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, firstname.lastname@example.org.
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
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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