ARTICLES
Plasma membrane translocation of trimerized MLKL
protein is required for TNF-induced necroptosis
Zhenyu Cai1,4 , Siriporn Jitkaew1,4 , Jie Zhao1,4 , Hsueh-Cheng Chiang2 , Swati Choksi1 , Jie Liu3 , Yvona Ward1 ,
Ling-gang Wu2 and Zheng-Gang Liu1,5
The mixed lineage kinase domain-like protein (MLKL) has recently been identified as a key RIP3 (receptor interacting protein 3)
downstream component of tumour necrosis factor (TNF)-induced necroptosis. MLKL is phosphorylated by RIP3 and is recruited to
the necrosome through its interaction with RIP3. However, it is still unknown how MLKL mediates TNF-induced necroptosis. Here,
we report that MLKL forms a homotrimer through its amino-terminal coiled-coil domain and locates to the cell plasma membrane
during TNF-induced necroptosis. By generating different MLKL mutants, we demonstrated that the plasma membrane localization
of trimerized MLKL is critical for mediating necroptosis. Importantly, we found that the membrane localization of MLKL is
essential for Ca2+ influx, which is an early event of TNF-induced necroptosis. Furthermore, we identified that TRPM7 (transient
receptor potential melastatin related 7) is a MLKL downstream target for the mediation of Ca2+ influx and TNF-induced
necroptosis. Hence, our study reveals a crucial mechanism of MLKL-mediated TNF-induced necroptosis.
Tumour necrosis factor (TNF) plays a critical role in diverse cellular
events including apoptosis and necroptosis1,2 . TNF is also a major
mediator of both inflammation and immunity and is involved in many
pathological conditions and autoimmune diseases3 . The molecular
mechanisms of TNF signalling have been significantly worked out.
For instance, it is known that TNF triggers the formation of a
TNFR1 signalling complex by recruiting several effectors such as
TRADD, RIP1 and TRAF2 to mediate different signalling pathways.
Importantly, under certain conditions, this TNFR1 signalling complex
(complex I) dissociates from the receptor and recruits other proteins to
form different secondary complexes for apoptosis and necroptosis4–6 .
Necroptosis needs the presence of RIP3 (receptor interacting protein
3) and MLKL (mixed lineage kinase-domain like) in the necrosome7–11 .
Other proteins including CYLD and SIRT2 are also suggested to play
a role in the formation of the necrosome12,13 . Apoptosis is primarily
initiated through the recruitment of the death domain protein FADD
(Fas-associated death domain protein) to form complex II. FADD
recruits and activates the initiator cysteine proteases caspases 8 and 10,
which drive apoptosis2,14 . Importantly, there is also cross-talk between
these two pathways15–17 .
Although the mechanism of TNF-induced apoptosis is well
elucidated, the signalling events that lead to TNF-initiated necroptosis
are still largely unknown. Caspase-independent necroptosis has been
proposed to involve the generation of reactive oxygen species (ROS)
derived from mitochondria18,19 . More recently, RIP3 has been found to
be essential for recruiting MLKL and the mitochondrial phosphatase
PGAM5 during TNF-induced necroptosis10,11,20 . Although it has been
suggested that PGAM5 is essential for mitochondrial fragmentation
during necroptosis, the mechanism of MLKL function in TNF-induced
necroptosis is still unknown.
Here we show that MLKL forms homotrimers and locates to the cell
plasma membrane during TNF-induced necroptosis. Importantly, we
found that MLKL membrane localization is critical for Ca2+ influx.
Furthermore, we identified TRPM7 as a MLKL downstream target to
mediate Ca2+ influx and TNF-induced necroptosis. Our study reveals a
key mechanism of MLKL-mediated necroptosis.
RESULTS
MLKL forms trimers on necroptosis induction
To explore how MLKL mediates TNF-induced necroptosis, we first investigated whether MLKL forms oligomers, because its N terminus contains two coiled-coil domains, predicted by the MultiCoil program21 .
Interestingly, these coiled-coil motifs are highly conserved among
the MLKL orthologues in different vertebrate species (Supplementary
Fig. 1a). Furthermore, the MultiCoil scores suggest that these two
coiled-coil motifs of MLKL most likely form three-stranded coiled-coils
1
Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. 2 Synaptic
Transmission Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA. 3 Center for Molecular
Medicine, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. 4 These authors contributed equally to this work.
5
Correspondence should be addressed to Z-G.L. (e-mail: zgliu@helix.nih.gov)
Received 20 February 2013; accepted 28 October 2013; published online 8 December 2013; DOI: 10.1038/ncb2883
NATURE CELL BIOLOGY VOLUME 16 | NUMBER 1 | JANUARY 2014
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ARTICLES
b
a
2ME
+
Mr (K) 250
150
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100
75
Monomer
50
IB: Anti-FLAG
Input IgG Anti-FLAG
Input IgG Anti-FLAG
GFP–MLKL +
FLAG–MLKL +
+
+
GFP–MLKL +
FLAG–MLKL +
+
+
Mr (K) 80
IB: Anti-GFP
54
c
IP
IP
–
+
+
+
+
80
Trimer
150
100
75
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Mr (K) 54
IB: Anti-FLAG
NEM – – + +
2ME – + – +
Mr (K) 250
Monomer
50
IB: Anti-GFP
IB: Anti-FLAG
d
Non-reducing
e
Reducing
MLKLMLKLHT29
shRNA
shRNA
TSZ 0 2 4 8 16 4 8 0 2 4 8 16 4
8 (h)
Mr (K) 250
HT29
HT29
Trimer
150
100
75
f
Mr (K) 250
IB: Anti-MLKL
Δ
IB: Anti-RIP3
43
43
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IB: Anti-actin
h
MEF
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Mr (K) 250
150
100
75
50
IB: Anti-actin
TSZ
4 8 16 (h)
Non-reducing gel
g
43
J774A.1
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(h)
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IB: Anti-actin
Non-reducing gel
Non-reducing gel
2
Monomer
Δ
IB: Anti-RIP3
57
43
0
50
57
Monomer
TS
4 8 16
Trimer
Monomer
U937
0 2 4 8 (h)
IB: Anti-MLKL
Trimer
2
150
100
75
50
JurkatFADD–/–
TSZ 0 2 4 8
Mr (K) 250
150
100
75
50
0
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100
75
8 (h)
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50
IB: Anti-MLKL
Monomer
43
IB: Anti-actin
Non-reducing gel
Figure 1 MLKL forms homotrimeric protein on necrosis induction.
(a) HEK293 cells were transfected with FLAG–MLKL. The cell lysates were
resolved on an SDS–PAGE gel with or without β-mercaptoethanol (2ME)
and analysed by immunoblotting with anti-FLAG antibody. (b) HEK293
cells were transfected with GFP–MLKL and FLAG–MLKL as indicated.
Cell lysates were immunoprecipitated either with anti-FLAG antibody (left
panel) or anti-GFP antibody (right panel) and analysed by immunoblotting
with the indicated antibodies. (c) HEK293 cells were transfected with
FLAG–MLKL, then lysed with or without 30 mM N -ethylmaleimide (NEM)
to block all reactive sulphydryl groups. The cell lysates were resolved
with or without 2ME and analysed by immunoblotting with anti-FLAG
antibody. (d,e) Control-shRNA or MLKL-shRNA HT29 cells were treated
with TSZ (TNF, Smac mimetic and the caspase inhibitor z-VAD-FMK) or
TS (TNF and Smac mimetic) as indicated. The cell lysates were resolved
either on reducing or non-reducing gel and analysed by immunoblotting
with the indicated antibodies. (f–h) JurkatFADD−/− , U937 (f), MEF (g)
and J774A.1 (h) cells were treated with TSZ at different time points
as indicated. The cell lysates were resolved on non-reducing gel and
analysed by immunoblotting with the indicated antibodies. 1 indicates
phosphorylated RIP3. All western data are representative of two or three
independent experiments. Uncropped images of western blots are shown
in Supplementary Fig. 8.
(Supplementary Fig. 1b). To determine whether MLKL protein can
oligomerize, we overexpressed MLKL in HEK293 cells and analysed
MLKL protein expression by SDS–polyacrylamide gel electrophoresis
under reducing and non-reducing conditions. Interestingly, although
MLKL protein was detected as a monomer at Mr 54K under reducing
conditions as observed previously10,11 , it was also spotted at about
Mr 160K, roughly threefold of a MLKL monomer (Fig. 1a). When
GFP–MLKL and FLAG–MLKL were co-expressed, GFP–MLKL was
co-precipitated with FLAG–MLKL protein and vice versa (Fig. 1b).
These results indicated that the MLKL protein indeed oligomerizes
and most likely forms homotrimers when it is overexpressed in
HEK293 cells. As reducing agents, such as dithiothreitol (DTT) or
β-mercaptoethanol (2ME), eliminated the trimeric form of MLKL,
the trimers of MLKL protein are probably stabilized by disulphide
bonds. When MLKL-transfected cells were lysed in the lysis buffer
with N -ethylmaleimide, which blocks reactive sulphydryl groups,
the trimeric band of MLKL disappeared even in the non-reducing
condition, suggesting that the disulphide bonds of the trimerized MLKL
proteins were formed by oxidation during cell lysis (Fig. 1c).
To examine whether the MLKL protein forms trimers in cells
induced to undergo necroptosis, we treated HT29 cells with a
combination of TNF, Smac mimetic, and the caspase inhibitor
z-VAD-FMK (TSZ). MLKL proteins were detected as monomers
at a relative molecular mass of 54,000 (Mr 54 K) under reducing
conditions, whereas MLKL proteins were detected as trimers as early
as 2 h after TSZ treatment under non-reducing conditions (Fig. 1d).
In particular, most of the MLKL protein existed as trimers at 4
and 8 h following TSZ treatment (Fig. 1d). The HT29 cells stably
transfected with a MLKL short hairpin RNA (shRNA) were used
as a control for the specificity of the anti-MLKL antibody (Fig. 1d).
Importantly, MLKL protein forms trimers in necrotic cells (TSZ
treatment), and not in apoptotic cells (TS treatment; Fig. 1e). The
observation of MLKL trimerization in necrotic cells seems to be a
general phenomenon because MLKL protein also forms trimers in
the necrotic JurkatFADD−/− , U937, mouse embryonic fibroblast (MEF)
and mouse J774A.1 cells, all of which are sensitive to TSZ-induced
necroptosis (Fig. 1f–h). Taken together, these results suggest that MLKL
protein trimerizes during necroptosis.
