Predicted glycosyltransferases promote development and prevent spurious cell clumping in the choanoflagellate S. rosetta
- University of California, Berkeley, United States
- Howard Hughes Medical Institute, University of California, Berkeley, United States
Figures
Figure 1 with 4 supplements
Mutant cells aggregate and fail to form rosettes.
(A) Wild type cells are unicellular or form linear chains in the absence of rosette inducing factors (RIFs) and develop into organized spherical rosettes when cultured with RIFs. Rosettes are …
Figure 1—figure supplement 1
Cell division is not required for clump formation in mutants.
Wild type, Jumble, and Couscous cells were vortexed and diluted to 1 × 105 cells/ml. Either no addition, DMSO, or 250 μM aphidicolin were added to wild type, Jumble, and Couscous. After 24 hr, cells …
Figure 1—figure supplement 2
Class C mutant growth curves.
Mutant and wild type cells were plated at a density of 1 × 104 cells/ml and counted every 12 hr to assess growth. Mean density plotted ±SD (n = 2 biological replicates with three technical …
Figure 1—figure supplement 3
Jumble and Couscous clumps formed in the absence or presence of RIFs are comparable in size.
Jumble and Couscous were cultured for 24 hr, either without RIFs or with RIFs. To perform the clumping assay, cells cultured either with or without RIFs were vortexed and then incubated for 30 min. …
Figure 1—figure supplement 4
Jumble and Couscous cells adhere to wild type cells.
Fluorescent mWasabi expressing wild type cells uninduced or induced to form rosettes by the addition of RIFs were mixed with either Jumble or Couscous cells and imaged after 30 min. Mutant cells …
Figure 2 with 4 supplements
Jumble maps to a predicted glycosyltransferase that localizes to the Golgi apparatus.
(A) Jumble has a predicted transmembrane domain (marked TM) and secondary structure (alpha helices marked by black rectangles). Structural homology algorithms predict that Jumble is structurally …
Figure 2—figure supplement 1
Mapping cross scheme.
Flow chart of the steps used in mapping cross and bulk segregant analysis.
Figure 2—figure supplement 2
Alignment of Jumble homologs and predicted structure.
(A) S. rosetta Jumble amino acid sequence was aligned to the predicted sequences encoded by homologs from nine other choanoflagellate species, first identified by best reciprocal BLAST using the …
Figure 2—figure supplement 3
Alignment of Jumble to fungal homologs.
S. rosetta Jumble protein sequence was aligned to predicted/unannotated protein sequences from four fungal species identified by best reciprocal BLAST: Saitoella complicata (NCBI accession …
Figure 2—figure supplement 4
Ultrastructure of S.rosetta and ER co-localization of Jumblelw1.
(A) A transmission electron micrograph shows the ultrastructure of S. rosetta. The Golgi apparatus has been pseudo-colored pink and labelled. Image provided courtesy of Kent McDonald and adapted …
Figure 3 with 1 supplement
Couscous maps to a predicted mannosyltransferase with a PAN/Apple domain.
(A) Couscous has a predicted signal sequence (S), a PAN/Apple domain (PAN), and a mannosyltransferase domain. The causative lesion is a 1-base pair deletion at nucleotide position 2447 that causes a …
Figure 3—figure supplement 1
Couscous homology and localization.
(A) The predicted mannosyltransferase domain from S. rosetta was aligned to the alpha-mannosyltransferase domain of MNN2 genes from S. cerevisiae (NCBI accession NP_009571.1) and C. albicans (NCBI …
Figure 4 with 3 supplements
Disruption of basal glycosylation patterns in Jumble and Couscous mutants.
FITC-labelled jacalin binds the apical and basal poles of wild type single cells (B) and becomes enriched in the ECM in the center of rosettes (A, B’ boxed region from A). Although FITC-jacalin …
Figure 4—figure supplement 1
Jacalin Western blot in cell lysates.
