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
brane is required for the accumulation of PIP2in the inner leaflet
of the cleavage furrow.
Depletion of SM inhibits the recruitment of RhoA to the
cleavage furrow. It has been shown in Saccharomyces cerevisiae
that PIP2is one factor that is required for the translocation of
RhoGTPase to the site of the contractile ring formation; the de-
pletion of PIP2results in a mislocalization of RhoGTPase in the
furrow (45). RhoA localization was observed by immunofluores-
cence microscopy in HeLa cells fixed with trichloroacetic acid
(Fig. 8A). As reported previously (44), RhoA was localized to the
cleavage furrow and the midbody in control cells. In contrast, in
SMase-treated cells, RhoA was no longer localized to the cleavage
furrow. To exclude the possibility that RhoA was not observed in
SMase-treated cells due to the trichloroacetic acid fixation, we
studied its localization in living cells. Since EGFP-tagged C. el-
egans RhoA (CeRhoA) was shown to be properly localized to the
cleavage furrow in mammalian cells (46), we analyzed the EGFP-
hoA was localized to the contractile ring (Fig. 8B, upper; also
see Movie S5 in the supplemental material). In the cells treated
with SMase, EGFP-CeRhoA was less concentrated in the cleav-
age furrow (Fig. 8B, lower; also see Movie S6 in the supplemen-
tal material). Quantitative analysis of fluorescence intensity
confirmed that smaller amounts of RhoA accumulated in the
cleavage furrow in SMase-treated cells than in control cells
In addition to PIP2, MKLP1 is reported to be required for the
translocation of RhoA to the cleavage furrow (18, 46). We thus
checked the possibility that RhoA did not localize to the furrow
due to the abnormal localization of MKLP1. Since we were not
able to obtain HeLa cells stably expressing EGFP-MKLP1, we sta-
bly expressed EGFP-MKLP1 in LLC-PK1 cells (Fig. 8D). As re-
ported previously (18, 26, 46), EGFP-MKLP1 was accumulated
at the central spindle at late anaphase. Furthermore, in SMase-
treated cells, EGFP-MKLP1 was still localized to the central spin-
of MKLP1. Taken together, the results suggest that in the SMase-
treated cells, RhoA accumulation to the cleavage furrow was hin-
dered due to the abnormal localization of PIP2and not the
Cholesterol is essential for SM accumulation in the cleavage
furrow. We examined how SM is accumulated in the cleavage
(Fig. 9A). It has been shown that the overexpression of the PH
overexpression of synaptojanin reduces the amount of PIP2(10).
In the cells transiently overexpressing one of these domains, we
the control level (n ? 3). Even in the cells expressing these pro-
teins, EGFP-lysenin staining persisted at the cleavage furrow.
These results indicate that PIP2is not required for the accumula-
tion of SM to the cleavage furrow. In these cells, EGFP-CeRhoA
was less concentrated in the cleavage furrow than in control cells
(Fig. 9B), indicating that PIP2is required for the accumulation of
RhoA to the cleavage furrow.
localization of SM, since cholesterol facilitates the formation of
dextrin (M?CD) removed cellular cholesterol and thus inhibited
the cellular staining with D4 (Fig. 10B). We found that treatment
with M?CD abolished the accumulation of mCherry-lysenin
reduced the amount of cholesterol (Fig. 10D), whereas it did not
significantly reduce the total amount of SM (Fig. 3C). Lysenin
binds SM only when the local concentration of SM is high (16).
These results suggest that cholesterol in the outer leaflet of the
plasma membrane is required for the SM accumulation in the
cleavage furrow. Consistently, the cleavage furrow was re-
observed after SMase treatment (Fig. 3A and B).
FIG 6 SM is not required for the accumulation of cholesterol in the cleavage
furrow. (A) Cholesterol-rich domains are concentrated in the outer leaflet of
the accumulation of cholesterol-rich domain. The cells described above were
incubated in the presence of SMase. Bars (A and B), 5 ?m. (C) Quantitative
analysis of fluorescence intensity. The fluorescence intensity of EGFP-D4 was
measured in the cleavage furrow (square) and the polar region (circles). The
? SD (n ? 20). (D) EGFP-D4 and mCherry-D4 bind specifically to choles-
terol. Lipid binding properties of purified protein of EGFP-D4 from E. coli
(green) and cell extracts from HeLa cells expressing mCherry-D4 (red) were
assayed by ELISA. Data are means ? SD (n ? 3).
