Fig 1.
Exocytosis is important for cytokinesis, and vesicles are delivered to the division site during contractile-ring maturation.
(A) Time course in min (middle focal plane; left panels) and quantification (right panel) of ring constriction and membrane invagination in cells expressing GFP-Psy1 and Rlc1-tdTomato (strain JW2402, see S1 Table for details) treated with either EtOH or BFA (see Materials and Methods). Time 0 marks the start of the movie. *, p < 0.05 compared with the control from two-tailed t test in this and other graphs. (B) Psy1 (green) concentrates to the medial cortex during ring (red) maturation, during which the diameter of a compact ring has no changes (indicated by arrowheads, compared to the cell in ring-assembly stage marked by an arrow). (C) Time course of the appearance and localization (arrowheads) of the v-SNARE Syb1 at the division site (sum projection) during ring maturation. (D) Time course (sum projection) and (E) plot (of sum intensity) of accumulation of β-glucan synthase Bgs1 at the division site during ring maturation. Arrowheads mark division-site localization and arrows mark Bgs1 vesicles in (D). Intensity increase rate (mean ± standard deviation [SD]) in min is indicated (n = 3 cells). (F) Histogram showing the intensity of single Bgs1 vesicles imaged with the same setting as for the cells in (D, E). Mean ± SD of the first peak (possibly a single vesicle) after fitting with a Gaussian distribution is shown. (G) Time course showing localization of the exocyst subunit Sec3 to the division site during ring maturation (marks with Rlc1 in the same cell) in the middle focal plane. The cell boundaries are marked with broken lines in this and some other figures. Bars, 5 μm.
Fig 2.
Vesicles and membrane are delivered to the cleavage furrow evenly during ring constriction.
(A) Sec3 labeled exocysts stay at the rim of the division plane during ring constriction. The vertical views are projections from z-stacks spaced at 0.2 μm. (B, C) An Syb1 vesicle is deposited just behind the ring at the division plane in the middle focal plane. The traveling vesicles are marked by arrowheads. (C) Enlarged images for boxed region in (B). (D, E) Trafficking of Syb1 vesicles to the division site in a single focal plane after photobleaching Syb1 signal within the white-boxed region in a cell with a partial septa (DIC) and a constricting Rlc1 ring. Two examples of traveling vesicles from the same cell are marked with arrowheads. The yellow arrowheads mark a vesicle remaining still for 1.5 s before signal spreading out, suggesting vesicle tethering/fusion. (E) Enlarged red- and blue-boxed regions in (D). (F) Distribution of final tractable deposition sites of Syb1 vesicles relative to the position of constricting ring (at the beginning of the movie) from 7 cells (color coded) in 2-min movies after Syb1 signal at the division site was bleached as in (D). X- and y-axes are along the division site and cell long axis, respectively. The ring position (the diameter marked by the color lines) is displaced along the y-axis away from its real position at y = 0 for clarity. (G) Numbers of tractable Syb1 vesicles per min at the middle focal plane when the division site Syb1 signal is unperturbed or bleached. (H) EM images (left) and diameters (right) of secretory vesicles (arrows) in wt and sec8-1 mutant. (I-K) Dynamics of Psy1 on the plasma membrane revealed by FRAP. Red boxes mark the bleached regions, which were bleached at time 0. (I) Psy1 dynamics at the non-growing side of an interphase cell (left panel) or the division site of a cell with constricting ring (right panel), which is marked by Rlc1 taken at -1 min. (J) Recovery of Psy1 intensity after photobleaching at time 0. Mean ± standard error of the mean (SEM) is plotted. (K) The center of the bleached region (arrows) at the division site (enlarged on the right) does not move in any directions in cells with a constricting ring. Bars, 5 μm.
Fig 3.
Actin filaments nucleated by the formin For3 and myosin-V motor Myo52 are important for vesicle delivery to the division site.
(A) Syb1 localization at the division site does not depend on microtubules revealed by MBC treatment. (B) Syb1 localization at the division site depends on For3 and Myo52. Yellow arrowheads mark examples of greatly reduced Syb1 signal at the division site in mutant cells compared with wt cells (red arrowheads). (C, D) For3 and Myo52 localize to the whole cleavage furrow as a washer or disk during ring constriction (labeled with myosin-II heavy chain Myo2 or light chain Rlc1). Localization of For3 or Myo52 in the middle focal plane, maximum-intensity projection of a z-stack spaced at 0.2 μm, or vertical view of the red-boxed regions of the same cell (and 4 min later for For3) or from another two Myo52 cells with different extent of ring constriction (blue boxes). Bars, 5 μm.
Fig 4.
Sites of endocytosis at different stages of cytokinesis.
