). Second, rab-7(ok511), a null mutation in rab-7, which encodes a Rab family small GTPase with fundamental functions in lysosomal biogenesis and function (35), partially suppressed the BMP signaling defect, the sma-9(0) suppression phenotype, of tsp-12(0) mutants [see SI Appendix, Fig. S1 B and C for details on the sma-9(0) suppression assay]. Taken together, our data indicate that TSP-12 and TSP-14 are required to maintain cell-surface levels of the DAF-4/BMPRII receptor, likely at the level of receptor recycling, preventing loss of receptors to degradative lysosomes.

Fig. 4.

tsp-12(0); tsp-14(0) double mutants have reduced levels of DAF-4/BMPRII. Western blotting analysis of worm lysates from 100 L3 animals of various genotypes, including wild type, tsp-12(0) single, tsp-14(0) single, and tsp-12(0); tsp-14(0) double mutants. “#1” and “#2” refer to independent isolates of the same genotype. (A) Strains carrying the endogenously tagged DAF-4::GFP::3xFLAG (jj177). (C) Strains carrying the intestinally expressed DAF-4::GFP (pwIs922). (E) Strains carrying the endogenously tagged SMA-6::GFP::3xFLAG (jj270). The ratio of GFP (or FLAG)/actin refers to the relative intensity of the GFP (or FLAG) band over that of the actin band, with the ratio in each wild-type control set as 1.0. Each experiment was repeated for an additional 2 times. An average of the normalized FLAG/actin or GFP/actin ratios from all of the experimental repeats is plotted in B (for jj177), D (for pwIs922), and F (for jj270), respectively. Error bars represent SEM. Statistical analysis was done using a 2-tailed t test with 95% CIs. tsp-12(0); tsp-14(0) double mutants have reduced levels of DAF-4::GFP (B and D) but not SMA-6::GFP (F). *P < 0.05.

TSP-12 and TSP-14 Share Overlapping Expression and Localization Patterns.

To further dissect the mechanism of how TSP-12 and TSP-14 regulate the proper localization of DAF-4/BMPRII, we examined the expression and localization patterns of endogenous TSP-12 and TSP-14. We used CRISPR/Cas9 and generated both N- and C-terminally tagged TSP-12 (TSP-12NT, TSP-12CT) and C-terminally tagged TSP-14 (TSP-14CT). As shown in SI Appendix, Fig. S1, TSP-14CT is fully functional, while TSP-12NT is mostly functional. We therefore used TSP-14CT and TSP-12NT for all subsequent analyses.

We have previously reported that endogenously tagged TSP-12 is localized to both the cell surface and intracellular vesicles in multiple cell types in hermaphrodites, including the germline, hypodermis, intestine, and nervous system, among others (23). Like TSP-12, TSP-14 is also widely expressed in multiple somatic tissues throughout hermaphrodite development, including the nose, pharynx, hypodermis, and developing vulva, as well as in the germline, in particular in the sperm (Fig. 5). Also like TSP-12, TSP-14 is localized to both the cell surface and intracellular vesicles (Fig. 5).

Fig. 5.

TSP-14 is localized to both the cell surface and intracellular vesicles in multiple cell types. Airyscan confocal fluorescent images (A–F) and the corresponding DIC images (A′–F′) showing the expression and localization of endogenous TSP-14::GFP::3xFLAG in different tissues in hermaphrodites. TSP-14::GFP::3xFLAG is detectable in the anterior sensory cilia (thick arrow in A), pharynx (thin arrow in A), developing hypodermis (B, L2 stage; C, L3 stage; D, L4 stage), vulva at the L4 Christmas tree stage (E), and adult gonad (F; arrow points to sperm cells). L3- and L4-stage worms appear to have more TSP-14–positive vesicles in the hypodermis as compared with L2 worms (B), and the TSP-14–positive vesicles in the L4 hypodermis appear to be more even in size and shape (D). (Scale bars, 10 µm.)

