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Research Article
1
Lipids regulate P2X7-receptor-dependent phagosome
actin assembly via ADP translocation and ATP
synthesis in the lumen
Mark. P. Kuehnel1, Vladimir Rybin1, Paras K. Anand1, Elsa Anes2 and Gareth Griffiths1,*
1
EMBL, Meyerhofstr. 1, 69117 Heidelberg, Germany
Molecular Pathogenesis Centre, Unit of Retrovirus and Associated Infections, Faculty of Pharmacy, University of Lisbon, Av. Forcas Armadas,
1600-083 Lisbon, Portugal
2
*Author for correspondence (e-mail: griffiths@embl.de)
Accepted 5 November 2008
Journal of Cell Science 122, 000-000 Published by The Company of Biologists 2009
doi:10.1242/jcs.034199
Summary
Latex bead phagosomes isolated from J774 macrophages
polymerize actin. We show here that five lipids –
phosphatidylinositol-4-phosphate, phosphatidylinositol-(4,5)bisphosphate, sphingosine-1-phosphate (S1P), ceramide-1phosphate and phosphatidic acid – stimulate both actin
assembly and transport of ADP across the phagosomal
membrane into the lumen. Once there, this ADP is converted
to ATP by adenylate kinase activity. High luminal ATP
concentrations correlated well with phagosome actin assembly
under different conditions. The ATP-binding P2X7 receptor was
detected in phagosomes. Although S1P stimulated actin
assembly by phagosomes from P2X7R-containing bone marrow
macrophages, S1P-stimulated actin assembly was inhibited in
Introduction
Phagocytosis is the process whereby large particles, most
prominently pathogens, are taken up by cells such as macrophages
into a membrane-enclosed organelle, called the phagosome. The
use of latex bead phagosomes (LBPs) has been a powerful model
system to analyze phagosomes in vitro and in vivo (Desjardins et
al., 1994; Desjardins and Griffiths, 2003). The assembly of actin
by membranes plays a crucial role in phagosome biogenesis, both
during the uptake process (Aderem and Underhill, 1999) and in the
fusion of phagosomes with late endosomes and lysosomes (Jahraus
et al., 2001; Kjeken et al., 2004; Anes et al., 2006). An in vitro
system was developed to monitor the membrane-dependent actin
assembly (Defacque et al., 2000a). During this in vitro actin
assembly, via barbed-end insertion, the filaments grow out
perpendicularly from the membrane, as monitored by fluorescence
microscopy (Defacque et al., 2000a). This in vitro reaction contains
no cytosol or GTP, requiring only rhodamine-G actin, thymosin B4
(a monomeric actin buffer), ATP and a buffer. The process requires
membrane-bound
ezrin
and/or
moesin
linked
to
phosphatidylinositol-(4,5)-bisphosphate (PIP2) (Defacque et al.,
2002) and is also regulated by gelsolin (Defacque et al., 2000b).
We recently showed that a number of lipids could stimulate the
assembly of actin by LBP, including phosphatidylinositol-4phosphate (PIP), PIP2, sphingosine-1-phosphate (S1P) and
arachidonic acid. The same lipids also enhanced this process on
phagosomes enclosing the non-pathogenic Mycobacterium
smegmatis and the pathogenic M. tuberculosis. Some of the lipids
that stimulated phagosome actin assembly in vitro, when added to
phagosomes from cells lacking P2X7R. We propose that luminal
ATP accumulates in response to selected lipids and activates
the P2X7R that signals across the phagosomal membrane to
trigger actin assembly on the cytoplasmic membrane surface.
In the accompanying paper, more evidence is provided in
support of this model from the analysis of actin assembly at the
plasma membrane of intact macrophages.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/122/??/????/DC1
Key words: ATP, Lipids, P2X7, Actin, Phagosomes
macrophages infected with pathogenic mycobacteria, were able to
enhance the fusion of the phagosomes with lysosomes and led to
further destruction of the pathogens (Anes et al., 2003).
We focus here on the unexpected observation that five
phosphorylated lipids, when incorporated into phagosomal
membranes, induce the synthesis of ATP by LBP. Our data argue
that these lipids open ‘channels’ in the phagosomal membrane that
allow ADP, but not ATP, to cross the membrane and enter the lumen.
There, a luminal adenylate kinase activity converts the ADP to ATP,
which appears to stimulate LBP actin assembly. The detection of
the P2X7 receptor (P2X7R; official symbol P2RX7) in phagosomes,
which is known to interact with actin (Kim et al., 2001) led to the
hypothesis that the luminal ATP binds to this receptor and stimulates
transmembrane signaling to the actin assembly machinery on the
cytoplasmic surface of phagosomes.