56
NATURE CELL BIOLOGY VOLUME 16 | NUMBER 1 | JANUARY 2014
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ARTICLES
a
–
–
+
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HT29
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150
100
75
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Ctrl-shRNA RIP3-shRNA
Trimer
Monomer
50
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50
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–
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100
75
50
IB: Anti-MLKL
8
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IB: Anti-MLKL
Monomer
57
IB: Anti-RIP3
IB: Anti-actin
d
RIP3–/–
TSZ 0
Mr (K) 150
100
75
50
2
4
0
2
4 (h)
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57
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Mr (K) 250
150
100
75
50
43
IB: Anti-actin
83
Monomer
e
Non-reducing Reducing
RIP3-D160N–YFP –
RIP3–YFP –
FLAG–MLKL +
–
+
+
+
–
+
–
–
+
–
+
+
+
–
+
AG
MEF
WT
FL
c
–M
LK
FL
L
AG
T3 –
57 ML
A/ KL
S3 58
A
43
IB: Anti-FLAG
Trimer
Monomer
Δ
IB: Anti-GFP
RIP3–YFP –
Mr (K) 250
150
100
75
50
83
+
– +
IB: Anti-FLAG
Trimer
Monomer
IB: Anti-GFP
Figure 2 RIP3 kinase activity is critical for MLKL trimerization (a) HT29
cells (left) or MEF cells (right) were pre-treated with or without necrostatin-1
(Nec-1) and then treated with TSZ for 4 h. The cell lysates were resolved
on non-reducing gel and analysed by immunoblotting with anti-MLKL
antibody. (b) Control-shRNA or RIP3-shRNA HT29 cells were treated
with TSZ as indicated. The cell lysates were resolved on non-reducing
gel and analysed by immunoblotting with anti-MLKL antibody. (c) WT or
RIP3-deficient MEF cells were treated with TSZ and cell lysates were resolved
on non-reducing gel and analysed by immunoblotting with the indicated
antibodies. (d) HEK293 cells were transfected with FLAG–MLKL, RIP3–YFP
or RIP3-D160N–YFP as indicated. The cell lysates were resolved either
on reducing or non-reducing gel and analysed by immunoblotting with the
indicated antibodies. (e) HEK293 cells were transfected with FLAG–MLKL or
FLAG–MLKL-T357A/S358A with or without RIP3–YFP as indicated. The cell
lysates were resolved on non-reducing gel and analysed by immunoblotting
with the indicated antibodies. 1 indicates phosphorylated RIP3. All western
data are representative of two or three independent experiments. Uncropped
images of western blots are shown in Supplementary Fig. 8.
RIP3 kinase is critical for MLKL trimerization
As the upstream regulators of TNF-induced necroptosis, both RIP1
and RIP3 are required for the trimerization of MLKL because treating
the HT29 and MEF cells with necrostatin-1, a RIP1 inhibitor22 ,
or knocking down RIP3 in HT29 cells or the deletion of RIP3
in MEF cells prevented the trimerization of MLKL (Fig. 2a–c). As
RIP3 phosphorylates MLKL (refs 10,11), we then examined whether
RIP3 facilitates MLKL trimerization. When wild-type (WT) or the
kinase-dead RIP3 was co-expressed with MLKL in HEK293 cells, the
WT RIP3, but not the mutant RIP3-D160N, markedly increased the
formation of MLKL trimers under non-reducing conditions (Fig. 2d).
In addition, when the two main RIP3-phosphorylation sites of MLKL
were mutated10 , RIP3 was no longer able to significantly promote
the trimerization of the mutant MLKL-T357A/S358A (Fig. 2e). These
results suggest that the kinase activity of RIP3 is critical for promoting
MLKL trimerization.
CC2 mutant protein failed to trimerize whereas MLKL CC1 mutant
protein was still able to form trimers efficiently as compared to WT
MLKL (Fig. 3b). These results indicated that the CC2 domain is
responsible for MLKL trimerization. As the carboxy-terminal kinase
domain of MLKL mediates its interaction with RIP3, we examined
whether these mutations in the coiled-coil domains affect MLKL
interaction with RIP3 and found that both MLKL CC1 and CC2
mutant proteins interacted with RIP3 normally (Fig. 3c). To check
whether these mutations in MLKL coiled-coil domains affect its
function to mediate necroptosis, we expressed these mutant MLKL
plasmids in HT29 MLKL-shRNA cells, in which the endogenous MLKL
expression was knocked down by a specific MLKL-shRNA targeting its
30 UTR. Whereas the WT MLKL protein partially restored TNF-induced
necroptosis in these cells, both MLKL CC1 and CC2 mutant proteins
failed to do so although they were expressed at similar levels to the WT
protein (Fig. 3d). It is surprising to see that the MLKL CC1 mutant
protein did not reconstitute the sensitivity of these cells to necroptosis
because it still trimerizes and interacts with RIP3. Therefore, it is
possible that the CC1 mutations altered some other features of MLKL
protein that is critical for its function in mediating necroptosis.
The coiled-coil domain 2 of MLKL is responsible for its
trimerization
We next investigated whether MLKL trimerization is mediated by the
two coiled-coil domains and whether the trimerization is important
for MLKL to mediate necroptosis. To address these questions, we
generated MLKL coiled-coiled domain 1 (CC1) and CC2 mutants by
substituting glycine for the conserved leucine or isoleucine residues in
these domains, CC1:L58G/I76G and CC2:L162G/L165G, respectively
(Fig. 3a). These mutations disrupt the α-helices of these two coiled-coil
domains and lead to a compromised secondary structure of these
domains as predicted by the MultiCoil program. When these two
mutant MLKL plasmids were expressed in HEK293 cells, MLKL
MLKL locates to plasma membrane in necroptosis
As protein localization is critical for function4 , we next examined
the cellular localizations of the WT and two mutant MLKL proteins.
To do so, we first overexpressed DsRed-tagged WT MLKL and CC1
and CC2 mutants in HEK293 cells. We found that all three MLKL
proteins localized evenly in the cytoplasm (Fig. 4a). However, when
these MLKL plasmids were co-transfected with FLAG–RIP3, which
increases the trimerization of the WT and the CC1 mutant MLKL
NATURE CELL BIOLOGY VOLUME 16 | NUMBER 1 | JANUARY 2014
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ARTICLES
a
1
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CC2
Protein kinase domain
471
MLKL CC1
L58G l76G
1
196
CC1
CC2
Protein kinase domain
471
MLKL CC2
L162G/L165G
b
c GFP–MLKL-CC2 –
CC1
–
+
WT
+
RIP3–YFP –
CC2
– +
Trimer
–
+
–
+
+
–
–
+
IB: Anti-GFP
IB: Anti-FLAG
Monomer
IB: Anti-GFP
83
IB: Anti-GFP
Input
50
d
–
–
+
+
IP: FLAG
IB: Anti-FLAG
Mr (K) 250
150
100
75
–
–
–
+
GFP–MLKL-CC1 –
GFP–MLKL-WT –
FLAG–RIP3 –
IB: Anti-FLAG
**
50
GFP–MLKL
IB: Anti-MLKL
MLKL
43
IB: Anti-actin
40
20
0
GFP
WT
CC1
CC2
CC2
Mr (K) 100
75
CC1
80
60
WT
MLKL-shRNA
100
GFP
120
HT29
Percentage of
GFP-positive cells
140
TSZ
Figure 3 The coiled-coil domain 2 of MLKL is responsible for its trimerization.
(a) Schematic of coiled-coil-defective mutant proteins of MLKL. The hatched
rectangles represent the predicted regions of the coiled-coil domains,
named CC1 and CC2. (b) HEK293 cells were transfected with FLAG–MLKL,
FLAG–MLKL-CC1 mutant or FLAG–MLKL-CC2 mutant with or without
RIP3–YFP as indicated. The cell lysates were resolved on non-reducing
gel and analysed by immunoblotting as indicated. (c) HEK293 cells were
transfected with GFP–MLKL, GFP–MLKL-CC1 or GFP–MLKL-CC2 with
FLAG–RIP3 as indicated. After 24 h, cell lysates were immunoprecipitated
with anti-FLAG antibody (IP: FLAG) and analysed by immunoblotting with
anti-FLAG and anti-GFP antibodies. (d) Left, HT29 MLKL-shRNA cells were
transfected with GFP, GFP–MLKL, GFP–MLKL-CC1 or GFP–MLKL-CC2
plasmids as indicated for 24 h, followed by treatment with TSZ for 24 h.
Survival was determined by counting GFP+ cells and normalized to untreated
cells. Results shown are averages ± s.d. from three independent experiments.
Right, immunoblot analysis of MLKL and its mutants’ levels in HT29 cells or
rescue MLKL-shRNA HT29 cells. All western data are representative of two
or three independent experiments. ∗∗ P < 0.01. Statistics source data for d
can be found in Supplementary Table 1. Uncropped images of western blots
are shown in Supplementary Fig. 8.
proteins (Fig. 2d), the two mutant MLKL proteins formed some
aggregates in the cytoplasm and surprisingly, most of the WT MLKL
moved to the edges of the transfected cells (Fig. 4a), indicating that
the WT MLKL proteins may translocate to the plasma membrane in
the presence of RIP3. As it is known that the RIP1/RIP3 necrosome
forms aggregates in the cytoplasm10,23 , we then re-performed these
experiments with RIP3–YFP instead of FLAG–RIP3 to examine whether
only MLKL or both MLKL and RIP3 proteins locate to the plasma
membrane. A CellLight Plasma Membrane–CFP was also included
in these experiments as a control of plasma membrane localization.
The overexpressed WT and the mutant MLKL proteins or RIP3 evenly
localized in cytoplasm as reported previously10 (Fig. 4b). When RIP3
and MLKL proteins are co-transfected, the CC1 and CC2 mutant
MLKL and RIP3 proteins remained in the cytoplasm and formed
aggregates (Fig. 4c). Again, surprisingly, we found that not only MLKL
but also RIP3 protein located to the plasma membrane as most of the
MLKL and RIP3 proteins co-localized with the CFP–plasma membrane
marker (Fig. 4c). To confirm that the plasma membrane translocation
of MLKL and RIP3 proteins occurs during TNF-induced necroptosis,
HT29 MLKL-shRNA cells were transfected with DsRed-tagged WT and
mutant MLKL plasmids alone or were co-transfected with RIP3–YFP
plasmid. Unlike in HEK293 cells, these proteins were expressed at levels
lower or similar to their respective endogenous proteins in HT29 cells
and do not form aggregates (Fig. 4d, upper panel and e). Whereas
the MLKL CC1 and CC2 mutants and RIP3 formed aggregates in the
cytoplasm following TSZ treatment, the WT MLKL alone or the WT
MLKL and RIP3 together localized to the plasma membrane in response
to TSZ (Fig. 4d, upper panel and 4f). To quantitatively determine the
translocation of MLKL to the plasma membrane on necrotic induction,
the ratio of the fluorescence intensity near the plasma membrane region
versus the cytosolic region was determined by using line intensity
profiles across selected cells of each sample24 (Supplementary Fig. 2).