Because TSP-12 and TSP-14 function redundantly to regulate BMP signaling, we asked whether the 2 proteins colocalize by examining strains carrying different combinations of TagRFP- or GFP-tagged TSP-12 and TSP-14 (SI Appendix, Table S1). We focused on hypodermal cells for these studies because these cells are where the BMP receptors and Smads are expressed and function to regulate body size (40, 41). In all cases, we observed partial colocalization between TSP-12 and TSP-14 (Fig. 6) in hypodermal cells. We then asked whether TSP-12 and TSP-14 regulate each other’s expression and/or localization. For this purpose, we generated tsp-12NT; tsp-14(0) and tsp-12(0); tsp-14CT animals (SI Appendix, Table S1), and examined the localization patterns of the tagged proteins by confocal microscopy (SI Appendix, Fig. S4 A–D) and the steady-state level of the tagged proteins by Western blot (SI Appendix, Fig. S4 E and F). We found that the localization pattern or expression level of TSP-12 was not altered in tsp-14(0) mutants, and vice versa, suggesting that TSP-12 and TSP-14 do not regulate each other’s expression or subcellular localization.

Fig. 6.

TSP-12 and TSP-14 partially colocalize in the hypodermis. Airyscan confocal images of early L4 hypodermal cells showing the localization of tagged TSP-12 (A and F), tagged TSP-14 (B and G), and the corresponding merged images (C and H) and their zoomed-in versions (D and I). Colocalization between each respective pair of tagged TSP-12 and TSP-14 proteins was quantified using 2 different methods and shown in E and J. MOC, Mander’s overlap coefficient; PCC, Pearson’s correlation coefficient. (E) Correlation of TagRFP::TSP-12 and TSP-14::GFP (PCC), and the fraction of TagRFP::TSP-12 that overlaps with TSP-14::GFP (MOC). (J) Correlation of GFP::TSP-12 and TSP-14::TagRFP (PCC), and the fraction of GFP::TSP-12 that overlaps with TSP-14::TagRFP (MOC). (Error bars in E and J represent SEM. Statistical analysis was done using a 2-tailed t test with 95% CIs. Scale bar, 5 µm.)

Both TSP-14 and TSP-12 Are Localized to Endosomes in Hypodermal Cells.

To determine if TSP-12 and TSP-14 are normally localized in the right time and place to directly affect DAF-4/BMPRII sorting, we crossed different TagRFP- or mCherry-tagged organelle markers (SI Appendix, Table S1) into the TSP-14::GFP::3xFLAG (TSP-14CT) background and measured colocalization using confocal microscopy with Airyscan. We focused on hypodermal cells of larval stage 4 (L4) worms, because 1) we have previously shown that TSP-12 and TSP-14 function redundantly in hypodermal cells to promote BMP signaling (23), 2) TSP-12 and TSP-14 partially colocalize with each other, and 3) hypodermal cells of L4 larvae exhibit the strongest TSP-12 and TSP-14 signals without any interference from the autofluorescent granules present in intestinal cells. We found that TSP-14 predominantly colocalizes with the early endosome markers EEA-1 and SNX-1 (35) (Fig. 7 A and B), late endosome/lysosome/phagosome marker RAB-7 (34) (Fig. 7C), and endosome/multivesicular body marker HGRS-1 (42) (Fig. 7D). TSP-14 also partially colocalizes with the endosome marker RME-8 (43) (Fig. 7E) and recycling endosome marker RAB-11 (34) (Fig. 7F). However, TSP-14 does not colocalize with the late Golgi marker SYN-16 (44) (Fig. 7H) or the endoplasmic reticulum (ER) marker TRAM (45) (Fig. 7I). Intriguingly, while TSP-14 does not colocalize with the retromer complex marker VPS-35 (42) (Fig. 7G), the 2 proteins appear to be in close juxtaposition and are often visible as pairs, suggesting that they represent different microdomains on the same endosomes. Taken together, these colocalization studies demonstrate that in larval hypodermal cells, intracellular TSP-14 is primarily localized to endosomes, including early, late, and recycling endosomes, where they could directly control DAF-4 intracellular sorting within the endosomal system.

Fig. 7.

TSP-14::GFP is localized to endosomes in the hypodermis. Airyscan confocal images of early L4 hypodermal cells showing localization of various intracellular markers in red (A–I) and the corresponding TSP-14::GFP images (A′–I′). Merged images are shown as unzoomed (A″–I″) and zoomed-in versions (A‴–I‴). (Scale bars, 5 µm.) Colocalization of each pair of red and green signals was quantified using the PCC. The MOC was used to calculate the fraction of TSP-14::GFP signals that overlap with each of the markers. Results of the quantification are shown (Right). Error bars represent SEM. Statistical analysis was done using a 2-tailed t test with 95% CIs.

