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 across the division site along the cell long axis in septated cells as in (A). (C) Time course and (D) quantification (in minutes) of Sec3 and Spn1 localizations and appearance timing (D) at the division site. SPB separation is defined as time 0. (E) 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 h. Yellow boxes, cells without septa; Red boxes, cells with septa. (F) Localization of Spn1 and the contractile-ring marker Rng8 in sec8-1 cells grown at 36°C for 4 h. (G) Spn1 intensities at the division site in WT and sec3-913 cells grown at 36°C for 4 h. Cells were grouped into no septum, forming septum, and closed septum stages. **, P < 0.01; ***, P < 0.001. (H) FRAP analyses (photobleached at time 0) of Spn1 at the division site in WT and sec3-913 cells grown at 36°C for 4 h. Mean ± SD. Bars, 5 μm.

Septin rings recruit or anchor the exocyst complex to the rim of the division plane during late stage of 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. (D and E) 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.

The 3D structural model of predicted interactions between Spn2 and Sec15 generated by AlphaFold. (A)

AlphaFold2_advanced predicted interaction between Spn2 and Sec15 in rank 1 model with pLDDT score of 81.6. (B, C) Inset of enlarged view of the predicted interactions. Spn2 is colored in yellow and Sec15 in magenta, contacts between interface residues with distance <4 Å are colored in cyan in (A, B). Residues in (C) are colored corresponding to their pLDDT scores as indicated in the legends below, contacts between interface residues with distance < 4 Å are colored in red. (D) Residue position scores of five predicted models for Spn2 and Sec15 interactions ranked according to pLDDT scores.

Septins and the exocyst interact physically and directly.

Reciprocal coimmunoprecipitation of Sec15 with Spn2 (A and B) and Spn1 (C and 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. Tagged proteins were detected on iBright Imager. Tubulin was used as a loading control. Asterisk (*) in D marks Spn1-13Myc. The vertical dashed lines mark the positions of protein ladders that was excised out. (E and F) Septins and the exocyst subunits 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 ONPG showing interactions between (E) Sec15 with Spn1, Spn2, and Spn4; and (F) Sec6 with Spn1 and its coil-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.

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). (B, D) 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.

Genetic interactions between septin and exocyst mutations

Genetic interactions between septin and exocyst mutations

.a

Septins are important for proper localization and distribution of secretory vesicles.

(A and B) 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 h. 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. (C and D) Localizations of Rab11 GTPase Ypt3 (C) and 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 full septa. Bars, 500 nm (A, left), 100 nm (A, right), and 5 μm (C and D).

Septins are important for localization and distribution of secretory cargos Bgs1 and Eng1. (A)

Localization (top) and intensity (bottom) of 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 h. ***, 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 the insets. Eng1 intensities (Middle, mean intensities and Right, individual cells) are from line scans at the middle focal plane. Bars, 5 μm (A and C) and 500 nm (B).

S. pombe strains used in this study.

Localization and division site levels of septin and exocyst subunits in mutants; and FRAP analyses of Spn1 and Sec3. (A and B)

Localization of Spn1 in WT and exocyst mutants at 25°C (A) and 4 h at 36°C (B). Arrows indicate cells with mislocalized Spn1 at the center of the division plane. (C and D) Quantifications of Spn1 intensities at the division site in WT and exocyst mutants at 25°C (C) and 4 h 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 h. Time-lapse images show recovery of Spn1 signals over time. Red box marks the region photobleached at time 0. (F and G) Localization of Exo70 (F) and Sec8 (G) in WT and spn1Δ cells. Yellow boxes, cells without a septum; Red boxes, cells with a closed septum. (H) Quantifications of Exo70 (left) and Sec8 (right) intensities at the division site in WT and spn1Δ cells. ***, P < 0.001. (I) FRAP analyses of Sec3 at the division site in WT and spn1Δ cells. Red box marks the region photobleached at time 0. Bars, 5 μm.

Sec3 and Spn1 localization in gef3, rho4, or gef3 rho4 mutants. (A)

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. (B) Spn1 localization in WT and gef3Δ rho4Δ cells. Bars, 5 μm.

The 3D structural models of septin-exocyst interactions generated by AlphaFold. (A, C, E, G, I)

Rank 1 model of AlphaFold2_advanced predicted interaction between Sec15 and Spn1 (A), Sec6 and Spn1 (C), Spn2 and Sec5 (E), Spn4 and Sec15 (G), and Spn4 and Sec3 (I). Septin subunits are colored in yellow and exocyst in magenta, contacts between interface residues with distance < 4 Å are colored in cyan. (B, D, F, H, J) pLDDT scores of five predicted models for Sec15 and Spn1 (B), Sec6 and Spn1 (D), Spn2 and Sec5 (F), Spn4 and Sec15 (H), and Spn4 and Sec3 (J).

Septins and 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. Tagged proteins were detected on iBright Imager. Tubulin was used as a loading control. Asterisk (*) in B marks Spn1-13Myc. The dashed vertical lines mark the positions of protein ladders which was excised out.