Nature Cell Biology
6, 1142 - 1144 (2004)
Published online: 1 November 2004; | doi:10.1038/ncb1189
Organization of a sterol-rich membrane domain by cdc15p during cytokinesis in fission yeastTetsuya Takeda, Toshimitsu Kawate
& Fred ChangDepartment of Microbiology, Columbia University College of Physicians and Surgeons, 701 West 168th St, New York, NY 10032, USA.
Correspondence should be addressed to Fred Chang fc99@columbia.eduMany membrane processes occur in discrete membrane domains containing lipid rafts1, but little is known about how these domains are organized and positioned. In the fission yeast Schizosaccharomyces pombe, a sterol-rich membrane domain forms at the cell-division site2. Here, we show that formation of this membrane domain is independent of the contractile actin ring, septation, mid1p and the septins, and also requires cdc15p3, an essential contractile ring protein that associates with lipid rafts. cdc15 mutants have membrane domains in the shape of spirals. Overexpression of cdc15p in interphase cells induces abnormal membrane domain formation in an actin-independent manner. We propose that cdc15p functions to organize lipid rafts at the cleavage site for cytokinesis.
Cytokinesis requires numerous membrane events, including membrane insertion, secretion, membrane fusion, and the assembly and anchoring of the contractile ring to the plasma membrane4. In S. pombe, sterol-rich membrane domains appear at growing cell tips in interphase and at the medial cell-division site in late anaphase, long after actin-ring assembly and just before septation2. This membrane domain begins as a ring at the cell surface overlying the contractile ring, and then spreads into the invaginating plasma membrane surrounding the septum. This ring can be visualized by staining cells with filipin, a polyene antibiotic that binds to sterols2 (Fig. 1a). Although filipin staining carries certain caveats, especially in animal cells, it is not as toxic to S. pombe (see Supplementary Information, Fig. S1). In addition, we also visualized membrane domains using an acylated green fluorescent protein (Ac−GFP), a marker for lipid rafts5 (Fig. 1d). The precise function of these membrane domains in cytokinesis is still not clear. Sterol depletion disturbs contractile ring morphology and causes cytokinesis defects2, but these treatments may be pleiotropic.
 | | Figure 1. Cdc15p affects the organization of a sterol-rich membrane domain during cytokinesis. | | |  | (a) Mutant S. pombe cells affected in different aspects of cytokinesis were stained with 5  g ml-1 of filipin, a sterol dye, and imaged live within 5 min of filipin addition. For temperature-sensitive mutants, exponential-phase cells were grown in YE5S medium at 25 °C and shifted to the restrictive temperature, 35.5 °C, for 3.5 h. In Lat-A cells, 100 M Latrunculin-A was added to cells for 10 min. (b) 3D-reconstructed images of filipin staining in a wild-type cell and a cdc15-287 cell. Two images with different viewing angles are shown for each cell. (c) Filipin staining in cdc15-null cells (cdc15::ura4+ germinated spore; see Supplementary Information, Methods). (d) Localization of acylated-GFP in wild-type and cdc15-287 cells. Cells carrying REP3X−Ac−GFP were grown in the absence of thiamine at 25 °C for 14 h and then shifted to 35.5 °C for 3.5 h. Arrowheads indicate the spiral pattern of Ac−GFP. (e) Filipin staining of cdc8-382 cdc15-287 and cdc12-299 cdc15-287 double-mutant cells incubated at 35.5 °C for 3.5 h. (f) Induction of membrane domains in G2-phase-arrested cells by cdc15+ overexpression. cdc25-22 cells with REP1−cdc15 were grown in EMM plus thiamine media, washed, and resuspended in EMM plus thiamine (uninduced) or EMM minus thiamine (induced) media, grown for 15 h at 25 °C, shifted to 36 °C for 3 h, and then stained with filipin before imaging. The arrest of cells in interphase was confirmed by DAPI staining (data not shown). Arrowheads show abnormal membrane domains on the sides of cells. (g) Actin-independent induction of medial membrane domains by cdc15+ overexpression in G2-phase-arrested cells. Cells were prepared as in f, but 100 M Lat A or control DMSO (0.5% final concentration) was added to cells 2 h after raising the temperature to 36 °C, before the formation of abnormal membrane domains. Scale bars represent 10 m.