Using similar approaches, we also identified the intracellular vesicles where TSP-12 is localized (see SI Appendix, Table S1 for strain information). Like TSP-14, intracellular TSP-12 is also localized to early endosomes, late endosomes, and recycling endosomes (Fig. 8 A–D), is in close juxtaposition with the retromer VPS-35 (Fig. 8E), and is absent in the ER (Fig. 8G). Unlike TSP-14, GFP::3xFLAG::TSP-12 exhibits some, but not extensive, colocalization with the late Golgi marker TagRFP::SYN-16 (Fig. 8F). Taken together, our data show that both TSP-12 and TSP-14 are localized to endosomes where they could directly affect DAF-4/BMPRII endosomal sorting.

Fig. 8.

TSP-12::GFP is localized to endosomes in the hypodermis. Airyscan confocal images of early L4 hypodermal cells showing localization of various intracellular markers in red (A–G) and the corresponding GFP::TSP-12 images (A′–G′). Merged images are shown as unzoomed (A″–G″) and zoomed-in versions (A‴–G‴). (Scale bars, 5 µm.) Colocalization of each pair of red and green signals was quantified using the PCC. The MOC was used to calculate the fraction of GFP::TSP-12 signals that overlap with each of the markers. Results of the quantification are shown (Right). Error bars represent SEM. Statistical analysis was done using a 2-tailed t test with 95% CIs.

tsp-12(0); tsp-14(0) Double Mutants Exhibit Altered Endosomal Morphology.

The endosomal localization of TSP-14::GFP and TSP-12::GFP and the presence of abnormally large DAF-4–positive late endosomes in tsp-12(0); tsp-14(0) double mutants prompted us to ask whether TSP-12 and TSP-14 maintain the structural integrity of the endosomal system in general. We examined the distribution of various endolysosomal proteins in the intestinal cells of tsp-12(0); tsp-14(0) double mutants, including RAB-5, RAB-7, RAB-10, RAB-11, and LMP-1.

The endosomal marker RAB-5 is normally concentrated as small puncta near the basolateral membrane and forms an interconnected network in intestinal cells of wild-type worms (Fig. 9A). tsp-12(0); tsp-14(0) double mutants showed a range of abnormal RAB-5 distribution patterns, ranging from slightly more dispersed enlarged puncta (Fig. 9E), to large aggregates, to large vacuole-like structures (Fig. 9 E′ and E″). Because of the wide range of defects observed, we grouped the worms into different classes based on the distribution patterns of RAB-5, class I (Fig. 9E), class II (Fig. 9E′), and class III (Fig. 9E″), and quantified the number of animals in each group. As shown in Fig. 9K, nearly 50% of tsp-12(0); tsp-14(0) mutants exhibited severe abnormality of GFP::RAB-5 distribution.

Fig. 9.

tsp-12(0); tsp-14(0) double mutants exhibit altered endosomal morphology. Airyscan confocal images of wild-type (A–D and I) and tsp-12(0); tsp-14(0) mutant (E–H″ and J) intestinal cells expressing GFP::RAB-5 (A and E–E″), GFP::RAB-7 (B and F–F″), TagRFP::RAB-10 (C and G–G″), RFP::RAB-11 (D and H–H″), and LMP-1::TagRFP (I and J). For each of the endosomal markers, a range of different morphologies was observed in tsp-12(0); tsp-14(0) double mutants and were arbitrarily grouped into 3 different classes based on severity, with class I being the least severe and very similar to wild-type patterns and class III being the most severe. (K) The percentage of animals exhibiting different classes of morphology was quantified and plotted. (Scale bars, 10 µm.) Mut, tsp-12(ok239); tsp-14(jj95) double mutant.

RAB-7, a late endosome and early lysosome marker (35), is localized to a small number of punctate structures and numerous small vesicles that are more enriched in the apical side of intestinal cells in wild-type worms (Fig. 9B). In a majority of tsp-12(0); tsp-14(0) double mutants, there is a reduction of apically localized RAB-7 and a significant increase in abnormally large RAB-7–positive vacuoles (Fig. 9 F–F″ and K). Similarly, the basolateral early endosome/recycling endosome marker RAB-10 (46, 47) and the apical recycling endosome marker RAB-11 also exhibited abnormal distribution patterns in tsp-12(0); tsp-14(0) double mutants (Fig. 9 G–G″, H–H″, and K) when compared with wild-type animals (Fig. 9 C, D, and K). Unlike these endosomal markers, the lysosome marker LMP-1 appeared to have a similar distribution pattern in the wild type and tsp-12(0); tsp-14(0) double mutants (Fig. 9 I–K).

