Septins function in exocytosis via physical interactions with the exocyst complex in fission yeast cytokinesis
- Department of Molecular Genetics, The Ohio State University, United States
- Department of Biological Chemistry and Pharmacology, The Ohio State University, United States
Figures
Figure 1 with 4 supplements
Septins and the exocyst colocalize at the division site and septins partially depend on the exocyst for localization.
(A) Co-localization of Spn1-mEGFP and Sec3-tdTomato at the division site in cells without (early) and with (late) septa. Sad1-mRFP1 marks the spindle pole body (SPB). (B) Line scans showing Spn1 and Sec3 intensities at the division site along the cell long axis in septated cells as in (A). (C) SoRa (Super resolution by Optical Re-Assignment) confocal microscopy of cells expressing both Spn1-mEGFP and Exo70-tdTomato showing their perfect colocalization in the middle focal plane. (D) Time course and (E) quantification (in minutes) of Sec3 and Spn1 localizations and appearance timing at the division site. SPB separation is defined as time 0. (F) Localization of Spn1 (Max intensity projection, Middle focal plane, and End-on view of the division site) in WT and sec3-913 cells grown at 36°C for 4 hr. Yellow boxes, cells without septa; red boxes, cells with septa. (G) Localization of Spn1 and the contractile-ring marker Rng8 in sec8-1 cells grown at 36°C for 4 hr. (H) Spn1 intensities at the division site in WT and sec3-913 cells grown at 36°C for 4 hr. Cells were grouped into no septum, forming septum, and closed septum stages. **p < 0.01; ***p < 0.001. (I) FRAP analyses (photobleached at time 0) of Spn1 at the division site in WT and sec3-913 cells grown at 36°C for 4 hr. Mean ± SD. Bars, 5 μm.
Figure 1—figure supplement 1
Localization, intensity, and dynamics of the septin Spn1 in exocyst mutants at the division site.
Localization of Spn1 in WT and exocyst mutants at 25°C (A) and 4 hr at 36°C (B). Arrows indicate cells with mislocalized Spn1 at the center of the division plane. Quantifications of Spn1 intensities at the division site in WT and exocyst mutants at 25°C (C) and 4 hr at 36°C (D). No septum: cells with Spn1 signal at the division site but no septum is visible under DIC; forming septum: septum with a visible gap in the middle; closed septum: no visible gap in the septum. *p < 0.05; **p < 0.01; ***p < 0.001. (E) FRAP analyses of Spn1 at the division site in WT and sec3-913 cells grown at 36°C for 4 hr. Time-lapse images show recovery of Spn1 signals over time. The red box marks the region photobleached at time 0.
Figure 1—video 1
Accumulation of the septin Spn1-mEGFP and the exocyst marked by Exo70-tdTomato to the division site.
The cell (strain JW9170) was imaged on a single-focal plane at the cell surface every 10 s in time-lapse TIRF microscopy (Nikon Ti Microscope). DIC image shows the cell had no septa at the beginning of the movie. Scale bar, 5 μm. Display rate: 10 frames per second (fps).
Figure 1—video 2
Dynamic localization of Exo70-tdTomato at the division site on a single-focal plane close to the cell surface.
The cell (strain JW9170) was imaged without delay (500 ms exposure) in time-lapse TIRF microscopy (Nikon Ti Microscope). DIC image shows the cell had no septa at the beginning of the movie. Scale bar, 5 μm. Display rate: 10 fps.
Figure 1—video 3
Dynamic localization of Exo70-tdTomato at the division site on the middle focal plane.
The cell (strain JW9170) was imaged without delay (500 ms exposure) in time-lapse microscopy (Nikon Ti Microscope). DIC image shows the cell had no septa at the beginning of the movie. Scale bar, 5 μm. Display rate: 5 fps.
Figure 2 with 1 supplement
Septin rings recruit or anchor the exocyst complex to the rim of the division plane during cytokinesis.