As shown in Fig. 4d, lower panel, the relative intensity ratio of WT
MLKL near the plasma membrane region versus the cytosolic region
was greatly increased after TSZ treatment. However, no increase of
the relative intensity ratio was observed in either MLKL-CC1- or
CC2-mutant-transfected cells (Fig. 4d, lower panel). Furthermore,
the trimerization of DsRed–MLKL and CC1 was also observed on
TSZ treatment in MLKL shRNA HT29 cells (Fig. 4g). Therefore, CC1
mutation disrupts MLKL plasma membrane localization. Importantly,
58
NATURE CELL BIOLOGY VOLUME 16 | NUMBER 1 | JANUARY 2014
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ARTICLES
Merged
YFP
CFP
Merged
c
TSZ 4 h
CC2
WT
CC1
HEK293
DsRed–MLKL
MLKL
DsRed–CC1
TSZ 0 h
Mr (K)
100
75
50
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83
IB: Anti-actin
43
50
1
CC1
IB: Anti-actin
Non-reducing
g
DsRed-tagged
TSZ
WT
0 4
CC1
0 4
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CC2
0 4
WT
CC1 CC2
0 4 0 4 0
4 (h)
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150
100
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j
DsRed–MLKLT357A/S358A
RIP3–
YFP
DsRed+
YFP+DIC
TS 4 h
IB: Anti-RIP3
TSZ 0 h
TSZ 0 h
TSZ 4 h
Monomer
IB: Anti-DsRed
RIP3-D160N– DsRed+
YFP
YFP+DIC
-
DsRed–MLKL RIP3–YFP DsRed+YFP+DIC
Trimer
57
DsRed–
MLKL
RIP3–YFP DsRed+YFP+DIC
h
k
RIP3–YFP
YFP+DIC
DsRed–MLKL RIP3–YFP DsRed+YFP+DIC
TSZ 4 h
i
CC2
IB: Anti-GFP
83
43
CC2
DsRed–CC2
MLKL
TNF 4 h
WT
DsRed–MLKL
NSA
TSZ 4 h
0
DsRed-tagged
TSZ 4 h
2
TSZ 0 h
100
75
RIP3–YFP DsRed+YFP+DIC
-
TSZ 4 h
3
CC1
Mr (K)
WT
TSZ 0 h
TSZ 4 h
**
CT
Relative ratio
( Ipm /Icyt)
4
HT29
HT29 MLKL-shRNA
5
Merged
TSZ 0 h
RIP3–YFP
e
DsRed–
CC2
CFP
DsRed–MLKL RIP3–YFP DsRed+YFP+DIC
f
CT
DsRed–
CC1
TSZ 4 h TSZ 0 h
DsRed–
MLKL
RIP3–YFP
CC2
CC1
CC2
CC1
CC2
d
DsRed
TSZ 4 h
DsRed DsRed+DAPI
CFP
MLKL
DsRed+DIC
MLKL
Dsred
DsRed
MLKL
b
FLAG–RIP3
CC1
a
Figure 4 MLKL locates to plasma membrane following necroptosis
induction. (a) HEK293 cells expressing DsRed–MLKL, DsRed–MLKL-CC1
or DsRed–MLKL-CC2 with or without FLAG–RIP3 were analysed for
subcellular localization by confocal imaging. (b,c) HEK293 cells expressing
DsRed–MLKL, DsRed–MLKL-CC1 or DsRed–MLKL-CC2 with (c) or
without (b) RIP3–YFP were analysed for co-localization with plasma marker
(CellLight Plasma Membrane–CFP). (d) MLKL-shRNA HT29 cells were
transfected with DsRed–MLKL, DsRed–MLKL-CC1 or DsRed–MLKL-CC2 and
treated with or without TSZ as indicated. Upper panel, subcellular localization
of MLKL and its mutants was imaged by confocal microscopy. Lower panel,
relative fluorescence intensity ratio of plasma-membrane-adjacent area
(Ipm ) versus average cytosolic area (Icyt ). Thirty transfected cells were
analysed from each sample. Results shown are averages ±s.e.m. from three
independent experiments. (e) Western blotting analysis of expression levels of
DsRed–MLKL and RIP3–YFP in HT29 and HEK293 cells. (f) Representative
images of MLKL-shRNA HT29 cells co-expressing DsRed–MLKL (upper
-
panel), DsRed–MLKL-CC1 (middle panel) and DsRed–MLKL-CC2 (lower
panel) with RIP3–YFP treated with TSZ. (g) Analysis of trimerization of
DsRed–MLKL and DsRed-CC1 in cells from d. The cell lysates were resolved
either on reducing or non-reducing gel and analysed by immunoblotting
with the indicated antibodies. (h–j) Confocal imaging of the subcellular
localization of MLKL and RIP3 in MLKL-shRNA HT29 cells co-expressing
RIP3–YFP and DsRed–MLKL treated with TNF (h, upper panel) or TS
(h, lower panel) or RIP3-D160N–YFP (i), or DsRed–MLKL-T357A/S358A
(j). (k) Confocal imaging of the subcellular localization of MLKL and RIP3
in MLKL-shRNA HT29 cells transfected with RIP3–YFP and treated with
TSZ (upper panel) or DsRed–MLKL plus RIP3–YFP and treated with TSZ
plus NSA (lower panel) as indicated. Scale bar, 5 µm; DIC, differential
interference contrast; DAPI was used as a nuclear staining marker;
∗∗
P < 0.01. Statistics source data for d can be found in Supplementary
Table 1. Scale bar, 10 µm. Uncropped images of western blots are shown in
Supplementary Fig. 8.
NATURE CELL BIOLOGY VOLUME 16 | NUMBER 1 | JANUARY 2014
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a
T C M
0 4 0 4 0 4
b
Reducing
1-3-2
TSZ
1-3-2
Non-reducing
Total
input
T C M
0 4 0 4 0 4 (h)
Mr (K) 250
150
100
75
TSZ
Mr (K) 54
Trimer
IB: Anti-MLKL
100
Δ
IB: Anti-RIP3
57
100
43
c
0
57
Monomer
50
4
0
Cell
surface
4 (h)
IB: Anti-MLKL
Δ
IB: Anti-RIP3
IB: Anti-Na+/K+ ATPase
43
IB: Anti-actin
IB: Anti-Na+/K+ ATPase
17
IB: Anti-COX IV
IB: Anti-actin
90
IB: Anti-calnexin
Total
Cell
input
surface
RIP3–YFP – + + – + +
FLAG–MLKL + – + + – +
Mr (K) 250
Trimer
150
100
IB: Anti-FLAG
75
50
Monomer
100
43
+
+
IB: Anti-Na /K ATPase
17
IB: Anti-actin
IB: Anti-COX IV
90
IB: Anti-calnexin
Figure 5 Plasma membrane translocation of trimerized MLKL in
response to necroptosis induction. (a) Control shRNA or MLKL shRNA
HT29 cells (clone 1-3-2) were treated with TSZ. The total, cytosolic
and crude membrane fractions were resolved either on reducing or
non-reducing gel and analysed by immunoblotting as indicated. T, total
cell lysate. C, cytosolic fraction. M, membrane fraction. (b) HT29
cells were treated with TSZ as indicated. Total cellular lysate and
the biotinylated, cell surface fraction were resolved on reducing gel
and analysed by immunoblotting as indicated. (c) HEK293 cells were
transfected as indicated. Total cellular lysate and the biotinylated, cell
surface fraction were resolved on non-reducing gel and analysed by
immunoblot as indicated. 1 indicates phosphorylated RIP3. Data shown
are representative of two independent experiments. Uncropped images of
western blots are shown in Supplementary Fig. 8.
MLKL and RIP3 membrane localization is specific to necroptosis
because TNF or TS treatment did not change their localizations
in the cytoplasm (Fig. 4h). As in the case of MLKL trimerization,
RIP3 kinase activity and MLKL phosphorylation are also essential
for the membrane localization of MLKL (Fig. 4i,j). In addition, RIP3
membrane translocation requires a functional MLKL because knocking
down MLKL or administering the chemical necrosulphonamide (NSA),
which targets MLKL, abolished RIP3 translocation (Fig. 4k)10 .
We then examined whether TSZ treatment leads to the translocation
of the endogenous MLKL and RIP3 proteins in HT29 cells by
immunohistochemistry. As shown in Supplementary Fig. 3, both
MLKL and RIP3 antibodies are not ideal for immunohistochemistry
staining owing to their high background staining in MLKL and RIP3
knockdown cells, respectively. Nevertheless, TSZ-induced plasma
membrane translocation of MLKL and RIP3 was detected in control
shRNA cells. (Supplementary Fig. 3).
To biochemically prove the translocation of MLKL to the plasma
membrane on necroptosis induction, we first separated the crude
membrane and cytosolic fractions from HT29 cells with or without
TSZ treatment. As shown in Fig. 5a, most MLKL detected in the crude
membrane fraction of treated cells is trimeric. Consistent with our
microscopy study, both MLKL and RIP3 are present in the crude
membrane fraction (Fig. 4a). As most cell membrane fractionation
approaches are contaminated with membrane-containing organelles
such as mitochondria or ER, to provide more specific biochemical
evidence, we then decided to use a cell surface protein isolation
approach to examine whether MLKL and RIP3 are recruited to the
plasma membrane. The rationale for this experimental design is that
MLKL and RIP3 most likely interact with some transmembrane/cell
surface proteins if they are recruited to the plasma membrane. As shown
in Fig. 5b, when cell surface proteins were isolated, both MLKL and the
phosphorylated RIP3 were detected in the sample of TSZ-treated cells
but not in the sample of non-treated cells. In this experiment, Na+ /K+
ATPase was used as a positive control for transmembrane/surface
protein and actin, COX IV and calnexin were used as a control for
cytosolic, mitochondrial and ER membrane proteins, respectively
(Fig. 5b). Importantly, the membrane localization of MLKL and RIP3
was also detected when cell surface proteins were isolated from necrotic
MEF and J774A.1 cells (Supplementary Fig. 4). When MLKL and
RIP3 were overexpressed in HEK293 cells and the cell surface proteins
were isolated and analysed under non-reducing conditions, only the
trimerized MLKL was detected although there is more monomer MLKL
protein detected in the total input control (Fig. 5c). This result further
indicates that the trimerized MLKL locates at the plasma membrane.
Taken together, these experiments suggest that the trimerized
MLKL and RIP3 locate to the plasma membrane during TNF-induced
necroptosis. These findings described in Figs 4 and 5 suggest that the
plasma membrane localization of MLKL may be critical for its function
to mediate TNF-induced necroptosis.
60
MLKL-mediated calcium influx is involved in plasma membrane
rupture during necroptosis
Next we investigated the potential mechanism of MLKL function
at the plasma membrane during necroptosis. One of the unique
features of necroptosis is the loss of plasma membrane integrity.