Collectively, the abnormal distribution of various endosomal markers observed in tsp-12(0); tsp-14(0) double mutants and the endosomal localization of TSP-14 and TSP-12 suggest that TSP-12 and TSP-14 play important roles in maintaining the structural and functional integrity of the endosomal system.

tsp-12(0); tsp-14(0) Double Mutants Do Not Appear to Broadly Affect Protein Trafficking.

Since tsp-12(0); tsp-14(0) double mutants exhibit defects in DAF-4/BMPRII localization and endosomal morphology, we asked whether other cargo proteins might also be mislocalized in tsp-12(0); tsp-14(0) double mutants. We first attempted to examine the localization of the BMP type I receptor SMA-6. Gleason and colleagues have reported that SMA-6/BMPRI recycles through a clathrin-dependent and retromer-mediated pathway, which is different from DAF-4/BMPRII, which recycles through an ARF-6– and RME-1–dependent pathway (7). Unfortunately, the endogenously tagged SMA-6::GFP::3xFLAG signal is too faint to detect via microscopy. Nevertheless, we were able to detect the SMA-6::GFP::3xFLAG protein on Western blots. Unlike DAF-4, where the steady-state levels are clearly reduced, we did not observe any change of steady-state level of the endogenously tagged SMA-6::GFP::3xFLAG in tsp-12(0); tsp-14(0) double mutants (Fig. 4 E and F).

Given that our data suggest a cargo-specific role for TSP-12 and TSP-14 in endosomal sorting, affecting DAF-4 but not SMA-6, we extended this analysis by examining the localization of 2 heterologous mammalian cargoes with well-understood recycling routes that have been well-established in the C. elegans intestine: hTfR::GFP (human transferrin receptor) and hTAC::GFP (human IL2-alpha chain), both of which recycle through recycling endosomes, but only 1 of which, hTAC::GFP, recycles via the ARF-6 pathway (46–48). We found that hTAC::GFP is mislocalized in the intestine in tsp-12(0); tsp-14(0) double mutants in a manner similar to that of DAF-4 (Fig. 10 G–J), but hTfR::GFP is localized normally in the intestine in tsp-12(0); tsp-14(0) double mutants (Fig. 10 A–F). Taking together the observations that the steady-state level of SMA-6 protein and the localization of hTfR::GFP are not altered in tsp-12(0); tsp-14(0) double mutants, we concluded that TSP-12 and TSP-14 regulate the recycling of a subset of cargo proteins and represent a cargo-specific recycling mechanism important for BMP signaling.

Fig. 10.

tsp-12(0); tsp-14(0) double mutants affect the localization of the artificial cargo hTAC::GFP but not the artificial cargo hTfR::GFP. (A–D) Airyscan confocal images showing the basolateral membrane localization (A and C) and apical and intracellular localization (B and D) of the artificial cargo hTfR::GFP in wild-type (A and B) and tsp-12(0); tsp-14(0) mutant (C and D) intestinal cells. The relative fluorescence intensity in the regions boxed with solid lines in A–D was quantified and plotted in E and F, respectively. The fluorescence intensity of 8 worms was measured for each genotype on each focal plane and averaged. Error bars denote SEM. n.s., not significant. Statistical analysis was done using a 2-tailed t test with 95% CIs. (G–J) Airyscan confocal images showing the apical and intracellular localization of the artificial cargo hTAC::GFP in wild-type (G) and 3 different tsp-12(0); tsp-14(0) mutant worms. (K) The relative intracellular fluorescence intensity of hTAC::GFP was measured and quantified in ways similar to that described for hTfR::GFP. ***P < 0.0001. (Scale bars, 20 µm.) Mut, tsp-12(ok239); tsp-14(jj95) double mutant.

