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First published online 25 September 2007
doi: 10.1242/jcs.007567

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Schizosaccharomyces pombe protein phosphatase 1 in mitosis, endocytosis and a partnership with Wsh3/Tea4 to control polarised growth

Isabel Alvarez-Tabarés^1,2,*, Agnes Grallert¹, Jose-Miguel Ortiz² and Iain M. Hagan^1,

¹ CRUK Cell Division Group, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK
² Departamento de Biología Molecular, Facultad de Medicina, Unidad de Biomedicina-CSIC, Universidad de Cantabria, 39011 Santander, Spain

View larger version (65K): [in this window] [in a new window]   Fig. 1. Dis2.NEGFP accumulates in nuclei, dots within the nucleus and at the cell periphery. dis2.NEGFP (IH2908) cells were grown to mid-log phase in EMM2-FS and mounted in EMM2-FS with (B) or without (A,C) 10 µg/ml Hoechst 33342. In A the inset shows Dis2.NEGFP on both sides of a primary septum. In B and C the insets show enlargements of the indicated nuclei. In C the green arrows indicate the accumulation of Dis2.NEGFP in the cytokinetic actin ring during anaphase. See also Movies 1 and 2 in supplementary material. Bars, 5 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 2. Dis2.NEGFP associates with centromeres. dis2.NEGFP pcp1.RFPC (IH2731; A,B) and dis2.NEGFP cnp1.Cherry (IH5283; C,D) cells were processed as for Fig. 1 with the exception that DMSO or DMSO containing CBZ at a final concentration of 25 µg/ml were included in the culture medium for imaging in A and B, respectively. (A,B) The Dis2.NEGFP signal does not colocalise with the Pcp1.RFPC signal of interphase cells. (C,D) The Dis2.NEGFP signal colocalises with the Cnp1.Cherry signal of interphase (C) and mitotic (D) cells. Inset in D shows enlargement of the nucleus marked with the asterisk. (E) For ChIP analysis whole cell extracts (wce) were prepared from the indicated strains for precipitation with magnetic beads to which either no antibody (–) or a rabbit antibody that recognised the Pk epitope (+) had been covalently attached. There was a clear enrichment of the signal arising from PCR with primers to the central but no other sequences in immunoprecipitates from dis2.NPk (lane 5), but not when antibodies were not conjugated to the beads (lane 4) or the strain did not contain the tagged dis2+ gene (lane 2). Bars, 5 µm.

View larger version (58K): [in this window] [in a new window]   Fig. 3. Sds21.NEGFP accumulates in nuclei, dots within the nucleus and at the cell periphery. Cells of the indicated strains were grown as for Fig. 1 with (A-C) or without (D,E) 10 µg/ml Hoechst 33342. In D and E the strains indicated in the central panel were transiently exposed to TRITC-lectin before being mixed and mounted with the deletion strain. (A,B) Sds21.NEGFP accumulates in nucleoli of dis2+ cells (IH2352) and is recruited to all sites normally occupied by Dis2.NEGFP when the dis2+ gene has been deleted (IH2635). (C) The distribution of Dis2.NEGFP is not affected by deletion of sds21+(IH2090). (D) Imaging sds21.NEGFP cells that have been dipped in red lectin (central micrograph) alongside sds21.NEGFP dis2. cells shows an increase in the intensity of the Sds21.NEGFP signal upon deletion of dis2+. (E) Imaging dis2.NEGFP cells that have been dipped in red lectin (central micrograph) alongside dis2.NEGFP sds21. cells shows no increase in the intensity of the Dis2.NEGFP signal upon deletion of sds21+. (F) Western blotting with antibodies to GFP establishes that the increase in Sds21.NEGFP signal intensity in dis2. arises from an increase in protein levels. A-E. Bar, 5 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 4. Dis2.NEGFP recruitment to cell tips requires microtubules. dis2.NEGFP (IH2908; A) and a mix of dis2.NEGFP (IH2908) and nmt81.GFP.atb2 (IH1741) cells (B-D) were processed as for Fig. 1 with the exception that DMSO containing CBZ, to a final concentration of 25 µg/ml, was included in the culture medium for mounting in C and D and an equivalent amount of DMSO was added to the culture in B. Whereas Dis2.NEGFP distribution was not affected by the addition of DMSO (B), disruption of the microtubule cytoskeleton (verified by the lack of any microtubule signals in the control nmt81.GFP.atb2 cells, asterisks) abolished the association of Dis2.NEGFP with cell tips (compare inset in B to that in D). See also Movies 3 and 4 in supplementary material. The Dis2.NEGFP dots that are seen away from the cell tip were not affected by microtubule depolymerisation. Bar, 5 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 5. Dis2.NEGFP in endocytosis. dis2.NEGFP (IH2908; A), dis2.NEGFP sla2.Cherry (IH5205; B), dis2.NEGFP sla2. (IH4722; C), a mix of dis2.NEGFP (IH2908) and crn1.GFP (IH3528) cells (D,E) and a mix of dis2.NEGFP sla2.Cherry (IH5205) and act1.GFP (IH4266) cells (F) were processed as for Fig. 1 with the exception that DMSO containing Lat A, to a final concentration of 50 µM, was included in the culture medium for mounting in E and F and an equivalent amount of DMSO added to the culture in D. (A) Kymograph of the area between the green lines shows the internalisation movement of Dis2.NEGFP. (B) Kymograph of the area in the rectangles shows the colocalisation of Dis2.NEGFP and Sla2.Cherry. Note that Sla2.Cherry signal appears at the cell cortex before Dis2.NEGFP (C) Kymograph showing that the internalisation of Dis2.NEGFP is avoided in sla2. cells grown at 33°C. (D,E) Whereas Dis2.NEGFP distribution was not affected by the addition of DMSO (D), disruption of the actin cytoskeleton (verified by the lack of any Crn1.GFP signals in the control crn1.GFP cells – asterisks) severely reduced the number of Dis2.NEGFP dots seen away from the cell tip, but did not affect the association of Dis2.NEGFP with cell tips (compare inset in D and E). Bars, 5 µm. (F) Kymograph showing that internalisation of Dis2.NEGFP and Sla2.CH foci was abolished by depolymerisation of the F-actin cytoskeleton.

