Caenorhabditis elegans num-1 negatively regulates endocytic recycling

Abstract

Much of the material taken into cells by endocytosis is rapidly returned to the plasma membrane by the endocytic recycling pathway. Although recycling is vital for the correct localization of cell membrane receptors and lipids, the molecular mechanisms that regulate recycling are only partially understood. Here we show that in Caenorhabditis elegans endocytic recycling is inhibited by NUM-1A, the nematode Numb homolog. NUM-1AGFP fusion protein is localized to the baso-lateral surfaces of many polarized epithelial cells, including the hypodermis and the intestine. We show that increased NUM-1A levels cause morphological defects in these cells similar to those caused by loss-of-function mutations in rme-1, a positive regulator of recycling in both C. elegans and mammals. We describe the isolation of worms lacking num-1A activity and show that, consistent with a model in which NUM-1A negatively regulates recycling in the intestine, loss of num-1A function bypasses the requirement for RME-1. Genetic epistasis analysis with rab-10, which is required at an early part of the recycling pathway, suggests that loss of num-1A function does not affect the uptake of material by endocytosis but rather inhibits baso-lateral recycling downstream of rab-10.

Figure 1.—

Organization of the num-1 locus and expression of num-1A and num-1B. (A) The organization of the exons and introns in num-1 is shown above the scale bar. Boxes represent exons. The lines connecting them show the splicing patterns of the primary transcripts determined by characterization of cDNAs and RT–PCR products. The region spanning parts of exons 3 and 4 of num-1A that encodes the PTB domain is shaded. The regions deleted in num-1(bc365) and num-1(ok433) are indicated by lines below the scale bar. (B) Transgenes used to study num-1 expression and function. Each line is a schematic of part of a plasmid construction used in this study. The plasmid pVB63LN contains an 8.4-kb XhoI–SpeI fragment that spans the entire num-1 locus. The other fragments encode different NUM-1∷GFP fusion proteins. In each case, the gfp gene has been inserted immediately prior to the stop codon of num-1. (C–H) Photomicrographs of hermaphrodite worms harboring a num-1A∷gfp transgene viewed with either Nomarski differential interference contrast (DIC) (C) or fluorescence (D–J) optics. The worms were viewed from either the left or the right. C–F show medial optical sections; for G, I, and J, the optical section was a plane just below the baso-lateral membrane of the intestine. The images in C–H were obtained with the aid of a Leica DMRB compound microscope. Images in I and J were obtained with the aid of Leica confocal microscope. The worms in C–I were of the genotype unc-4(e120); svIs27 [num-1A∷gfp unc-4 (+)]. The svIs27 array harbors the plasmid pVB68LN, which lacks sequences necessary for the expression of num-1B. The worm in J was wild-type N2 control. The arrows in C and D indicate the basal (b) and apical (a) cell membranes of the intestine. The arrowhead in D indicates the position of an adherens junction separating adjacent intestinal cells. The arrow in E indicates the intestine primordium in the embryo. The arrows in F indicate the pharynx (p) and nerve ring (n), respectively. The arrows in G indicate two coelomocytes (c). Note also the punctate pattern within the intestine in this focal plane. The arrow in H points to the adherens junctions separating the apical and basal domains of the seam cells. (I) Confocal micrograph of the intestine of an svIs27 hermaphrodite showing the punctate staining of NUM-1A∷GFP close to the baso-lateral membrane of the intestine. (J) A confocal micrograph of a wild-type hermaphrodite taken with identical settings. Bars in C–H are 20 μm; in I and J, 4 μm. (K–N) Photomicrographs of L4 hermaphrodite worms harboring a num-1B∷gfp transgene viewed with either Nomarski DIC (K and M) or fluorescence (L and N) optics. “can” denotes the excretory canal. The uv1 cells are four neurosecretory cells in the uterus; two of the four are seen in this focal plane. (O) Diagram showing the arrangement of the intestine, which is shaded. At left is shown a medial saggital section viewed from the left. A, anterior; P, posterior; D, dorsal; V, ventral. The baso-lateral and apical membranes of the intestine are indicated. (Right) A transverse section viewed from the anterior. (P) Schematic of C. elegans, Drosophila, and human Numb proteins. All three contain both a PTB domain and at least one DPF motif. Human and Drosophila Numb proteins also harbor the NPF motif.

Figure 2.—

Defects associated with elevated levels of num-1 transcripts. (A and B) Micrographs of wild-type N2 (A) and svIs23[num-1A(++)] (B) young adult hermaphrodites viewed with Nomarski DIC optics. Vacuoles (indicated by large and small arrows) are present in the intestine of num-1A(++) worms. (C and D) Micrographs of an adult svIs27[num-1A∷gfp] hermaphrodite worm viewed with Nomarski DIC (C) or fluorescence (D) optics. Two large vacuoles, indicated by arrows, are present in the intestine. GFP fluorescence is seen around the perimeter of both. (E and F) Micrographs of the intestines of rme-1 and rab-10 mutant hermaphrodites for comparison. (G and H) Micrographs of an svIs30[num-1B∷gfp] adult worm viewed with Nomarski DIC (G) or fluorescence (H) optics. The tip of a canal running posteriorly is indicated by an arrow. In wild-type worms, the canal runs almost the entire length of the animal. In the animal shown, the canal extends only to the mid-body region. (I and J) The same worm at higher magnification.