However, considering that the plasma membrane rupture is a
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60
40
20
Fluo4
DMSO
80
40
20
0
DMSO
c
HT29 Ctrl-shRNA
T357A/S358A
4h
HT29 MLKL-shRNA
0h
4h
4
3
2
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0
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CC1
CC2
T357A/S358A
f
DMSO
TCZ
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MEF MLKL–/–
100
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10 10 10 10 10
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DMSO
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80
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60
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20
0
100 101 102 103 104
Fluo4
20
0
0h
Fluo4
5
Counts
100
4h
Merged
CC2
CC1
Relative fluorescence ratio
(transfected/non-transfected)
WT
TSZ
120
0h
TS
Fluo4+
DsRED+DIC
MEF
4h
60
TSZ
DsRed
e
PI-negative (%)
100
0h
Fluo4
80
0
d
**
100
Ca2+-free DMEM
DMEM
TSZ
120
Merged
PI-negative (%)
120
b
DMEM
Ca2+-free DMEM
PI-negative (%)
a
Control Calcium-free BAPTA-AM
Figure 6 MLKL-mediated calcium influx is involved in plasma membrane
rupture during necroptosis. (a) HT29 cells were cultured in DMEM
with or without calcium and treated with dimethylsulphoxide (DMSO)
control, TSZ for 24 h or TS for 48 h. Cell survival was determined by PI
staining. Results shown are averages ± s.e.m. from three independent
experiments. (b) HT29 cells were loaded with Fluo4 AM and then treated
with TSZ in the presence of normal medium or calcium-free medium
for 4 h. Representative confocal images of live HT29 cells are shown.
(c) HT29 shRNA control cells or MLKL shRNA cells were loaded with
Fluo4 AM and then treated with TSZ for 4 h. Representative confocal
images of live HT29 cells are shown. (d) Left, HT29 MLKL-shRNA
cells were transfected with DsRed–MLKL-WT, DsRed–MLKL-CC1,
DsRed–MLKL-CC2 or DsRed–MLKL-T357A/S358A as indicated. After
24 h transfection, cells were loaded with Fluo4 and then treated with TSZ
for 4 h. The representative confocal images are shown. Right, ratiometric
measurement of Fluo4 mean fluorescence intensity between transfected
and non-transfected cells (20 cells from each part). Results shown are
averages ± s.e.m. from three independent experiments. (e) MEF cells
were cultured with or without calcium or pre-treated with BAPTA-AM
(10 µm) for 30 min and then treated with TCZ for 13 h. Cell survival was
determined by PI staining. Results shown are averages ± s.e.m. from three
independent experiments. (f) WT, MLKL-deficient or RIP3-deficient MEF
cells were treated with TSZ or TCZ for 6 h. Cells were collected and loaded
with Fluo4. Fluo4 fluorescent cells were determined by FACS analysis.
∗∗
P < 0.01. Scale bar, 10 µm. Statistics source data for this figure can be
found in Supplementary Table 1.
relatively late event, for example, at 8 h after TSZ treatment in
HT29 cells, compared with MLKL plasma membrane translocation
(3–4) h following necroptosis induction, we explored the possible
immediate downstream event of MLKL translocation. As extracellular
calcium influx has been suggested to play a role in necroptosis19 , we
decided to examine whether MLKL plasma membrane localization is
required for calcium influx. We first confirmed that calcium influx
is involved in necroptosis in HT29 cells as most of these cells were
negative for propidium iodide (PI) staining under necroptosis but not
apoptosis when they were cultured in calcium-free medium (Fig. 6a).
To prove there is calcium influx during necroptosis, HT29 cells
were loaded with the calcium indicator Fluo4. We found that Fluo4
fluorescence was markedly increased after 4 h of TSZ treatment in
cells cultured in regular medium but not in calcium-free medium
(Fig. 6b). Importantly, this calcium influx during necroptosis in
HT29 cells requires MLKL because it was completely blocked in
MLKL-shRNA cells or in NSA-treated cells (Fig. 6c and Supplementary
Fig. 5a). The involvement of MLKL-mediated calcium influx in
TSZ-induced membrane damage is further confirmed in JurkatFADD−/−
and mouse J774A.1 cells (Supplementary Fig. 6). Furthermore, treating
the cells with necrostatin-1 or knocking down RIP3 also blocked
TSZ-induced calcium influx, suggesting that both RIP1 and RIP3 are
required for calcium influx (Supplementary Fig. 5a). The defect of
calcium influx in MLKL-shRNA cells following TSZ treatment was
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a
HT29
120
DMSO
TSZ
*
PI-negative (%)
60
40
Control
Nifedipine
NiCl2
c
U937
120
60
40
LaCl3
0
2-APB
DMSO
Control
d
NAC
LaCl3
JurkatFADD–/–
TSZ
**
100
LaCl3 + NAC
DMSO
TSZ
**
100
80
PI-negative (%)
PI-negative (%)
80
20
20
80
60
40
60
40
20
20
0
TSZ
**
100
80
0
JurkatFADD–/–
120
**
100
PI-negative (%)
b
DMSO
Control
LaCl3
NAC
LaCl3 + NAC
0
Control
LaCl3
(100 µm)
LaCl3
(200 µm)
Figure 7 Calcium channels are involved in necroptosis. (a) HT29 cells
were treated with nifedipine (100 µm), NiCl2 (1 mM), LaCl3 (100 µm) or
2-APB (50 µm) and TSZ for 24 h. Cell survival was determined by flow
cytometry of PI staining. (b,c) JurkatFADD−/− cells (b) or U937 cells (c)
were pre-treated with LaCl3 (100 µM), NAC (5 mM) or LaCl3 plus NAC.
Then, the cells were treated with TSZ as indicated. After 10 h, cell survival
was determined by PI staining. (d) JurkatFADD−/− cells were pre-treated
with the increased concentration of LaCl3 (200 µM) and then treated with
TSZ as indicated. After 10 h, cell survival was determined by PI staining.
Results shown are averages ± s.e.m. from three independent experiments.
∗
P < 0.05, ∗∗ P < 0.01. Statistics source data for this figure can be found
in Supplementary Table 1.
restored by the ectopic expression of the WT MLKL, but not the
MLKL CC1, CC2 and MLKL T357A/S358A mutants (Fig. 6d). As the
trimerization and the plasma membrane localization of MLKL and
the phosphorylation of RIP3 were not affected under calcium-free
conditions in TSZ-treated cells (Supplementary Fig. 5b,c), these
results indicated that calcium influx is a downstream event of MLKL
trimerization and membrane localization.
In our earlier study, we demonstrated that an important downstream
event of MLKL during necroptosis is the generation of ROS (ref. 11).
We now demonstrated that calcium influx is another important
downstream event of MLKL during TNF-induced necroptosis.
Although ROS plays a minimal role in necroptosis in HT29 cells,
calcium influx is involved in plasma membrane damage of these cells.
In other types of cell that are sensitive to TNF-induced necroptosis,
such as U937 cells, both calcium influx and ROS are critical for
MLKL to mediate necroptosis (Supplementary Fig. 7). In addition,
the intracellular calcium release also contributes to TSZ-induced
necroptosis in some types of cell, such as MEFs, because the deletion of
extracellular calcium only marginally blocked TCZ-induced membrane
damage in MEFs, but the cell-permeable calcium chelator BAPTA-AM,
which decreases the total level of intracellular Ca2+ , blocks membrane
damage more efficiently (Fig. 6e). Nevertheless, MLKL and RIP3 are
required for both extracellular calcium influx and the intracellular
calcium release in MEFs (Fig. 6f).
The calcium influx during necroptosis suggests that calcium channels
in the plasma membrane may play a role in necroptosis. Therefore, we
then tested which type of calcium channel is involved in necroptosis19 .
When we treated HT29 cells with the L-type voltage-dependent
calcium channel blocker nifedipine, the T-type voltage-dependent
calcium channel blocker NiCl2 or the non-voltage-sensitive channel
blockers LaCl3 and 2-APB before administration of TSZ, we found
that only the two non-voltage-sensitive channel blockers, LaCl3 and
2-APB, protected cells from TSZ-induced necroptosis (Fig. 7a). The
non-voltage-sensitive channel blocker LaCl3 had a similar effect on
TSZ-induced necroptosis in JurkatFADD−/− and U937 cells (Fig. 7b–d).
These results indicated that the non-voltage-sensitive channels may
function downstream of MLKL.
62
TRPM7 is involved in MLKL-mediated necroptosis
Among the known non-voltage-sensitive channels, TRPM7 has
previously been implicated in necroptosis25–27 . As TRPM7 is expressed
in HT29 cells, we then tested whether TRMP7 is involved in
TSZ-induced necroptosis and found that TRPM7 knockdown
significantly protected HT29 cells from TSZ-induced necroptosis
(Fig. 8a). Furthermore, the same protective effect was also observed in
TRPM7-shRNA JurkatFADD−/− and TRPM7-shRNA J774A.1 cells on
necrotic induction (Fig. 8b,c). Importantly, knocking down TRPM7
did not affect RIP3 phosphorylation and MLKL trimerization and
NATURE CELL BIOLOGY VOLUME 16 | NUMBER 1 | JANUARY 2014
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ARTICLES
a
*
80
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60
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0
shRNA: Ctrl
TRPM7- TRPM7shRNA1 shRNA2
60
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TRPM7shRNA1
43
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4
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mTRPM7shRNA1
mTRPM7shRNA2
IP: TRPM7
IB: TRPM7
IB: Anti-actin
Mr (K) 220
43
HT29
TSZ 0
Trimer
2
4
TRPM7-shRNA1
Mr (K) 57
8 (h)
Δ
IB: Anti-RIP3
74
IB: Anti-RIP1
43
IB: Anti-actin
Monomer
50
43
Ctrl-shRNA
8 (h)
150
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IP: TRPM7
IB: TRPM7
IB: Anti-actin
Mr (K) 220
e
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2
J774A.1
TRPM7shRNA1
Ctrl-shRNA
TRPM7shRNA1
TRPM7shRNA2
Ctrl-shRNA
IP: Anti-TRPM7
IB: Anti-TRPM7
IB: Anti-actin
TSZ 0
Mr (K) 250
80
JurkatFADD–/–
HT29
d
*
100
PI-negative (%)
PI-negative (%)
100
Mr (K) 220
DMSO
TSZ
J774A.1
**
100
**
0
shRNA: Ctrl
c
DMSO
TSZ
JurkatFADD–/–
*
120
PI-negative (%)
b
DMSO
TSZ
HT29
8
0
2
4
IB: Anti-MLKL
f
g
HT29 TRPM7-shRNA1
RIP3 DsRed+YFP+DIC
GCaMP3 ΔF/F0
TSZ 4 h
1,400
Ctrl-shRNA + DMSO
Ctrl-shRNA + TSZ
TRPM7-shRNA1 + DMSO
TRPM7-shRNA1 + TSZ
–100
1.0
0.5
0
–0.5
–1.0
h
–60
HT29 Ctrl-shRNA
HT29 TRPM7-shRNA1
HT29 calcium-free
1.5
TSZ 0 h
MLKL
pA
0
1
2
i
3
4
IP:
TSZ
1,000
Mr (K) 54
600
57
200
220
–20
–200
20
60
Figure 8 TRPM7 is involved in MLKL-mediated necroptosis.