Discussion

In this study, we showed 2 redundant tetraspanins, TSP-12 and TSP-14, to be critical for intracellular trafficking of the BMP type II receptor in C. elegans. Both endogenous TSP-12 and TSP-14 are localized to endosomal compartments where they could directly mediate cargo sorting. Lack of TSP-12 and TSP-14 led to misrouting of DAF-4/BMPRII to lysosomes and late endosomes, resulting in reduced cell-surface localization and decreased steady-state level of DAF-4/BMPRII protein. We further showed that this effect appears to be relatively specific to DAF-4/BMPRII or to cargoes that are recycled by ARF-6–dependent mechanisms, as tsp-12(0); tsp-14(0) mutants did not exhibit any reduction in the level of SMA-6/BMPRI protein or abnormal distribution of an artificial cargo, hTfR::GFP.

Our findings are consistent with the notion that BMPRI and BMPRII utilize distinct sorting and recycling pathways (see Fig. 11 for a model). Previous work showed that SMA-6/BMPRI depends on clathrin-dependent endocytosis and recycles back to the cell surface via a retromer-dependent recycling pathway that includes the retromer component VPS-35, while DAF-4/BMPRII relies on clathrin-independent endocytosis and its recycling is mediated by the small GTPase ARF-6 (7). Intriguingly, we found that TSP-12/TSP-14 and the retromer component VPS-35 are localized in close juxtaposition but show limited to no colocalization (Figs. 7 and 8). Since SMA-6/BMPRI requires VPS-35 for recycling and DAF-4/BMPRII requires TSP-12/TSP-14 for recycling, and tetraspanins are known to organize membranes into tetraspanin-enriched microdomains and regulate the lateral clustering or trafficking of various membrane or membrane-associated proteins (29, 30), we postulate that TSP-12/TSP-14 may organize endosomes into specialized compartments/domains such that different intracellular cargoes can be trafficked into different subcellular destinations. A role for TSP-12/TSP-14 in organizing and stabilizing specialized domains in endosomes is supported by our observation that the morphology of various endosomal compartments is disorganized in tsp-12(0); tsp-14(0) double mutants. This disorganization does not completely disrupt broad protein trafficking, because we did not detect any abnormal localization of the artificial cargo hTfR::GFP, and there does not appear to be any reduction of SMA-6/BMPRI protein level in tsp-12(0); tsp-14(0) double mutants.

Fig. 11.

Model for how TSP-12 and TSP-14 function to regulate DAF-4/BMPRII trafficking. This model is based on this work and work from Gleason et al. (7). DAF-4/BMPRII and SMA-6/BMPRI use distinct endocytic and recycling pathways. While SMA-6/BMPRI utilizes clathrin-dependent endocytosis and retromer-mediated retrograde trafficking for its recycling, DAF-4/BMPRII relies on clathrin-independent endocytosis and its recycling depends on TSP-12/TSP-14 function. At present, the relationship between TSP-12/TSP-14 and SMA-6/BMPRI and DAF-4/BMPRII at the cell surface is unknown. PM, plasma membrane. See text for detailed discussion.

Endosomes play central roles in the trafficking of cell-surface receptor proteins (9, 49, 50). Recent work has identified distinct and complementary microdomains on endosomes: a recycling microdomain that contains retromer-associated sorting nexin 1 (SNX-1) and its binding partner J-domain protein RME-8, and a degradative microdomain that is enriched in the transport ESCRT-0 component Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate HGRS-1) (42). Furthermore, an E2 ubiquitin-conjugating enzyme, UBC-13, has been found to play an important role in the separation of retromer and ESCRT microdomains on endosomes (51). It is unknown at this time if ARF-6–dependent cargo sorting requires a specific microdomain for its recycling function. Future work will be needed to determine the relationships between TSP-12/TSP-14 and these previously identified endosome microdomains, the precise composition of TSP-12/TSP-14–enriched membrane microdomains, and how they regulate endosomal morphology and function.

Previous work has shown that the TGFβ receptors are constantly recycled in the presence and absence of ligand (52, 53) and that receptors internalized to EEA1-positive early endosomes are active for signaling (52). At present, it is unclear whether or not TSP-12/TSP-14–mediated recycling of DAF-4/BMPRII involves ligand-bound DAF-4/BMPRII and/or actively signaling DAF-4/BMPRII. Since TSP-12 and TSP-14 function to promote BMP signaling (23), the reduction of cell-surface level of DAF-4/BMPRII and its accumulation in lysosomes and lysosome-related organelles in tsp-12(0); tsp-14(0) double mutants suggest that TSP-12 and TSP-14 may function after or independent of R-Smad activation to recycle DAF-4/BMPRII receptor back to the cell surface and allow repeated signaling. This is consistent with our observations that a null mutation in RAB-7, which compromises lysosomal function, can partially rescue the sma-9 suppression phenotype, a BMP signaling defect, of tsp-12(0) mutants. It is also consistent with recent findings showing that blocking the ESCRT complex, which is involved in degradation of the TGFβ receptors, can lead to up-regulation of the transcriptional and phenotypic outputs of TGFβ signaling (8).

