Contractile ring stability in S. pombe depends on F-BAR protein Cdc15p and Bgs1p transport from the Golgi complex

Abstract

Cdc15p is known to contribute to cytokinesis in fission yeast; however, the protein is not required to assemble the contractile ring of actin and myosin, but it helps to anchor the ring to the plasma membrane. Cdc15p has a lipid-binding F-BAR domain, suggesting that it provides a physical link between the plasma membrane and contractile ring proteins. However, we find that a more important function of Cdc15p during cytokinesis is to help deliver a transmembrane enzyme, Bgs1p (also called Cps1p), from the Golgi apparatus to the plasma membrane, where it appears to anchor the contractile ring. Bgs1p synthesizes the cell wall in the cleavage furrow, but its enzyme activity is not required to anchor the contractile ring. We estimate that ∼ 2,000 Bgs1p molecules are required to anchor the ring. Without Bgs1p anchors, contractile rings slide along the plasma membrane, a phenomenon that depends on an unconventional type II myosin called Myp2p.

Figure 1. Comparison of cytokinesis in three mutant strains

Imaging in all figures was done at 25°C on 25% gelatin pads in EMM5S unless specified otherwise. Time zero in all figures is defined by the separation of the spindle pole bodies. (A) A DIC image of 41xnmt1cdc15 cells depleted of Cdc15p by incubation with 2.8 µM thiamine in EMM5S for 24 h at 25°C and with 2.8 µM thiamine in the gelatin pad. A white arrow highlights a cell with two septa and black arrows highlight branched cells. Scale bar 5 µm. (B) Histogram of the positions of septa measured as the percent offset from the cell center: (white bars) wild type cells (n = 24); (black bars) cells depleted of Cdc15p (n = 41); (dark grey bars) cdc15ΔSH3 cells with Cdc15p lacking the SH3 domain (n = 80); and (light grey bars) Δclp1 cells (n = 31). (C) Time series of fluorescence micrographs (maximum intensity projections of 19 z-slices, 4 min intervals) of cells expressing Rlc1p-tdTomato. First panel, a wild type cell with a centrally located contractile ring that began to constrict at +24 min. Second panel, a cell depleted of Cdc15p with a contractile ring that moved toward the lower pole beginning at +16 min. Third panel, a cdc15ΔSH3 cell with a contractile ring that moved toward the lower pole at +20 min. Fourth panel, a Δclp1 cell with a contractile ring that moved toward the lower pole beginning at time 20 min. Vertical lines at the end of time series mark the lengths of the daughter cells Scale bar 2 µm. (D – F) Time courses of (Ο, ●) appearance of cortical nodes tracked with Rlc1p-tdTomato, (□, ■) completion of contractile rings, (Δ,▲) onset of ring constriction and (▼) ring sliding. (Open symbols) wild type cells (same data used in D-F) and (filled symbols) mutant cells. (D) Wild type (Ο, □, n = 24 and Δ, n = 29) and cells depleted of Cdc15p (●, ■, n = 10, ▲, n = 9, ▼ n = 17). (E) Wild type and cdc15ΔSH3 cells (●, ■, ▼ n = 32; ▲, n = 24). (F) Wild type and Δclp1 cells (●, ■,▲, ▼, n = 34). (G) Time course of contractile ring constriction measured as the circumference of Rlc1ptdTomato fluorescence in (Ο, n = 12) wild type cells, (□, n = 9) cells depleted of Cdc15p, (Δ, n = 9) cdc15ΔSH3 and (●, n = 21) Δclp1 cells.

Figure 2. Recruitment of formin Cdc12p to nodes and contractile rings

(A) Time series of fluorescence micrographs (maximum intensity projections of 20 z-slices, 4 min intervals) of cells expressing Cdc12p-3YFP. Top panel, a wild type cell with a centrally located contractile ring that began to constrict at time 24 min. Middle panel, a cell depleted of Cdc15p with a contractile ring that constricted completely. Bottom panel, a cell depleted of Cdc15p with a contractile ring that moved toward the bottom pole beginning at time 18 min, failed to constrict and then disassembled. Scale bar 2 µm. (B and C) Time courses of the accumulation and loss of Cdc12p-3YFP molecules (mean ± 1 SD) at the cleavage site at 25°C. (black Ο) wild type cells (n = 13). (B) (red □) whole sample of 41xnmt1cdc15 (n = 10) cells depleted of Cdc15p. (C) (blue □ with dashed line) one cell depleted of Cdc15p with a ring that disassembled after failing to constrict and (red □ with dashed line) one Cdc15p depleted cell with a constricting ring.