(a–c) HT29 (a), JurkatFADD−/− (b) or J774A.1 (c) cells expressing
control-shRNA or TRPM7-shRNAs were treated with or without TSZ for
24 h. Cell survival was determined by PI staining. Upper panel, results
shown are averages ± s.e.m. from three independent experiments. Lower
panel, TRPM7 protein levels were determined by immunoprecipitation
and immunoblotting with anti-TRPM7 antibody. (d,e) Control-shRNA
or TRPM7-shRNA HT29 cells were treated with TSZ as indicated. The
cell lysates were resolved either on non-reducing (d) or reducing gel (e)
and analysed by immunoblotting as indicated. (f) Representative confocal
images of live TRPM7-shRNA HT29 cells co-expressing DsRed–MLKL and
RIP3–YFP treated with TSZ at time 0 and 4 h. Scale bar, 5 µm. (g) The
changes of GCaMP3 fluorescence in control-shRNA, MLKL-shRNA HT29
100
mV
43
5
6
IgG
0
8 (h)
7
TRPM7
4
0
4
Input
0
4
(h)
IB: Anti-MLKL
Δ
IB: Anti-RIP3
IB: Anti-TRPM7
IB: Anti-actin
cells or HT29 cells cultured in calcium-free medium during TSZ treatment.
Data are expressed as change of GCaMP3 fluorescence 1F over basal
fluorescence F0 at any given time. Results shown are averages ± s.e.m.
in 10 cells. (h) Recording TSZ-induced current in control-shRNA or
TRPM7-shRNA HT29 cells. I–V curve measurements were obtained
after a 4 h TSZ treatment and averaged from the 10 cells in each
experiment. (i) HT29 cells were treated with TSZ for 4 h. Cell lysates were
immunoprecipitated with anti-TRPM7 antibody. The immunoprecipitated
complexes were analysed by immunoblotting as indicated. 1 indicates
phosphorylated RIP3. Data shown are representative of three independent
experiments. ∗ P < 0.05, ∗∗ P < 0.01. Scale bar, 10 µm. Statistics source
data for this figure can be found in Supplementary Table 1. Uncropped
images of western blots are shown in Supplementary Fig. 8.
NATURE CELL BIOLOGY VOLUME 16 | NUMBER 1 | JANUARY 2014
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plasma membrane localization following TSZ treatment (Fig. 8d–f).
The decreased levels of RIP1 and RIP3 at 8 h after TSZ treatment
in Fig. 8d are due to proteasome-mediated degradation of these two
proteins. To confirm that TRMP7 is responsible for the calcium influx
during necroptosis, a genetically encoded Ca2+ indicator, GCaMP3
(ref. 28), was used to monitor the Ca2+ level in live HT29 cells treated
with TSZ. We found that the GCaMP3 fluorescent signal was markedly
increased at 4 h after TSZ treatment in the control HT29 cells; however,
no change of GCaMP3 fluorescent signal was detected in TRPM7
knockdown cells or in cells cultured in calcium-free medium (Fig. 8g).
The actual representative images of these experiments are shown in
Supplementary Videos 1–3. To further prove that a TRPM7-mediated
current was induced by TSZ treatment, we performed patch-clamp
recordings in both control shRNA and TRPM7 shRNA HT29 cells
after 4 h of TSZ treatment. As shown in Fig. 8h, control shRNA cells
treated with TSZ exhibited robust TSZ-induced currents, whereas
TRPM7 knockdown significantly inhibited the increase of currents.
These results indicated that TSZ triggered a TRPM7-dependent current
in HT29 cells. As MLKL and RIP3 localize to the plasma membrane
where the TRPM7 resides, we then examined whether MLKL and RIP3
are in the same complex with TRPM7 on necroptosis induction. By
performing immunoprecipitation experiments with a TRPM7 specific
antibody, we found that both MLKL and the phosphorylated RIP3
co-precipitated with TRPM7 after 4 hrs of TSZ treatment (Fig. 8i).
Taken together, these results suggest that TRPM7 is a downstream
component of MLKL and is responsible for the calcium influx and
subsequent plasma membrane damage.
DISCUSSION
The mechanism of TNF signalling has been intensively studied and, in
particular, the regulation of TNF-induced apoptosis is well understood.
However, the machinery of TNF-initiated necroptosis is still largely
unknown. Our present data provide a mechanism of MLKL-mediated
TNF-induced necroptosis, by which MLKL targets the RIP3 necrosome
to the plasma membrane and regulates calcium influx through TRPM7.
Our results demonstrate that MLKL needs to form trimers and to
localize to the plasma membrane to mediate TNF-induced necroptosis.
A recent study showed the structure of MLKL (ref. 29), which supports
our finding that MLKL functions as a trimer.
Necroptosis is characterized by a rise in cytosolic Ca2+ , increased
ROS, intracellular acidification, a depletion of ATP, and ultimately
plasma membrane rupture19 . Our study demonstrates that the
trimerized MLKL locates at the plasma membrane and causes TRPM7mediated calcium influx. As TRPM7 was known as a cation channel, it
is important to test whether other ions such as Na+ and Mg2+ may also
play a role in necroptosis, particularly in cells that are not protected
well by calcium-free medium, such as MEFs. Interestingly, a recent
study reported that cations, particularly Ca2+ , function as switches
of amyloid-mediated membrane disruption30 . As RIP1 and RIP3
form an amyloid-like complex23 , it is possible that MLKL-mediated
cations/Ca2+ influx triggers the necrosome to disrupt the plasma
membrane. A previous study showed that the intracellular C terminus
of TRPM7 was cleaved by caspase 8 during apoptosis31 . It reported
that the cleavage of TRPM7 increases its activity as a cation channel,
but did not affect the Ca2+ influx. In our study, we showed that TSZ
treatment leads to a marked increase of Ca2+ influx, which is regulated
64
by MLKL/RIP3. Therefore, it is critical to study how RIP3/MLKL
regulates TRPM7 activity in a future study.
PGAM5 was identified as a key mediator of necroptosis as it is
involved in calcium ionophore-, ROS- and TNF-induced necroptosis20 .
However, because RIP3 and MLKL are needed for TNF-induced
necroptosis, it is possible that PGAM5 is a downstream component of
calcium influx and ROS generation in the necrotic pathway.
Taken together, our present results provide evidence that MLKL
connects the RIP3 necrosome with the changes of the plasma membrane
and the Ca2+ influx through a cation channel, TRPM7, in TNF-induced
necroptosis.
METHODS
Methods and any associated references are available in the online
version of the paper.
Note: Supplementary Information is available in the online version of the paper
ACKNOWLEDGEMENTS
We thank J. Han for MLKL-deficient MEF cells and MLKL antibody, and L. Looger
for GCaMP3 plasmid. This research was supported by the Intramural Research
Program of the Center for Cancer Research, National Cancer Institute, National
Institutes of Health.
AUTHOR CONTRIBUTIONS
Z.C. and Z-G.L. conceived the study. Z.C. and S.J. did most of the experimental
work. J.Z. did some confocal imaging experiments and cell death assays using MEF
cells and TRPM7 shRNA JurkatFADD−/− cells. L-g.W. and H-C.C. designed and
performed whole-cell patch clamp recording. Y.W. helped with confocal microscopy.
J.L. generated MEF cells from RIP3-deficient mice and performed analysis. Z.C., S.C.
and Z-G.L. wrote the paper. Z-G.L. supervised the study. All authors discussed the
results and implications and commented on the manuscript at all stages.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at www.nature.com/doifinder/10.1038/ncb2883
Reprints and permissions information is available online at www.nature.com/reprints
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PGAM5 functions at the convergence point of multiple necrotic death pathways. Cell
148, 228–243 (2012).
21. Wolf, E., Kim, P. S. & Berger, B. MultiCoil: a program for predicting two- and
three-stranded coiled coils. Protein Sci. 6, 1179–1189 (1997).
22. Degterev, A. et al. Chemical inhibitor of nonapoptotic cell death with therapeutic
potential for ischemic brain injury. Nat. Chem. Biol. 1, 112–119 (2005).
23. Li, J. et al. The RIP1/RIP3 necrosome forms a functional amyloid signaling complex
required for programmed necrosis. Cell 150, 339–350 (2012).
24. Oancea, E., Teruel, M. N., Quest, A. F. & Meyer, T. Green fluorescent protein
(GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent
indicators for diacylglycerol signaling in living cells. J. Cell Biol. 140,
485–498 (1998).
25. Aarts, M. et al. A key role for TRPM7 channels in anoxic neuronal death. Cell 115,
863–877 (2003).
26. McNulty, S. & Fonfria, E. The role of TRPM channels in cell death. Pflugers Arch.
451, 235–242 (2005).
27. Bates-Withers, C., Sah, R. & Clapham, D.E. TRPM7, the Mg(2+) inhibited channel
and kinase. Adv. Exp. Med. Biol. 704, 173–183 (2011).
28. Tian, L. et al. Imaging neural activity in worms, flies and mice with improved GCaMP
calcium indicators. Nat. Methods 6, 875–881 (2009).
29. Murphy, J. M. et al. The pseudokinase MLKL mediates necroptosis via a molecular
switch mechanism. Immunity 39, 443–453 (2013).
30. Sciacca, M. F.
et al. Cations as switches of amyloid-mediated
membrane disruption mechanisms: calcium and IAPP. Biophys. J. 104,
173–184 (2013).
31. Desai, B. N. et al. Cleavage of TRPM7 releases the kinase domain from the ion
channel and regulates its participation in Fas-induced apoptosis. Dev. Cell 22,
1149–1162 (2012).