Formation of endocytic patches labeled with fimbrin Fim1 at the division site during ring (Rlc1) maturation (A–C), ring constriction (D–F), and septum maturation (G–H). (A, D, G) Montages showing endocytic patch assembly (arrowheads) viewed in the middle focal plane. (B, E) Sum intensity projection of 498 Fim1 images in the middle focal plane in a 2-min movie along with Rlc1, Fim1, and DIC images at time 0. The right panel in E: line scans (color-coded) of Fim1 intensity as marked on the sum image to the left. (C, F, H) Distribution of endocytic patch assembly sites from multiple cells (color-coded) during ring maturation, ring constriction, and septum maturation. The dashed line at y = 0 marks the division site. (F) The ring (the diameter marked by the color lines) is displaced along the y-axis away from its real position at y = 0 for clarity. (I) Numbers of assembled Fim1 patches in the middle focal plane per minute at different stages of cell division. *, p < 0.05 compared with ring maturation stage. Bars, 5 μm.
Fig 5.
The TRAPP-II complex colocalizes with post-Golgi vesicles and travels to the division site during cytokinesis.
(A) Trs120 localization to cell tips (arrowheads) and division site (arrow) during the cell cycle marked with spindle pole body protein Sad1. Images of DIC, max intensity projection, and middle focal plane of cells are shown along with vertical views on the right of the regions marked by short red lines on the numbered cells. (B) Trs120 colocalizes with Sec72 and Bgs4 (examples marked by white arrows) but not with Anp1 labeled cis-Golgi structures. Red arrows point out the division-site signal of Trs120. (C) Trs120 colocalizes with Syb1 labeled vesicles/compartments (arrows) and travels to the division site with Syb1 (arrowheads) during cytokinesis. (D) A Trs120 punctum travels with Bgs4 to the cell tip in an interphase cell. (E) Percentage of Trs120 puncta containing Anp1, Sec72, or Syb1. (F) Trs120 accumulation at the division site (arrows) is dramatically reduced or undetectable in for3Δ. Quantification of septating cells (with partially or fully formed septa) with detectable Trs120 concentration at the division site is shown on the right. (G) Trs120 puncta (arrowheads) move to the division site during ring maturation (left) and constriction (right). The dashed horizontal line is to aid tracking Trs120. (H) Distribution of final tractable docking sites of Trs120 labeled puncta in 2-min movies during ring constriction (top) and septum maturation (bottom). The data are plotted as in Fig 4F and 4H. Bars, 5 μm.
Fig 6.
Phenotypes of TRAPP-II mutants and synthetic interactions between TRAPP-II and exocyst mutations.
(A, B) Time courses (A) and quantification (B) of the contractile-ring assembly (from the appearance of cytokinesis nodes to formation of a compact ring without lagging nodes) and constriction (from the start of ring contraction until it constricts to a dot with the highest Rlc1 intensity) in wt and trs120Δ cells observed with tetrad fluorescence microscopy. (B) The rings of ~50% trs120Δ cells did not or only partially constricted during the 14-h movies, so the quantification includes only those fully constricted ring. (C) Morphological defects of three trs120 mutants in YE5S medium at 36°C. Wt, trs120-ts1, trs120-M1, and 81nmt1-trs120 cells were cultured at 36°C for 6, 4, 2, and 6 h, respectively. (D, E) Synthetic genetic interactions between TRAPP-II and exocyst mutations. (D) The predicted trs120-M1 sec8-1 mutant (circles) cannot form colonies on YE5S plate at 25°C. (E) DIC images showing the synthetic interaction between trs120-ts1 and sec8-1 mutations at 25°C. (F) Growth of trs120-ts1, exo70Δ, and the double mutant at 32°C on YE5S medium with phloxin B (PB), which accumulates in dead cells [131]. Bars, 5 μm.
Fig 7.
Exocyst and TRAPP-II mutants affect cargo delivery to the division site differently.
(A) Syb1 accumulates at the division site or cell tips in trs120-ts1 and sec8-1 mutants grown in YE5S medium at 36°C for 2 h. (B–D) EM images (B, C) and quantification (D) showing abnormal accumulation of vesicles and other secretory compartments at the division site or cell tips in trs120-M1 or ypt3-i5 cells grown at 36°C for 4 h. (D) Quantification of secretory vesicles or round-shaped vesicle-like structures (diameter <150 nm without engulfed membrane-bound materials) accumulated in each longitude EM thin section. (E) Localization of the glucanase Eng1 to the division site in trs120-M1 and sec8-1 cells. Arrowheads mark the center localization of Eng1 at the division plane in wt cells. Arrows mark the remaining Eng1 at the rim in trs120-M1. Vertical views of the boxed regions are shown at the bottom corner. (F, G) Localization of glucan synthase Bgs1 in the middle focal plane at the division site in trs120-M1 and sec8-1 cells during septum maturation. (G) Line scans of Bgs1 intensity along the division plane (marked by arrows in F). Top, Mean ± SD from multiple cells. Bottom, individual wt and trs120-M1 cells. Bars in A, E, F, 5 μm.
