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First published online 12 August 2008
doi: 10.1242/jcs.030122

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btn1 affects cytokinesis and cell-wall deposition by independent mechanisms, one of which is linked to dysregulation of vacuole pH

¹ MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT
² Department of Genetics, Evolution and Environment, University College London, Gower Street, London WC1E 6BT
³ MRC Cell Biology Unit, Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT
⁴ General and Adolescent Paediatric Unit, UCL Institute of Child Health, University College London, Gower Street, London WC1E 6BT

View larger version (27K): [in this window] [in a new window]   Fig. 1. btn1 cells have an increased septation index compared with wild-type at 25°C, and this is increased even further at 37°C. (A) Graph of septation indices of wild-type 972 and btn1 cells grown at 25°C, or for 18 hours at 37°C. n=300 (three replicates). (B) btn1 cells have multiple septa at 37°C. Calcofluor-stained septa of wild-type 972 and btn1 cells grown for 18 hours at 37°C. Scale bars: 10 µm.

View larger version (78K): [in this window] [in a new window]   Fig. 2. btn1 cells have defects in septa and the cell wall that are worse at 37°C than at 25°C. (A) btn1 cells at 25°C have thicker septa compared with wild-type cells. (i) High-pressure freezing EM of wild-type 972 and btn1 cells, showing a cross-section through the outer third of the division septum (arrowheads indicate `white' primary septum, arrows show outer secondary septum). Scale bars: 100 nm. (ii) Graph of septum thickness in nm (wild-type 972, n=5; btn1, n=6; **P<0.01). (B) btn1 cells at 25°C have thicker cell walls compared with wild-type cells. High-pressure freezing EM of the cell-wall cross-section of wild-type 972 and btn1 cells at 25°C. Scale bars: 100 nm. (C) btn1 cells have thicker septa and cell walls at 37°C compared with wild-type cells. (i) High-pressure freezing EM representative of septated wild-type 972 and btn1 cells after 18 hours at 37°C (arrowheads indicate primary septa; arrows show the secondary septum; L, lipid droplets; V, vacuole; N, nucleus). Scale bars: 2 µm. (ii) Graph of septum thickness in nm (wild-type 972, n=6; btn1, n=6; ***P<0.001). (iii) High-pressure freezing EM representative of interphase wild-type 972 and btn1 cells after 18 hours at 37°C (arrow indicate excess cell-wall deposition). (D) Growth of btn1 cells is rescued by sorbitol. (i) Wild-type 972 and btn1 cells grown on YES agar or YES + 1 M sorbitol agar at 37°C for 3 days. (ii) Phase-contrast images of the indicated cells grown in YES + 1 M sorbitol for 18 hours at 37°C. Scale bars: 10 µm. (E) The septation index of btn1 cells, which is increased at 25°C compared with wild-type cells, is restored to near wild-type levels by growth in sorbitol. Graph of septation indices of wild-type 972 and btn1 cells grown in the indicated medium. n=300 (three replicates).

View larger version (12K): [in this window] [in a new window]   Fig. 3. btn1 cells are sensitive to zymolyase digestion. Zymolyase sensitivity over time of wild-type 972 cells compared with btn1 cells at (A) 25°C and (B) 37°C. Cell lysis was monitored by measuring OD600 nm over time (minutes). Typical results are presented.

