Fig 1.
A tissue-specific degradation system to deplete early essential proteins.
(A, B) Cartoons depicting tissue-specific protein degradation scheme (adapted from [29]). In the presence of endogenous ZIF-1 (zif-1(+)), ZF-tagged targets are degraded in somatic cells, leading to an early arrest in the case of early essential proteins. zif-1(−) mutants (zif-1(gk117)) fail to degrade endogenous ZF-tagged targets. (C, E, G) PIE-1::GFP, a natural target of ZIF-1, and SEC-5::ZF::GFP and ZF::GFP::GIP-1, two heterologous ZIF-1 targets, are degraded in somatic cells but not in the germ cell precursors. Dashed white lines outline embryos. (D, F, H) In zif-1(−) embryos, ZF-containing targets are not degraded. (I, L, O) Bean-stage zif-1(−) mutants, in which indicated endogenous loci have been tagged with ZF::GFP but ZIF-1 is not expressed (“gut(+)”). Note the localization to the apical surfaces of intestinal cells (“M,” arrowhead). (J, M, P) Expression of ZIF-1 in intestinal cells (magenta dashed lines) results in tissue-specific degradation (“gut(−)”). Scale bar is 10 μm. (K, N, Q) Average 10 μm line intensity profiles across the apical midline (“M,” arrowhead) of control embryos (green lines show mean; lighter green shading indicates standard deviation) show a peak in GFP signal intensity in GIP-1gut(+) (n = 12), MZT-1gut(+) (n = 11), and AIR-1gut(+) (n = 13) embryos, and no peak and reduced cytoplasmic signal upon degradation of tagged proteins (magenta lines show mean; lighter magenta shading indicates standard deviation) in GIP-1gut(−) (n = 12), MZT-1gut(−) (n = 10), and AIR-1gut(−) (n = 15) embryos. See S1 Fig, S2 Fig and S3 Fig for details. a.u., arbitrary unit; GFP, Green Fluorescent Protein; ZF, zinc finger domain 1.
Fig 2.
Degradation of γ-TuRC components and/or AIR-1 results in intestinal nuclear number defects.
(A) Top: cartoon depicting the organization of a wild-type bean-stage embryonic intestinal primordium, showing the dorsal and ventral tiers of cells. Below: bean-stage embryos of indicated genotypes expressing intestine-specific histone::mCherry. Nuclei are organized into a dorsal (left) and ventral (right) tier of cells. Scale bar is 5 μm. (B) Expression of ZIF-1 from an early intestinal promoter (elt-2p, “early gut(−),” magenta dots) or late intestinal promoter (ifb-2p, “late gut(−),” gray dots) to degrade MZT-1, GIP-1, AIR-1, or GIP-1;AIR-1 perturbs intestinal nuclear number as compared to control embryos (black dots), which have ZF::GFP-tagged GIP-1 and AIR-1 but lack elt-2p::zif-1. “AIR-1*” embryos are from air-1(0/[AIR-1::ZF::GFP]) mothers and carry zero, one, or two copies of air-1(0). The distribution of nuclear number in AIR-1 and AIR-1* embryos is not significantly different (two-tailed t test, p = 0.81). Dot size indicates number of embryos. Early gut(−): control, n = 28; MZT-1, n = 38; GIP-1, n = 21; AIR-1, n = 28; AIR-1*, n = 39; GIP-1;AIR-1, n = 20. Late gut(−): GIP-1, n = 28; AIR-1, n = 22; GIP-1;AIR-1, n = 18. GFP, Green Fluorescent Protein; ZF, zinc finger domain 1; γ-TuRC, γ-tubulin ring complex.
Fig 3.
W03G9.8 is a MZT1 ortholog but, unlike GIP-1, is not required for γ-TuRC localization to the apical ncMTOC.
