Novel LOTUS-domain proteins are organizational hubs that recruit C. elegans Vasa to germ granules
- Center for Genomics and Systems Biology, Department of Biology, New York University, United States
- NYU Abu Dhabi Center for Genomics and Systems Biology, New York University Abu Dhabi, United Arab Emirates
- Goodman Cancer Research Centre and Department of Biochemistry, McGill University, Canada
- Max Delbrück Center for Molecular Medicine, Germany
Figures
Figure 1 with 1 supplement
MIP-1 and MIP-2 are novel LOTUS-domain proteins.
(A) Proteins that co-immunoprecipitate with MEG-3 from embryo extracts include known components of P granules (orange), as well as two previously uncharacterized paralogs (aqua): C38D4.4 (MIP-1) and …
Figure 1—figure supplement 1
Sequence alignments and 3D structural models for MIP LOTUS domains.
(A) Multiple sequence alignment showing conserved positions of predicted LOTUS domains in MIPs together with known LOTUS domains from other species: T. thermophilus B-box zinc finger protein with …
Figure 2 with 2 supplements
Phenotypes caused by reduction of MIP-1 and MIP-2 in the adult germ line.
Quantification and examples of increasing phenotypic severity across P0, F1, and F2 generations upon continuous mip-1(RNAi);mip-2(RNAi) versus L4440(RNAi) controls. (A) Proportion of treated animals …
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Figure 2—source data 1
Phenotypes produced by simultaneous depletion of MIP-1 and MIP-2 by RNAi.
- https://cdn.elifesciences.org/articles/60833/elife-60833-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Schematic of protocol for RNAi treatment and scoring of phenotypes for data in Figure 2 (see also Materials and methods).
Figure 2—figure supplement 2
Double mip null mutants show a similar array of germline phenotypes as double mip RNAi.
(A) Phenotypes observed in the germ line of homozygous mip-1(uae1);mip-2(uae-2) double null mutants cultured at 25°C. Description of phenotypes in Supplementary file 1d (Table S4). (B) DIC and …
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Figure 2—figure supplement 2—source data 1
Double mip null mutants show a mortal germline phenotype.
- https://cdn.elifesciences.org/articles/60833/elife-60833-fig2-figsupp2-data1-v2.xlsx
Figure 3 with 1 supplement
MIP-1 and MIP-2 form granules in the germ cell lineage and germ line throughout development.
Micrographs of fixed samples harboring MIP-1::GFP and MIP-2::mCherry. (A) MIP-1 and MIP-2 colocalize in granular structures in the germline precursor lineage in the embryo; beginning in P4 the …
Figure 3—figure supplement 1
MIP-1 and MIP-2 colocalize in live embryos.
Super-resolution micrographs of MIP-1 and MIP-2 granule segregation in live early embryos. (A) MIP-1::GFP; MIP-2::mCherry embryos from 1 cell to 28 cells. (B) Free-floating granules in the P1 cell …
Figure 4 with 1 supplement
MIP-1 and MIP-2 are P granule components.
(A-B) MIP-1::GFP (green) and PGL-1::mCherry (fuschia) colocalize in (A) embryos and (B) adult germ line. (A) Left: P granules in P2 cell of a live four-cell embryo (anterior to the left). Some …
Figure 4—figure supplement 1
MIP-2 and MEG-3 colocalize throughout the volume of P granules in the early embryo.
Sequential images of Z-stacks through granules in P3 cells labeled with (A) GFP::MEG-3 and (B) MEG-3::Cerulean;MIP-2::mCherry. Settings: (A) 155 nm Z-step, 2.8 µm Z-section; (B) 75 nm Z-step, 1.36 …
Figure 5 with 6 supplements
MIP-1 and MIP-2 are required for assembly of core P granule components.
(A) Simultaneous depletion of MIP-1 and MIP-2 by RNAi affects the normal coalescence of GFP::PGL-3, GLH-1::GFP, and GFP::MEG-3 granules in the embryonic P lineage. Embryos shown are at the four-cell …
Figure 5—figure supplement 1
Localization of MIP-1 and MIP-2 in the adult germ line is not dependent on pgl-1, pgl-3, or meg-3.
