Fig 1.
The C. reinhardtii genome encodes the proteins required for peptidergic signaling.
A. Potential prepropeptides with predicted prohormone convertase (blue circle) or furin (brown circle) cleavage sites were identified previously [13]; the number that could generate one or more amidated product(s) is indicated by sub-circles. Proteins with potential C-terminal amidation sites were subdivided into those that could be amidated without (pink) or with (red) the participation of a carboxypeptidase B–like enzyme. B. The C. reinhardtii genome encodes 21 subtilisin-like S8 domain-containing proteases that were categorized based on the predicted presence (+) or absence (−) of a signal sequence (Sig) and/or TMH. C. A gene annotation screen identified 146 C. reinhardtii receptors, which were classified into 12 groups on the basis of their putative structure/function. ER, endoplasmic reticulum; GPCR, G protein–coupled receptor; TMH, transmembrane helix; TNF, tumor necrosis factor; TRP, transient receptor potential.
Fig 2.
CrPAM expression varies during the sexual life cycle.
A. Immunoblot of detergent soluble (S) and insoluble (P) fractions of minus and plus vegetative cells (V−, V+), minus and plus resting gametes (G−, G+), and samples taken 10 minutes and 1 hour after mixing minus and plus gametes. Equal amounts of protein (30 μg) were loaded (Coomassie-stained segment is shown) and affinity-purified luminal domain antibody was used to identify CrPAM. B. Quantification of immunoblot data revealed that CrPAM protein levels were significantly higher (*P = 0.018) in plus gametes compared with minus and plus vegetative cells; mean ± SEM (n = 4). C. CrPHM and CrPAL specific activities in detergent soluble fractions used for immunoblot analysis. PAL activity was higher in plus gametes than in minus vegetative cells (*P = 0.011); mean ± SEM (n = 5). D. Maximal projection confocal images of minus and plus vegetative cells and gametes stained with antibodies against acetylated tubulin (Ac tub, red) and the CrPAM luminal domain (CrPAM lu, green). The boxed regions are contrast enhanced and enlarged in the CrPAM channel to reveal the punctate CrPAM staining. Scale bar = 5 μm. E. Quantification of CrPAM immunofluorescence integrated intensity in the cell bodies of vegetative cells and resting gametes. CrPAM intensity was higher in gametes than in vegetative cells (***P < 0.0001; n = 20–24 cells for each cell type; one-way ANOVAs). F. Quantification of the number of PAM-positive puncta in cilia (#, ##, **P < 0.001, ***P < 0.0001; 30–70 cilia were analyzed for each group ± SEM). The underlying numeric data for this figure can be found in S1 Data. PAL, peptidyl-α-hydroxyglycine α-amidating lyase; PAM, peptidylglycine α-amidating monooxygenase; PHM, peptidylglycine α-hydroxylating monooxygenase.
Fig 3.
Biochemical characterization of ectosome-rich pellets.
A. Comparison of ectosome-rich pellets (2 μg protein/lane) prepared from vegetative cells incubated for 4 hours and from gametes mixed and allowed to mate for 1 hour. Proteins were visualized using silver stain. Std, molecular weight markers. B. Comparison of proteins in the mating gamete low-speed pellet (LSP, 2 μg protein), ectosome-rich pellet (E, 2 μg protein), and soluble secretome (S, 40 μL corresponds to approximately 80 μg cell protein); proteins were visualized using silver stain. C. CrPHM and CrPAL activities were measured in the 20,000g supernatant (20Kg sup), ectosome-rich pellet (E), and soluble secretome (S); specific activities are the average of three independent experiments ± SEM (***P < 0.0001; one-way ANOVAs). D. Negatively stained electron microscope image of the ectosome-rich pellet prepared from 1-hour mating medium of mixed minus and plus gametes. E. Histogram illustrating the size distribution of isolated ectosomes (mean ± SEM = 170 ± 14 nm; n = 400). F. Mating ectosome-rich pellets prepared from biological triplicates two independent times were subjected to mass spectrometric analysis. All six samples were separated by SDS-PAGE; after visualization with colloidal Coomassie, each gel lane was sliced into four fragments as shown. Proteins and amidated peptides were identified by mass spectrometric analysis (S2 Table). The underlying numeric data for this figure can be found in S1 Data.