TSP-12 and TSP-14 are paralogs, with TSP-14 belonging to the TspanC8 subfamily of tetraspanins (22, 54). Mammalian TspanC8 tetraspanins are known to bind directly to ADAM10 and promote its maturation and trafficking from the endoplasmic reticulum to the cell surface (55, 56). In C. elegans, TSP-12 and TSP-14 function redundantly to regulate both Notch signaling and BMP signaling (22, 23, 54). We have previously shown that TSP-12 and TSP-14 can bind to the ADAM10 ortholog SUP-17 in yeast and that TSP-12, but not TSP-14, functions in the early embryo to promote the cell-surface localization of SUP-17/ADAM10 (23). Surprisingly, in this study, we detected little or no endogenous TSP-12 or TSP-14 in the ER, and only some Golgi localization for TSP-12, but not TSP-14, in hypodermal cells. Instead, we showed that TSP-12 and TSP-14 are endosomally localized proteins that function to regulate the recycling of the BMP type II receptor DAF-4 to the cell surface. These findings raise the possibility that the mammalian counterparts of TSP-12 and TSP-14 may function in similar ways to regulate transmembrane protein recycling to the cell surface after endocytosis. At present, we cannot rule out the possibility that TSP-12 and/or TSP-14 may be localized to and function in the ER in other cell types. Similarly, since TSP-12 and TSP-14 are both localized to endosomes as well as on the cell surface, we cannot rule out the possibility that either or both proteins may also exert functions at the cell surface.

While TSP-12 and TSP-14 share functional redundancy in regulating both BMP and Notch signaling, their localization patterns and functions do not completely overlap. First, in addition to shared expression in multiple cell types, TSP-12 and TSP-14 also exhibit gene-specific expression patterns. For example, TSP-14, but not TSP-12, is localized to the anterior sensory cilia and on sperm (Fig. 5). Second, TSP-12 and TSP-14 exhibit protein-specific subcellular localization patterns. TSP-12 is localized to the basolateral side of the developing vulva (23), while TSP-14 is localized to both the apical and basolateral sides of the developing vulva (Fig. 5). Moreover, TSP-12 and TSP-14 do not completely colocalize in hypodermal cells (Fig. 6). TSP-14, but not TSP-12, showed much more pronounced colocalization with early endosome markers, while TSP-12, but not TSP-14, showed limited colocalization with the Golgi marker SYN-16 (compare Figs. 7 and 8). Third, TSP-12, but not TSP-14, functions in the early embryo to promote the cell-surface localization of SUP-17/ADAM10 (23). Future work will be needed to uncover additional distinct and shared functions of TSP-12 and TSP-14, and to determine how these 2 proteins function individually or together to regulate the trafficking of DAF-4/BMPRII, SUP-17/ADAM10, and other unidentified proteins.

In summary, we have identified 2 redundant tetraspanins, TSP-12 and TSP-14, that play critical roles in the intracellular trafficking of the type II receptor in the BMP pathway. Our work highlights the importance of tetraspanin-mediated intracellular trafficking in the regulation of BMP signaling in vivo. Because mutations in tetraspanins and abnormal BMP signaling are associated with a variety of human diseases, including cancer (3, 31, 32, 57), future work delineating how tetraspanins regulate the intracellular trafficking of different players in the BMP pathway may provide new therapeutic avenues for some of these diseases.

Materials and Methods

All C. elegans strains used in this study were derived from the Bristol N2 strain, which was used as the wild-type control. Strains generated and used in this study are listed in SI Appendix, Table S1. Worms were cultured at 20 °C (unless otherwise noted) and genetically manipulated according to standard protocols as described by Brenner (58). Oligonucleotides and plasmids used in this study are listed in SI Appendix, Table S2. All plasmids were verified by sequencing. Additional methods are described in SI Appendix, Materials and Methods

CRISPR Experiments.