NATURE CELL BIOLOGY VOLUME 16 | NUMBER 1 | JANUARY 2014
© 2014 Macmillan Publishers Limited. All rights reserved
65
METHODS
DOI: 10.1038/ncb2883
METHODS
Constructs, shRNAs, reagents and antibodies. FLAG epitope-tagged human
MLKL was described previously11 . pEGFP-N1-RIP3 and pEGFP-N1-RIP3-D160N
were provided by F. K. Ming Chan (University of Massachusetts Medical School,
USA). FLAG–RIP3 was provided by J. Han (Xiamen University, China). pEYFPN1-RIP3 and pEYFP-N1-RIP3-D160N were generated by subcloning RIP3 into
pEYFP-N1 from RIP3–EGFP constructs. MLKL point mutants were generated using
site-directed mutagenesis and confirmed by sequencing analysis. These mutants
were inserted into pCMV-Tag2A(FLAG), pDsRed-Monomer-C1 (DsRed) and
pEGFP-C3(GFP) plasmids. GCaMP3 plasmid was a gift from L. Looger (Addgene
plasmid #22692). All of the plasmid constructs were confirmed by DNA sequencing.
The shRNA lentiviral plasmids were purchased from Sigma-Aldrich. The shRNA
against MLKL corresponds to the 30 untranslated region 1,936–1,956 relative to
the first nucleotide of the start codon of human MLKL (GenBank accession
number NM_152649). The shRNA MLKL 1-3-2 HT29 clone has been reported
previously11 . The shRNAs against human TRPM7 corresponded to the coding
regions 1,312–1,332 and 5,948–5,968 (TRPM7-sh#1and TRPM7-sh#2, respectively)
relative to the first nucleotide of the start codon of human TRPM7 (GenBank
accession number NM_017672). The shRNAs against mouse TRPM7 corresponded
to the coding regions 4,107–4,127 and 4,538–4,558 (TRPM7-sh#1 and TRPM7sh#2, respectively) relative to the first nucleotide of the start codon of mouse
TRPM7 (GenBank accession number NM_021450). The shRNA against human
RIP3 corresponds to the coding region 1,490–1,510 relative to the first nucleotide
of the start codon of human MLKL (GenBank accession number NM_006871).
TNFα and z-VAD-fmk were purchased from R&D. Cycloheximide was
purchased from Sigma. Smac mimetic was a gift from S. Wang (University of
Michigan, Ann Arbor, Michigan, USA). Fluo4 was purchased from Invitrogen.
NSA (necrosulphonamide) was purchased from ChemBridge. Antibodies were from
commercial sources: anti-V5 (catalogue number: R960-25, Invitrogen); anti-FLAG
(catalogue number, F9291; clone number, M2), anti-actin (catalogue number,
A3853; clone number, AC-40), anti-GFP (catalogue number, G1544) and antiMLKL (catalogue number, M6687) from Sigma; anti-DsRed (catalogue number,
632496; Clontech); anti-TRPM7 (catalogue number, 75-114; clone number, N74/25;
NeuroMab); anti-RIP3 (catalogue number, ab72106; Abcam); anti-RIP1 (catalogue
number, 610459; clone number, 38; BD Biosciences). Antibody against mouse
MLKL was provided by J. Han (Xiamen University, China).
Cell culture. HT-29, HEK293, mouse J774A.1 and MEF cells were cultured in
DMEM. JurkatFADD−/− and U937 cells were cultured in RPMI1640. WT and
RIP3-deficient MEF cells were generated from day 14 embryos of the same litter
of RIP3 heterozygous breeding32 . WT and MLKL-deficient MEF cells were provided
by J. Han (Xiamen University, China)33 . All media were supplemented with 10%
FBS (vol/vol), 2 mM l-glutamine, and 100 units ml−1 penicillin/streptomycin. The
stable HT29 MLKL-shRNA 1-3-2 clone was described previously11 .
Lentivirus infection. HEK293T cells were co-transfected with pCMV-VSV-G and
pCMV-dr8.2-dvpr and either non-targeting or MLKL-shRNA or TRPM7-shRNA
plasmids. After 24 h, supernatant was collected and this lentiviral preparation was
used to infect cells. After 24 h of infection, cells were selected with puromycin for a
further 48 h.
Necroptosis induction and calcium-free medium treatment. Necroptosis was
induced by pretreatment with z-VAD-fmk (20 µM) and Smac mimetic (10 nM)
or cycloheximide (10 µg ml−1 ) for 30 min and followed by TNFα (30 ng ml−1 )
for the indicated time periods. Apoptosis was induced by TNFα(30 ng ml−1 ) and
Smac (10 nM) for the indicated time periods. Calcium-free DMEM was purchased
from Invitrogen. Calcium-free RPMI1640 was purchased from USbiological. For
calcium-free medium treatment, HT29, MEF and J774A.1 cells were pre-incubated
with calcium-free medium together with z-VAD-fmk and Smac mimetic for 30 min
and followed by TNFα for the indicated time periods. For JurkatFADD−/− and U937
cells, cells were pre-incubated with calcium-free medium overnight (12 h) and then
treated with z-VAD-fmk, Smac mimetic and TNFα for the indicated time periods.
Crude cell membrane fraction. HT-29 cells (2.5 × 106 cells) were detached from
the culture plates with a non-enzymatic lifting solution consisting of HBSS with
1 mM EDTA and washed twice with PBS. Cells were re-suspended in 1 ml of
the fractionation buffer (250 mM sucrose, 20 mM HEPES at pH 7.4, 10 mM KCl,
1.5 mM MgCl2 , 1 mM EDTA and 1 mM EGTA) and placed on ice for 10 min. Cells
were disrupted by freezing with liquid nitrogen for 5 min and then were thawed
on ice, repeated three times. The lysates were passed through a 25 G needle (BD
Biosciences) ten times. Nuclei and unbroken cells were removed by centrifugation
at 750g for 5 min. The supernatant was collected and centrifuged again at 10,000g
for 5 min. The supernatant was then centrifuged at 100,000g in an Optima TLX
Ultracentrifuge (Beckman Coulter) for 1 h at 4 ◦ C. The supernatant containing
cytosolic proteins were concentrated using an acetone precipitation method. The
pellets containing membrane proteins were washed with the fractionation buffer
and were re-centrifuged at 100,000g for 45 min. The pellets were resuspended in M2
buffer and resolved in 4–20% SDS–polyacrylamide gels for western blot analysis.
Cell surface protein isolation. Surface biotinylation was performed according to
the manufacturer’s instructions with modifications using the Cell Surface Protein
Isolation Kit (Pierce). Briefly, HT-29 cells (3 × 107 cells) were washed three times
with ice-cold PBS solution and incubated with a crosslinking reagent (DSP, 2 mM)
in PBS for 30 min at room temperature, followed by incubation in 10 mM Tris,
pH 7.5, buffer for 15 min to quench the reaction. Then, a membrane-impermeable
biotinylation reagent sulpho-NHS-SS-biotin (0.2 mg ml−1 in PBS buffer) was added
and incubated for 30 min on ice with gentle shaking. The reaction was stopped,
cells were lysed in lysis buffer containing 0.1% SDS, and biotinylated proteins
from the cell surface were isolated from lysates by incubation with immobilized
streptavidin–agarose beads. A sample of the initial cell lysate was retained for
analysis of total proteins. The biotinylated proteins were eluted from the beads using
SDS–PAGE sample buffer containing 50 mM DTT. All of the eluted proteins and
0.2% of the total proteins were separated on SDS–PAGE for immunoblot analysis.
For HEK293 cell surface biotinylation, the cells (1 × 107 cells) were washed and
directly incubated with biotinylation reagent for 30 min on ice with gentle shaking.
Cells were lysed without SDS and incubated with immobilized streptavidin–agarose
beads. The biotinylated proteins were removed from the beads by boiling in
SDS–PAGE sample buffer without DTT for 5 min. All of the eluted proteins and
0.2% of the total proteins were separated on SDS–PAGE under non-reducing
conditions, and proteins were quantified by immunoblot analysis.
Immunoblotting, crosslinking and immunoprecipitation. Cells were collected
and lysed in M2 buffer (20 mM Tris, pH 7, 0.5% NP40, 250 mM NaCl, 3 mM
EDTA, 3 mM EGTA, 2 mM DTT, 0.5 mM phenylmethylsulphonyl fluoride, 20 mM
glycerol phosphate, 1 mM sodium vanadate and 1 µg ml−1 leupeptin). Cell lysates
were separated by SDS–PAGE and analysed by immunoblotting. For non-reducing
gel analysis, cells were lysed in M2 buffer without DTT and separated by SDS–PAGE
without β-mercaptoethanol. The dilution ratio of the antibodies used for western
blotting is 1:1,000. The proteins were visualized by enhanced chemiluminescence,
according to the manufacturer’s (Amersham) instructions.
For immunoprecipitation, lysates were precipitated with antibody (1 µg) and
protein G-agarose beads by incubation at 4 ◦ C overnight. Beads were washed four
to six times with 1 ml M2 buffer, and the bound proteins were removed by boiling
in SDS buffer and resolved in 4–20% SDS–polyacrylamide gels for western blot
analysis.
For TRPM7 endogenous immunoprecipitation, crosslinking of cellular proteins
was performed before the cell lysis. Cells were washed three times with ice-cold PBS
solution and incubated with a crosslinking reagent (DSP, 2 mM) in PBS for 30 min at
room temperature, followed by incubation in 10 mM Tris, pH 7.5, buffer for 15 min
to quench the reaction. Then, cells were lysed in RIPA buffer containing 0.1% SDS
and the samples were used in regular immunoprecipitation procedure as described
above. All western data are representative of two or three independent experiments.
Confocal imaging and analysis. HT29 or HEK293 cells were cultured in a
4-well chamber slide (Nunc Lab-Tek II Chamber Slide system) for 24 h and
transfected with 0.2 µg pDsRed–MLKL and 0.2 µg pEYFP–RIP3 or FLAG–RIP3
by Lipofectamine 2000 (Invitrogen). After 24 h post-transfection, HT29 cells were
treated with TNFα/Smac mimetic/ z-VAD-fmk and plated in a 37◦ imaging
station chamber. For imaging WT and mutant DsRed–MLKL with RIP3–YFP in
HEK293 cells, transfected cells were directly visualized by confocal microscopy.
For membrane localization analysis, 5–10 random fields (based on total counted
cell number) were selected in each slide and a total of 30 cells were counted for
membrane localization analysis. To quantify the fluorescence intensity ratio between
the plasma membrane and cytosol in HT29 cells, a line (5 µm) was drawn across each
one of the cells. The line intensity profiles were obtained by using a linescan program
(MetaMorph). The results shown are averages ± s.e.m. from three independent
experiments.