Fig 8.
Trs120 co-localizes with Rab11 GTPase Ypt3 and the TRAPP-II complex works preferentially with Ypt3 in cytokinesis.
(A, B) Micrographs (left) and quantification (right) of co-localization (arrowheads) of Ypt3 with Trs120 (A, n = 439 puncta) or Rab8 GTPase Ypt2 (B, n = 141 puncta) in the puncta. Best focal planes for the boxed regions are enlarged and shown on the right. (C) Ypt3 puncta travel to the division site during cytokinesis and to cell tips during interphase alone (arrows) or with Ypt2 (boxes) imaged in single focal plane. (D-G) Analyses of trafficking and docking of Ypt3 labeled secretory vesicles/compartments to the division site after photobleaching Ypt3 signal within the boxed region at time 0 as illustrated in (D). (D) Time course of Ypt3 trafficking during ring constriction in a single focal plane. The yellow and red arrowheads mark a vesicle travelling to the leading edge and the rim of the cleavage furrow, respectively. (E) Left, sum projection of the 3-min movie for the cell in (D). Middle panels, sum projection of Ypt3 signal for another cell during septum maturation along with Rlc1 and DIC images at time 0 and Ypt3 images at time -1 and 0 min. Right panels, kymographs along the color-coded lines marked on the sum projections on the left (to avoid blocking Ypt3 signal, the lines were moved upward slightly) showing different docking/tethering times for Ypt3 puncta at the leading edge (or center) or the rim of the division site. Pairs of red and yellow arrowheads mark Ypt3 with long and short dwell time, respectively. (F) Docking/tethering time for Ypt3 puncta at the center or the rim of the division site of 10 cells during late stage of ring constriction or early stage of septum maturation. (G) Distribution of final tractable docking sites of Ypt3 labeled puncta in 3-min movies during ring constriction (left) and septum maturation (right, numbers of vesicles/compartments delivered to each region are shown). The data are plotted as in Fig 4F and 4H. Bars, 5 μm.
Fig 9.
Mislocalized TRAPP-II complex but not Ypt3 can ectopically target vesicle cargos to mitochondria.
(A) Micrographs of cells expressing Tom20-GBP Trs120-3GFP RFP-Bgs4. Colocalization of Trs120 with vesicle cargo Bgs4 on some mitochondrial structure is marked by arrows. (B) Mislocalization of Ypt3 to mitochondrial structure (arrowheads) by Trs120. (C) Mistargeted Ypt3 cannot recruit Bgs4 to mitochondria (arrows), although Ypt3 at its native location still colocalizes with Bgs4 at the division site and cell tips (asterisks). (D) Mistargeted Trs120 does not ectopically target the exocyst to mitochondria (arrows). Bars, 5 μm.
Fig 10.
Mislocalized TRAPP-II complex can ectopically target vesicles and tubulovesicular membrane structures to mitochondria.
(A–D) EM images (A–C) and quantification (D) showing post-Golgi secretory vesicles (arrowheads) or elongated tubulovesicular membrane structures (yellow arrows, maybe specialized late-Golgi cisternae or recycling endosomes) mis-targeted and tethered to mitochondria (M) in cells expressing Tom20-GBP Sec3-GFP (B) or Tom20-GBP Trs120-3GFP (C) but not Tom20-GBP alone (A). Some free vesicles (asterisks) or tubulovesicular structures (red arrows) accumulated in the cytoplasm or near the active growth sites were marked. N = Nucleus, S = septum. (D) Quantification of the mitochondria associated with mis-targeted secretory vesicles or tubulovesicular structures. (E) A working model for the roles of the exocyst and TRAPP-II complexes in vesicle trafficking and membrane deposition at the cleavage furrow during cytokinesis. The events 1–6 are symmetrical at the division site and omitted at some locations for clarity. 1. New membrane is deposited throughout the cleavage furrow. 2. Endocytic vesicles are mostly generated at the rim of the division plane or the adjacent regions and may become tubulovesicular structures like recycling endosomes. 3. Myosin-Vs transport secretory vesicles or recycling endosome equivalents along actin cables to the division plane. 4. Secretory vesicles can also reach the division site by actin-independent random walk. 5. Exocyst complexes localize to the rim of the cleavage furrow and preferentially tether 90-nm secretory vesicles probably through interaction with Rab8 GTPase Ypt2. 6. TRAPP-II complexes localize along the cleavage furrow (slightly biased to the leading edge) to directly tether or indirectly promote the tethering of Rab11 GTPase Ypt3-labeled recycling endosome equivalents or 90-nm secretory vesicles.