A spatiotemporal reconstruction of the C. elegans pharyngeal cuticle reveals a structure rich in phase-separating proteins
- Department of Molecular Genetics, University of Toronto, Canada
- The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Canada
- Department of Pharmacology and Toxicology, University of Toronto, Canada
- Molecular Medicine Program, The Hospital for Sick Children, Canada
- Wroclaw University of Science and Technology, Faculty of Fundamental Problems of Technology, Department of Biomedical Engineering, Poland
- Department of Cell and Molecular Biology, St. Jude Children’s Research Hospital, United States
- PIT Bioinformatics Group, Institute of Mathematics, Eötvös University, Hungary
- Center for Quantitative Biology, Academy for Advanced Interdisciplinary Studies, Peking University, China
- Department of Computer Science and Technology, Tsinghua University, China
- Department of Biochemistry, University of Toronto, Canada
Figures
Figure 1
The pharyngeal cuticle and surrounding cells.
(A) A schematic of the relative position of the C. elegans pharynx (green). (B) A schematic of the pharynx. The image of the outer cells is transparent, revealing the pharyngeal cuticle underneath. …
Figure 2 with 1 supplement
Pulse-chase and cuticle mutant experiments show dye association with the cuticle.
(A) Schematic showing the pulse-chase assay. Synchronized populations of L3 or adult worms were incubated with a dye for 3 hr (the ‘pulse’), after which worms were washed with M9 and run on normal …
Figure 2—figure supplement 1
The fluorescence and filter controls for dye staining.
Images of young adult wildtype worms incubated with the indicated dyes for 3 hr at the following concentrations: calcofluor white (CFW) (0.01% w/v), eosin Y (EY) (0.15 mg/mL), Congo Red (CR) (0.02% …
Figure 3
Probing pharyngeal cuticle composition with characterized dyes.
(A–D) Images of the buccal and mc1 channel cuticles and surrounding cells. The dyes or GFP-fusion protein examined is indicated. DIC, differential interference contrast; CFW, calcofluor white; ThS, …
Figure 4 with 1 supplement
An informatic reconstruction of the pharyngeal cuticle.
(A) A chart of 2716 genes whose expression oscillates over larval development with a periodicity that corresponds to larval stages. See text for details. Each row represents a single gene. Rows are …
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Figure 4—source data 1
This is the master file with all relevant data for the spatiotemporal map.
- https://cdn.elifesciences.org/articles/79396/elife-79396-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Tissue-enriched expression levels of tissue-enriched classes of genes.
Each plot shows the expression level (as measured in single-cell sequencing analyses; Cao et al., 2017) (y-axes) of the tissue-enriched set of genes in each tissue type (x-axis). Each dot represents …
Figure 5 with 1 supplement
The spatiotemporal map has predictive power.
(A) Average gene expression in each of the indicated tissue types plotted as a function of developmental time. In the first hour of the time course, for example, 219 genes peak in expression and the …
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Figure 5—source data 1
Supporting information for the network diagram in Figure 5C and related insights.
- https://cdn.elifesciences.org/articles/79396/elife-79396-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
The pharynx secretome has a dense protein–protein interaction (PPIs) network relative to other secretomes.
(A) The PPIs among gene products whose corresponding mRNAs are enriched in the indicated tissue (as derived from the L2 single-cell sequencing dataset; Cao et al., 2017) and secreted (as defined by …
Figure 6 with 1 supplement
The localization of six fluorescently tagged pharynx cuticle components.
Each of the six large horizontal boxes contain data about the six predicted gene products indicated on the left. In each box, the four images on the left are of the head of a single worm, imaged …
Figure 6—figure supplement 1
A comparison of the tagged ABU-14 and IDPC-1 localization patterns.
(A) The C. elegans RP3439 trIs113[Pabu-14:abu-14:sfGFP; rol-6(d); unc-119(+)] strain. The lower panels show a magnified view of the areas boxed in the upper lefthand panel to illustrate how the …
Figure 7
The pharynx secretome is enriched with intrinsically disordered proteins with phase separation capability.