(A) Localization of Sec3 at the division site in WT and spn1Δ cells. Yellow boxes, cells without a septum; red boxes, cells with a closed septum. (B) Sec3 intensity at the division site in WT and spn1Δ cells. ***p < 0.001. (C) FRAP analyses of Sec3 at the division site in WT and spn1Δ cells. Mean ± SEM. End-on views (D) and kymographs (E) of Sec8 and the contractile ring marker Rlc1 at the division site in WT and spn1Δ cells. Bars, 5 μm.
Figure 2—figure supplement 1
Localization, intensity, and dynamics of the exocyst subunits in spn1Δ cells at the division site; and Sec3 and Spn1 localization in rho4, gef3, or gef3 rho4 mutants.
Localization of Exo70 (A) and Sec8 (B) in WT and spn1Δ cells. Yellow boxes, cells without a septum; red boxes, cells with a closed septum. (C) Quantifications of Exo70 (left) and Sec8 (right) intensities at the division site in WT and spn1Δ cells. ***p < 0.001. (D) FRAP analyses of Sec3 at the division site in WT and spn1Δ cells. Red box marks the region photobleached at time 0. (E) Spn1 localization in WT and gef3Δ rho4Δ cells. (F) Sec3 localization in WT, rho4Δ, gef3Δ, and gef3Δ rho4Δ cells. Arrowheads mark examples of the cells with mislocalized Sec3 at the center of the division plane in mutant but not WT cells. End-on views of the division plane of cells with a closed septum are shown on the last column. Bars, 5 μm.
Figure 3 with 7 supplements
The 3D structural model of predicted interactions between Spn2 and Sec15 generated by AlphaFold.
(A, B) AlphaFold2_advanced predicted interaction between Spn2 and Sec15 in rank 1 model with predicted local-distance difference test (pLDDT) score of 81.6. The pTM value = 0.51. Spn2 is colored in yellow and Sec15 in magenta. (B) Inset of enlarged view of the predicted interactions, contacts between interface residues with distance <4 Å are colored in red (those in cyan in A). Residues are colored corresponding to their pLDDT scores as indicated in the legends below. (C) Residue position scores of five predicted models for Spn2 and Sec15 interactions ranked according to pLDDT scores. (D) PAE (Predicted Alignment Error) plot for the top-ranked model shown in (A–C), where colors represent confidence in the relative positioning of residues across the two proteins. Lower values (blue) represent high confidence while higher values (red) show low confidence in domain–domain interactions.
Figure 3—figure supplement 1
The 3D structural models of septin–exocyst interactions generated by AlphaFold.
Rank 1 model of AlphaFold2_advanced predicted interaction between Sec15 and Spn1 (A, pTM score = 0.47), Sec6 and Spn1 (C, pTM = 0.45), Spn2 and Sec5 (E, pTM = 0.37), Spn4 and Sec15 (G, pTM = 0.48), and Spn4 and Sec3 (I, pTM = 0.43). Septin subunits are colored in yellow and the exocyst in magenta, contacts between interface residues with distance <4 Å are colored in cyan. Predicted local-distance difference test (pLDDT) scores of five predicted models (left) and the PAE plot of rank 1 model (right) for Sec15 and Spn1 (B), Sec6 and Spn1 (D), Spn2 and Sec5 (F), Spn4 and Sec15 (H), and Spn4 and Sec3 (J).
Figure 3—figure supplement 2
The predicted 3D structural model of S. pombe exocyst complex by AlphaFold3, highlighting the residues that interact with septins (also see Figure 3—video 1).
Individual subunits are colored distinctly and labeled (ipTM: 0.56, pTM: 0.60). Surface-exposed residues previously identified as putative septin-interacting sites are highlighted in yellow. The model demonstrates that ~84% predicted septin-interacting residues are accessible on the outer surface of the assembled complex. As AlphaFold3 limits to fit the whole complex in 5000 tokens, full-length Sec5, Exo70, and Ex84 were used, but only amino acids 1–500 for Sec3, 1–546 for Sec6, 1–865 for Sec8, 1–620 for Sec10, and 1–396 for Sec15 were used. These truncations were selected based on the budding yeast exocyst cryo-EM structure (PDB: 5YFP), which shows that these regions are sufficient for stable inter-subunit interactions and are unlikely to interfere with septin binding based on our modeling.
