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. 1996 Nov 12;93(23):12867-72.
doi: 10.1073/pnas.93.23.12867.

Redistribution of Phosphatidylethanolamine at the Cleavage Furrow of Dividing Cells During Cytokinesis

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Free PMC article

Redistribution of Phosphatidylethanolamine at the Cleavage Furrow of Dividing Cells During Cytokinesis

K Emoto et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Ro09-0198 is a tetracyclic polypeptide of 19 amino acids that recognizes strictly the structure of phosphatidylethanolamine (PE) and forms a tight equimolar complex with PE on biological membranes. Using the cyclic peptide coupled with fluorescence-labeled streptavidin, we have analyzed the cell surface localization of PE in dividing Chinese hamster ovary cells. We found that PE was exposed on the cell surface specifically at the cleavage furrow during the late telophase of cytokinesis. PE was exposed on the cell surface only during the late telophase and no alteration in the distribution of the plasma membrane-bound cyclic peptide was observed during the cytokinesis, suggesting that the surface exposure of PE reflects the enhanced scrambling of PE at the cleavage furrow. Furthermore, cell surface immobilization of PE induced by adding the cyclic peptide coupled with streptavidin to prometaphase cells effectively blocked the cytokinesis at late telophase. The peptide-streptavidin complex treatment had no effect on furrowing, rearrangement of microtubules, and nuclear reconstitution, but specifically inhibited both actin filament disassembly at the cleavage furrow and subsequent membrane fusion. These results suggest that the redistribution of the plasma membrane phospholipids is a crucial step for cytokinesis and the cell surface PE may play a pivotal role in mediating a coordinate movement between the contractile ring and plasma membrane to achieve successful cell division.

Figures

Figure 1
Figure 1
Specific binding of SA-Ro complex to PE. (A) Ro conjugated with 125I-SA-Ro bound selectively to PE-containing liposomes. 125I-SA-Ro was incubated with various concentrations of liposomes composed of egg yolk phosphatidylcholine (PC), dicetyl phosphate and cholesterol (respective molar ratios, 0.75/0.1/1.0) without (•) or with PE (○), phosphatidylserine (PS, □), phosphatidylinositol (PI, ▪), phosphatidic acid (PA, ▴), or cardiolipin (CL, ▵) each at a molar ratio to egg yolk phosphatidylcholine of 0.25. (B and C) Ro conjugated with FL-SA stained permeabilized erythrocyte membranes, but not intact erythrocytes. FL-SA-Ro was added to intact (B) or SLO-treated erythrocytes (C) at 4°C. The intact erythrocytes were permeabilized with SLO after FL-SA-Ro treatment and both the samples were washed and photographed. FL-SA-Ro fluorescence and phase contrast of same specimens, respectively, are shown. Intact erythrocytes were virtually unstained, whereas permeabilized membranes were labeled uniformly. (D) Binding of 125I-SA-Ro to intact and permeabilized erythrocytes. Permeabilized erythrocytes bound 10 times more 125I-SA-Ro than intact erythrocytes and preadsorption of 125I-SA-Ro with PE-containing liposomes abolished this binding (n = 3). (Bar = 10 μm.)
Figure 2
Figure 2
FL-SA-Ro selectively labeled the cleavage furrows of intact CHO cells during cytokinesis. Cells at prometaphase (A), anaphase (B), late telophase (C), and G1 phase (D) were incubated with FL-SA-Ro for 30 min at 4°C, then washed and photographed. Phase contrast micrographs, FL-SA-Ro staining and 4′-6-diamino-2-phenylindole (DAPI) fluorescence of the same specimens, respectively, are shown. (E) Late telophase cells were double-labeled with FL-SA-Ro and TRITC-phalloidin. Note that strong fluorescence was observed only at the cleavage furrow during cytokinesis; indicated by arrows in C. (Bar = 10 mm.)
Figure 3
Figure 3
Blockade of cell division by SA-Ro. SA (A) or SA-Ro (B), both 7.2 μM, was added to prometaphase cells, and then the cells were incubated at 37°C for 2 h, washed, and photographed. Cells in telophase/late telophase accumulated after SA-Ro treatment. (Bar = 30 μm.) (C) Concentration-dependent cell division arrest by SA-Ro. SA-Ro, ○; SA without Ro, •; 7.2 μM SA-Ro preabsorbed with PE-containing liposomes, (▵).
Figure 4
Figure 4
Localization of F-actin and microtubules in SA-Ro-arrested cells. Prometaphase cells were incubated for either 45 min (A and B) or 120 min (C and D) in the presence of SA-Ro, and the SA-Ro-treated cells were fixed and stained with TRITC-phalloidin (A and C) or rat antiyeast tubulin monoclonal antibody (B and D). (Bar = 10 μm.)

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