(A–E) An analysis of the entire proteome for the indicated properties. The tissue type examined, as well as the number of genes in each bin, is indicated at the bottom of the graph in (E) …
Figure 8
Cuticle proteins can likely phase separate and are enriched with protofilament but not amyloidogenic sequence.
(A) In vitro purified maltose-binding protein (MBP) control (15% Ficoll) or fusions with the FUS-positive control (5% Ficoll) and IDPC-2 (15% Ficoll) phase separate into spheres upon cleaving off …
Figure 9 with 1 supplement
Properties of the low-complexity protein families that are likely secreted into the developing cuticle.
(A) Average percent amino acid composition of the proteins within the indicated tissue type. The percentages along a single row sum to 100. The color scale indicates the range of values within a …
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Figure 9—source data 1
Supporting information for the chart diagram in Figure 9C.
- https://cdn.elifesciences.org/articles/79396/elife-79396-fig9-data1-v2.xlsx
Figure 9—figure supplement 1
Consensus sequence for select low-complexity families.
Sequence logos based on full-length protein sequence alignments (Wozniak and Kotulska, 2015) of the APPGs (12 sequences), IDPAs (3 sequences), IDPBs (5 sequences), and IDPCs (7 sequences). Sequences …
Figure 10 with 2 supplements
Expression of pharynx-enriched genes in distinct cell types.
(A) A UMAP of 1675 pharynx cells modified with permission from Packer et al., 2019's online tool. The clusters are numbered according to Packer et al., 2019. The cell type identities are partially …
Figure 10—figure supplement 1
Identity assignment of the pharynx UMAP clusters.
(A) The cluster map and cluster numbers were derived from Packer et al., 2019 using the single-cell L2 sequencing data from Cao et al., 2017. Note the cropped Y-axis for reasons of graphical …
Figure 10—figure supplement 2
UMAP plots of the gold standard genes used to assign identity to the pharynx UMAP reference cluster.
The UMAP plots of the genes listed in Figure 10—figure supplement 1 were extracted from the Packer et al. dataset (see https://cello.shinyapps.io/celegans_L2/) and show the published markers used to …
Additional files
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Supplementary file 1
Genes of special interest and evidence of pharynx expression.
Note that the data within this table is also available in the Figure 4—source data 1 file so as to put it in context with other data for each gene. Also note that the column indicators below are named after the column in Supplementary File 1. (C) All 78 gene products called out in Figure 4A are shown, along with all 226 genes represented in Figure 4A, in addition to 17 genes that are of special interest (including additional members of the idpp gene class referred to elsewhere in the text). (B) The APPGs that have higher sequence similarity to one another and have more similar temporal expression patterns are described as APPG family (#1) members to distinguish them from more divergent APPGs. (E) The Name Status indicates the 41 new WormBase-approved gene assignments. (H) The indicated hour and degree is with respect to Figure 4A. (J) In some cases, the updated Signal P algorithm will identify a signal peptide when ParaSite did not, as indicated with a ‘no, but likley SS.’ (L) The spatial expression patterns of the indicated clones can be inspected at http://nematode.lab.nig.ac.jp/dbest/srchbyclone.html. A green color indicates confirmation of the expected expression pattern (enriched in pharynx); ‘no signal’ indicates little to no signal anywhere in photo micrographs. In two cases indicated in pink, signal could be observed in the animal, but the pharynx lacked signal. (M, N) The PubMed ID number (PMID) is shown for the publication that provides additional spatial expression information for the gene. The nature of the data is either from a transgene (transg), an antibody (Ab), or is sequence-based (seq). A green color indicates confirmation of the expected expression pattern (enriched in pharynx).
- https://cdn.elifesciences.org/articles/79396/elife-79396-supp1-v2.xlsx
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Supplementary file 2
Transcript levels of six low-complexity protein families within pharynx cells.
The data is extracted from the data presented in Figure 10B.
- https://cdn.elifesciences.org/articles/79396/elife-79396-supp2-v2.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/79396/elife-79396-mdarchecklist1-v2.pdf