Role for Sphingomyelin in Cytokinesis
April 2012 Volume 32 Number 8mcb.asm.org 1401
A transbilayer colocalization between the SM-rich domains
in the outer leaflet and PIP2-rich domains in the inner leaflet.
Several observations suggest that a significant pool of PIP2associ-
ates with the detergent-resistant membrane (DRM) fraction (4,
colocalize during the course of the whole cell cycle. However,
there is no direct evidence that SM-rich domains in the outer
leaflet overlap PIP2in the inner leaflet of the plasma membrane,
since the sizes of these domains are below the resolution of con-
used photoactivated localization microscopy (PALM) and direct
served the lipids at the apical face of the plasma membrane at
interphase (Fig. 11A). The Dronpa-labeled PH domain was ex-
pressed in LLC-PK1 cells to visualize PIP2at the inner leaflet, and
SM at the outer leaflet was stained by the exogenous addition of
Alexa 647-labeled lysenin. At the apical plasma membrane, small
clusters of SM and PIP2were observed which were colocalized
(Fig. 11A, left). We then estimated the clusters of SM and PIP2by
spatial statistical analysis (Fig. 11B, left). The curves of the L(t)
value were found to be above the higher confidence lines, with
peaks at approximately 250 nm for both stainings, indicating that
SM and PIP2form similar-sized domains. Both domains disap-
peak of the L(t) curve of PIP2was not observed (Fig. 11B, center).
It has been reported that treatment with HPA12, an inhibitor of
CERT, selectively decreased the amount of cellular SM (43). Al-
though HPA12 inhibited normal cell growth, we found that the
cleavage furrows were also regressed when the cells were treated
of PIP2by PALM after HPA12 treatment (Fig. 11A, right). We
found that HPA12 treatment prevented the formation of PIP2
We confirmed that under this condition, the amount of SM was
decreased to 22.5% ? 3.8% of the control level (n ? 3).
We then examined whether the addition of exogenous SM re-
exogenous SM after SMase treatment (Fig. 11C, center). Further-
appeared in the inner leaflet just beneath the SM clusters that
located in the outer leaflet of the plasma membrane. We con-
firmed that the addition of exogenous PC did not induce the for-
mation of the PIP2clusters in the SMase-treated cells (Fig. 11C,
and PIP2are colocalized at the plasma membrane and that SM is
required for the formation of the PIP2clusters.
We next observed the distribution of SM and PIP2in the cells
undergoing cytokinesis. We expressed PAmCherry1-PH in LLC-
PK1 cells and stained the cells with Alexa 647-labeled lysenin.
FIG 7 Depletion of SM abolishes PIP2accumulation in the cleavage furrow. (A) Treatment with SMase does not concentrate PIP2into the furrow. Cells
expressing mCherry-PH (red) were incubated in the absence (upper) or presence (lower) of SMase, and then pictures were taken at the indicated times. Bars, 5
then pictures were taken at the indicated times. Bar, 5 ?m. (C) Fluorescence intensity of EGFP-lysenin (green) and mCherry-PH (red) was measured along the
line. (D) Quantitative analysis of fluorescence intensity. The fluorescence intensity of mCherry-PH was measured in the cleavage furrow (square) and the polar
region (circles). The intensities were normalized to the intensity at the polar region. Data are means ? SD (n ? 20). (E) SMase treatment does not decrease the
amount of PIP2. Cells were labeled with [33P]orthophosphoric acid. The mitotic cells were incubated with or without SMase for 1 h.
Abe et al.
mcb.asm.org Molecular and Cellular Biology
Using PALM/dSTORM analysis, we found that PH and lysenin
stainings were localized in the cleavage furrow (Fig. 12B, left).
Since these proteins were highly concentrated, individual clusters
were not identified in the midsection. On the other hand, we
found several clusters of CeRhoA, PH, and lysenin that were co-
localized around the cleavage furrow in the apical section (Fig.