Micrographs of MIP-1 and MIP-2 granules in germ lines from live animals. (A) MIP-1::GFP;MIP-2::mCherry germ lines from animals treated with RNAi of pgl-1, pgl-3, meg-3 and control RNAi (L4440). MIP …
Figure 5—video 1
Normal formation of PGL-3 granules is affected in the early embryo when mips are depleted.
Time lapse acquisition of the first two rounds of cell division in embryos from animals carrying GFP::PGL-3 and treated with L4440 control (top) or with mip-1 and mip-2 double RNAi (bottom).
Figure 5—video 2
Normal formation of GLH-1 granules is affected in the early embryo when mips are depleted.
Time lapse acquisition of the first two rounds of cell division in embryos from animals carrying GLH-1::GFP and treated with L4440 control (top) or with mip-1 and mip-2 double RNAi (bottom).
Figure 5—video 3
Normal formation of MEG-3 granules is affected in the early embryo when mips are depleted.
Time lapse acquisition of the first two rounds of cell division in embryos from animals carrying MEG-3::GFP and treated with L4440 control (top) or with mip-1 and mip-2 double RNAi (bottom).
Figure 5—video 4
Normal formation of MIPs granules is affected in the early embryo of a meg-3 null mutant.
Time lapse acquisition of early embryonic development of embryos from animals carrying MIP-1::GFP and MIP-2::mCherry in a WT genetic background (top) and a meg-3 null mutant background (bottom).
Figure 5—video 5
MEG-3 localizes to P granules in the embryo but not in the adult germ line.
Z-stack acquisition through a gonad arm including a few embryos in a strain carrying MEG-3::mCerulean and MIP-2::mCherry.
Figure 6
Biophysical properties of P granule components.
(A) Dissected MIP-1::GFP, MIP-2::GFP, and GLH-1::GFP adult germ lines treated with 5% 1,6-hexanediol (HD) or egg buffer as a control (EB), imaged at the last experimental time point (190 s …
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Figure 6—source data 1
Biophysical properties of MIPs and other germ granule proteins in the germ line and early embryos.
- https://cdn.elifesciences.org/articles/60833/elife-60833-fig6-data1-v2.xlsx
Figure 7
MIP-1 and MIP-2 physically interact.
(A-B) Cartoons indicating prey and bait protein fragments used in co-immunoprecipitation experiments. LOTUS domains are depicted in cyan. (C–E) Co-immunoprecipitation of full-length 6xHis-tagged …
Figure 8
MIP-1 and MIP-2 interact directly with GLH-1, a Vasa helicase ortholog.
(A) 3D structural models showing the overlap between Vasa helicase bound to the Oskar LOTUS domain (gray ribbons) and GLH-1 helicase N-terminal (orange) and C-terminal (salmon) helicase domains …
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Figure 8—source data 1
The mobility of GLH-1 granules depends on the presence of MIP-1 in P3 cells.
- https://cdn.elifesciences.org/articles/60833/elife-60833-fig8-data1-v2.xlsx
Figure 9 with 3 supplements
MIP-1 and MIP-2 have opposing effects on granule growth and distribution in the adult germ line.
(A) Identification of granules in the −3, –4, and −5 oocytes using Imaris image analysis software. Examples of typical micrographs of dissected germ lines used as input for the software are paired …
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Figure 9—source data 1
MIP-1 and MIP-2 affect each other’s localization and granule size in the germline.
- https://cdn.elifesciences.org/articles/60833/elife-60833-fig9-data1-v2.xlsx
Figure 9—figure supplement 1
MIPs affect each other’s condensation in embryos.
(A) MIP-2::mCherry four-cell embryos (left), and MIP-1::GFP four-cell embryos (right). (B) Quantification of MIP granule volume in four-cell embryos comparing mip deletion strains with their …
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Figure 9—figure supplement 1—source data 1
MIP-1 and MIP-2 affect each other’s granule size in early embryos.
- https://cdn.elifesciences.org/articles/60833/elife-60833-fig9-figsupp1-data1-v2.xlsx
Figure 9—video 1
MIP-2 granule formation depends on mip-1 in the early embryo.