Cas9 target sites were selected using the CRISPR online design tool CHOPCHOP (http://chopchop.cbu.uib.no). We usually chose 2 adjacent target sites for each gene to increase the efficiency of our CRISPR experiments. The single-guide (sg)RNA sequences used in this study are listed in SI Appendix, Table S2. Vector pRB1017 (59) was used to generate all of the plasmids for sgRNA expression. All plasmids containing the homologous repair templates used in the CRISPR/Cas9 knockin experiments were generated by following the method described by Dickinson et al. (60). Specifically, 500- to 600-bp homology arms for each target gene were amplified from N2 genomic DNA using PCR and then inserted into the GFP^SEC^3xFlag vector pDD282 or the TagRFP^SEC^3xMyc vector pDD286 by Gibson Assembly (New England BioLabs). To tag a protein with GFP at the N terminus, amplified homology arms were assembled with ClaI- and SpeI-digested pDD282 fragments. To tag a protein with GFP at the C terminus, amplified homology arms were assembled with AvrII- and SpeI-digested pDD282 fragments. Similarly, amplified homology arms were assembled with ClaI- and NgoMIV-digested pDD286 fragments, or with AvrII- and NgoMIV-digested pDD286 fragments, in order to generate N- or C-terminally TagRFP-tagged proteins, respectively.

For each knockin experiment, a mixture of 50 ng/µL Cas9 plasmid pDD162, 50 ng/µL sgRNA plasmid (25 ng/µL of each of the 2 sgRNA plasmids), 20 ng/µL repair template plasmid, and 3 red fluorescent coinjection markers [10 ng/µL pJKL724(myo-3p::mrfp::unc-54 3′UTR), 10 ng/µL LiuFD236(rab-3p::2xnls::Tagrfp), gift from Oliver Hobert (Columbia University, New York, NY), and 4 ng/µL pCFJ90(myo-2p::mcherry::unc-54 3′UTR)] (61) was injected into the gonad of young adult N2 worms. The selection strategy described in Dickinson et al. (60) was then used to establish knockin lines. To confirm the knockins, genomic DNA sequences flanking the inserted tags were amplified and subjected to DNA sequencing.

Body Size Measurement, sma-9(0) Suppression Assay, and Statistical Analysis.

Body size measurement was performed following the protocol described in Tian et al. (20). Hypochlorite-bleached eggs were collected and then incubated in M9 buffer at 15 °C for 24 h to synchronize L1 worms. Synchronized L1 worms were transferred onto fresh nematode growth medium (NGM) agar plates and allowed to develop at 20 °C for another 2 or 3 d to the L4 stage. L4 worms at the Christmas tree stage were then washed off the plates with M9 buffer, treated with 0.3% sodium azide, and mounted onto 2% agarose pads. Images of the straight worms were taken using a Leica DMR2 compound microscope equipped with a Hamamatsu Orca-03G camera using iVision software (BioVision Technologies). The body length of each worm was measured using open-source Fiji software, and a 2-tailed t test with 95% CIs was performed using GraphPad Prism.

Mutations in the BMP signaling pathway can suppress the postembryonic coelomocyte phenotype of sma-9(0) mutants (17, 22, 23). The sma-9(0) suppression assay (SI Appendix, Fig. S1B) was therefore used to test the functionality of any endogenously tagged BMP pathway component. For the sma-9(0) suppression (Susm) assay, we generated strai15s (SI Appendix, Table S1) expressing the CC::gfp marker arIs37(secreted CC::gfp) or ccIs4438(intrinsic CC::gfp) in a specific mutant background. Worms with GFP-labeled coelomocytes (CCs) were visualized under a Nikon SMZ1500 stereo zoom microscope equipped with a Sola light engine (Lumencor).

LysoTracker Red Staining.

The indicator LysoTracker (Molecular Probes) was used to label acidic compartments in the intestine of worms. L2- or L3-stage worms were placed on OP50-seeded NGM plates containing 4 µM LysoTracker Red and cultured on these plates for 16 to 24 h in the dark at 20 °C. Before imaging using the confocal microscope, worms were placed on nonseeded NGM plates for 20 min to remove the stained bacteria in the intestine lumen of worms.

Live Imaging and Image Analysis.

