A Role for Sphingomyelin-Rich Lipid Domains in the Accumulation
of Phosphatidylinositol-4,5-Bisphosphate to the Cleavage Furrow
Mitsuhiro Abe,aAsami Makino,aFrançoise Hullin-Matsuda,a,bKeiju Kamijo,cYoshiko Ohno-Iwashita,dKentaro Hanada,e
Hideaki Mizuno,fAtsushi Miyawaki,fand Toshihide Kobayashia,b
Lipid Biology Laboratory, RIKEN Advanced Science Institute, Wako, Saitama, Japana; INSERM U1060, Université Lyon 1, INSA Lyon, Villeurbanne, Franceb; Department of
Stem Cell Biology and Histology, Tohoku University School of Medicine, Sendai, Miyagi, Japanc; Faculty of Pharmacy, Iwaki Meisei University, Iwaki, Fukushima, Japand;
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japane; and Laboratory for Cell Function and Dynamics, RIKEN
Brain Science Institute, Wako, Saitama, Japanf
brane, followed by separation into two cells. Several proteins are
required for the formation and ingression of the cleavage furrow.
The Rho-type GTPase RhoA is a key regulator of the furrow for-
mation and ingression. RhoA regulates the ingression of the con-
tractile ring and the completion of cytokinesis by activating its
effectors (13). The translocation and activity of RhoA are regu-
lated by the ECT2-MKLP1 complex in a microtubule-dependent
manner (18, 46).
in cytokinesis (5, 30, 39). Phosphatidylinositol-4,5-bisphosphate
ing cytokinesis in mammalian cells (10), whereas phosphatidyle-
PIP2production blocks the recruitment of RhoGTPase to the site
terol is concentrated at the cleavage furrow during cytokinesis in
animal cells (31). The depletion of cholesterol or the inhibition of
its synthesis impairs cytokinesis (8, 9). Sphingolipids are also in-
volved in cytokinesis. The inhibition of sphingolipid biosynthesis
induces the formation of multinuclear cells due to a defect in
cytokinesis in yeast (38). Sphingolipids are required for the com-
32). However, little is known about the role of sphingolipids in
this cytokinetic event.
Sphingomyelin (SM) is a major sphingolipid, comprising ap-
proximately 10% of the total phospholipids in mammalian cells.
Together with cholesterol, SM forms specific liquid-ordered lipid
domains in model membranes (24, 25). The existence and func-
tion of these domains in biological membranes are a matter of
debate (17, 23). Recently, we developed methods for observing
fter chromosome segregation, the cell divides by the forma-
tion and ingression of a cleavage furrow at the plasma mem-
SM in vivo using lysenin, an earthworm protein that binds specif-
ically to SM-rich domains (16, 19, 42).
the outer leaflet are required for the enrichment of PIP2in the
for proper cytokinesis.
MATERIALS AND METHODS
Lipid probes. pQE30-EGFP-lysenin-161-297, expressing the nontoxic
EGFP-lysenin, was constructed by replacing Venus in pQE30-Venus-lys-
enin-161-297 (19) with PCR-amplified enhanced green fluorescent pro-
tein (EGFP). pQE30-lysenin-161-297, expressing the nontoxic lysenin,
pQE30-EGFP-PH, expressing the EGFP-PH domain, was constructed by
replacing lysenin-161-297 in pQE30-EGFP-lysenin-161-297 with the PH
domain of human PLC? 1, which was obtained from HeLa cell cDNA by
PCR amplification. Recombinant proteins were expressed in Escherichia
coli strain JM109 and purified using HisTrap FF crude columns (GE
647 labeling kit (Invitrogen, CA). Enzyme-linked immunosorbent assay
(ELISA) was carried out as described previously (19). Anti-mCherry an-
tibody (TaKaRa Bio, Japan) and anti-His antibody (Qiagen, CA) were
used as primary antibodies for ELISA.
Received 11 August 2011 Returned for modification 24 October 2011
Accepted 30 January 2012
Published ahead of print 13 February 2012
Address correspondence to Toshihide Kobayashi, email@example.com.