View larger version (112K): [in this window] [in a new window]   Fig. 6. Dis2.NEGFP associates with both growing and non-growing tips. dis2.NEGFP (IH2908; A,B), dis2.NEGFP cdc10.129 (IH2345; C) and dis2.NEGFP cdc25.22 (IH2356; D) cells were processed as for Fig. 1. (A) Prior to mounting, dis2.NEGFP cells were re-suspended in TRITC-lectin so that the entire cell surface became fluorescent. Washing this lectin out by repeated changes of medium and subsequent growth resulted in dark non-stained tips, identifying those tips that were actively growing and those that were not (stained tips). (B) Dis2.NEGFP foci associated with the cell tips during interphase, mitosis and immediately after cytokinesis. Note the lower Dis2.NEGFP signal intensity at the tips of mitotic cells. (C,D) Dis2.NEGFP associated with both cell tips in cells that are arrested before NETO at START (C) and after NETO at the G2-M boundary (D). Bars, 5 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 7. Abolishing Tea1, Tea2 and Mal3 function abolishes Dis2.NEGFP tip association. Cells of the indicated strains were processed as described for Fig. 1. (A-C) Deletion of pom1+(IH2349; A), bud6+ (IH3092; B) or tea3+ (IH3202; C) did not affect Dis2.NEGFP association with the cell tips. (D-G) Deletion of tea1+ (IH3201; E), mal3+ (IH3155; F) or tip1+ (IH3757; G), and the presence of tea2.1 (IH2859) grown at 36°C (D) abolished the association of Dis2.NEGFP with cell tips. (H) Quantification of Dis2.NEGFP cap formation. Bars, 5 µm.

View larger version (58K): [in this window] [in a new window]   Fig. 8. Transit through NETO and Tea1 function control DEAF distribution. (A) dis2.NEGFP tea1 cells (IH3210) were processed as in Fig. 6A. Deletion of tea1+ resulted in a polarised distribution of DEAFs towards the tip, at which new cell wall had been added above the TRITC-lectin coating, indicating that it was the growing tip. Bar, 5 µm. (B) Quantification of DEAF distribution in the indicated strains shows that Tea1 is required to ensure that endocytosis occurs to a similar degree on both sides of the interphase nucleus. The figures on the y axis are a ratio of the number of dots seen on one side of the nucleus against those on the other.

