PI(3,4)P 2 levels at the midbody as well as elon-
gated intercellular bridges (Fig. 4, F and G,
and fig. S14A). Removal of TSG101 together
with PI3K-C2asynergized and almost com-
pletely abolished VPS36 recruitment to the
midbody (fig. S13G). Thus, PI3K-C2aÐderived
PI(3,4)P 2 facilitates VPS36 recruitment and
ESCRT-III assembly. In agreement, PI4KA
inhibition reduced the ESCRT-III subunit
CHMP4B at the midbody (fig. S14B), but supres-
sion of CHMP4B had no effect on VPS36 lo-
calization (Fig. 4E), confirming that VPS36
is recruited before ESCRT-III during abscis-
sion. At the midbody, depletion of either PI3K-
C2aor VPS36 reduced CHMP4B enrichment
Gulluniet al.,Science 374 , eabk0410 (2021) 10 December 2021 6 of 14
1.25
GFP-VPS36
RFP-α-tubulin
DNA VPS36 PI(3,4)P2
midbody
C D
1um 2um
00:15
00:05 00:25
00:10 00:30
00:35
00:00 00:20
E
150 -
100 -
20 -
Head Lens
PI3K-C2α
ALIX
α-Cry
AB
H
CHMP4B norm fluorescence
intensity at midbody (AU)
0
ctrl
PI3K-C 2 α
siRNA
VPS36
siRNA
1.0 2.0 3.0
***
***
α-tubulin CHMP4B Merge
Midbody section
I
0
0.5
1.0
1.5
2.0
2.5
3.0
(^) e
cn
ec
se
ro
ulf
mr
on
(^6)
(^3) S
PV
ody (AU)b
di
m (^) t
a
yti
sn
et
ni
- wt kd c3
***
3.5
GFP-C2α
siRNA C2α ++++
--
** **
DNA PI3K-C2α
lens
DNA C2α Merge
lens lens
DNA ALIX DNA ALIX Merge
lens
lens
VPS36
Vinculin
50 -
100 -
1
0.0625
0.5
8
Log2 Head/Lens
2
0.25
ALIX
PI3K-C2
α
VPS36
4
0.125
**
**
lens
VPS36 norm fluorescence
intensity at midbody (AU)
0
ctrl
PI 3 K-C2α
siRNA
CHMP4B
siRNA
1.0 2.0 3.0
***
α-tubulin VPS36 Merge
Midbody section
F
0
0.75
1.00
1.25
1.50
0.25
0.50
(^) e
cn
ec
se
ro
ulf
mr
on
(^6)
3
S
P
V
)
U
A(
yd
ob
di
m (^) t
a
yti
sn
et
ni
+/+ --/
Fam-I Fam-II
--/ --/ --/
α-tubulin VPS36
--/
Fam-III
α-tubulin PI(3,4)P2
0
0.50
0.75
1.00
1.50
0.25
PI(3,4)P2 norm fluorescence
intensity at midbody (AU)
G
+/+ --/
Fam-I Fam-II
--/ --/ --/ --/
Fam-III
Fig. 4. PI3K-C2a/VPS36 axis is an ALIX alternative pathway to recruit
CHMP4B to the midbody in lens.(A) Immunoblot analysis (left) and
quantification plot (right) showing Log2 of head/lens protein level for PI3K-C2a,
ALIX, and VPS36 in adult zebrafish. (B) Immunofluorescence showing (top) ALIX
or (bottom) PI3K-C2aexpression in 72-hpf zebrafish lens. (C) Immuno-
fluorescence staining showing colocalization between endogenous VPS36 and
PI(3,4)P 2 at the midbody ring. (D) Snapshots taken from time-lapse imaging of
GFP-VPS36 during cytokinesis in HeLa cells stably expressing red fluorescent
protein (RFP)–a-tubulin. Arrowheads indicate enrichment of VPS36 at the
abscission site. (E) Quantification of VPS36 levels at midbody in control and
siRNA-treated cells.n≥200 cells, mean ± SD. (FandG) Immunofluorescence
staining and quantification of endogenous (F) VPS36 and (G) PI(3,4)P 2 levels at the
midbody in fibroblasts derived from patients with homozygous deletion of
PI3K-C2a. +/+ genotypes were pulled together.n≥50 cells, mean ± SD.
(H) Quantification of CHMP4B levels at midbody in control and siRNA-treated
HeLa cells.n≥120 cells, mean ± SD. (I) Quantification of VPS36 levels at
midbody in control and siRNA-treated cells upon transfection of siRNA-resistant
WT, KD, and PI(3)P–producing (C3) forms of PI3K-C2a.n≥30 cells, mean ± SD.
If not previously specified, all results are shown as mean or representative picture
of at least three independent experiments ± SEM. **P< 0.01; ***P< 0.001.
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