Figure 3—figure supplement 3
The predicted 3D structural models of S. pombe septin complexes by AlphaFold3, highlighting the exocyst-interacting residues (also see Figure 3—video 2; Figure 3—video 3; Figure 3—video 4).
(A–C) Two subunits of each septin were used to construct the octameric or hexameric complex. Different subunits are colored distinctly and labeled. Surface-exposed residues previously identified as putative exocyst-interacting sites are highlighted in yellow. (A) The octameric model (ipTM: 0.43, pTM: 0.48) of Spn1 to Spn4 demonstrates that ~96% predicted exocyst-interacting residues are accessible on the outer surface of the assembled complex. (B) The hexameric complex of two subunits of each Spn1, Spn2, and Spn4 (ipTM: 0.53, pTM: 0.58) shows 92%, (C) of each Spn1, Spn3, and Spn4 (ipTM: 0.54, pTM: 0.57) shows 86% exocyst-interacting residues are available on outer surface of assembled complex.
Figure 3—video 1
The predicted 3D structural model of S. pombe exocyst complex by AlphaFold3, highlighting the residues that interact with septins.
Individual subunits are colored distinctly and labeled. Surface-exposed residues previously predicted as putative septin-interacting sites are highlighted in yellow.
Figure 3—video 2
The predicted 3D structural model of S. pombe septin octameric complex by AlphaFold3, highlighting the exocyst-interacting residues.
Two subunits of each septin Spn1–Spn4 are used to construct the octameric complex. Different subunits are colored distinctly and labeled. Surface-exposed residues previously predicted as putative exocyst-interacting sites are highlighted in yellow.
Figure 3—video 3
The predicted 3D structural model of S. pombe septins Spn1, Spn2, and Spn4 hexameric complex by AlphaFold3, highlighting the exocyst-interacting residues.
Two subunits of each Spn1, Spn2, and Spn4 are used to construct the hexameric complex. Different subunits are colored distinctly and labeled. Surface-exposed residues previously predicted as putative exocyst-interacting sites are highlighted in yellow.
Figure 3—video 4
The predicted 3D structural model of S. pombe septins Spn1, Spn3, and Spn4 hexameric complex by AlphaFold3, highlighting the exocyst-interacting residues.
Two subunits of each Spn1, Spn3, and Spn4 are used to construct the hexameric complex. Different subunits are colored distinctly and labeled. Surface-exposed residues previously predicted as putative exocyst-interacting sites are highlighted in yellow.
Figure 4 with 1 supplement
Septins and the exocyst interact physically.
Reciprocal co-immunoprecipitation of Sec15 with Spn2 (A, B) and Spn1 (C, D). Septin or exocyst subunits tagged with mEGFP or 13Myc were immunoprecipitated using antibodies against GFP from cell lysates, separated on SDS–PAGE, and incubated with appropriate antibodies. Tubulin was used as a loading control. Asterisk (*) in (D) marks Spn1-13Myc. The vertical dashed lines mark the positions of protein ladders that were excised out. n = 3. (E, F) Septins and the exocyst subunits may interact directly, revealed by the yeast two-hybrid assays. X-gal overlay results (insets on the top of the columns) and quantification of β-galactosidase activities using o-nitrophenyl-β-D-galactopyranoside (ONPG) showing interactions between (E) Sec15 with Spn1, Spn2, and Spn4; and (F) Sec6 with Spn1 and its coiled–coil motif Spn1(300–469). Data is shown in Mean ± SD, n = 3 (in E) or 4 (in F). ***p ≤ 0.0001, **p ≤ 0.001, *p ≤ 0.01 compared with their respective controls in one-way ANOVA with Tukey’s post hoc test.
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Figure 4—source data 1
Raw western blot images unlabeled.
- https://cdn.elifesciences.org/articles/101113/elife-101113-fig4-data1-v1.zip
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Figure 4—source data 2
Raw western blot images labeled.
- https://cdn.elifesciences.org/articles/101113/elife-101113-fig4-data2-v1.zip
Figure 4—figure supplement 1
Septins and the exocyst interact physically.