Figure 3. Bgs1p localization and its role in contractile ring anchoring

(A) Fluorescence micrographs (maximum intensity projections of 18 confocal z-slices) of five different wild type cells expressing (first column) GFP-Bgs1p, (second column) Sec72p-mCherry. The third column is a merge of the two fluorescence images. Fourth column is cross-section views of GFP-Bgs1p in the middle of the top cell and the cleavage furrows of the other cells. Cell 1, interphase; Cell 2, early cytokinesis; Cell 3, late cytokinesis; Cell 4, newly divided daughter cells; Cell 5, daughter cells growing at old ends. (B and C) Time course of cytokinesis tracked with Rlc1p-tdTomato in (open symbols) wild type cells and (filled symbols) temperature sensitive Bgs1p mutant cps1–191. (Ο, ●) appearance of Rlc1p-tdTomato in nodes; (□, ■) completion of the contractile ring; (Δ, ▲) onset of contractile ring constriction; and (▼) onset of ring sliding. (B) Time course of cytokinesis at 25°C (Ο, n = 14; ●, n = 16); (□, n = 14; ■ n = 16); (Δ, n = 9;▲, n = 16) and (▼ n=11). (C) Time course of cytokinesis at 34°C: (Ο, n = 14; ●, n = 13); (□, n = 14; ■ n = 13); (Δ, n = 9;▲, n = 13) and (▼ n=13). (D) Time series of fluorescence micrographs (maximum intensity projection of 18 confocal z-slices, 4 min intervals) at 34°C of a cps1–191 cell expressing Rlc1p-tdTomato and mCherry-Atb2p (tubulin). Spindle microtubules appear at time 0 min. The contractile ring slid toward the lower pole at time 14 min. Scale bar 2 µm.

Figure 4. Bgs1p accumulates slowly around the equator of cdc15 mutant cells

(A) Time series of fluorescence micrographs (maximum intensity projections of 18 confocal z-slices, 2 min intervals) of four strains expressing (upper panels) Rlc1p-tdTomato to mark contractile rings, and (lower panels) GFP-Bgs1p to mark primary septa and Sad1p-GFP to mark spindle pole bodies. Each image is a 40 × 40 pixel ROI of the cell center. First panel, wild type cells; second panel, cdc15ΔSH3 cells; third panel, cells depleted of Cdc15p; and fourth panel, Δclp1 cells. Boxes mark when GFP-Bgs1p formed a ring. (B) Time courses of appearance of Myp2p-YFP in contractile rings and numbers of GFP-Bgs1p molecules in cleavage furrows. (Left Y-axis, filled symbols) Appearance of Myp2p-YFP in contractile rings of (●, n = 17) wild type cells, (■, n = 11) Cdc15p depleted cells, (▲, n = 17) cdc15ΔSH3 cells and (◆, n = 19) Δclp1 cells. (Right Y-axis, open symbols) Average numbers of GFP-Bgs1p molecules around the equators of (Ο, n = 15) wild type cells, (□, n = 12) cells depleted of Cdc15p, (Δ, n = 19) cdc15ΔSH3 cells, (◊, n = 12) Δclp1 cells and (▽, n=12) wild type cells incubated with 320 µM Aculeacin A. GFP-Bgs1p fluorescence intensities were measured in 20-optical sections at 2 min intervals for 2 h, normalized and corrected for background. (C – E) Time courses of (Ο, ●) appearance of Golgi vesicles containing GFP-Bgs1p at the cell center, (□, ■) appearance of GFP-Bgs1p rings and (Δ, ▲) onset of contractile ring constriction tracked with Rlc1p-tdTomato. (Open symbols) all three graphs use the same data for wild type cells (n = 19). (Filled symbols) mutant cells. (C) Wild type and cells depleted of Cdc15p (n = 14). (D) Wild type and cdc15ΔSH3 cells lacking the C-terminal SH3 domain (n = 19). (E) Wild type and Δclp1 cells (n = 13). (F–I) Effects of Aculeacin A on cytokinesis. (F) Time course of cytokinesis in wild type cells expressing Rlc1p-tdTomato, GFP-Bgs1p and Sad1p-GFP preincubated for 15 min with (open symbols) 380 mM dimethyl sulfoxide (DMSO) or (filled symbols) with 320 µM Aculeacin A in DMSO prior to imaging with DMSO or Aculeacin A. Events: (Ο, n = 14; ●, n = 21) appearance of Rlc1p-tdTomato in nodes; (□, n = 14; ■, n=21) contractile ring formation; (◇, n = 15; ◆, n = 21) appearance of a band of GFP-Bgs1p around the equator; and (Δ, n = 14) onset of contractile ring constriction. (G) Time course of contractile ring constriction measured as the circumference of Rlc1p-tdTomato fluorescence in wild type cells (Ο, n = 12) with 380 mM DMSO or (□, n = 21) 320 µM Aculeacin A in DMSO. (H – I) Time series of (upper panel) DIC images and (middle and lower panels) maximum intensity projected fluorescent images of wild type cells expressing GFP-Bgs1p and Sad1p-GFP and Rlc1p-tdTomato showing the formation of (middle panel) a septum and (lower panel) a contractile ring from nodes. (H) Wild type cells treated with 380 mM DMSO. Scale bar 2 µm. (I) Wild type cells treated with 320 µM Aculeacin A in DMSO. Scale bar 2 µm.