 Full Figure and legend (98K) |
| |
Here, we initiated a genetic analysis to identify factors involved in membrane domain organization during cytokinesis. We screened a set of representative cytokinesis mutants6,
7 by filipin staining (Fig. 1a; also see Supplementary Information, Fig. S1). Of ells defective in actin ring assembly, cdc3-313 (profilin), cdc8-382 (tropomyosin), cdc12-299 (formin), myo2-E1 (myosin type II heavy chain), cdc4-377 (myosin light chain) and rng2-346 (IQGAP) mutants, as well as Latrunculin-A-treated cells2, still exhibited a medial band of filipin-staining membrane. This band was often broader than the wild-type band, suggesting that the actin ring is not necessary for formation of this medial membrane band; however, it may restrict its width. The membrane domain seemed normal in the ring-positioning mutant mid1-366 (also in mid1 ), the septin mutant spn4 8, an arp2-1 mutant9, and an imp2 (PCH) mutant. It also formed normally in cdc11-119 and cdc14-118 mutants, which are defective in septum formation and the SIN pathway10, suggesting that localized secretion and endocytosis involved in septation were not required. Therefore, membrane domain formation in cytokinesis is a process largely independent of the actin ring, septin ring and septation.
Cdc15p, a member of a conserved family of PCH proteins11 (including Saccharomyces cerevisiae Hof1 (also known as Cyk2) and murine PSTPIP), is essential for multiple processes in cytokinesis, including actin ring formation and septation, and interacts with the formin cdc12p and type-I myosin myo1p3,
6,
12. Notably, cdc15-mutant cells often formed a spiral of filipin-staining membrane instead of a ring (Fig. 1b; also see Supplementary Information, Fig. S1). When imaged in a single focal plane, regions with filipin staining appeared as irregular broad patches on the plasma membrane. Three-dimensional (3D) reconstruction images revealed that these regions often formed a continuous spiral pattern on the cell surface that extended from cell tip to cell tip (Fig. 1b). Ac−GFP also localized in a similar pattern, showing that this pattern was not an artefact of filipin treatment (Fig. 1d). In addition to conditional cdc15 alleles, cdc15-null cells (germinating spores) also formed membrane spirals (Fig. 1c; also see Supplementary Information, Fig. S1). Other actin-ring mutants occasionally showed spiral-like membrane domains, but at lower frequencies than cdc15 mutants (see Supplementary Information, Fig. S1).
We tested whether abnormal actin organization is responsible for the spiral membrane domains in these recessive cdc15 mutants. Although mutants with severe defects in actin-ring formation (tropomyosin cdc8 and formin cdc12 mutants) generally exhibited a broad filipin-staining band (Fig. 1a), cdc8-382 cdc15-287 and cdc12-299 cdc15-287 double mutants formed membrane spirals (>60% of cells; n > 120) much like cdc15-287 mutants (Fig. 1e). Thus, this cdc15 effect cannot be explained solely by loss of the actin ring.
Overexpression of cdc15+ induced abnormal membrane distribution in interphase cells. We expressed cdc15+ from a thiamine-represssible nmt1 promoter in G2-arrested cdc25-22 cells. In 76% (n = 49) of these cells, filipin staining revealed abnormal sterol-rich membrane domains in patches (or spirals) on the sides of cells. (Fig. 1f). During the induction time course, the addition of 100 M Latrunculin A at a time point just before the appearance of these abnormal membrane domains did not affect their formation (75% cells; n = 108; Fig. 1g). Thus, cdc15p was sufficient to organize membrane domains in an actin-independent manner.
Consistent with its role in membrane organization, cdc15−GFP localized at the contractile ring and in dots at the septum at sites overlapping with the sterol-rich membrane domains (see Supplementary Information, Fig. S1)3,
12. Cdc15p ring localization preceded membrane domain formation and was actin-independent (see Supplementary Information, Fig. S1)13. Biochemical experiments showed that cdc15p is a peripheral plasma membrane protein that fractionates in the detergent-resistant membrane fraction, a biochemical test for lipid-raft association (see Supplementary Information, Fig. S2).