(A) Alignment of W03G9.8 (renamed MZT-1) with Caenorhabditis, Drosophila, Arabidopsis, human and Schizosaccharomyces pombe orthologs. Sequences are also in S1 Text. (B) Projected optical sections (embryos and germline) or a single optical section (seam cells) showing localization of endogenous tagRFP::GIP-1 and ZF::GFP::MZT-1 at centrosomes in the two-cell embryo, the apical ncMTOC of the pharynx and intestinal primordia in a 1.4-fold embryo, the cell junctions of larval seam cells, and the membrane and centrosomes of the adult germline. Scale bar is 10 μm; insets are 2× magnifications with channels shown separately. (C–N) Images are projected optical sections through the midline of live bean-stage embryos. The intestinal primordium is outlined by white dashed lines. Embryos are expressing a GFP-tagged version of the indicated protein from either the endogenous locus (GFP::MZT-1 in C, D; GFP::GIP-1 in F, H), an extrachromosomal array (GFP::GIP-2 in I–K), or a maternally expressed single-copy insertion (TBG-1::GFP in L–N). Control zif-1(−) embryos expressing elt-2p::zif-1 but lacking ZF-tagged CRISPR alleles are shown in (C, F, I, L), GIP-1gut(−) embryos in (D, G, J, M), and MZT-1gut(−) embryos in (E, H, K, N). Apical GFP::MZT-1 was observed in 29/29 control and in 0/29 GIP-1gut(−) embryos; apical GFP::GIP-1 was observed in 17/17 control and 28/28 MZT-1gut(−) embryos; apical GFP::GIP-2 was observed in 37/37 control, 1/24 GIP-1gut(−), and 19/21 MZT-1gut(−) embryos; and apical GFP::TBG-1 was observed in 19/19 control, 3/16 GIP-1gut(−), and 14/15 MZT-1gut(−) embryos. ZF::GFP::MZT-1 depletion is shown in (E), ZF::GFP::GIP-1 depletion is shown in (G), and gut granules are visible in (D). Note that in GIP-1gut(−) and MZT-1gut(−) embryos, the non-degraded ZF::GFP-tagged protein is still present outside of the intestinal primordium. Scale bar is 5 μm. CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; GFP, Green Fluorescent Protein; ncMTOC, non-centrosomal microtubule organizing center; tagRFP, a variant of Red Fluorescent Protein; ZF, zinc finger domain 1; γ-TuRC, γ-tubulin ring complex.
Fig 4.
MZT-1 is required to localize GIP-1 to the centrosome but not to the centriole or apical ncMTOC.
Images are projected optical sections through the intestinal primordium of live zif-1(−) control (A–C) and MZT-1gut(−) (D–F) embryos as they develop from the E8–E16 divisions (A, D) through polarization (B, C, and E, F). The intestinal primordium is outlined by white dashed lines. Embryos express endogenous GFP::GIP-1 (green, single channel shown below each panel) and intestine-specific mCherry::TBA-1/α-tubulin (red). In addition, MZT-1gut(−) embryos have non-degraded ZF::GFP::MZT-1 in non-intestinal cells. Note that the divisions in A are not completely synchronous; anterior (leftmost) cells are in mitosis, while more posterior (rightmost) cells have not yet started to divide. Top inset to the right of A and D shows a metaphase spindle. zif-1(−) control embryos (n = 24 centrosomes) recruit a large amount of GIP-1 to the PCM, as compared to MZT-1gut(−) embryos (n = 18 centrosomes, white joined arrows). MZT-1gut(−) intestinal cells have abnormal numbers of centrioles and multipolar spindles. (B, E) After division, centrosomes shed their PCM (blue joined arrows in bottom inset at left). (C, F) In polarized intestinal primordia, GIP-1 localizes to the apical surfaces at the midline (“M”). Centriole and apical localization of GIP-1 appears unaffected in MZT-1gut(−) embryos (n = 28/28). Scale bar is 5 μm or 2.5 μm in insets. GFP, Green Fluorescent Protein; ncMTOC, non-centrosomal microtubule organizing center; PCM, pericentriolar material; ZF, zinc finger domain 1.
Fig 5.