Supplemental material for this article may be found at http://mcb.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
mcb.asm.org0270-7306/12/$12.00Molecular and Cellular Biologyp. 1396–1407
Cell culture and drug treatments. HeLa cells were grown at 37°C in
Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, CA) supple-
mented with 10% fetal bovine serum. For synchronizing cells, HeLa cells
were synchronized with 40 ng/ml nocodazole (Sigma-Aldrich, MO) for 3
h, and mitotic cells were harvested by shake-off. The harvested cells were
plated in a poly-D-lysine-coated dish (BD, NJ) and incubated in the pres-
ence of 40 ng/ml nocodazole for an additional 30 min. Nocodazole then
was washed out, and the cells were incubated under various conditions.
For SMase experiments, after the nocodazole wash, HeLa cells were
treated with 2.5 IU/ml of Staphylococcus aureus SMase (Sigma-Aldrich,
MO). Treatment with the CERT inhibitor HPA12 was done as described
previously (43). LLC-PK1 cells were grown at 37°C in Medium 199 (In-
vitrogen, CA) supplemented with 5% fetal bovine serum.
bated in DMEM supplemented with 10% lipoprotein-deficient serum
containing 10 ?g/ml of nontoxic EGFP-lysenin. To label the cholesterol-
rich domain, HeLa cells were incubated in DMEM supplemented with
10% fetal bovine serum containing 10 ?g/ml of EGFP-domain 4 of theta
toxin (D4). For the immunostaining of RhoA, cells were fixed with 10%
trichloroacetic acid as described previously (18).
Expression of histone H2B, MKLP1, PH, synaptojanin, TubbyC,
cells. The coding sequences for human histone H2B, MKLP1, the PH
domain of PLC? 1, synaptojanin, the Tubby domain (TubbyC), and
phosphatidylinositol 4-phosphate 5-kinase ? (PIP5K?) were obtained
from HeLa cell cDNA by PCR amplification. The coding region of
Caenorhabditis elegans RhoA was obtained from the C. elegans cDNA
library by PCR amplification. Amplified EGFP, mCherry, Dronpa
(1), and PAmCherry1 (37) were cloned into expression vector pcDNA-
DEST40-Dronpa, and DEST40-PAmCherry1, respectively. The coding
sequences were cloned into pDsRed-Monomer-N1, pEGFP-N1 (TaKaRa
Bio, Japan), DEST40-EGFP, DEST40-mCherry, DEST40-Dronpa, or
DEST40-PAmCherry1. Fragments of lysenin and D4 were cloned into
DEST40-mCherry. For expressing EGFP-tubulin and EGFP-myosin II
regulatory light chain (MRLC), pEGFP-Tub (TaKaRa Bio, Japan) and
pEGFP-MRLC (18) were used, respectively. HeLa or LLC-PK1 cells were
transfected with the expression vectors by Lipofectamine LTX (Invitro-
gen, CA) and were cultured in the presence of 1 mg/ml G418 (Nacalai
Tesque, Japan) for 14 days. Stable clones were selected.
Confocal microscopy. Time-lapse microscopic observation was car-
ried out on the FV 1000 confocal microscope with a 60?, 1.1-numerical-
aperture PlanApo objective lens (Olympus, Japan) equipped with an en-
vironmental chamber maintained with humidity at 37°C and 5% CO2.
Images were captured using FV10-ASW software (Olympus, Japan).
direct stochastic optical reconstruction microscopy (PALM/dSTORM) im-
bated in Medium 199 supplemented with 5% lipoprotein-deficient serum
min and fixed with 4% paraformaldehyde and 0.2% glutaraldehyde for 30
Polyphosphoinositide analysis. Cells were labeled for 4 h with 0.1
?Ci/ml [33P]orthophosphoric acid in phosphate-free DMEM containing
40 ng/ml nocodazole, and mitotic cells were harvested by shake-off. The
harvested cells were plated in a poly-D-lysine-coated dish (BD, NJ) and
incubated a further 30 min. Cells were washed twice with ice-cold phos-
Lipid extraction was performed as described previously (21). The lipids
were separated on high-performance thin-layer chromatography
(HPTLC) plates that were first premigrated in a methanolic solution of
potassium oxalate (1%, wt/vol) using the solvent system chloroform-ac-
etone-methanol-acetic acid-water (80:30:26:24:14, vol/vol). The radioac-
tive spots identified by comparison with lipid standards were quantified
fetal bovine serum containing L-[U-14C]serine (1 ?Ci/ml). For the last 4
h, 40 ng/ml of nocodazole was added to synchronize cells. Cells were
washed twice with cold PBS and then scraped on ice in 2 mM EDTA.