In summary, we identify cdc15p as a protein that affects the organization of the membrane domain at the cell division site. We propose that cdc15p is part of a protein-based structure that anchors lipid rafts at the plasma membrane to the underlying contractile ring. These discrete membrane domains are a conserved feature of cytokinesis, as cytokinesis in sea urchin embryos requires a similar sterol-rich membrane ring at the cleavage furrow (D. Burgess, personal communication), and proteins found in lipid rafts contribute to animal cell cytokinesis14. Spirals seen in cdc15 mutants raise interesting biophysical questions as to how such patterns are formed in biological systems. Actin−like and tubulin-like proteins can form spirals in bacteria15,
16. As cytoskeletal proteins (or the mutant cdc15p protein) do not form spirals in cdc15 cells (data not shown), membrane spirals may result from self organization of distinct membrane domains (with limited spatial cues from other cellular components) into large-order patterns17.
Note: Supplementary Information is available on the Nature Cell Biology website.
Received 9 August 2004; Accepted 22 September 2004; Published online: 1 November 2004.
REFERENCES
-
Mukherjee, S. & Maxfield, F. R. Annu. Rev. Cell Dev. Biol. 20, 839−866 (2004). | Article | PubMed |
-
Wachtler, V., Rajagopalan, S. & Balasubramanian, M. K. J. Cell Sci. 116, 867−874 (2003). | Article | PubMed | ISI | ChemPort |
-
Fankhauser, C. et al. Cell 82, 435−444 (1995). | Article | PubMed | ISI | ChemPort |
-
O'Halloran, T. J. Traffic 1, 921−926 (2000). | Article | PubMed | ChemPort |
-
Zacharias, D. A., Violin, J. D., Newton, A. C. & Tsien, R. Y. Science 296, 913−916 (2002). | Article | PubMed | ISI | ChemPort |
-
Chang, F., Woollard, A. & Nurse, P. J. Cell Sci. 109, 131−142 (1996). | PubMed | ISI | ChemPort |
-
Le Goff, X., Utzig, S. & Simanis, V. Curr. Genet. 35, 571−584 (1999). | Article | PubMed | ISI | ChemPort |
-
Berlin, A., Paoletti, A. & Chang, F. J. Cell Biol. 160, 1083−1092 (2003). | Article | PubMed | ISI | ChemPort |
-
Morrell, J. L., Morphew, M. & Gould, K. L. Mol. Biol. Cell 10, 4201−4215 (1999). | PubMed | ISI | ChemPort |
-
Krapp, A., Gulli, M. P. & Simanis, V. Curr. Biol. 14, R722−R730 (2004). | Article | PubMed | ISI | ChemPort |
-
Lippincott, J. & Li, R. Microsc. Res. Tech. 49, 168−172 (2000). | Article | PubMed | ISI | ChemPort |
-
Carnahan, R. H. & Gould, K. L. J. Cell Biol. 162, 851−862 (2003). | Article | PubMed | ISI | ChemPort |
-
Wu, J. Q., Kuhn, J. R., Kovar, D. R. & Pollard, T. D. Dev. Cell 5, 723−734 (2003). | Article | PubMed | ISI | ChemPort |
-
Skop, A. R., Liu, H., Yates, J., Meyer, B. J. & Heald, R. Science 305, 61−66 (2004). | Article | PubMed | ISI | ChemPort |
-
Jones, L. J., Carballido-Lopez, R. & Errington, J. Cell 104, 913−922 (2001). | Article | PubMed | ISI | ChemPort |
-
Ben-Yehuda, S. & Losick, R. Cell 109, 257−266 (2002). | Article | PubMed | ISI | ChemPort |
-
Baumgart, T., Hess, S. T. & Webb, W. W. Nature 425, 821−824 (2003). | Article | PubMed | ISI | ChemPort |
Acknowledgements
We thank A. Berlin, R. Lustig and B. Enloe for technical support; V. Simanis, A. Chang, A. Pidoux, K. Gould, J. Pringle and P. Crews for valuable reagents; D. Burgess, M. Ng, B. Lauring, F. Maxfield, S. Mukherjee, P. Tran, B. Feierbach, A. Rafael, R. Daga, A. Yonetani and other members of our lab for discussion and advice. This work was supported by National Institutes of Health grant R01 GM056836 to F.C. and Postdoctoral Fellowships from TOYOBO Biotechnology Foundation and Uehara Memorial Foundation to T.T.
Competing interests statement:
The authors declare that they have no competing financial interests. |