View larger version (43K): [in this window] [in a new window]   Fig. 9. Wsh3/Tea4 recruits Dis2.NEGFP to cell tips. (A) dis2.NEGFP wsh3+ (IH2089) cells were dipped in red lectin and mounted alongside dis2.NEGFP wsh3. (IH5722) cells. Continuous imaging of GFP fluorescence in three consecutive z slices (compressed into a single maximum projection) showed that the Dis2.NEGFP cap structure seen at the end of dis2.NEGFP wsh3+ cells was absent when wsh3+ was deleted (three left panels, see Movie 5 in supplementary material). Depolymerisation of microtubules by the addition of 25 µg/ml CBZ abolished Dis2.NEGFP cap signals in both the wsh3+ and the wsh3. backgrounds (right panel). (B) Consecutive green and red images of dis2.NEGFP wsh3.C2tdTom (IH5726) cells were continuously taken in three consecutive z stacks to reveal the dynamics of the association of Dis2.NEGFP with Wsh3.C2tdTom at cell tips. See Movie 6 in supplementary material. Bars, 5 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 10. Recruitment of PP1 to cell tips by Wsh3/Tea4 modulates cell polarity without affecting the recruitment of Wsh3/Tea4 or Tea1. (A) Epitope-tagged wsh3 genes were induced by derepression of a leu1::nmt41wsh3.PkCX transgene in a wsh3. background. Extracts were prepared and Pk immunoprecipitates were isolated and blotted with antibodies to recognise the Pk or GFP epitopes. Lane 1: wsh3. leu1::nmt41wsh3.PkC dis2+(IH5740); 2: wsh3. leu1+ dis2.NEGFP (IH5722); 3: wsh3. leu1::nmt41wsh3.PkC dis2.NEGFP (IH5742); 4: wsh3.leu1::nmt41wsh3.PkC.V223A dis2.NEGFP (IH5743); 5: wsh3.leu1::nmt41wsh3.PkC.F225A dis2.NEGFP (IH5744). (B) wsh3+, wsh3.V223A or wsh3.F225A genes were integrated under the control of the nmt41 promoter at the leu1 locus. Cells were grown to early log phase in the presence of 20 µM thiamine to repress the expression of the transgene. At time 0 all three cultures were then washed three times in thiamine-free medium to relieve the repression of the transgenes. Cells were then imaged over the indicated time frames after induction to identify Dis2.NEGFP (three left panels) and stained with calcofluor white 20 hours after induction (right panel). Dis2.NEGFP failed to associate with cell tips of any strain when the expression was fully repressed at time 0. Induction of wild-type wsh3+ gene for 12 hours resulted in recruitment of Dis2.NEGFP to cell tips, whereas similar induction of Wsh3.V223A and Wsh3.F225A did not. The enhanced protein levels 20 hours after induction (see supplementary material Fig. S3) led to complete delocalisation of Dis2.NEGFP and alteration of cell morphology. This enhanced induction of Wsh3 sequestered Dis2.NEGFP away from the nucleus. No adverse effects upon cell morphology or Dis2.NEGFP distribution were apparent when either mutant allele was induced for 20 hours despite the fact that the mutant proteins accumulated to the same degree as the wild type Wsh3 molecules (see supplementary material Fig. S3). (C) Anti-Pk immunofluorescence of cells in which Pk-tagged wild-type or mutant Wsh3 had been induced for 12 hours in leu1::nmt41wsh3.PkCX wsh3. (IH 5742, 5743, 5744) cells. (D) wsh3. leu1::nmt41wsh3.X cells (IH 5737, 5738, 5739) were grown in the presence of the indicated levels of thiamine, subjected to osmotic shock by the addition of an equal volume of pre-warmed medium containing 2.4 M sorbitol and the proportion of cells with morphological defects was scored 3 hours later. (E) The temperature of cdc11.132 (IH127) and cdc11.132 wsh3. (IH5755) (upper panel) cdc11.132 wsh3. leu1::nmt41wsh3.X (IH5761, 5762, 5763; lower panels) cultures was shifted to 36°C in the presence of 0.15 µM thiamine to induce the expression of Wsh3 proteins. (F) wsh3. tea1.PkGFP (IH5751; left panel), or wsh3. tea1.PkGFP leu1::nmt41wsh3.X (IH5752, 5753, 5754; remaining panels) cells 12 hours after induction of transgene expression. (G) Rhodamine phalloidin staining of wsh3. cells in which expression of the indicated wsh3 allele was induced from a leu1::nmt41wsh3.X transgene (IH5737, 5738, 5739) by removal of thiamine for 24 hours. Bars, 5 µm.