Fig 4.
Proteomic analysis of mating ectosome-rich pellets reveals the presence of peptidergic signaling machinery.
A. Predicted prepropeptides (S7 Table) identified in mating ectosomes were aligned using CLUSTALW. A rooted phylogenetic tree was generated using UPGMA hierarchical clustering; groupings are delineated by colored nodes. The Cre03.g204500 chemotactic peptide precursor and the most abundant amidated product precursor (Cre12.g487700) are indicated in red; the twelve most abundant precursors are shown in blue. B. Transcriptomic analysis of the four subtilisin-like Type II membrane endoproteases identified in mating ectosomes (data from [30]). The underlying numeric data for this figure can be found in S1 Data. C. Electron micrograph of an ectosome budding from a C. reinhardtii cilium. The orientation expected of a Type II membrane protein is illustrated. D. Fifteen of the 146 C. reinhardtii receptors (S1 Table) were identified in mating ectosomes. Asyn, asynchronous; db, dibutyryl; FPKM, fragments per kilobase of transcript per million mapped reads; GPCR, G protein–coupled receptor; Syn, synchronous; TNF, tumor necrosis factor; UPGMA, unweighted paired group method with arithmetic mean; VLE1, vegetative lytic enzyme 1.
Fig 5.
Identification of amidated products in mating ectosome-rich pellets.
A. Sequences of the three amidated tryptic peptides identified are shown in boxes; the amidated residue identified is indicated in green. For each identified amidated tryptic peptide, a schematic diagram of the protein that could have generated it is shown; the sequence of each of these larger proteins (shown in blue) includes a cleavage site and a glycine residue. Thick black lines mark tryptic peptides that were identified throughout the proprotein. The domain organization and potential C-terminal amidation sites of Cre02.g077800 and Cre02.g077850, signal peptide–containing prepropeptide precursors (S7 Table) that resemble amidated protein precursor Cre03.g204500, are shown; neither of these potential amidated peptides was detected. B and C. Total spectral counts (n = 6; ± SEM) for the identified amidated protein precursors and the average number of spectral counts in gel slices encompassing proteins of different apparent molecular weight ranges are indicated. The underlying numeric data for this figure can be found in S1 Data. aa, amino acid.
Fig 6.
GATI-amide acts as a chemotactic modulator for C. reinhardtii gametes.
A. Agarose block assay. CC124 minus gametes were uniformly spread on the surface of a Petri dish containing 1% agar in M-N medium. Agarose blocks containing 10 μM GATI-amide, 10 μM GATI-OH, M-N medium, or M-N medium containing 0.1% DMSO were placed onto the Petri dish; after 30 minutes in the dark, minus gametes had accumulated around the agarose block containing GATI-amide (red arrow). Similar results were obtained in three independent experiments. B. Microfluidic chemotaxis assay. Microfluidic channel slides were used for chemoattractant gradient formation. An image of a gradient of red food dye is shown. For each treatment, the number of cells in regions 0, I, and 2 were counted over time. C. Chemotaxis assay of minus gametes in microchannel slide. The chemotaxis index (CI) was measured over a period of 4 hours in response to 10 μM GATI-amide, GATI-OH, GATI-Gly, M-N medium, and vehicle control. The CI in the presence of GATI-amide was significantly higher at 3 hours (**P < 0.01) and 4 hours (*P < 0.05) compared to 0.1% DMSO. D. Dose dependence of the response of minus gametes to GATI-amide. A 4-hour incubation period was used; 0.2% DMSO was used as the vehicle control to accommodate the use of 20 μM peptide. The CI of 5 μM GATI-amide was significantly higher than the control at 3 hours (**P < 0.01); for 10 μM GATI-amide, the CI was significantly higher at 3 hours (**P < 0.01) and 4 hours (*P < 0.05), while for 20 μM GATI-amide it was significantly higher at 2 hours (*P < 0.05), 3 hours (***P < 0.01), and 4 hours (*P < 0.05). The only CI to ever exceed 1.00 was for GATI-amide. E. Chemotactic response. The response of minus and plus gametes to 10 μM GATI-amide and 0.1% DMSO. The number of cells for each treatment was counted along the entire channel length, and the ratio of the number of cells in each slice/number of cells in slice 0 (nslice X/nslice 0) was plotted for the 4-hour time point. The response of gametic CC124 cells to GATI-amide was significantly (**P < 0.001) different from gametic CC125 cells. Data are the average of three independent experiments ± SEM. F. Trajectory plots. Plots are shown for minus and plus gametes. Data are representative of three independent experiments; all plots shown are from one experiment. G. Population response. The center of mass was calculated for each population from trajectory plots like those shown in F. Results are the average of three independent experiments ± SEM. H. Motile behavior. The swimming velocities of minus and plus gametes were not altered by the presence of the amidated peptide. Results are the average of three independent experiments ± SEM. One-way and two-way ANOVAs were used as appropriate. The underlying numeric data for this figure can be found in S1 Data. GATI-amide, VLYPNDPAAYAAYAPGTGGGATI-amide; M-N medium, gametic medium.