Figure 5. Cellular distribution and domain analysis of Cdc15p

(A) Sedimentation velocity ultracentrifugation of homogenates of wild type cells expressing either cis-Golgi marker Anp1p-mCherry, mEGFP-Cdc15p, GFP-Bgs1p, clathrin light chain Clc1p-mCherry or trans-Golgi marker Sec72p-mEGFP. Homogenate samples of 1 ml were centrifuged on 12 ml gradients of 18%–60% sucrose for 140 min at 38,000 rpm at 4°C. Fractions of 250 µl were (fraction 1 is the top of the gradient) analyzed by SDS-PAGE and quantitative immunoblotting: (■) GFP-Bgs1p; (□) Sec72p-mEGFP; (Ο) mEGFP-Cdc15p; (▲) Clc1p-mCherry and (Δ) Anp1p-mCherry. (B–I) Domain analysis using truncation mutations of Cdc15p expressed in cells depleted of Cdc15p: mCherry-Cdc15pMDSH3_298–927 lacking the F-BAR domain; mCherry-Cdc15pFBD_1–297 consisting of only the F-BAR domain; and mCherry-Cdc15pMD_298–868 consisting of only the middle domain and Cdc15pΔSH3-mCherry consisting of the F-BAR domain and the middle domain expressed in wild type cells. (B) Sedimentation velocity ultracentrifugation as in (A) except that fractions were 500 µl. Symbols: (Ο) mCherry-Cdc15p; (□) Cdc15pΔSH3-mCherry; (Δ) mCherry-Cdc15pMDSH3_298–927; (●) mCherry-Cdc15pMD_298–868; and (■) mCherry-Cdc15pFBD_1–297. (C – E) Maximum intensity projections of images of 20 confocal z-slices (Δz = 0. 36) of mCherry fluorescence from truncation mutants of Cdc15p. (C) Cells depleted of Cdc15p and expressing mCherry-Cdc15p-FBD_1–297. White arrows highlight fluorescent bands. Nodes are not visible over the high background fluorescence. (D) Cells depleted of Cdc15p expressing mCherry-Cdc15p-MD_298–868. White arrows highlight broad zones of fluorescence in mitotic cells and arrowheads mark interphase nuclei. (E) Cells expressing Cdc15pΔSH3-mCherry and mEGFP-Bgs1p (not shown). Arrows highlight fluorescence in contractile rings. (F) Time courses of (Ο, ●) onset of contractile ring constriction tracked with Rlc1p-tdTomato, and formation of rings of (□, ■) GFP-Bgs1p, (▲) mCherry-Cdc15p FBD_1–297 and (▼) mCherry-Cdc15p-MD_298–868. (Open symbols) wild type cells (n = 29) and (filled symbols) cells depleted of Cdc15p (●, ■ n = 10, ▲ n = 12 and ▼ n = 11). (G–I) Time series of fluorescence micrographs (maximum intensity projections of 20 z-slices, 2 min intervals) of cells depleted of Cdc15p expressing fluorescent fusion proteins. The contrast was digitally enhanced to visualize the weak signals. Scale bars 2 µm. (G) Sad1p-mEGFP and mCherry-Cdc15p FBD_1–297. (H) (Upper row) Rlc1p-mEGFP and (lower row) Sad1p-mCherry and mCherry-Cdc15p-MD_298–868. (I) (Upper row) mEGFP-Bgs1p and (lower row) mCherry-Cdc15p-MD_298–868.

