12 research outputs found
Integrity of Narrow Epithelial Tubes in the <i>C</i>. <i>elegans</i> Excretory System Requires a Transient Luminal Matrix
<div><p>Most epithelial cells secrete a glycoprotein-rich apical extracellular matrix that can have diverse but still poorly understood roles in development and physiology. Zona Pellucida (ZP) domain glycoproteins are common constituents of these matrices, and their loss in humans is associated with a number of diseases. Understanding of the functions, organization and regulation of apical matrices has been hampered by difficulties in imaging them both <i>in vivo</i> and <i>ex vivo</i>. We identified the PAN-Apple, mucin and ZP domain glycoprotein LET-653 as an early and transient apical matrix component that shapes developing epithelia in <i>C</i>. <i>elegans</i>. LET-653 has modest effects on shaping of the vulva and epidermis, but is essential to prevent lumen fragmentation in the very narrow, unicellular excretory duct tube. We were able to image the transient LET-653 matrix by both live confocal imaging and transmission electron microscopy. Structure/function and fluorescence recovery after photobleaching studies revealed that LET-653 exists in two separate luminal matrix pools, a loose fibrillar matrix in the central core of the lumen, to which it binds dynamically via its PAN domains, and an apical-membrane-associated matrix, to which it binds stably via its ZP domain. The PAN domains are both necessary and sufficient to confer a cyclic pattern of duct lumen localization that precedes each molt, while the ZP domain is required for lumen integrity. Ectopic expression of full-length LET-653, but not the PAN domains alone, could expand lumen diameter in the developing gut tube, where LET-653 is not normally expressed. Together, these data support a model in which the PAN domains regulate the ability of the LET-653 ZP domain to interact with other factors at the apical membrane, and this ZP domain interaction promotes expansion and maintenance of lumen diameter. These data identify a transient apical matrix component present prior to cuticle secretion in <i>C</i>. <i>elegans</i>, demonstrate critical roles for this matrix component in supporting lumen integrity within narrow bore tubes such as those found in the mammalian microvasculature, and reveal functional importance of the evolutionarily conserved ZP domain in this tube protecting activity.</p></div
LET-653 is sufficient to expand the gut lumen.
<p>(A-D) Representative 1.5-fold embryos after heat shock overexpression of full-length LET-653 (A), LET-653(PAN) (B), LET-653(PAN+ZP-N) (C) or LET-653(PAN+ZP-N+mucin) (D) fusions under the <i>hsp16</i>.<i>41</i> promoter. Gut lumen expansion is visible by DIC in panel A. (Aā-Dā) GFP expression. Tagged protein was expressed and secreted to similar degrees for the first three genotypes, but secreted more variably for the final genotype. White brackets flank lumen edges at the widest point along the gut length. Scale bar, 5 Ī¼m. (Aā-Dā) Schematic outlines of the entire embryo and gut (lumen shaded dark gray). Narrow regions alternate with wide ābulbsā (red arrows), suggesting a local expansion effect. (E) Individual plotted measurements of gut lumen width in microns. n = 13 for full-length LET-653 and nā„21 for the others. Median, 25th percentile, and 75th percentile are shown for each. *** p<0.001, Studentās <i>t</i>-test, two-tailed.</p
<i>let-653</i> is required for proper alae formation and vulva lumen shaping.
<p>(A,B) Comparison of body shape in WT and <i>let-653(cs178)</i> L4 larvae rescued to viability with a duct-specific <i>lin-48pro</i>::LET-653::SfGFP transgene. The mutants are slightly shorter and fatter than WT. (C,D) TEM images of alae in WT and <i>let-653(s1733)</i> L1 larvae. Transverse sections through the head region at the posterior bulb of the pharynx are shown. Boxed regions are magnified in adjacent panels. Arrowed bars indicate width of the alae. (Cā, Dā) <i>let-653</i> mutants have alae and cuticle organization defects. White dotted line indicates edge of the epidermal cell layer. In <i>let-653</i> mutants, the striated layer of the cuticle (black bracket) appears thin or absent, and the space between this layer and the underlying epidermis is expanded and filled with diffuse, lightly staining material. (E) The permeability barrier is intact in <i>let-653</i> mutants. L1 larvae were incubated in 2 ug/ml Hoechst dye in M9 for 15 minutes and then rinsed and scored for nuclear fluorescence. <i>let-4(mn105)</i> was used as a positive control for permeability [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006205#pgen.1006205.ref038" target="_blank">38</a>]. Data from three replicates shown, n>15 per genotype for each experiment. No significant difference found between <i>let-653</i> and WT permeability by Studentās <i>t-</i>test, two-tailed. (F-I) Comparison of L4 vulva lumen morphology in WT (F), duct-rescued <i>let-653</i> mutants (G,H) and a hypomorphic chondroitin biosynthesis mutant <i>sqv-5(k175)</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006205#pgen.1006205.ref101" target="_blank">101</a>]. Animals were staged as mid-L4 based on fusion of the anchor cell and horizontal orientation of the ventral vulva āfingersā. Modest vulval expansion defects or asymmetries were observed in 11/31 of mutant larvae.</p
Summary and model for the role of LET-653 in tube morphogenesis.