Aliquots of cell extract were taken for protein quantification, and lipid
extraction was performed according to Bligh and Dyer (3). Lipids were
separated on HPTLC plates with a solvent mixture of methyl acetate-n-
Radioactive lipids were quantified with a BAS 5000 Bioimaging Analyzer.
Cholesterol analysis. Aliquots of cell extract were taken for protein
under UV after samples were sprayed with primuline solution, were col-
lected and then extracted with a mixture of methanol-water-hexane (2:1:
2). After centrifugation, the upper hexane phase was collected, dried un-
der nitrogen, and then analyzed with a Shimadzu GC-14AH gas
ate Inc., IL) capillary column (30 m by 0.32 mm; 0.25 ?m) was used with
to 320°C at 2°C/min and with isothermal holding at 320°C for 10 min.
Cholesterol was quantified using stigmasterol as the internal standard.
SM-rich domains are concentrated in the outer leaflet of the
localization of SM during cytokinesis, we stained the cells with a la-
beled lysenin. First, we stained the SM in the outer leaflet of the
plasma membrane with the exogenously added EGFP-lysenin. We
found that the fluorescence intensity of EGFP-lysenin increased
around the region of the contractile ring and the midbody (Fig. 1A;
sibility that the increased fluorescence intensity is due to the closely
apposed membranes in the furrow region, we compared EGFP-lys-
enin staining to that of a lipophilic dye. We stained the mitotic
cells with 1,1=-dioctadecyl-3,3,3=,3=-tetramethylindocarbocyanine per-
chlorate (DiIC18) as a nonspecific membrane probe. We found that
DiIC18was evenly distributed on the plasma membrane (Fig. 1B).
Quantitative analysis of the fluorescence intensity indicates that,
compared to DiIC18, EGFP-lysenin is significantly accumulated in
the cleavage furrow (Fig. 1B and C). We examined whether SM was
nesis by expressing mCherry-lysenin in HeLa cells (Fig. 1D). Al-
though intracellular dots and faint cytoplasmic staining were ob-
evident staining. ELISA confirmed that both EGFP-lysenin and
mCherry-lysenin specifically bound to SM (Fig. 1E). These results
concentrated to the outer leaflet of the cleavage furrow during cyto-
We estimated the concentration of SM at the cleavage furrow.
First, we made uniform giant unilamellar vesicles (GUVs) con-
taining several concentrations of SM (0 to 60%) and egg phos-
phatidylcholine (PC) as standards. To make a standard curve, we
stained the GUVs with EGFP-lysenin and quantified the fluores-
cence intensity. We found that the fluorescence intensity linearly
increased when the SM concentration was increased from 0 to
avoid overestimation due to the apposed two membranes, we
Role for Sphingomyelin in Cytokinesis
April 2012 Volume 32 Number 8mcb.asm.org 1397
centrations of SM in the outer leaflet at the cleavage furrow and
tively (Fig. 2B).
To address the possibility that the accumulation of SM is re-
quired for cytokinesis, we depleted SM by sphingomyelinase
(SMase) treatment. We found that in 50% of cells (n ? 30) the
cleavage furrows did not progress properly but regressed, re-
sulting in binucleated cells in 3 h (Fig. 3A and B; also see Movie
S2 in the supplemental material). In control cells, no defect in
cytokinesis was observed, suggesting that the addition of
EGFP-lysenin did not affect cytokinesis. This treatment de-
creased SM to 19.7% ? 2.6% of the control level (n ? 3) (Fig.
