Dynamic regulation of caveolin-1 trafficking in the germ line and embryo of Caenorhabditis elegans

Abstract

Caveolin is the major protein component required for the formation of caveolae on the plasma membrane. Here we show that trafficking of Caenorhabditis elegans caveolin-1 (CAV-1) is dynamically regulated during development of the germ line and embryo. In oocytes a CAV-1-green fluorescent protein (GFP) fusion protein is found on the plasma membrane and in large vesicles (CAV-1 bodies). After ovulation and fertilization the CAV-1 bodies fuse with the plasma membrane in a manner reminiscent of cortical granule exocytosis as described in other species. Fusion of CAV-1 bodies with the plasma membrane appears to be regulated by the advancing cell cycle, and not fertilization per se, because fusion can proceed in spe-9 fertilization mutants but is blocked by RNA interference-mediated knockdown of an anaphase-promoting complex component (EMB-27). After exocytosis, most CAV-1-GFP is rapidly endocytosed and degraded within one cell cycle. CAV-1 bodies in oocytes appear to be produced by the Golgi apparatus in an ARF-1-dependent, clathrin-independent, mechanism. Conversely endocytosis and degradation of CAV-1-GFP in embryos requires clathrin, dynamin, and RAB-5. Our results demonstrate that the distribution of CAV-1 is highly dynamic during development and provides new insights into the sorting mechanisms that regulate CAV-1 localization.

Figure 1.

Dynamic behavior of CAV-1-GFP during development. (A–D) Subcellular localization of CAV-1-GFP during oogenesis. CAV-1-GFP–labeled ring-like structures (CAV-1 bodies) and puncta. CAV-1-GFP puncta first appear in early oocytes in the bend region of gonad, near the plasma membrane and deeper in the cytoplasm (A and B). (C) An enlargement of the square indicated in A. (D) A Nomarski image of the oocytes. (E) Degradation of CAV-1-GFP after fertilization. After fertilization, CAV-1-GFP accumulates on the plasma membrane and then largely disappears before the first cell division of the embryo (E). CAV-1-GFP partitioned to the polar body of embryos remains highly fluorescent and does not disappear (arrowhead). (F) A Nomarski image of the embryos. (G–I) CAV-1-GFP changes localization dynamically, accumulating in the cortex and clustering around the nucleus (H). Perinuclear CAV-1-GFP is lost during nuclear breakdown (I). (J–L) Internalization of GFP-CAV-1 in the embryo. Plasma membrane localized GFP-CAV-1 (J, 0′) is then internalized (K and L). Oo, oocyte; emb, embryo; sp, spematheca. Arrows indicate the direction of maturation of oocytes and embryos. Scale bars, 10 μm. (M–O) CAV-1-GFP on the plasma membrane and in CAV-1 bodies was photobleached, and the recovery of fluorescence was followed as a function of time. From left to right, before (Prebleach), immediately after (0 min), and 36 min after bleaching are shown. The squares show the photobleached areas. Note that little fluorescence recovery occurred in CAV-1 bodies. Partial recovery occurred on the plasma membrane.

Figure 2.

CAV-1-GFP accumulates on the plasma membrane in rme-2 mutant oocytes. Subcellular localization of CAV-1-GFP in wild-type (A) and rme-2(b1008) (B) worms. CAV-1-GFP localizes to the CAV-1 body and the plasma membrane in oocytes of wild-type worms (A). In rme-2(b1008), CAV-1-GFP mainly localized to the plasma membrane, with the number and size of CAV-1-GFP labeled-CAV-1 bodies greatly reduced (B). Arrows indicate the direction of maturation of oocytes and embryos. Oo, oocyte; Sp, spematheca. Scale bars, 10 μm.

Figure 3.