<p>(A) Model for LET-653 aECM assembly. Upon LET-653 secretion, PAN-mediated interactions with the luminal matrix (purple) capture LET-653 and prevent its rapid outflow from the lumen. The ZP-C and/or C-terminal regions then interact with a membrane-associated factor (pink rectangle) that anchors LET-653 more tightly. A membrane-associated protease (scissors) cleaves LET-653 at the CCS, allowing ZP-N dependent polymerization and/or recruitment of additional matrix factors; however, the C-terminal peptide (black squiggle) remains tightly affiliated with the matrix. Arrangement of LET-653 polymers is hypothetical; see text for alternative models. (B) Role of LET-653 in duct tube maintenance. During phase I of duct tube morphogenesis, membrane-associated LET-653(ZP) (blue) resists (T-bars) stretching and/or constrictive forces (arrows) to maintain a uniform lumen diameter and allow fluid passage. During phase II, LET-653 is gradually cleared from the lumen, and a LET-653-dependent cuticle structure begins to form and take over the lumen shaping role. Finally, once tube morphogenesis is complete, the cuticle matures. In <i>let-653</i> mutants, the lumen becomes too narrow during elongation and fragments into separate compartments (arrow). Upstream of the fragmentation, the lumen dilates (asterisk) as a secondary consequence of fluid accumulation.</p
LET-653 PAN and ZP domains confer distinct localization patterns and mediate duct lumen cycling and protection, respectively.
<p>(A-C) LET-653 fusions lacking the C-terminal, ZP or PAN domains showed differential functions and localization in the duct. Only LET-653(ZP) rescued mutant defects. (D,G,J) LET-653(āC) accumulated intracellularly rather than extracellularly, suggesting a failure in secretion. (E-F) LET-653(PAN) and LET-653(ZP) fusions localized normally to the duct in 1.5 fold embryos. (H-I) LET-653(PAN) accumulated in the late L1 larval duct lumen, but LET-653(ZP) did not. Confocal slices. (Gā,Hā,I,) <i>lin-48pro</i>::mRFP marks the duct cell. (K) LET-653(PAN) associated with fibrous material in the center of the lumen (arrow) (L) LET-653(ZP) associated with the dorsal apical membrane region. Dā,Eā,Jā,Kā,Lā show DIC images for comparison. Scale bars, 5 Ī¼m.</p
<i>let-653</i> functions in parallel to <i>lpr-1</i>.
<p>(A) Comparative timecourse of lumen dilation and pore AJ loss in <i>let-4(mn105)</i>, <i>lpr-1(cs73)</i>, and <i>let-653(cs178)</i> mutants. <i>let-653</i> single mutant data are reproduced from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006205#pgen.1006205.g002" target="_blank">Fig 2A</a>. n at least 17 animals per genotype for each timepoint. (B) <i>let-4(mn105)</i> and (C) <i>lpr-1(cs207)</i> embryos bearing the LET-653::SfGFP transgene. LET-653::SfGFP is visible in the duct/pore lumen (lines). Scale bars, 10 Ī¼m. (D) Double mutant analysis. ** p<0.002, *p<0.01, Fisherās Exact test, compared to single mutants at same timepoint.</p
LET-653 is cleaved at its C-terminus.
<p>Anti-GFP Western blot performed on 1.5-fold transgenic embryos. Full-length LET-653 fusions were expected to be found at 100kD, or higher if post-translationally modified. These were not detected, but fusions lacking the PAN domains were readily observed. C-terminally tagged LET-653 yields a band just below 40kD (arrowhead), indicating that SfGFP and about 80 amino acids of LET-653ās C-terminus have been cleaved from the full-length protein, consistent with cleavage at the predicted CCS. The loading control antigen is UNC-15/paramyosin. Blot shown is representative of n = 11.</p
Dynamic PAN- and stable ZP-mediated interactions in the duct lumen.
<p>Fluorescence recovery after photobleaching (FRAP) of LET-653 translational fusions in the duct lumen. (A-C) Pre-bleach, bleach, and post-bleach frames taken from FRAP experiments on the indicated fusions in 1.5-fold embryos (A-Aā) or L1 larvae (B-Bā,C-Cā). For each experiment, the post-bleach frame chosen corresponds to a time point after recovery plateau had been reached. Mean fluorescence intensity measurements were taken within the bleach ROI (white box) and, in L1 experiments, an adjacent ROI (purple box). Scale bars, 5 Ī¼m. (D-F) Fluorescence recovery curves plotting mean and SE for nā„5 replicates per stage for ssSfGFP::LET-653 and n = 2 replicates for ssSfGFP::(PAN). t = 0s represents the first post-bleach frame; raw values for each experiment were normalized to the highest measurement taken. (G, H) Comparisons of mobile fractions and recovery half-times. Full-length LET-653 (D,E) exhibited a significantly lower average mobile fraction than LET-653(PAN) (F). However, the half-time of recovery (t<sub>1/2</sub>) was not significantly different between the two fusion proteins. ** p<0.01, * p<0.05, Studentās <i>t</i>-test, two-tailed.</p
<i>let-653</i> acts in the excretory duct and pore cells, not the canal cell.