Time-lapse acquisition of the first two rounds of cell division in embryos from animals carrying MIP-2::GFP in a WT genetic background (top) and a mip-1 null mutant background (bottom).
Figure 9—video 2
MIP-1 granule formation depends on mip-2 in the early embryo.
Time-lapse acquisition of the first two rounds of cell division in embryos from animals carrying MIP-1::GFP in a WT genetic background (top) and a mip-2 null mutant background (bottom).
Figure 10 with 2 supplements
MIP-1 and MIP-2 are required for proper GLH-1 localization in vivo.
(A-B) Localization of GLH-1::GFP in (A) fixed dissected gonads and (B) live animals of different genetic backgrounds: WT (strain DUP64), mip-1(uae1) null allele (strain GKC551), and mip-2(uae2) null …
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Figure 10—source data 1
GLH-1 localization and granule size in the germline depends on MIP-1 and MIP-2.
- https://cdn.elifesciences.org/articles/60833/elife-60833-fig10-data1-v2.xlsx
Figure 10—figure supplement 1
MIP-1 and MIP-2 are required for the proper localization of PGL-3 granules.
Localization of GFP::PGL-3 in oocytes from adult dissected gonads of different genetic backgrounds: Wild-type strain (JH2017) (top), mip-1(uae1) null (strain GKC525) (middle), and mip-2(uae2) null …
Figure 10—video 1
Localization of GLH-1 granules is affected in germ cells when individual mips are removed.
Z-stack acquisition through a section of the gonad of animals carrying GLH-1::GFP in a WT background (left), a mip-1 null background (middle), or a mip-2 null background (right). Pachytene germ …
Figure 11
Conceptual illustration of MIP-1 and MIP-2 function in the C.elegans germ line.
The MIPs form homo- and heterodimers and bind Vasa helicases through their LOTUS domains to nucleate and scaffold RNP complexes. These associations are likely enhanced by additional interactions …
Additional files
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Supplementary file 1
An Excel file containing multiple tables in different tabs.
(a) Table S1. Proteins enriched in GFP::MEG-3 pulldowns. Proteins are sorted according to their p-value from the t SAM statistic as previously described (Chen et al., 2016). (b) Table S2. Similarity measures between predicted MIP LOTUS domain structures and LOTUS domains from other metazoans. Protein domains: M1, MIP-1; M2, MIP-2; L1, LOTUS1; L2, LOTUS2. SeqID, sequence identity; GDT, Global Distance Test parameters for best templates; PDB ID, protein databank identifier of the best template; RMSD, backbone root mean square distances in Å, with number of aligned residues in parentheses. Published structures used for comparison: D. melanogaster Oskar (PBD ID 5nt7), H. sapiens TDRD5 (PBD ID 3s93). (c) Table S3. Pairwise MIP LOTUS structural similarity analysis. Values shown are backbone rmsd values in Å and number of aligned residues (in parentheses). M1, MIP-1, M2, MIP-2; L1, LOTUS1; L2, LOTUS2. (d) Table S4. Description of MIP depletion phenotypes. (e) Table S5. Pairwise predicted binding affinities between MIP LOTUS domains and between MIP LOTUS domains and GLH-1. Affinities are in kcal/mol. M1, MIP-1; M2, MIP-2; L1, LOTUS1; L2, LOTUS2. Predictions for GLH-1 binding considered only the helicase CTD domain. Predictions for combinations with no values given were highly unfavorable (>0 kcal/mol). Predicted binding affinities for the native Drosophila complexes: Oskar LOTUS homodimer = −54.5 kcal/mol; Oskar LOTUS—Vasa helicase complex = −53.5 kcal/mol. (f) Table S6. Strains produced and used in this study. (g) Table S7. Guide RNA sequences, repair templates, and screening primers for CRISPR strains produced in this study. (h) Table S8. Plasmid DNA constructs for in vitro pulldown experiments.
- https://cdn.elifesciences.org/articles/60833/elife-60833-supp1-v2.xlsx
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Transparent reporting form
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