For immunofluorescence staining of endogenous MLKL and RIP3, HT29 cells
were plated on coverslips in 24-well plates in complete media. The next day, cells
were washed three times with PBS followed by fixation with 4% paraformaldehyde
(in PBS) for 12 min at room temperature. The cells were washed three times with PBS
after fixation. Cells were permeabilized with 1% Triton X-100 (in 0.02% BSA/PBS)
for 2 min at room temperature and washed again three times with PBS. The cells
were incubated for 10 min with blocking reagent (20% goat serum in 2% BSA/PBS)
then incubated with the indicated primary antibodies for 1 h: rabbit polyclonal
anti-human MLKL (1:50), or rabbit polyclonal anti-human RIP3 (1:250). The cells
were washed three times in PBS, and then incubated with the FITC-conjugated
goat anti-rabbit secondary antibodies and rhodamine-conjugated phalloidin for
NATURE CELL BIOLOGY
© 2014 Macmillan Publishers Limited. All rights reserved
METHODS
DOI: 10.1038/ncb2883
30 min, followed by DAPI for 5 min. Cells were washed three times with PBS and
mounted on slides using Fluorogel (Electron Microscopy Sciences). Stained cells
were observed on a Zeiss AxioObserver.Z1 microscope equipped with a PlanApo
×63/1.4 oil DIC III objective. Representative confocal images were generated on
a Zeiss LSM510 META laser scanning microscope (Zeiss). All confocal images are
representative of three independent experiments.
Fluo4 and GCaMP3
Ca2+
imaging. Cells were loaded with 2 µM Fluo4 AM in
the HEPES-buffered saline solution (HBSS) containing (in mM): 134 NaCl, 5.4 KCl,
1.0 MgSO4 , 1.0 NaH2 PO4 , 1.8 CaCl2 , 20 HEPES and 5 d-glucose (pH 7.4) at 37 ◦ C
for 30 min. For cells cultured in calcium-free medium, CaCl2 was depleted from
HBSS solution. For fluorescence imaging, cells were excited at 488 nm, emission was
detected at 505–550 nm and a DIC transmission image was acquired simultaneously.
A Zeiss LSM 510 Meta confocal microscope with a ×63 oil objective was used for
imaging. The Fluo4 fluorescence intensity was measured by ImageJ (NIH). For Fluo4
flow cytometry analysis, Fluo4-loaded cells were detached with a non-enzymatic
lifting solution consisting of HBSS with 1 mM EDTA and washed twice with HBSS.
Cells were suspended in HBSS buffer with or without calcium and analysed by flow
cytometry on a FACS Calibur (Becton Dickinson) at an excitation wavelength of
488 nm and with a 530 nm detection channel.
The stabilized GCaMP3 HT29 cells were established by transfecting HT29 cells
with GCaMP3 construct and selecting with G418 (1.5 mg ml−1 ) for 1 week. The
fluorescence intensity at 488 nm (F488 ) was monitored using a Zeiss LSM 510 Meta
confocal microscope with an incubator chamber. Time-lapse Ca2+ imaging was
carried out for 8 h with 10 min intervals. Three independent experiments were
performed and representative data in one experiment were expressed as the change
in GCaMP3 fluorescence 1F over the basal fluorescence F0 at any given time. All
confocal images are representative of three independent experiments.
Electrophysiology. Control shRNA or TRPM7 shRNA HT29 cells were treated
with dimethylsulphoxide (DMSO) or z-VAD-FMK (TSZ) for 4 h. Whole-cell
patch-clamp experiments were performed at 4 h after TSZ treatment. The I –V
relationship was obtained with a 500 ms voltage ramp pulse applied from −100 mV
to +100 mV. The holding potential was 0 mV. The extracellular solution (ECF)
contained (in mM): 140 Gluta-NaCl, 5.4 KCl, 2 CaCl2 , 25 HEPES and 33 glucose.
The pH was adjusted to 7.4 with NaOH. The pipette solution contained (in mM):
145 Cs-methanesulphonate, 8 NaCl, 5 ATP, 1 MgCl2 , 10 EGTA, 4.1 CaCl2 and 10
HEPES. The pH was adjusted to 7.3 with CsOH.
Cell rescue and survival assay . For cell rescue assay, HT29 cells were infected
with MLKL-shRNA against the 30 untranslated region of MLKL. After selection with
puromycin for a further 48 h, these HT29 MLKL shRNA cells were transfected with
the GFP-tagged MLKL, CC1 or CC2 plasmids (0.2 µg per well in a 6-well plate)
as indicated in Fig. 3d and then treated with TSZ or DMSO for a further 24 h.
All GFP-positive cells were counted by flow cytometry from 10,000 events in each
transfection. Cell survival was determined by comparing GFP-positive cells from
TSZ-treated wells with the DMSO-treated control wells in each transfection. The
normalization equation is as follows:
GFP-positive cells (%) =
Number of GFP-positive cells with TSZ treatment
Number of GFP-positive cells with DMSO treatment
Data represent three independent experiments.
For cell death analysis, cells were treated with different reagents as indicated and
then stained with PI (1 µg ml−1 ) for 5 min. A total of 10,000 cells were counted by PI
flow cytometry and cell survival was determined as PI-negative population. Three
independent experiments were performed.
Statistical analysis. A Student’s t -test and one-way analysis of variance were used
for comparison among all different groups. P < 0.05 was considered statistically
significant.
32. Newton, K., Sun, X. & Dixit, V. M. Kinase RIP3 is dispensable for normal NF-kappa
Bs, signaling by the B-cell and T-cell receptors, tumor necrosis factor receptor 1,
and Toll-like receptors 2 and 4. Mol. Cell Biol. 24, 1464–1469 (2004).
33. Wu, J. et al. Mlkl knockout mice demonstrate the indispensable role of Mlkl in
necroptosis. Cell Res. 23, 994–1006 (2013).
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S U P P L E M E N TA R Y I N F O R M AT I O N
DOI: 10.1038/ncb2883
a
Coiled-coil domain 1
H.sapiens
P.troglodytes
M.musculus
R.norvegicus
B.taurus
Coiled-coil domain 2
H.sapiens
P.troglodytes
M.musculus
R.norvegicus
B.taurus
MultiCoil score
b
Residue position
Supplementary Figure 1 Structure determination and assembly pattern
prediction of coiled-coil domain, related to Figure 1. (a) Sequence
alignment of the N-terminal coiled-coil domain of MLKL from vertebrate
species. Two distinctive heptad repeat pattern (abcdefg)n characteristic
of a coiled-coil architecture are indicated above the alignment. Coiledcoil residues occupying hydrophobic ‘a’ and ‘d’ position are denoted by
yellow and red. These coiled-coil motifs are commonly shared among
the MLKL orthologs in different vertebrate species. (b) Prediction of
oligomerization states of coiled-coil domain of MLKL by Multicoil program
(http://groups.csail.mit.edu/cb/multicoil/cgi-bin/multicoil.cgi). The score
for potential coiled-coils with a dimeric probability is indicated by blue
line and a trimeric probability is indicated by red line. The sum of these
two probabilities is the total probability for forming a coiled-coil, which is
indicated by black line.
Supplementary Figure 1
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S U P P L E M E N TA R Y I N F O R M AT I O N
a
b
DsRed-WT
DAPI
TSZ 4h
c
DsRed-CC1
DAPI
TSZ 0h
DsRed-CC1
DsRed-MLKL
TSZ 0h
TSZ 4h
d
TSZ 0h
DsRed-CC2
DAPI
DsRed-CC2
R=
Ipm
Icyt
TSZ 4h
Ipm
Icyt
Supplementary Figure 2 Quantification of plasma membrane translocation
of DsRed-MLKL in response to TSZ treatment, related to Figure 4d.
(a-c) MLKL shRNA HT29 cells transfected with DsRed tagged WT
MLKL (a), CC1 (b) or CC2 (c) mutants and then treated with TSZ as
indicated. A line intensity profile across the cell was obtained in a given
image. Representative intensity profiles are shown in the right. Y-axis,
Gray level; X-axis, Line distance. (d) The image shown in (a) was used
for a representative example to show how to calculate a relative ratio
of flurorescence intensity between plasma membrane adjacent area
and cytosolic area. The ratio [R] was calculated by dividing the plasma
membrane intensity [Ipm] by the average cytosolic fluorescence intensity
[Icyt]. Scale bar, 5µm.
Supplementary Figure 2
2
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S U P P L E M E N TA R Y I N F O R M AT I O N
a
HT29-MLKL-shRNA
HT29-CT-shRNA
Phalloidin
RIP3
Phalloidin
DAPI
Merged
MLKL
Phalloidin
RIP3
Phalloidin
DAPI
Merged
TSZ 4h
TSZ 4h
TSZ 0h
TSZ 0h
MLKL
b
HT29-RIP3-shRNA
HT29-CT-shRNA
DAPI
Merged
TSZ 0h
Merged
TSZ 4h
TSZ 4h
TSZ 0h
DAPI
Supplementary Figure 3 Subcellular localization of endogenous MLKL
and RIP3 in HT29 cells. Control shRNA (left panel) or MLKL shRNA
(right panel) HT29 cells were treated with TSZ as indicated. Subcellular
localization of (a) MLKL or (b) RIP3 was detected by confocal imaging
of anti-MLKL antibody or anti-RIP3 antibody staining, respectively. Cell
morphology was shown by F-actin probe phalloidin staining. DAPI was used
for nuclear staining. White arrows indicate membrane localization. Scale
bar, 10µm.
Supplementary Figure 3
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S U P P L E M E N TA R Y I N F O R M AT I O N
a
b
MEF
Total input
TSZ 0
4
Cell surface
4 ( hours )
0
J774A.1
Total input
TSZ 0
8
Cell surface
0
8 ( hours )
54kDa
IB: Anti-MLKL
54kDa
IB: Anti-MLKL
57kDa
IB: Anti-RIP3
57kDa
IB: Anti-RIP3
100kDa
IB: Anti-Na+/K+ ATPase
43kDa
IB: Anti-Actin
17kDa
IB: Anti-COX IV
90kDa
IB: Anti-Calnexin
Supplementary Figure 4 Cell surface protein isolation in MEF cells and
J774A.1 cells. MEF cells (a) or J774A.1 cells (b) were treated with TSZ as
indicated. Total cellular lysate and the biotinylated, cell surface fraction
were resolved on reducing gel and analyzed by immunoblot as indicated.
IB: Anti-Na+/K+ ATPase
100kDa
43kDa
IB: Anti-Actin
17kDa
IB: Anti-COX IV
90kDa
IB: Anti-Calnexin
Na+/K+ ATPase was used as a positive control for transmembrane/surface
protein while Actin, COX IV and Calnexin were used as a positive control
for cytosolic, mitochondrial and ER membrane proteins, respectively.
Uncropped images of western blots are shown in Supplementary Fig.8.