Reciprocal co-immunoprecipitation between Spn1 with Sec6 (A, B); Spn2 with Sec5 (C, D); Spn4 with Sec15 (E, F); and Spn4 with Sec3 (G, H). Septin or exocyst subunits tagged with mEGFP, GFP, mYFP, or 13Myc were immunoprecipitated, separated on SDS–PAGE, and incubated with appropriate antibodies. Tubulin was used as a loading control. Asterisk (*) in B marks Spn1-13Myc. The dashed vertical lines mark the positions of protein ladders which were excised out. Spn4 and Sec3 may not interact with each other because they only Co-IP in one direction with a weak band. n = 3.
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Figure 4—figure supplement 1—source data 1
Raw western blot images unlabeled.
- https://cdn.elifesciences.org/articles/101113/elife-101113-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
Raw western blot images labeled.
- https://cdn.elifesciences.org/articles/101113/elife-101113-fig4-figsupp1-data2-v1.zip
Figure 5 with 3 supplements
Localization patterns of both Sec15 and Sec5 at the division site depend on septins.
Localization of (A, B) Sec15 and (C, D) Sec5 at the division site in WT and septin mutant cells. Yellow boxes, cells without a septum; red boxes, cells with a closed septum in (A, C). Quantification of cells with intact and mislocalized Sec15 (B) and Sec5 (D) signals in WT and septin mutant cells with obvious septa. Scale bars, 5 µm.
Figure 5—video 1
Localization of Sec15 as a ring at the rim of the division plane during cytokinesis in WT cells.
Sec15-mEGFP cells (strain JW9726) were imaged at 3 min intervals for 2 hr in time-lapse confocal microscopy (UltraVIEW Vox CSUX1; PerkinElmer). 3D projections of fluorescence images from 14 slices spaced at 0.4 µm at each time point are shown. Display rate: 50 fps.
Figure 5—video 2
Mislocalization of Sec15 as a disk at the division site in spn1Δ cells.
sec15mEGFP spn1Δ cells (strain JW9852) were imaged at 3 min interval for 3 hr in time-lapse confocal microscopy (UltraVIEW Vox CSUX1; PerkinElmer). 3D projections of fluorescence images from 14 slices spaced at 0.4 µm at each time point are shown. Display rate: 50 fps.
Figure 5—video 3
Mislocalization of Sec15 as a disk at the division site in spn4Δ cells.
sec15mEGFP spn4D cells (strain JW9853) were imaged at 3 min interval for 3 hr in time-lapse confocal microscopy (UltraVIEW Vox CSUX1; PerkinElmer). 3D projections of fluorescence images from 14 slices spaced at 0.4 µm at each time point are shown. Display rate: 50 fps.
Figure 6
Septins are important for proper localization and distribution of secretory vesicles.
EM thin-section images (A) and quantifications of secretory vesicles (B) in WT, sec8-1, and spn1Δ cells with forming or closed septa. Cells were grown at 36°C for 4 hr. Red boxes indicate the enlarged regions on the right. Arrowheads mark secretory vesicles. *p < 0.05; **p < 0.001; ***p < 0.0001 compared to WT. n = numbers of thin sections. Localizations of the Rab11 GTPase Ypt3 (C) and the v-SNARE Syb1 and Rlc1 (D) in WT and spn1Δ cells. Arrows mark examples of cells with closed septa. Syb1 intensities at the division site (D, right) from line scans at the middle focal plane of cells with closed septa (at the end of ring constriction indicated by an Rlc1 dot at the center of the division plane). Bars, 500 nm (A, left), 100 nm (A, right), and 5 μm (C, D).
Figure 7
Septins are important for localization and distribution of secretory cargos Bgs1 and Eng1.
(A) Localization (top) and intensity (bottom) of the glucan synthase Bgs1 in WT and spn1Δ cells. Arrows mark examples of cells with a closed septum. Bgs1 intensities from line scans across the division site at the middle focal plane were compared in cells with closed septa. (B) EM thin-section images (left) and septum thickness (right) of WT, spn1Δ, and sec8-1 cells with closed septa. Cells were grown at 36°C for 4 hr. ***p < 0.0001 compared to WT. (C) Localization (left) and intensity (middle and right) of Eng1-GFP in WT and spn1Δ cells. The end-on views of Eng1 at the division site in cells with closed septa are shown as insets. Eng1 intensities (middle, mean intensities; and right, individual cells) are from line scans at the middle focal plane. Bars, 5 μm (A, C) and 500 nm (B).