For live-worm imaging, animals were mounted on 2% agarose pads with 5 mM levamisole (CAS 16595-80-5; Sigma-Aldrich). To improve the resolution and signal-to-noise ratio, fluorescent images were taken using the Zeiss LSM i880 microscope with Airyscan and processed and viewed with Zen software. A 40× Fluar objective (N.A. 1.3) was used with Immersol 518F oil (Carl Zeiss) to capture both the differential interference contrast (DIC) and fluorescent images. The excitation for GFP was 488 nm, and the excitation for RFP, TagRFP, or mCherry was 561 nm. The emission of GFP was collected using the filter BP495-550, and the emission of RFP, TagRFP, or mCherry was collected using the filter LP570. The pinhole was set at 100 µm, master gain at 805, and digital gain at 1.00. For imaging TSP-14::GFP(jj219), GFP::TSP-12(jj181), TagRFP::TSP-12(jj257), TSP-12::TagRFP(jj231), or TSP-14::TagRFP(jj265), laser power was set at 20%, and for imaging DAF-4::GFP(jj177), laser power was set at 15%. For various hypodermal or intestinal endosomal markers such as GFP::RAB-5, GFP::RAB-7, TagRFP::RAB-10, RFP::RAB-11, and LMP-1::TagRFP and the intestinally overexpressed DAF-4::GFP and hTfR::GFP, laser power was set at 10%.

To determine the subcellular localization of TSP-14 and TSP-12, and the colocalization of TSP-14::GFP with different subcellular markers, quantitative colocalization analysis was performed using the JACoP plugin of open-source Fiji software (62). For each image, the Costes threshold regression was used as the reference to establish a threshold, 3 randomly selected square regions were chosen, and the colocalization analysis was conducted by calculating both Pearson’s correlation coefficient and Mander’s overlap coefficient (63). To measure the fluorescence intensity of DAF-4::GFP in the intestine, which is composed of 20 individual epithelial cells forming a long tube around the lumen, images were collected from both the basolateral plane of the intestine, which captures the top basolateral surface of the intestinal tube, and the midsagittal plane, which captures the cross-section along the apical lumen of the intestine. To examine the mislocalization of intestinal DAF-4::GFP in tsp-12(0); tsp-14(0) double mutants, the focal plane where the abnormally enlarged vesicles accumulated was chosen for further imaging analysis. The basolateral plane of intestinal cells was chosen for imaging and quantification of the hTfR::GFP and hTAC::GFP artificial cargoes.

Western Blot Analysis.

For the Western blotting experiments, 100 L3 worms were hand-picked into 20 µL ddH₂O, immediately flash-frozen in liquid nitrogen, and kept frozen for 20 min. Then, 5 µL 5× SDS sample buffer (0.2 M Tris⋅HCl, pH 6.8, 20% glycerol, 10% SDS, 0.25% bromophenol blue, 10% β-mercaptoethanol) was added to each sample. The samples were then boiled at 95 °C for 10 min, centrifuged at 13,000 rpm for 20 min, and stored at −20 °C until gel electrophoresis. Proteins were separated using 8% SDS/PAGE, and then transferred onto Immobilon-P PVDF membrane (MilliporeSigma). The membrane was incubated in 10% skim milk in PBST (3.2 mM Na₂HPO₄, 0.5 mM KH₂PO₄, 1.3 mM KCl, 135 mM NaCl, 0.05% Tween 20, pH 7.4) for at least 30 min at room temperature, and then incubated at 4 °C overnight in primary antibodies diluted in 5% skim milk/PBST. Primary antibodies used included goat anti-GFP IgG polyclonal antibody (Rockland Immunochemicals; RL600-101-215M; diluted 1:1,000), mouse anti-FLAG IgG monoclonal M2 antibody (Sigma-Aldrich; F3165; diluted 1:5,000), and mouse anti-actin IgM JLA20 monoclonal antibody (Developmental Studies Hybridoma Bank; diluted 1:10,000). Secondary antibodies used included peroxidase-conjugated donkey anti-goat IgG, peroxidase-conjugated goat anti-mouse IgG, and peroxidase-conjugated goat anti-mouse IgM (all from Jackson ImmunoResearch; diluted 1:10,000). Enhanced chemiluminescence was detected using the Western Blotting Luminol Reagent (Santa Cruz Biotechnology; sc-2048). Each Western blot experiment was repeated 3 times using different biological samples. Open-source Fiji software was used to quantify Western blotting images.

Data Availability.

All data and associated protocols and materials are included in the manuscript and SI Appendix. All data will be made freely available.