SMase treatment may alter the gross integrity of the plasma
membrane. To rule out the possibility that the abnormal cytoki-
nesis was due to effects other than the decrease of SM, we tested
whether the SMase-induced regression was recovered by adding
back exogenous SM. As observed above (Fig. 3B), the cleavage
adding exogenous PC after SMase treatment. The cleavage fur-
rows were still regressed in 44% of the cells (n ? 40) (Fig. 4B and
and D). These results suggest that the abnormal cytokinesis is
induced by the decrease of SM. We also confirmed that the in-
cells and observed the phenotype. The addition of ceramide did
not affect the ingress of the cleavage furrow (Fig. 4E), suggesting
that the ceramide increase does not cause the abnormal cytokine-
FIG1 SM-rich domains are concentrated in the outer leaflet of the cleavage furrow. (A) SM is concentrated in the cleavage furrow. HeLa cells stably expressing
Quantitative analysis of fluorescence intensity. The fluorescence intensity of EGFP-lysenin and DiIC18was measured in the cleavage furrow and the polar
membrane. The intensities were normalized to the intensity at the polar region. Data are means ? SD (n ? 20). (D) SM-rich domains are localized in the outer
leaflet of the cleavage furrow. HeLa cells stably expressing mCherry-lysenin (red) were incubated with purified EGFP-lysenin (green). Bar, 5 ?m. (E) EGFP-
lysenin and mCherry-lysenin bind specifically to SM. Purified protein of EGFP-lysenin from E. coli (green) and cell lysate of HeLa cells expressing mCherry-
lysenin (red) were assayed for ELISA. Data are means ? SD (n ? 3).
Abe et al.
mcb.asm.org Molecular and Cellular Biology
sis. From these results, we conclude that SM is required for cyto-
kinesis in the cells.
We next observed the cytoskeleton in the SMase-treated cells.
Cytokinesis involves cleavage furrow formation followed by the
ine whether the midbody is formed before regression in the
expressing EGFP-tubulin. In control cells, EGFP-tubulin was lo-
calized to the central spindle at anaphase (Fig. 5A, 30 min) and to
the midbody at a late stage of cytokinesis (Fig. 5A, 100 min). Sim-
ilarly to control cells, EGFP-tubulin localized to the central spin-
dle in the SMase-treated cells. However, during further incuba-
tion, EGFP-tubulin did not concentrate in the midbody.
We also examined myosin II by stably expressing EGFP-fused
myosin II regulatory light chain (MRLC) in HeLa cells. Both in
control and SMase-treated cells, EGFP-MRLC was accumulated
pletion of the midbody formation in SMase-treated cells.
Accumulation of PIP2, but not cholesterol, is abolished by
depletion of SM. SM is postulated to form specific lipid do-
mains together with cholesterol (17, 23, 36). Cholesterol has
been shown to accumulate in the cleavage furrow in sea urchin
eggs (31). We next examined whether cholesterol is concen-
trated at the cleavage furrow in the cells treated with SMase.
Using domain 4 of theta toxin (D4) (35), we stained the cho-
lesterol-rich domain in both the outer and inner leaflets of the
plasma membrane in living cells during cytokinesis (Fig. 6A).
To observe the cholesterol-rich domains in the outer leaflet of
the plasma membrane, exogenous recombinant EGFP-D4 was
added to the cells. For the staining of the cholesterol-rich do-
mains at the inner leaflet of the plasma membrane, we ex-
pressed mCherry-D4 in the cells after plasmid transfection.
The cholesterol-rich domains stained with EGFP-D4 on the
outer leaflet were accumulated at the site of the furrow ingres-
sion in control cells (Fig. 6A, upper). In contrast, mCherry-D4
fluorescence was evenly distributed to the inner leaflet of the
plasma membrane throughout cytokinesis (Fig. 6A, lower).
These results indicate that the cholesterol-rich domains in the
FIG 2 Estimation of the concentration of SM at the cleavage furrow. (A)
Fluorescence intensity of EGFP-lysenin in giant unilamellar vesicles (GUVs).
GUVs containing several concentrations of palmitoyl SM (0 to 60%) and egg
PC were made and stained with EGFP-lysenin. Fluorescence intensity was
intensity of EGFP-lysenin in cells. HeLa cells stably expressing histone H2B-
DsRed (red) were stained with EGFP-lysenin (green). The fluorescence inten-
polar region (circle). Data are means ? SD (n ? 50).