Figure 3.—

Electron microscope analysis of worms with aberrant num-1 expression. Electron micrographs of transverse sections of the intestine of wild-type, num-1(bc365), NUM-1A-overexpressing svIs23[num-1A(++)] and rme-1(b1045) animals. Whereas num-1(bc365) mutants display a normal intestinal organelle distribution when compared to wild type, NUM-1 overexpression in svIs23[num-1A(++)] animals leads to aberrant accumulation of large vacuoles, indicated by arrows, similar to those observed in rme-1(b1045) mutants. The apical and baso-lateral membranes of the intestine are indicated by black and white dotted lines, respectively. Bars, 5 μm.

Figure 4.—

The intestinal vacuoles in num-1A(++) worms receive input from the baso-lateral endocytosis pathway. (A and B) Micrographs of an adult svIs23[num-1(++)] hermaphrodite into the body cavity (pseudocoelom) of which rhodamine–dextran, a fluid-phase marker for endocytosis, has been injected. In B, the worm is viewed with fluorescence optics 30 min post-injection. The dye has accumulated in the vacuoles, indicating that the vacuoles receive cargo from the baso-lateral endocytosis pathway. (C and D) A wild-type N2 worm injected as a control. The dye fails to accumulate in the intestine. (E and F) Micrographs of an adult svIs23[num-1(++)] hermaphrodite fed rhodamine–dextran. In F, the worm is viewed with fluorescence optics. The dye accumulates in the lumen of the intestine but not in the vacuoles, indicating that the vacuoles do not receive input from the apical endocytosis pathway. The worm shown had been fed rhodamine–dextran for 30 min. The vacuoles remain unlabeled even when worms are fed for extended periods of time (10 hr). (G and H) Micrographs of an svIs23[num-1(++)] worm carrying a transgenic array, arIs37, encoding a form of GFP, which is secreted from muscles into the pseudocoelom (Fares and Greenwald 2001a). The GFP accumulates in the intestinal vacuoles, indicated by arrows. (I and J) arIs37 hermaphrodite worm for comparison. GFP does not accumulate in the intestine.

Figure 5.—

The membranes of intestinal vacuoles present in num-1(++) animals are labeled by markers for recycling endosomes. Confocal fluorescence micrographs of the intestines of adult hermaphrodite worms harboring GFP markers for endosomal organelles. num-1(++) indicates svIs23[num-1(++)]. The transgenes used were pwIs87[P_vha-6∷gfp∷rme-1], pwIs112[P_vha-6∷hTAC∷gfp], pwIs249[P_vha-6∷gfp∷rab-5], and pwIs253[P_vha-6∷gfp∷rab-7] (Chen et al. 2006). The fusion proteins are under control of an intestine-specific promoter from the vha-6 gene (Chen et al. 2006).

Figure 6.—

num-1(bc365) affects recycling but not internalization of a transmembrane endocytosis marker. (A–F) Confocal fluorescence micrographs of the intestines of adult hermaphrodites expressing the hTAC∷GFP marker for endocytic recycling. In rme-1 or rab-10 mutants, the marker is present on the rim of intestinal vacuoles, indicated by arrows in B and E. In the rme-1 num-1A double mutant, there are no vacuoles but some hTAC∷GFP is present intracellularly. In the rab-10; num-1A double mutant, vacuoles are still present, and the hTAC∷GFP marker is clearly visible around the rims (indicated by arrows in F) as well as in other parts of the cytoplasm. The mutations used were rab-10(dx2), rme-1(b1045), and num-1(bc365).

Figure 7.—

Aberrant num-1A expression causes defects in other tissues. (A and B) Confocal fluorescence micrographs showing vacuoles in coelomocytes containing ssGFP. See the legend to Figure 4 for an explanation of ssGFP. The arrows indicate organelles in the coelomocytes containing GFP. GFP accumulates to higher levels within coelomocytes in animals with increased num-1A gene dosage (B) than in animals with normal num-1 expression (A). Bars, 10 μm. (C) Micrograph of a part of the hypodermis viewed with Nomarski DIC optics. The animal is an adult hermaphrodite with increased num-1A gene dosage. The presence of large vacuoles is indicated by arrows. Bar, 20 μm. (D–H) Electron micrographs of transverse sections of adult hermaphrodite worms showing parts of hypodermal cells, the overlying cuticle, and alae. D, F, G, I, and J show parts of the seam cells and the cuticle above. E, H, and K show part of hyp7 and the cuticle above. The top black dotted lines indicate the outer limit of the cuticle. The lower black dotted lines indicate the apical membrane of hypodermal cells. The asterisks in D, F, and J indicate the alae that run along the left and right. The alae and cuticle in the num-1(bc365) mutant (F) are not different from wild type (D). In contrast, the alae in num-1(++) animals (G and I) are flattened compared to wild type (D). (H) Part of the cuticle above hyp7 in num-1(++) worms; the cuticle is much thinner than in wild type (E) and lacks the typical three-layered structure. num-1(++) denotes the integrated array, svIs23[num-1(++)]. Bars, 1 μm.