Figure 6. Sliding of contractile rings in cells depleted of Cdc15p depends on Myp2p

(A) Growth of serial dilutions of wild type and mutant strains Δmyp2, cdc15–140 and Δmyp2 cdc15–140 for 3 d at 25°C, 32°C or 36°C. (B) Time series of fluorescence micrographs (maximum intensity projection of 19 confocal z-slices at 3 min intervals) of a cell expressing Myp2p-YFP and Sad1p-CFP (not shown). (C) Time course of the accumulation and disappearance of Myp2p-YFP molecules (mean numbers) in contractile rings of (Ο, n = 17) wild type cells, (□, n = 11) cells depleted of Cdc15p, (Δ, n = 17) cdc15ΔSH3 cells and (◇, n = 19) Δclp1 cells also expressing Sad1p-CFP. (D) Time series of fluorescence micrographs showing elongated structures containing myosins separating from contractile rings in cdc15 mutants. Images of 20 Z sections were collected every 3 min and reconstructed into sections across the mid plane of the each cell. First row: wild type cells expressing Myp2p-YFP and Sad1p-CFP. Second row: Δclp1 cells expressing Myp2p-YFP and Sad1p-CFP. Third row: cdc15ΔSH3 cells expressing Myp2p-YFP and Sad1p-CFP. Fourth row: cells depleted of Cdc15p expressing Myp2p-YFP and Sad1p-CFP. Fifth row: cells depleted of Cdc15p expressing Rlc1p-TdTomato and Sad1p-GFP. (E) Time series of fluorescence micrographs (maximum intensity projection of 19 confocal z-slices, 8 min intervals) of cells expressing Rlc1p-tdTomato and Sad1p-GFP and released from the S-phase block by hydroxyurea 2 h prior to imaging. (Upper panel) cell depleted of Cdc15p, (lower panel) a cell depleted of Cdc15p and lacking Myp2p. Scale bars 2 µm. (F) Time course of cytokinesis tracked with Rlc1p-tdTomato in (filled symbols) cells depleted of Cdc15p and (open symbols) cells depleted of Cdc15p and lacking Myp2p: (Ο, n = 14) appearance of Rlc1p-tdTomato in nodes; (□, n = 14) contractile ring completed; and (▲, n = 14; Δ, n = 9) onset of contractile ring constriction. Data from every 12^th min was represented.

Figure 7. Cytokinesis defects in Δmyp2 deletion mutants

(A) Time series of fluorescence micrographs of (upper panel) wild type cells and (lower panel) Δmyp2 mutant cells expressing Rlc1p-tdTomato and Sad1p-GFP. These are maximum intensity projections of 19 confocal z-slices at 4 min time intervals. Scale bar 2 µm. (B) Time course of contractile ring constriction measured as the average circumference of Rlc1p-tdTomato contractile rings ±1 SD in (Ο, n = 12) wild type cells and (□, n = 18) Δmyp2 cells. (C) Time course of the ratio between circumference of Rlc1p-tdTomato contractile rings and the circumference of the cleavage furrow (GFP-Bgs1p) in (Ο, n = 8) wild type cells and (●, n = 8) Δmyp2 mutant cells expressing Rlc1p-tdTomato, GFP-Bgs1p and Sad1p-GFP and imaged in 19 z-slices with 0.36 µm spacing every 2 min for 2 h at 25°C. (D) Time series of merged fluorescence micrographs (maximum intensity projection of 19 confocal z-slices, 4 min intervals) of Δmyp2 mutant cells expressing Rlc1p-tdTomato to mark contractile rings, Sad1p-GFP to mark spindle pole bodies and GFP-Bgs1p to mark primary septa (n = 51). Images are the middles (45 × 45 pixels) of cells with the long axis horizontal. The upper panel is representative of 43% of cells with coordinated rings and septa. The middle panel is representative of 33% of cells with the contractile ring and associated Bgs1p displaced to one side of the furrow where the septum grew and closed ahead of the contractile ring. The lower panel is representative of 24% of cells where the septum appeared to close and separate the daughter cells, while the ring regressed. The first image in each time series is at arbitrary time 0 min. The red arrows indicate the first appearance of asymmetry in the GFP-Bgs1p distribution. Scale bar 2 µm.