<p>(A-D) <i>let-653</i> mutants have morphological defects in the duct and pore. (A,B) Schematics of excretory system morphology in <i>WT</i> (A) and <i>let-653</i> mutants (B) at the early L1 stage. G1 pore cell is shown in blue, duct cell in yellow, canal cell in red, and G2 and W epidermal cells in pink. White regions represent lumens. Heavy black lines represent apical junctions. Black arrow, pore cell autocellular junction (AJ); red arrow, missing AJ in <i>let-653</i> mutant. Arrowheads, duct-canal intercellular junction (IJ), *, duct lumen dilation. Anterior is to the left and ventral is down in all images. (C, D) Duct (d) and pore (p) morphology in WT and <i>let-653(cs178)</i> mutants. Cell bodies (red in Cā, Dā) and all apical junctions (green in Cā, Dā) are marked as labeled. DIC, differential interference contrast of head; box shows region magnified in subsequent panels. c, canal cell nucleus. ph, pharynx. (E) LET-653b protein schematic and locations of mutant allele lesions. SP, signal peptide. PAN, PAN-Apple domains. ZP, zona pellucida domain. Conserved ZP domain cysteines are indicated above in black; additional cysteines are indicted in grey. PTS, Proline/Threonine/Serine-rich region is predicted by Net-O-Glyc [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006205#pgen.1006205.ref099" target="_blank">99</a>] to be highly O-glycosylated. LET-653b is the shortest of three protein isoforms produced from the <i>let-653</i> locus by alternative splicing; the other isoforms contain a larger central PTS domain (<a href="http://www.wormbase.org/" target="_blank">www.wormbase.org</a>). CCS, consensus furin cleavage site (R-X-R/K-R) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006205#pgen.1006205.ref100" target="_blank">100</a>]. (F) Tissue-specific expression of <i>let-653b</i> cDNA in the duct and/or pore efficiently rescued <i>let-653(s1733)</i> lethality. Data from multiple independent transgenic lines per construct are shown. *, p<0.01, Fisherās Exact test, compared to non-transgenic siblings.</p
<i>let-653</i> is required to maintain lumen integrity during duct tube elongation and narrowing.
<p>(A) Time course of duct lumen dilation and G1 pore AJ disappearance in <i>let-653(cs178)</i> mutants. Lumen dilation was visualized by DIC as a small bubble at least 1 Ī¼m in diameter adjacent to the canal nucleus, and the AJ was visualized with AJM-1::GFP as in panel B. n<u>ā„</u>30 for each timepoint. (B) Lumen dilation initiates near the duct-canal junction. DIC and inverted grayscale AJM-1::GFP images of a <i>let-653(cs178)</i> 3-fold embryo at early stage of lumen dilation. Note presence of pore AJ (arrow) and duct-canal junction (arrowhead). (C) Duct lumen measurements reveal two distinct phases of lumen growthāelongation and widening (I), followed by elongation with narrowing (II). Duct lumen length and width measurements in WT (black or green) and <i>let-653(cs178)</i> mutants (red). Each dot represents a single animal containing RDY-2::GFP and AJM-1::GFP markers as in D-I, except 1.5-fold measurements based on LET-653::SfGFP marker, and green dots based on <i>WT</i> TEM specimens. For <i>let-653</i>, only young 3-fold embryos with an intact duct lumen were scored. The appearance of lumen fragmentation and dilation prevented accurate measurements in older animals. (D-I) In <i>let-653</i> mutants, the distal duct lumen fragments during elongation and narrowing. Bracket indicates duct lumen. Black arrow indicates pore AJ. Arrowhead indicates duct-canal junction. Red arrows point to lumen or junction discontinuities. Asterisks indicate duct lumen dilation. Inverted grayscale fluorescent images of staged WT (D-F) and <i>let-653(cs178)</i> (G-I) animals expressing the apical membrane marker RDY-2::GFP and junction marker AJM-1::GFP. RDY-2::GFP also marks the canal lumen, which shows up prominently near the duct-canal junction in some images. F and I also include the cytoplasmic duct marker <i>lin-48pro</i>::mRFP. (J-N) The pore loses its AJ and intercellular junctions in <i>let-653(cs178)</i> L1 larvae. (J,K) F-actin visualization using confocal microscopy of VAB-10(ABD)::GFP. F-actin localizes near apical membranes and junctions in both WT (J) and <i>let-653</i> mutants (K). (L-N) Pore AJ loss and duct-pore separation after hatch. Inverted grayscale images of <i>let-653</i> mutants expressing the cytoplasmic marker <i>dct-5pro</i>::mCherry and junction marker AJM-1::GFP. Scale bars, 5 Ī¼m.</p