Results
Five phosphorylated lipids that enhance phagosome actin
assembly stimulate ATP accumulation in the phagosome lumen
The LBP actin assay allows a constitutive assembly of actin under
ischemic levels of ATP (0.2 mM) (Defacque et al., 2000a). However,
the process is inhibited under physiological levels of ATP (1-5 mM),
but under these conditions can be activated by a number of lipids,
especially the phosphorylated lipids PIP, PIP2, S1P, ceramide-1phosphate and phosphatidic acid (PA) (Anes et al., 2003) (Fig. 1C,
lower panel). For S1P and PIP, our recent experiments using 32Plabeled lipids, confirm that the lipids are incorporated into the LBP
membrane (Kuehnel et al., 2009).
2
Journal of Cell Science 122 ()
Fig. 1. (A) Procedure for
quantification of luminal
phagosome ATP. (B) Comparison
of LBP luminal ATP concentrations
estimated by luciferin fluorescence;
red line indicates the average basal
ATP level in unstimulated LBPs.
(C) Luminal ATP levels from LBPs
treated with different lipids in the
presence of 1 mM ADP and ATP.
Lower panel summarizes actin
assembly data (Anes et al., 2003).
Inset shows the effects of ceramide1-phosphate (C1P) on actin
assembly at 0.2 mM (low) and 5
mM (high) [ATP]; P values below
0.05. (D) LBP actin assembly with
1 mM ATP (ATP), 1 mM ATP plus
1 mM ADP (ATP + ADP) and the
phagosomes containing apyrasecoated beads and control avidincoated beads incubated with 1 mM
ATP and 1 mM ADP or ATP alone.
(E) Luminal ATP levels of LBP of
different maturation ages, with and
without PIP stimulation. Ctr,
control containing solvents but no
lipids. Ctr 0, LBP analysed
immediately after thawing from
frozen stocks. Ctr 30, incubation
for 30 minutes at 37°C. TX, Triton
X-100 treatment. Statistical
significance was determined by the
Student’s t-test. P values given for
the effects of lipids relative to the
average values seen with
unstimulated LBP. In all figures,
mean ± s.d. is shown.
The preliminary use of a luciferase assay (in the absence of
exogenous ATP) to detect ATP revealed that these five lipids
stimulated ATP synthesis by LBP. For a long period we followed
the (wrong) hypothesis that the ATP was synthesized on the
cytoplasmic side of the phagosome membrane. However,
experiments using Triton X-100 to lyze LBP membranes eventually
indicated that the ATP accumulated in the phagosome lumen; the
‘release’ of ATP detected in the early experiments came from the
lumen of broken phagosomes. For more definitive experiments we
needed to set up a gentle assay in which phagosomes stayed intact
in the presence of physiological levels of ATP to monitor ATP that
accumulated in the organelle lumen (Fig. 1A, assay scheme).
As shown in Fig. 1B, using this assay, a low level of ATP could
be detected in the lumen of untreated phagosomes, and its
concentration was increased by the addition of 1 mM ADP or ATP
alone. However, the Triton-releasable pool of ATP was slightly, but
not significantly, enhanced when LBPs were treated with both
nucleotides [ATP is needed for many LBP kinases (Emans et al.,
1996) whereas the role of ADP will become evident below]. The
levels of luminal ATP were considerably increased when
phagosomes were additionally treated with PIP or S1P. A significant
elevation of LBP lumenal ATP was seen when ATP alone was
present on the cytoplasmic surface during the incubation with PIP
or S1P; however, a much higher signal was seen when both ATP
and ADP were present in combination with these lipids (Fig. 1B).
Four lipids, PIP, PIP2, S1P and PA are known to stimulate LBP
actin assembly at physiological (1-5 mM) ATP; these are thus
classified here as (+) lipids (Anes et al., 2003) (Fig. 1C, lower panel).
We confirm in Fig. 1C, inset, that ceramide-1-phosphate (Cer1P)
also has notable effects, it inhibits the (constitutive) actin assembly
at low [ATP] while stimulating the (normally switched off) system
with high [ATP]. According to these results, Cer1P is thus also a
(+) lipid.
As seen in Fig. 1C (main panel), when PIP, PIP2, S1P, Cer1P or
PA were co-incubated with 1 mM ADP and 1 mM ATP, as well as
the different lipids, all the five (+) lipids induced significant
Lipids regulate actin
elevation of ATP in the phagosomal lumen. By contrast, lumenal
ATP was not detected when the corresponding dephosphorylated
species of these lipids – sphingosine (Sph), phosphatidylinositol
(PI), ceramide or diacylglycerol (DAG) – were used. These lipids
were also unable to stimulate LBP actin assembly with 5 mM ATP
(Fig. 1C, lower panel). Not all phospholipids stimulated ATP
accumulation in the lumen, because phosphatidylcholine (PC),
which inhibits actin assembly (Anes et al., 2003; Treede et al., 2007),
had no effect. Similarly, not all lipids that stimulate LBP actin
assembly induced ATP synthesis, because arachidonic acid, which
potently stimulates this actin assembly (Anes et al., 2003) failed to
elevate LBP ATP levels (unpublished data).