Fig 7.
Release of CrPAM in ciliary ectosomes is developmentally regulated.
A. HAP2 minus and CC125 plus gametes, mating cells (0- or 1-hour), and ectosome-rich pellets prepared from the 1-hour media were analyzed. Equal amounts of protein (30 μg) were fractionated and subjected to immunoblot analysis for ARF1, FMG1, and CrPAM. Quantification of FMG1 and CrPAM protein levels is shown in the lower panels; results are the average of two independent experiments—error bars indicate the range. B. Cell lysates and ectosomes prepared from wild-type CC124−/CC125+ gametes were analyzed as described for panel A. Results are the average of six independent experiments; error bars indicate ± SEM. Asterisks indicate a statistically significant difference between two groups (*P < 0.01). C. and D. Immunoblot analysis of cells and ectosomes harvested from vegetative CC124− (C) and CC125+ (D) cells; quantification of FMG1 and CrPAM levels is shown below. Results are the average of three experiments; error bars indicate ± SEM. CrPAM and FMG1 levels in vegetative ectosomes differed significantly from levels in cells (**P = 0.0065, ***P < 0.0001). E. CrPHM and CrPAL activities were assayed in cells and ectosomes released by vegetative CC124− and CC125+ cells, mating HAP2−/CC125+ cells, and mating CC124−/CC125+ cells. Both activities were significantly higher in ectosomes released by mating gametes (*P < 0.01, **P < 0.001, ***P < 0.0001; one-way ANOVAs). F. Immunogold-electron microscopy negative stain image showing localization of CrPAM on mating ectosomes with antibody against CrPAM luminal domain. Negative control, ectosomes incubated with gold-tagged secondary antibody alone. The underlying numeric data for this figure can be found in S1 Data. ARF1, ADP ribosylation factor 1; FMG1, flagellar membrane glycoprotein 1.
Fig 8.
CrPAM is rapidly released in mating ectosomes.
A. Maximal projection confocal images of minus and plus gametes fixed at the indicated times after mixing; permeabilized cells were stained using antibodies against the luminal domain of CrPAM (green) and Ac tub (red). CrPAM-positive ectosomes are marked by white arrows, and CrPAM-positive vesicles in the cell body are indicated by white arrowheads. B. Quantification of cell body CrPAM staining as a function of time. Data are averages of 28–37 cells ± SEM (*P < 0.01, ***P < 0.0001). C. Cells and ectosome-rich pellets (20 μg protein) prepared 15 and 60 minutes after gametes were mixed were subjected to immunoblot analysis using the CrPAM luminal antibody. D. Data for CrPAM levels in cells and their ectosome-rich pellets 15 and 60 minutes after mixing were quantified. Data are the average of duplicates, with error bars indicating the range. E. Maximal projection confocal images of cells fixed 5 minutes after minus and plus gametes were mixed, showing release of ciliary ectosomes; arrows mark puncta positive for CrPAM (green), FMG1 (red), or both FMG1 and CrPAM (yellow) (see expanded image). Scale bar = 5 μm. F. Surface intensity plots of CrPAM and FMG1 staining along the cilium outlined in panel E. The underlying numeric data for this figure can be found in S1 Data. Ac tub, acetylated tubulin; FMG1, flagellar membrane glycoprotein 1.