FIG 3 SM is required for proper cytokinesis. (A) Depletion of SM results in
regression of the cleavage furrow. HeLa cells stably expressing histone H2B-
were observed for 3 h (n ? 30) and classified into 3 groups. Gray, black, and
white colors indicate cells with normal cytokinesis, cells without nuclear divi-
sion, and cells with a regressed furrow, respectively. (C) SMase treatment
decreases the amount of SM in HeLa cells. Cells were labeled with
for the last 4 h. The mitotic cells were collected and incubated with 2.5 IU/ml
of SMase for 1 h. The lipids were extracted and separated on HPTLC plates.
Role for Sphingomyelin in Cytokinesis
April 2012 Volume 32 Number 8 mcb.asm.org 1399
outer, but not the inner, leaflet of the plasma membrane selec-
tively accumulate in the cleavage furrow during cytokinesis.
We tested whether treatment with SMase affects the enrich-
ment of cholesterol in the outer leaflet of the cleavage furrow
(Fig. 6B). In the SMase-treated cells, the cholesterol-rich do-
mains stained with EGFP-D4 were still concentrated in the
cleavage furrow before regression, whereas mCherry-D4
stained all around the inner leaflet of the plasma membrane, as
observed in the control cells. Quantitative analysis confirmed
that EGFP-D4 was concentrated to the cleavage furrow in both
control cells and SMase-treated cells (Fig. 6C). We confirmed
by ELISA analysis that both EGFP-D4 and mCherry-D4 selec-
tively bound to cholesterol (Fig. 6D). These results suggest that
SM is not required for the accumulation of cholesterol in the
outer leaflet of the cleavage furrow.
PIP2accumulates in the inner leaflet of the cleavage furrow
during cytokinesis in mammalian cells (6, 10). We observed the
domain of PLC? 1 (6, 10) fused to mCherry. As reported, PIP2
stained with mCherry-PH was highly concentrated in the inner
leaflet of the cleavage furrow (Fig. 7A, upper; also see Movie S3 in
the supplemental material). In addition, SM in the outer leaflet
and PIP2in the inner leaflet are colocalized during cytokinesis
(Fig. 7B and C). Interestingly, such PIP2accumulation was not
cence intensity confirmed that PIP2was not accumulated in the
furrow in the SMase-treated cells (Fig. 7D). We were not able to
when the cells were treated with SMase. Quantitative analysis of
PIP2indicates that the total amount of PIP2was not significantly
3) (Fig. 7E). These results suggest that SM at the plasma mem-
FIG 4 Abnormal cytokinesis is due to the SM decrease. (A) The cleavage
H2B-DsRed were incubated with 2.5 IU/ml of SMase for 1 h. The cells were
washed and incubated in DMEM supplemented with 10% lipoprotein-defi-
cient serum for an additional 3 h. (B) The regression phenotype is not sup-
pressed by adding exogenous PC. After treatment with SMase for 1 h, the cells
were washed and incubated in medium containing 20 ?M egg PC (Avanti
Polar Lipids, AL) for an additional 3 h at 37°C. (C) The regression phenotype
is suppressed by adding exogenous SM. After treatment with SMase, the cells
were washed and incubated in the medium containing 20 ?M brain SM
(Avanti Polar Lipids, AL) for an additional 3 h at 37°C. (D) Quantification of
the phenotype. Gray, black, and white colors indicate cells with normal cyto-
brain ceramide (Avanti Polar Lipids, AL). Bars, 5 ?m.
FIG 5 Cytoskeleton dynamics in the SMase-treated cells. (A) The cleavage
furrow is regressed before forming the midbody in the SMase-treated cells.
(green) were observed in the absence (upper) or presence (lower) of SMase.
(B) EGFP-MRLC is accumulated in the cleavage furrow. HeLa cells stably
expressing both histone H2B-DsRed (red) and EGFP-MRLC (green) were
observed in the absence (left) or presence (right) of SMase. Bars, 5 ?m.
Abe et al.
mcb.asm.org Molecular and Cellular Biology