AIR-1 promotes GIP-1 and TAC-1 localization to centrosomes but not to the apical ncMTOC.
Main images are projected optical sections through a portion of the intestinal primordium of zif-1(−) control (A, D, G, I) and AIR-1gut(−) (B, E, H, J) embryos. Inset images are single optical sections. (A–F) Intestine-specific mCherry::TBA-1/α-tubulin marks active centrosomes (enlarged in inset) in dividing E8–E16 cells. Images show endogenous GFP::GIP-1 (A, B) and transgene-expressed GFP::TAC-1 (D, E). Average centrosomal fluorescence intensity is quantified in (C, F, GFP::GIP-1: control, n = 17; AIR-1gut(−), n = 23 centrosomes; GFP::TAC-1: control, n = 32, AIR-1gut(−), n = 23 centrosomes). Note that AIR-1gut(−) images are uncorrected for the high levels of non-degraded AIR-1::ZF::GFP in tissues neighboring the intestinal primordium (see Materials and methods, S3 Fig), seen here as a green haze and making the significant reduction of GIP-1 and TAC-1 at the centrosome in AIR-1gut(−) cells an underestimate. Asterisks indicate a significant difference from control by two-tailed t test (p < 1 × 10−5). (G–J) Images show GFP::GIP-1 (G, H) and GFP::TAC-1 (I, J) in control (G, I) and AIR-1gut(−) (H, J) E16 polarized intestinal primordia. Apical GFP::GIP-1 was observed in 36/36 control and 47/47 AIR-1gut(−) embryos; apical GFP::TAC-1 was observed in 32/32 control and 40/44 AIR-1gut(−) embryos. Apical localization of GFP::TAC-1 is most evident in later comma-stage embryos (I, J). Arrowheads and “M” indicate the apical midline, and asterisks denote AIR-1::ZF::GFP fluorescence in primordial germ cells (H, J). Scale bar is 5 μm in main panels and 2 μm in insets. a.u., arbitrary unit; GFP, Green Fluorescent Protein; MTOC, microtubule organizing center; ncMTOC, non-centrosomal microtubule organizing center; ZF, zinc finger domain 1.
Fig 6.
Apical microtubules persist following depletion of GIP-1, MZT-1, and/or AIR-1 and other microtubule regulators.
(A) Still images from S1 Movie. Images show the final mitotic divisions before intestinal polarization and the appearance of the apical ncMTOC in a GIP-1gut(−) embryo. Centrosomes (asterisks) and sister cells (double-headed arrows) are indicated. Note that the posterior of this intestinal primordium contains one rather than four cells prior to division, yet apical microtubules still form at the midline (arrowhead and “M”). Time stamp indicates Hours:Minutes:Seconds (B, E, H) Projected 1.5 μm optical sections through the E16 intestinal primordium of embryos, with indicated genotypes expressing intestine-specific (B) or maternally expressed (E, H) mCherry::TBA-1/α-tubulin. (C) Cartoon summarizing measurements for intensity in D, E, I, and for apical enrichment in F, G, and J. (D, E, I) Normalized average TBA-1 pixel intensities along 10-μm line segments drawn across the apical midline of polarized intestinal primordia (n ≥ 10 embryos) for each indicated genotype with early gut(−) (D) and late gut(−) (E), and for control-treated (DMSO) and nocodazole-treated embryos (I). Light shaded regions show standard deviation. zif-1(−) control profiles are included in gray for reference (“C”). (F, G, J) Ratio of apical TBA-1 signal as compared to cytoplasmic levels for each indicated genotype with early gut(−) (F), late gut(−) (G), and nocodazole treatment (J). Each dot represents a single embryo of the indicated genotype and black bars indicate mean values for each genotype. No significant differences are observed between genotypes, except for a significant reduction in ZYG-9gut(−) embryos (p = 0.002) and a significant increase in [GIP-1;AIR-1]late gut(−) (p = 0.004), as compared to control. (H, I, J) Images are 1.5 μm projected optical sections through the E16 intestinal primordium of embryos treated with DMSO (control), or with 10 μg/mL or 30 μg/mL nocodazole (noco(10) and noco(30)) within 1 minute of treatment (T1) and 10 minutes later (T2). mCherry::TBA-1 intensity (I) and apical enrichment (J) are indicated for each case. Each dot represents a single embryo of the indicated genotype and black bars indicate mean values for each genotype. Comparing T1 to T2: DMSO, p = 0.54; noco(10), p = 0.002; noco(30), p = 0.0004. Comparing T2 control to T2 treatment: noco(10), p = 0.0003; noco(30), p = 0.0002. In F, G, and J, *p < 0.01, **p < 0.001 by two-tailed t test. Scale bar is 5 μm for all images. A, AIR-1gut(−); C, zif-1(−) control; G, GIP-1gut(−); GA, [GIP-1; AIR-1]gut(−); GNP, [GIP-1; NOCA-1]gut(−); ptrn-1(0); GZ, [GIP-1; ZYG-9]gut(−); M, MZT-1gut(−); ncMTOC, non-centrosomal microtubule organizing center; T, TPXL-1gut(−); Z, ZYG-9gut(−).