The ability of the (+) lipids to stimulate LBP actin assembly
correlates precisely with their ability to induce ATP accumulation
in the organelle lumen (Fig. 1C). Further evidence of a link between
these processes was demonstrated when LBP prepared from
internalized beads bound to apyrase (to break down luminal ATP
and ADP) were inhibited in actin assembly (Fig. 1D). Fig. 1D also
shows that in the presence of 1 mM ATP, the addition of 1 mM
ADP stimulates the assembly of actin by LBPs (Fig. 1D), correlating
with the ability of ADP to enhance ATP accumulation in the LBP
lumen (Fig. 1B). The possibility that contamination of the LBP
preparation with mitochondria that could synthesize ATP was ruled
out by the use of the mitochondrial respiratory inhibitor rotenone
(20nM) and sodium cyanide (1 μM), which had no effect on the
accumulation of LBP luminal ATP in response to S1P or PIP (Table
1).
An additional striking correlation between ATP synthesis and
LBP actin assembly emerged from a comparison of phagosomes
of different maturation stages. LBPs made after 2 hours or 24 hours
are active in actin assembly, as are M. smegmatis phagosomes;
however, those made at 12 hours are inactive (Defacque et al.,
2000a; Anes et al., 2006). For LBPs, this pattern correlates precisely
with overall kinase activity (Emans et al., 1996) and PIP2 levels
(Defacque et al., 2002). This pattern was also seen when we tested
the capacity of 2, 12, and 24 hour LBPs to synthesize luminal ATP
in response to PIP (Fig. 1E) or S1P (supplementary material Fig.
S1). Only the active, 2 hour and 24 hour stages revealed this activity.
Collectively, the data show that the lipid-stimulated accumulation
of luminal ATP by phagosomes correlated precisely with their ability
to nucleate actin, arguing that the ATP in the lumen was somehow
transmitting a signal to the actin assembly machinery on the other
side of the phagosome membrane.
ADP, but not ATP, is transported into the phagosomal lumen
We next sought direct evidence that ATP can be transported across
the phagosomal membrane using tritium-labeled ATP or ADP in
the medium containing the LBPs. Since both ADP and ATP need
to be added to the outside (cytoplasmic surface) of phagosomes to
achieve the highest levels of ATP in the lumen (Fig. 1B), we
compared the incorporation of [3H]ADP or [3H]ATP into the lumen
in the presence of unlabeled (1 mM) ADP and ATP in the bathing
medium, using HPLC analysis of total nucleotides, combined with
3
H radioactivity counts. Contrary to our expectations, when the
tritium label was in ATP, only a background level of radioactivity
could be detected in the LBP lumen incorporated into AMP, ADP
or ATP, and this level was affected little by addition of S1P or PIP
(Fig. 3A). By contrast, when LBPs were incubated with [3H]ADP
for 30 minutes in the presence of 1 mM ADP and ATP (but without
lipid stimulation) there was a significant accumulation of Tritonreleasable (luminal) counts in all three nucleotides: highest for AMP,
3
Fig. 2. (A) HPLC analysis of 3H-labeled nucleotide compositions [ATP (T),
ADP (D) and AMP (M)] within the lumen of control phagosomes (C0, no
incubation;C30, 30 minute incubation at 37°C) compared with phagosomes
stimulated by S1P or PIP for 30 minutes. All control and lysed conditions had
1 mM ADP and 1 mM ATP; one set of conditions contained 10 μCi [3H]ADP,
the other 10 μCi [3H]ATP. (B) Testing of adenylate kinase activity in extracts
of phagosomes. LBPs were treated with Triton X-100 and then incubated with
either tritium-labeled ADP (left) or AMP plus ATP (right) and nucleotide
levels were analyzed by HPLC after 30 minutes of incubation. The mass
action concentrations at the beginning and end of the 30 minute reaction are
given for a representative experiment. (C) Estimation of [ATP] by luciferase
fluorescence of phagosomal extracts incubated with 1 mM ADP and
sphingosine kinase inhibitor (DHS), phosphoinositide 4-kinase inhibitor
(adenosine) or adenylate kinase inhibitor (Ad2P5).
less for ADP and lowest for ATP. The addition of either S1P or PIP
led to a significant elevation of the labeled ATP pool, whereas the
labeled ADP stayed constant; the AMP rose slightly with PIP, but
not with S1P (Fig. 2A). These data demonstrate that it is not ATP,
but rather ADP that transports across the phagosomal membrane,
and that enzyme(s) in the lumen convert the ADP into AMP and
ATP.