Supplementary Figure 4
4
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S U P P L E M E N TA R Y I N F O R M AT I O N
a
HT29 CT-shRNA HT29 MLKL-shRNA HT29 RIP3-shRNA
DMSO
Counts
TSZ 4h
HT29 CT-shRNA
Nec-1 (20 µM)
HT29 CT-shRNA
NSA (0.5 µM)
Fluo4
TSZ
0
2
4
c
Ca2+ free DMEM
DMEM
8
0
2
4
8
( hours )
IB: Anti-MLKL
250kDa
Trimer
150kDa
TSZ 0h
100kDa
Ca2+ free DMEM
DsRed-MLKL RIP3-YFP DsRed+YFP+DIC
75kDa
50kDa
Monomer
57kDa
IB: Anti-RIP3
*
76kDa
TSZ 4h
b
IB: Anti-RIP1
43kDa
IB: Anti-Actin
Supplementary Figure 5 MLKL-mediated Calcium influx is involved in
plasma membrane rupture during necroptosis, related to Figure 6. (a) Flow
cytometric analysis of intracellular Ca2+ of control shRNA, MLKL shRNA
or RIP3 shRNA HT29 cells after treated with TSZ for 4 h with/out NSA or
Nec-1 as indicated. Cells were harvested and the Fluo4 fluorescent cells
were determined by FACS analysis using Fluo4. (b) HT29 cells were treated
with TSZ either in normal DMEM or calcium free DMEM at different time
points as indicated. The cell lysates were resolved on non-reducing gel and
analyzed by immunoblot with anti-MLKL, anti RIP3, anti-RIP1 or antiActin antibodies. * indicates phosphorylated RIP3. (c) Representative
image of live HT29 MLKL shRNA cells co-expressing DsRed-MLKL together
with RIP3-EYFP treated with TSZ at time 0 and 4 hours in calcium free
DMEM. Scale bar, 5µm. Uncropped images of western blots are shown in
Supplementary Fig.8.
Supplementary Figure 5
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S U P P L E M E N TA R Y I N F O R M AT I O N
a
b
Jurkat
FADD-/-
% PI Negative
100
80
Jurkat FADD-/-
DMEM
Ca2+ free DMEM
CT-shRNA
MLKL-shRNA
CT-shRNA
Calcium free medium
DMSO
*
TSZ 4h
Counts
60
40
20
0
DMSO
TSZ 10 h
Fluo4
d
c
J774A.1
DMEM
Ca2+ free DMEM
CT-shRNA
**
80
MLKL-shRNA
CT-shRNA
Calcium free medium
DMSO
TSZ 8h
Counts
60
40
20
Fluo4
f
Jurkat FADD-/-
shRNA:
J774A.1
shRNA:
MLKL
e
TSZ 24 h
Ctrl
DMSO
MLKL
0
Ctrl
% PI Negative
100
J774A.1
54 kDa
IB: Anti-MLKL
54 kDa
IB: Anti-MLKL
43 kDa
IB: Anti-Actin
43 kDa
IB: Anti-Actin
Supplementary Figure 6 Extracellular calcium is involved in necroptosis
in Jurkat FADD-/- and J774A.1 cells. Jurkat FADD-/- or J774A.1 cells were
cultured with/out calcium and treated with DMSO control or TSZ as
indicated. (a), (c) Cell survival was determined by PI staining. Results
shown are averages±S.E.M. from three independent experiments.*, p
< 0.05, **, p < 0.01. Statistics source data for Supplementary Figure
6 can be found in Table S2. (b), (d) Jurkat FADD-/- or J774A.1 cells
expressing control shRNA, MLKL shRNA or cells cultured in calcium
free medium were treated with TSZ as indicated. Cells were harvested
and the Fluo4 fluorescent cells were determined by FACS analysis using
Fluo4. (e), (f) Immunoblot analysis of Juarkat FADD-/- (e) or J774A.1 (f)
clones expressing control-shRNA or MLKL-shRNA with anti-MLKL or
anti-Actin antibodies. Uncropped images of western blots are shown in
Supplementary Fig.8.
Supplementary Figure 6
6
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S U P P L E M E N TA R Y I N F O R M AT I O N
a
DMSO
U937
TSZ
120
**
% Survival
100
80
60
40
20
0 Control Calcium free
Calcium free
+NAC
b
U937
CT-shRNA
MLKL-shRNA
CT-shRNA
Calcium free medium
DMSO
Counts
TSZ 4h
Fluo4
c
MLKL
shRNA:
Ctrl
U937
54 kDa
*IB: Anti-MLKL
43 kDa
IB: Anti-Actin
Supplementary Figure 7 ROS and calcium are both required for necroptosis
in U937 cells. (a) U937 cells were either cultured in calcium free medium
or cultured in calcium free medium and pre-treated with NAC (5 mM). Then,
the cells were treated with TSZ. After 10 hours, cell survival was determined
by PI staining. Results shown are averages of triplicates ±SEM. (b) U937
cells cultured in calcium free DMEM were treated with TSZ as indicated.
Cells were harvested and the Fluo4 fluorescent cells were determined
by FACS analysis using Fluo4. (c) Immunoblot analysis of U937 clones
expressing control-shRNA or MLKL-shRNA with anti-MLKL or anti-Actin
antibodies. * indicates non-specific band. * *, P<0.01. Statistics source
data for Supplementary Figure 7 can be found in Supplementary Table 1.
Uncropped images of western blots are shown in Supplementary Fig.8.
Supplementary Figure 7
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S U P P L E M E N TA R Y I N F O R M AT I O N
250kDa
150kDa
100kDa
75kDa
250kDa
50kDa
150kDa
37kDa
26kDa
20kDa
100kDa
15kDa
37kDa
250kDa
150kDa
100kDa
75kDa
75kDa
50kDa
50kDa
37kDa
26kDa
20kDa
15kDa
26kDa
Figure 1a
20kDa
15kDa
250kDa
150kDa
100kDa
75kDa
250kDa
150kDa
250kDa
100kDa
150kDa
100kDa
75kDa
50kDa
37kDa
75kDa
50kDa
50kDa
37kDa
37kDa
26kDa
20kDa
20kDa
26kDa
20kDa
15kDa
26kDa
15kDa
Figure 1b, right panel
Anti-FLAG
Figure 1b, left panel
Figure 1c
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
250kDa
26kDa
20kDa
150kDa
100kDa
75kDa
75kDa
50kDa
50kDa
37kDa
37kDa
Actin
Figure 1f
50kDa
37kDa
Actin
Figure 1d
Supplementary Figure 8
Supplementary Figure 8 Original scans of western blots. Black boxes indicate cropped region.
8
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S U P P L E M E N TA R Y I N F O R M AT I O N
250kDa
150kDa
100kDa
75kDa
50kDa
250kDa
150kDa
100kDa
75kDa
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
50kDa
37kDa
250kDa
150kDa
100kDa
75kDa
Figure 1h
50kDa
Figure 2a, left panel
37kDa
26kDa
20kDa
Figure 1g
250kDa
150kDa
100kDa
75kDa
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
50kDa
37kDa
26kDa
250kDa
150kDa
100kDa
75kDa
50kDa
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
250kDa
150kDa
100kDa
75kDa
37kDa
26kDa
20kDa
50kDa
Figure 2a, right panel
100kDa
75kDa
50kDa
37kDa
100kDa
75kDa
50kDa
37kDa
26kDa
26kDa
Figure 2c
Figure 2b
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
26kDa
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
26kDa
20kDa
15kDa
26kDa
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
26kDa
26kDa
Figure 2d
Anti-GFP
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
26kDa
20kDa
15kDa
Figure 2e
Figure 3b
Supplementary Figure 8 continued
Supplementary Figure 8 continued
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S U P P L E M E N TA R Y I N F O R M AT I O N
100kDa
75kDa
50kDa
37kDa
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
26kDa
20kDa
15kDa
50kDa
37kDa
26kDa
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
26kDa
20kDa
15kDa
75kDa
50kDa
37kDa
26kDa
250kDa
100kDa
75kDa
50kDa
37kDa
26kDa
75kDa
50kDa
37kDa
26kDa
50kDa
37kDa
Figure 4e
Figure 3c
150kDa
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
100kDa
75kDa
50kDa
37kDa
26kDa
Anti-MLKL
150kDa
100kDa
75kDa
50kDa
37kDa
26kDa
Anti-Actin
Figure 3d, right
26kDa
20kDa
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
26kDa
20kDa
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
26kDa
20kDa
Figure 4g
Supplementary Figure 8 continued
Supplementary Figure 8 continued
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S U P P L E M E N TA R Y I N F O R M AT I O N
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
26kDa
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
Figure 5b
26kDa
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
26kDa
150kDa
100kDa
75kDa
50kDa
37kDa
26kDa
Figure 5a
Figure 5c
250kDa
150kDa
250kDa
100kDa
75kDa
150kDa
100kDa
75kDa
50kDa
50kDa
37kDa
Figure 8a, lower panel
37kDa
26kDa
20kDa
15kDa
Figure 8b, lower panel
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
26kDa
Actin
Figure 8c, lower panel
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
26kDa
Figure 8d
Supplementary Figure 8 continued
Supplementary Figure 8 continued
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S U P P L E M E N TA R Y I N F O R M AT I O N
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
26kDa
250kDa
150kDa
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
26kDa
15kDa
75kDa
50kDa
37kDa
26kDa
250kDa
150kDa
100kDa
75kDa
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
26kDa
20kDa
250kDa
150kDa
75kDa
50kDa
37kDa
50kDa
37kDa
26kDa
Figure 8i
26kDa
250kDa
150kDa
100kDa
75kDa
Figure 8e
50kDa
37kDa
26kDa
250kDa
150kDa
100kDa
75kDa
50kDa
37kDa
26kDa
75kDa
50kDa
37kDa
100kDa
75kDa
Actin
Supplementary Fig. 5b
CT
MLKL
shRNA:
CT
Supplementary Figure 4b
MLKL
Supplementary Figure 4a
250kDa
150kDa
100kDa
75kDa
37kDa
100kDa
75kDa
50kDa
26kDa
37kDa
50kDa
150kDa
100kDa
75kDa
150kDa
50kDa
37kDa
MLKL
Actin
26kDa
20kDa
Supplementary Figure 6f
Supplementary Figure 6e
Supplementary Figure 7c
Supplementary Figure 8 continued
Supplementary Figure 8 continued
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S U P P L E M E N TA R Y I N F O R M AT I O N
Supplementary Table 1 Statistics source data.
Supplementary video 1 Representative time-lapse GCaMP3 fluorescence imaging of control-shRNA HT29 cells treated with TSZ for 8 hours. Left, GFP
channel; Middle, differential interference contrast channel (DIC); Right, Merged two channels.
Supplementary video 2 Representative time-lapse GCaMP3 fluorescence imaging of TRPM7-sh#1RNA HT29 cells treated with TSZ for 8 hours. Left, GFP
channel; Middle, differential interference contrast channel (DIC); Right, Merged two channels.
Supplementary video 3 Representative time-lapse GCaMP3 fluorescence imaging of control-shRNA HT29 cells cultured in calcium free DMEM and treated
with TSZ for 8 hours. Left, GFP channel; Middle, differential interference contrast channel (DIC); Right, Merged two channels.
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