Tables
Table 1
Viability of double mutants of the septin and exocyst from tetrad dissection at 25°C.
| Parent 1 | Parent 2 | Viable double mutants (%) at 25°C* | Total number of tetrads |
|---|---|---|---|
| spn1Δ | sec3-916 | 78 | 14 |
| spn1Δ | sec3-913 | 100 | 13 |
| spn1Δ | sec8-1 | 100 | 12 |
| spn1Δ | exo70Δ | 100 | 14 |
| spn1Δ | trs120-M1 | 100 | 27 |
| spn1Δ | trs120-ts1 | 95 | 18 |
| spn2Δ | sec3-916 | 83 | 10 |
| spn2Δ | sec3-913 | 100 | 10 |
| spn3Δ | sec3-916 | 100 | 12 |
| spn3Δ | sec3-913 | 75 | 11 |
| spn4Δ | sec3-916 | 80 | 10 |
| spn4Δ | sec3-913 | 100 | 14 |
| spn4Δ | sec8-1 | 100 | 11 |
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*
Percentage of viable double mutant colonies after tetrad dissection and grown at 25°C.
Table 2
Genetic interactions between septin and exocyst mutations at various temperatures*.
| Mutations | 25°C | 30°C | 32°C | 36°C |
|---|---|---|---|---|
| sec3-916 | +++† | ++‡ | +§ | -¶ |
| sec3-913 | +++ | +++ | ++ | - |
| sec8-1 | +++ | ++ | + | - |
| spn1Δ | ++ | ++ | ++ | ++ |
| spn1Δ sec3-916 | ++ | + | - | - |
| spn1Δ sec3-913 | ++ | ++ | ++ | - |
| spn1Δ sec8-1 | ++ | ++ | + | - |
| exo70Δ | +++ | +++ | ++ | - |
| spn1Δ exo70Δ | ++ | ++ | ++ | - |
| trs120-M1 | ++ | - | - | - |
| spn1Δ trs120-M1 | ++ | - | - | - |
| trs120-ts1 | +++ | +++ | + | - |
| spn1Δ trs120-ts1 | ++ | ++ | + | - |
| spn2Δ | +++ | +++ | +++ | +++ |
| spn2Δ sec3-916 | ++ | + | + | - |
| spn2Δ sec3-913 | +++ | ++ | + | - |
| spn3Δ | +++ | +++ | +++ | +++ |
| spn3Δ sec3-916 | ++ | ++ | + | - |
| spn3Δ sec3-913 | +++ | ++ | ++ | - |
| spn4Δ | ++ | ++ | ++ | ++ |
| spn4Δ sec3-916 | ++ | + | - | - |
| spn4Δ sec3-913 | ++ | ++ | ++ | - |
| spn4Δ sec8-1 | ++ | ++ | ++ | - |
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*
Cells were freshly grown on YE5S and YE5S + Phloxin B (which accumulates in dead cells) plates before checking the growth and morphology under DIC at different temperatures. The defects in cytokinesis and cell integrity compared with the parent strains were classified as follows:
-
†
+++, comparable to wt.
-
‡
++, some cell lysis or cytokinesis defects.
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§
+, severe cytokinesis defects with reduced growth.
-
¶
-, inviable.
Additional files
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Supplementary file 1
S. pombe strains used in this study.
- https://cdn.elifesciences.org/articles/101113/elife-101113-supp1-v1.docx
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Supplementary file 2
DNA oligos used in this study.
- https://cdn.elifesciences.org/articles/101113/elife-101113-supp2-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/101113/elife-101113-mdarchecklist1-v1.docx
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Septins function in exocytosis via physical interactions with the exocyst complex in fission yeast cytokinesis
eLife 13:RP101113.
https://doi.org/10.7554/eLife.101113.3