Fusion of CAV-1 bodies with the plasma membrane occurs after anaphase. (A and B) CAV-1-GFP in a fertilization-defective mutant, spe-9(hc52). L4 hermaphrodites of spe-9(hc52)-expressing CAV-1-GFP were incubated at 25°C for 24 h and examined by fluorescence microscopy. CAV-1 body formation was normal in oocytes (A), and degradation of CAV-1-GFP occurred with normal kinetics in unfertilized eggs (B). (C and D) CAV-1-GFP in a sperm-deficient mutant, fog-2(q71). CAV-1 body formation was normal in oocytes (C), and the degradation of CAV-1-GFP occurred with normal kinetics in ovulated but unfertilized eggs (D) in fog-2(q71). (E-H) CAV-1-GFP in embryos defective in the metaphase-to-anaphase transition. L4 hermaphrodites expressing CAV-1-GFP were incubated at 20°C for 48 h. emb-27(RNAi) embryos are arrested in metaphase of meiosis I. In emb-27(RNAi) hermaphrodites expressing CAV-1-GFP, CAV-1 body formation appears normal in oocytes (E), but degradation of CAV-1-GFP in embryos was completely blocked (F). CAV-1 bodies were observed underneath the plasma membrane (G). (H) An enlargement of the square indicated in G. Oo, oocyte; Emb, embryo; Sp, spematheca. Arrows indicate the direction of maturation of oocytes and embryos. Scale bars, 10 μm.

Figure 4.

CAV-1-GFP is degraded via clathrin- and RAB-5–dependent endocytosis after fertilization. The subcellular localization of CAV-1-GFP was determined in chc-1(RNAi) (C and D), dyn-1(RNAi) (E and F), and rab-5(RNAi) worms (G and H) by a confocal microscopy. The localization of CAV-1-GFP was normal in mock RNAi-treated animals fed bacteria harboring the empty RNAi vector L4440 (A and B). In the chc-1(RNAi), dyn-1(RNAi), and rab-5(RNAi) worms, CAV-1 body formation appeared normal (C, E, and G), but degradation of CAV-1-GFP was blocked (D, F, and H). Arrows indicate the direction of maturation of oocytes and embryos. Scale bars, 10 μm.

Figure 5.

CAV-1-GFP transport via the biosynthetic pathway. The subcellular localization of CAV-1-GFP was determined in rab-1(RNAi) (C and D) and sar-1(RNAi) (E and F) worms by confocal laser microscopy. The localization of CAV-1-GFP was normal in mock RNAi-treated animals fed bacteria harboring the empty RNAi vector L4440 (A and B). Arrows indicate the direction of maturation of oocytes and embryos. Scale bars, 10 μm.

Figure 6.

ARF-1–dependent transport of CAV-1-GFP to the CAV-1 body. CAV-1-GFP was normally localized to the CAV-1 body, and the plasma membrane in oocytes of mock RNAi-treated animals fed bacteria harboring the empty RNAi vector L4440 (A and B). In arf-1(RNAi) worms, the number of CAV-1 bodies was reduced (C) and large CAV-1-GFP–labeled aggregations were often observed (D, arrowheads). arf-1(ok796) displayed a very similar phenotype to arf-1(RNAi) (E and F). RNAi of agef-1, which encodes the predicted orthologue of a known Arf1 GEF, resulted in a severe defect in CAV-1 body formation (G and H). Scale bars, 10 μm.

Figure 7.

ARF-1 is required for export of CAV-1-GFP from the Golgi. CAV-1-GFP–expressing wild-type (A–C) and arf-1(ok796) (D–F) strains were stained with an antibody against a Golgi membrane protein SQV-8. CAV-1-GFP failed to colocalize with SQV-8 in the wild-type strain (C, inset). Deletion of arf-1 caused a significant alteration in Golgi morphology and a loss of CAV-1 bodies (D–F). Colocalization of CAV-1-GFP with SQV-8 was often observed in arf-1 mutants (F, inset). Scale bar, 20 μm. The insets (bottom right) are 3× enlargements of the indicated regions in the merged panels. (G) Stylized drawing of one gonad arm connected to the spermatheca and the uterus. Schematic localization of CAV-1-GFP is also indicated with green. In growing oocytes, CAV-1-GFP is transported to the CAV-1 bodies in an ARF-1–dependent manner. After fertilization, CAV-1-GFP is internalized via clathrin-dependent endocytosis.