Whole cell lysates from E. pacifica (co-cultured prey bacteria), wild type S. rosetta, Couscous, and Jumble were probed with jacalin. No clear differences in banding pattern were observed among the S…
Figure 4—figure supplement 2
Transgenic rescue restores jacalin staining at the center of complemented rosettes.
(A–F) Biotinylated-jacalin labelled with streptavidin Alexa Fluor 647 has the same localization pattern in the absence (A, C, E) and in the presence (B, D, F) of RIFs as that observed with …
Figure 4—figure supplement 3
Rosetteless staining in wild type and mutant cells.
Jumble and Couscous cells grown without and with RIFs were stained for Rosetteless (magenta), tubulin (gray), and actin (cyan). In uninduced Jumble and Couscous cells, Rosetteless staining localizes …
Figure 5
Model for promiscuous clumping in rosette defective Class C mutants.
Wild type S. rosetta has a glycosylated basal patch of ECM (red) as marked by the lectin jacalin that becomes enriched during the course of rosette formation. The Rosetteless protein, required for …
Tables
Table 1
Phenotypes of wild type and Class C mutants
https://doi.org/10.7554/eLife.41482.007| Strain | % cells in rosettes | Cell interactions | Successful outcross? | |
|---|---|---|---|---|
| wild type | 87.7 | Non-clumping | Yes | |
| Seafoam | 0 | Clumping | No | |
| Soapsuds | 0 | Clumping | No | |
| Couscous | 0 | Clumping | Yes | |
| Jumble | 0 | Clumping | Yes | |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information | |
|---|---|---|---|---|---|
| Gene (Salpingoeca rosetta) | jumble | NA | GenBank accession EGD72416/NCBI accession XM_004998928; | ||
| Gene (S. rosetta) | couscous | NA | GenBank accession EGD77026/NCBI accession XM_004990809 | ||
| Strain, strain background (S. rosetta) | wt | PMID: 24139741 | ATCC PRA-390; accession number SRX365844 | ||
| Strain, strain background (S. rosetta) | Mapping Strain | PMID: 24139741 | accession numberSRX365839 | ||
| Strain, strain background (S. rosetta) | Jumble | PMID: 25299189 | accession number SRR7866767 | ||
| Strain, strain background (S. rosetta) | Couscous | PMID: 25299189 | accession number SRR7866768 | Previously named Branched | |
| Strain, strain background (S. rosetta) | Seafoam | PMID: 25299189 | accession number SRR8263910 | ||
| Strain, strain background (S. rosetta) | Soapsuds | PMID: 25299189 | accession number SRR8263909 | ||
| Strain, strain background (Algoriphagus macihipongenesis) | Algoriphagus macihipongenesis | PMID: 22368173 | ATCC BAA-2233 | ||
| Strain, strain background (Echinicola pacifica) | Echinicola pacifica | PMID: 16627637 | DSM 19836 | ||
| Strain, strain background (Vibrio fishceri) | Vibrio fishceri ES114 | PMID: 15703294 | ATCC 700601 | ||
| Antibody | anti-Rosetteless | PMID: 25299189 | (1:400) | ||
| Recombinant DNA reagent | mCherry plasma membrane marker | PMID: 30281390 | RRID:Addgene_109094; Addgene ID NK624 | ||
| Recombinant DNA reagent | mCherry ER marker | PMID: 30281390 | RRID:Addgene_109096; Addgene ID NK644 | ||
| Recombinant DNA reagent | pEFl5'-Actin3'::jumble-mWasabi | this paper | Addgene ID NK690 | pUC19 backbone with 5’ S. rosetta elongation factor L (efl) promoter, jumble, mWasabi, and 3’ UTR from actin; assembled by Gibson assembly | |