View larger version (39K): [in this window] [in a new window]   Fig. 4. Defective vacuole and/or endocytic acidification causes a cell-wall defect. (A) vma1, vma3, btn1and btn1vma1 cells are sensitive to zymolyase. Enzyme sensitivity over time (minutes) of the indicated cells grown at 25°C. Cell lysis was monitored by measuring OD600 nm. (B) Acidic medium rescues zymolyase sensitivity and vacuole pH of btn1 cells. (i) Zymolyase sensitivity of wild-type cells or btn1 cells grown at 25°C in medium at pH 6 or pH 4. (ii) Vacuole pH as monitored by CDCFDA fluorescence. btn1 cells grown at 25°C in a medium of pH 6 are marked by post-staining with DAPI, those grown at pH 4 are unmarked. (C) Growth in acidic medium acidifies the vacuoles but differentially affects the zymolyase sensitivity of btn1vma1 cells and those deleted for vma1 or vma3. (i) Zymolyase sensitivity after 90 minutes and (ii) zymolyase sensitivity over time of indicated cells grown in medium of pH 4 or pH 6 at 25°C. Unpaired Student's t-tests were performed (n=3, *P<0.05, **P<0.01, ***P<0.001). (D) Vacuole pH as monitored by CDCFDA fluorescence of the indicated cells grown in a medium of pH 4 or pH 6 at 25°C. Typical results are presented for A, Bi and Cii. Scale bars: 10 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 5. Defective endocytic acidification does not cause all defects in btn1 cells. (A) Acidic medium does not rescue the cytokinesis defects of btn1 cells. Graph of septation indices of btn1 cells grown at 25°C in YES medium at pH 6, pH 4, or pH 6 + 1 M sorbitol. n=200 for each group (at least two replicates). (B) Acidic medium does not rescue septum thickness. Graph of septum thickness of btn1 cells grown at the indicated pH (n=6 for each) as determined by EM. (C) Acidic medium does not rescue cell death and/or lysis of btn1 cells at 37°C. (i) Phase-contrast images of btn1 cells grown for 18 hours in YES medium at pH 4. (ii) Graph of the number of cell cycles completed by 18 hours by btn1 cells grown in YES medium at pH 6, pH 4, or pH 6 + 1 M sorbitol. n=200 for each group (at least two replicates). (D) vma1 cells grow slowly but do not have cytokinesis defects or a swollen phenotype at 37°C. (i) YES plate showing wild-type, vma1 and btn1vma1 cells grown for 3 days at 37°C. (ii) Phase-contrast images of the same vma1 cells as in Di. (E) btn1vma1 cells have a severe cytokinesis defect. (i) Graph of septation indices of cells grown at the indicated temperatures. n=200 for each group (at least two replicates). (ii) Calcofluor-stained septa of vma1 and btn1vma1 cells grown at 37°C for 4 hours. Note the multiple septa in btn1vma1 cells. Scale bars: 10 µm.

View larger version (61K): [in this window] [in a new window]   Fig. 6. Ectopic expression of Btn1p rescues zymolyase defects and acts as a multicopy suppressor of v-ATPase-null cells. (A) Effect of ectopic expression of GFP-Btn1p, GFP-CLN3 and mutant Btn1p proteins (GFP-Btn1E240K, GFP-Btn1G136A) on the zymolyase sensitivity of btn1 cells grown in MM. Cell lysis was monitored after 90 minutes by measuring absorbance at OD600 nm. Unpaired Student's t-tests were performed (n>3, **P<0.01). (B) Ectopic expression of GFP-Btn1p in vma1 and vma3 cells rescues their sensitivity to zymolyase. Zymolyase sensitivity of the indicated cells (expressing GFP or GFP-Btn1p) after 90 minutes at 25°C. Unpaired Student's t-tests were performed (n=3, *P<0.05). (C) Ectopic expression of GFP-Btn1p in vma1 or vma3 cells reduces their vacuole luminal pH. (i) CDCFDA fluorescence of the indicated cells, expressing either GFP (bottom panel) or GFP-Btn1p (top panel). Images were captured at 0.1 seconds, an exposure at which minimal GFP fluorescence was observed. (ii,iii) Typical fluorescence images of vma3 cells expressing GFP-Btn1p showing (ii) Btn1p localising to cytoplasmic vesicles and the vacuole membrane, but excluded from the vacuole lumen (indicated by arrowheads), and (iii) CDCFDA fluorescence emanating from the vacuole lumen of a single cell from panel Ci. (D) Ectopic expression of GFP-Btn1p in vma1 and vma3 cells rescues their sensitivity to high pH. (i) Ectopic expression of GFP-Btn1p in vma3 cells rescues their defect in vacuole size. Phase-contrast images of vma3 cells expressing GFP or GFP-Btn1p, showing a reduction in vacuole size (arrowheads). (ii) The indicated cells, expressing either GFP or GFP-Btn1p, were grown for 3 days on MM agar plates, pH 6.5, at 25°C. Scale bars: 10 µm.

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