Fig 7.
GIP-1 is required for a subset of dynamic microtubules at the apical ncMTOC.
(A) Projected 1.5 μm optical sections through the E16 intestinal primordium show EBP-2/EB1::GFP localization in live bean-stage embryos of indicated genotypes. Intestinal primordium is outlined by yellow dashed lines; “M” and arrowhead indicate the apical midline. Scale bar is 5 μm. B) Average EBP-2::GFP pixel intensities along line segments drawn through the midline of polarized intestinal primordia (n ≥ 10, see S3 Fig), with abbreviated genotypes indicated (see Fig 6 legend). (C) Apical enrichment of EBP-2::GFP signal. Apical EBP-2::GFP is significantly reduced only in GIP-1gut(−) embryos compared to control (p = 0.022, two-tailed t test), and in [GIP-1;AIR-1]gut(−) compared to AIR-1gut(−) (p = 0.001, two-tailed t test). Apical enrichment of EBP-2::GFP was compared between most genotypes; because of high AIR-1::ZF::GFP background, apical enrichment of EBP-2::GFP in AIR-1gut(−) is only compared to other genotypes that include AIR-1gut(−) (see Materials and methods). (D–F) Rapid time-lapse imaging was used to measure comet speed and number in the intestinal primordia of live embryos of the genotypes indicated. D) Kymographs of EBP-2::GFP comets in indicated structures and stages. Scale bar is 2 seconds (t) by 2 μm (d). (E) Individual dots show total number of comets crossing two 5 μm lines drawn 3 μm from either side of the apical midline in an individual embryo. (F) Average speeds of comets growing from either individual centrosomes (“2-cell”; “E8”) or an apical ncMTOC from a single embryo (“C,” “G,” “A,” “GA”) of the indicated genotype and stage. Total number of comets analyzed per genotype ≥30. Comet speed is significantly increased in “G,” “GA,” and “GNP” compared with control embryos (p < 0.0005, two-tailed t test, see Materials and methods). In (C–F), *p < 0.01, **p < 0.001 by two-tailed t test. (G) Limiting factor model: loss of γ-TuRC leads to fewer growing microtubules, freeing up a limiting factor that promotes faster microtubule growth at remaining dynamic microtubules. A, AIR-1gut(−);C, zif-1(−) control; G, GIP-1gut(−); GA, [GIP-1; AIR-1]gut(−); GFP, Green Fluorescent Protein; GNP, [GIP-1; NOCA-1]gut(−); ptrn-1(0); M, MZT-1gut(−);ncMTOC, non-centrosomal microtubule organizing center; n.s., not significant; ZF, zinc finger domain 1; γ-TuRC, γ-tubulin ring complex.
Table 1.
Caenorhabditis elegans strains used in this study.
Table 2.
Extrachromosomal arrays used in this study.