12B, right). These results suggest that the clusters of SM and PIP2
remain colocalized at the plasma membrane during cytokinesis.
PALM/dSTORM observation indicates that PIP2is localized
(A) RhoA is not concentrated at the cleavage furrow in SMase-treated cells.
Cells were incubated in the absence (left) or presence (right) of SMase for 60
min. Cells were fixed with trichloroacetic acid and stained with anti-RhoA
antibody. (B) EGFP-CeRhoA weakly localizes in the cleavage furrow after
SMase treatment. HeLa cells stably expressing both histone H2B-DsRed (red)
and EGFP-CeRhoA (green) were observed in the absence (upper) or presence
(lower) of SMase. (C) Quantitative analysis of fluorescence intensity. The flu-
orescence intensity of EGFP-CeRhoA was measured in the cleavage furrow
sity at the cytosolic region. Data are means ? SD (n ? 20). (D) MKLP1 is
stably expressing histone H2B-DsRed (red) and EGFP-MKLP1 (green) were
observed in the absence (upper) or presence (lower) of SMase. The pictures
were taken at the indicated times after the SMase incubation. Bars, 5 ?m.
FIG 9 PIP2is required for the accumulation of RhoA but not SM. (A) EGFP-
was stained with EGFP-lysenin (green) in the cells overexpressing
mCherry-PH (red), mCherry-synaptojanin (red), or mCherry-TubbyC (red).
The intensities were normalized to the intensity at the polar region. Data are
cleavage furrow. mCherry-PH (red), mCherry-synaptojanin (red), or
mCherry-TubbyC (red) was overexpressed in HeLa cells stably expressing
cytosolic region. Data are means ? SD (n ? 20). Bars, 5 ?m.
Role for Sphingomyelin in Cytokinesis
April 2012 Volume 32 Number 8mcb.asm.org 1403
just beneath the SM-rich domains. This result raised the possibil-
5-kinase ? (PIP5K?), which produces PIP2from phosphatidyl-
inositol 4-phosphate (6, 41). PALM/dSTORM analysis revealed
that PIP5K? formed small domains in the inner leaflet of the
to the inner leaflet on the opposite side of SM-rich domains (Fig.
13, left). In the SMase-treated cells, neither domain was observed
(Fig. 13, right). These results suggest that PIP5K? is localized on
the cleavage furrow at cytokinesis. The depletion of SM from the
plasma membrane inhibits the completion of cytokinesis. In the
SM-depleted cells, PIP2is no longer concentrated to the furrow.
We analyzed the distribution of SM and PIP2by high-resolution
microscopy. We found that the clusters of both outer leaflet SM
and inner leaflet PIP2are colocalized at the plasma membrane,
and that SM is required for the formation of the PIP2clusters.
From these results, we conclude that the SM-rich domain in the
outer leaflet of the plasma membrane is required for the enrich-
needed for the recruitment of factors required for cytokinesis,
such as RhoA.
We found that cholesterol in the outer leaflet of the plasma
membrane is required for the SM accumulation at the cleavage
furrow. This observation is consistent with the results using
model membranes in which cholesterol facilitates the forma-
tion of SM-rich lipid domains (40). Cholesterol is accumulated
to the cleavage furrow in the SMase-treated cells (Fig. 10C),
suggesting that SM is not required for cholesterol accumula-
tion in the furrow. Since we could not detect the cholesterol
accumulation before the onset of anaphase, factors involved in
cell cycle progression or cytoskeleton may be needed for the
The accumulation of RhoGTPase to the cleavage furrow is
induced both by MKLP1 (18, 46) and PIP2(45). We found that
SMase treatment did not affect the localization of MKLP1 (Fig.
(Fig. 7A and D) in the treated cells. We also found that RhoA
was less concentrated in the cleavage furrow when PIP2was
interfered with (Fig. 9B). These results confirm the importance
of PIP2in the proper localization of RhoA but do not exclude
the possibility that MKLP1 is also involved in the translocation
of RhoA. One possibility is that MKLP1 determines the site of
RhoA translocation and PIP2works as a platform for the stable
attachment of RhoA to the plasma membrane and the comple-
tion of cytokinesis.
In PALM images, some clusters of SM localized next to the
clusters of PIP2or did not include any clusters of PIP2. We spec-
ulate that this phenomenon is caused by the following reasons.