| Recombinant DNA reagent | pEFl5'-Actin3'::jumblelw1-mWasabi | this paper | Addgene ID NK691 | pUC19 backbone with 5’S. rosetta elongation factor L (efl) promoter, jumblelw1, mWasabi, and 3’ UTR from actin; assembled by Gibson assembly | |
| Recombinant DNA reagent | pEFl5'-Actin3'::couscous-mWasabi | this paper | Addgene ID NK692 | pUC19 backbone with 5’ S. rosetta elongation factor L (efl) promoter, couscous, mWasabi, and 3’ UTR from actin; assembled by Gibson assembly | |
| Recombinant DNA reagent | pEFl5'-Actin3'::pac-P2A-mTFP | this paper | Addgene ID NK676 | pUC19 backbone with 5’ S. rosetta elongation factor L (efl) promoter, S. rosetta codon optimized puromycin resistance gene (pac), mTFP, and 3’ UTR from actin; assembled by Gibson assembly | |
| Recombinant DNA reagent | pEFl5'-Actin3'::pac-P2A-jumble-mTFP | this paper | Addgene ID NK694 | Parent vector: pEFl5'- Actin3'::pac-P2A-mTFP; jumble inserted using Gibson assembly | |
| Recombinant DNA reagent | pEFl5'-Actin3'::pac-P2A-jumblelw1-mTFP | this paper | Addgene ID NK695 | Parent vector: pEFl5'- Actin3'::pac-P2A-mTFP; jumblelw1 inserted using Gibson assembly | |
| Recombinant DNA reagent | pEFl5'-Actin3'::pac-P2A-mTFP-jumble | this paper | Addgene ID NK696 | Parent vector: pEFl5'- Actin3'::pac-P2A-mTFP; jumble inserted using Gibson assembly | |
| Recombinant DNA reagent | pEFl5'-Actin3'::pac-P2A-mTFP-jumblelw1 | this paper | Addgene ID NK697 | Parent vector: pEFl5'- Actin3'::pac-P2A-mTFP; jumblelw1 inserted using Gibson assembly | |
| Recombinant DNA reagent | pEFl5'-Actin3'::pac-P2A-couscous-mTFP, | this paper | Addgene ID NK698 | Parent vector: pEFl5'- Actin3'::pac-P2A-mTFP; couscous inserted using Gibson assembly | |
| Recombinant DNA reagent | pEFl5'-Actin3'::pac-P2A-couscouslw1-mTFP | this paper | Addgene ID NK699 | Parent vector: pEFl5'- Actin3'::pac-P2A-mTFP; couscouslw1 inserted using Gibson assembly | |
| Recombinant DNA reagent | pEFl5'-Actin3'::pac-P2A-mTFP-couscous | this paper | Addgene ID NK700 | Parent vector: pEFl5'-Actin3':: pac-P2A-mTFP; couscous inserted using Gibson assembly | |
| Recombinant DNA reagent | pEFl5'-Actin3'::pac-P2A-mTFP-couscouslw1 | this paper | Addgene ID NK701 | Parent vector: pEFl5'-Actin3':: pac-P2A-mTFP; couscouslw1 inserted using Gibson assembly | |
| Other | FITC-labelled jacalin | Vector Labs | RRID:AB_2336460; Vector Labs: Cat. No.FLK-4100 | (1:400) | |
| Other | biotinylated jacalin | Vector Labs | RRID:AB_2336541; Vector Labs: Cat. No. B-1155 | ||
| Other | Streptavidin Alexa Fluor 647 conjugate | Thermo Fisher Scientific | Thermo Fisher Scientific: Cat. No. 32357 | ||
Additional files
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Supplementary file 1
Supplementary tables.
(1) Table S1. Phenotypic classes of mutants isolated in this study and in the Levin et al. (2014) screen. (2) Table S2. Segregating variants in Rosetteless mapping cross. (3) Table S3. Segregating variants in Jumble mapping cross. (4) Table S4. Segregating variants in Couscous mapping cross. (5) Table S5. Fluorescent lectins tested.
- https://doi.org/10.7554/eLife.41482.020
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Transparent reporting form
- https://doi.org/10.7554/eLife.41482.021