Dronpa-lysenin and Alexa 647-lysenin simultaneously, SM-rich
domains stained with Dronpa-lysenin and Alexa 647-labeled lys-
enin were adjacent to one another. This result implies that the
problem is caused by the chromatic aberration and suggests that
the low expression of Dronpa-PH. When we transiently overex-
pressed Dronpa-PH, most clusters of Alexa 647-lysenin included
Dronpa-PH. This result implies that some clusters of SM could
not be detected with clusters of PIP2by PALM due to the low
expression of Dronpa-PH in the stable cell line.
Both SM- and cholesterol-rich domains are concentrated in the cleavage fur-
row. HeLa cells stably expressing histone H2B-DsRed (red) were incubated
with EGFP-D4 (green) and mCherry-lysenin (red). (B) Depletion of choles-
terol with M?CD reduces the staining of SM- and cholesterol-rich domain-
specific probes. The cells were incubated in 10 mM M?CD, and the pictures
were taken at the indicated times. (C) Depletion of SM with SMase does not
reduce the staining of cholesterol-rich domain-specific probes. The cells were
(A to C), 5 ?M. (D) Depletion of cholesterol with M?CD reduces the total
Data are means ? SD (n ? 3).
Abe et al.
mcb.asm.orgMolecular and Cellular Biology
Previously we showed by using immunoelectron micros-
copy that SM forms small domains on the outer leaflet of the
plasma membrane (19). Recently the detailed characterization
of PIP2domains in the inner leaflet of the plasma membrane
was reported (12). However, the transbilayer colocalization of
SM domains and PIP2domains has not been achieved. Using
superresolution fluorescence microscopy, we demonstrate
SM clusters in the outer leaflet of the plasma membrane (Fig.
localized to the inner leaflet on the opposite side of SM-rich
domains (Fig. 13). These results suggest that PIP2clusters are
the inner leaflet of the SM clusters.
We found that the localization of PIP2is restricted around the
clusters of SM and PIP5K? (Fig. 11 and 13). There are several
FIG 11 Transbilayer colocalization between the SM-rich domains in the outer leaflet and PIP2-rich domains in the inner leaflet of the plasma membrane. (A)
PALM/dSTORM images of Dronpa-PH (green) and Alexa 647-labeled lysenin (red). LLC-PK1 cells expressing Dronpa-PH were stained with Alexa Fluor
647-labeled lysenin. (Center) Cells were treated with SMase for 1 h. (Right) Cells were treated with HPA12 for 48 h. (Lower) Magnified images. (B) Ripley’s
respectively. (C) PALM/dSTORM images of Dronpa-PH (green)- and Alexa Fluor 647-labeled lysenin (red). LLC-PK1 cells expressing Dronpa-PH were
incubated with 2.5 IU/ml of SMase for 1 h at 37°C. The cells were washed and incubated for 1 h at 37°C. The cells were incubated for an additional 1 h at 37°C
647-labeled lysenin. The lower panels show magnified images. Bars, 2 ?m.
Role for Sphingomyelin in Cytokinesis
April 2012 Volume 32 Number 8mcb.asm.org 1405
may have direct or indirect interactions. Due to interactions, the
diffusion of PIP2may be restricted in the membrane. Second,
PIP5K? is activated by RhoGTPase, which is located in the PIP2
domain. It has been shown that RhoGTPase positively regulates
the activity of PIP5K? (41). These regulations may enhance the
In fact, several lines of evidence suggest that there are diffusion
barriers in the plasma membrane (20, 29, 34). Fluorescence cor-
relation spectroscopy (FCS) and fluorescence recovery after
bleaching (FRAP) analyses suggest that a protein fence limits the
diffusion of PIP2(14). Further experiments are needed to under-
stand how PIP2remains in the clusters.
Perry for software of spatial statistical analysis, and V. V. Verkhusha for
pPAmCherry1. We are grateful to R. Nakazawa and Y. Ichikawa for DNA
System Program of RIKEN and the Grant-in-Aid for Scientific Research
21113530 and 22390018 (to T.K.) from the Ministry of Education, Cul-
ture, Sports, Science, and Technology of Japan.
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