Adenylate kinase in the phagosomal lumen synthesises ATP
from ADP
It now seemed likely that an adenylate kinase in the lumen could
be involved in converting ADP to ATP, because this enzyme carries
out the reversible equilibrium reaction: 2ADP=ATP + AMP
(Yegutkin et al., 2001). We therefore estimated the molar ratios of
the 3H nucleotides under conditions in which luminal ATP
accumulates. When either ADP (Fig. 2B, left) or ATP plus AMP
(Fig. 2B, right) were initially added to Triton-treated phagosomes,
the reaction approached the equilibrium level of 1, which is
expected for adenylate kinase after a 30 minute incubation.
We next tested the specific inhibitor of adenylate kinase Ad2P5,
as well as inhibitors of sphingosine kinase or phosphoinositide 4kinase, the enzymes that synthesize the stimulatory lipids S1P and
4
Journal of Cell Science 122 ()
Fig. 3. (A) Actin nucleation assay performed with
phagosomes from control (WT) mice and P2X7R-knockout
(–/–) mice. The mean fraction of phagosomes nucleating
actin filaments is given, along with s.d. Asterisks indicate
significance below P<0.05 for the indicated comparisons.
The equivalent comparisons for the P2X7R-knockout
phagosomes were not statistically significant.
(B) Immunofluorescence microscopy of P2X7R on control
(WT), P2X7R-knockout (ko) and J774 LBPs in vitro.
Green, P2X7; Red, LBP (false coloration of the phasecontrast images). (C) Western blot for P2X7R in spleen
tissue from wild-type and P2X7R-knockout mice. Inset
shows a western blot for P2X7R in 2 hour LBPs from J774
macrophages.
PIP, on the accumulation of ATP in Triton-treated LBP in a
luciferase assay. A small, but not significant inhibition in the levels
of luminal ATP was seen with the lipid kinase inhibitors, whereas
the adenylate kinase inhibitor blocked over 90% of the ATP signal;
no extra inhibition was seen with the addition of the lipid kinase
inhibitors (Fig. 2C). A comparison of intact phagosomes (no Triton
X-100) relative to Triton-permeable LBPs showed that the adenylate
kinase activity was significantly higher when phagosomes were
permeabilized compared with intact LBPs (supplementary material
Fig. S2). Collectively, these data show that under conditions in
which phagosomes assemble actin, these organelles have
mechanisms for transporting ADP into their lumen and convert it
to ATP via luminally localized adenylate kinase activity.
Role of the P2X7 receptor in phagosome actin assembly
We hypothesized that the ATP made by this enzyme activity binds
ATP-binding receptor(s) that signal back across the membrane to
actin assembly machinery, presumably involving ezrin or moesin
on the cytoplasmic side of the membrane. An attractive candidate
for this is the P2X7 receptor, which could be detected in the J774
(2 hour stage) phagosome proteome (M. Desjardins, personal
commununication). This receptor, a member of the purinergic
receptor family has been especially well characterized in
macrophages (Burnstock, 2006; North, 2002). It has been shown
to posses both a low-affinity ATP-binding site that responds to mM
levels of ATP and a second, high-affinity site that is activated by
low μM levels of ATP (Klapperstück et al., 2001). Many links have
been made between the P2X7R and the actin cytoskeleton (Chen
et al., 2006; Li and Wu, 2003; Li et al., 2003; Pfeiffer et al., 2004).
Moreover, P2X7R has been found in a physical complex with actin,
phosphoinositide 4-kinase [which is essential for LBP actin
assembly (Defacque et al., 2002)] and ten other proteins in a
complex (Kim et al., 2001). In Fig. 3C, inset, we show that the
P2X7R can be detected on 2 hour LBPs by western blotting.
In order to analyze the potential role of P2X7R in more detail,
we used bone marrow macrophages (BMMs) from wild-type mice
and from mice lacking the P2X7 receptor (Solle et al., 2001). To
both cell types, latex beads were added for a 1 hour pulse and 1
hour chase. No difference was seen in the rate of phagocytosis or
in the total number of phagosomes formed between the two cell
types (data not shown). The LBPs were isolated from both cells
and compared in an in vitro actin assembly assay. For this, we first
compared both high (5 mM) ATP and ischemic low (0.2 mM)
concentrations of ATP (Fig. 3); under ischemic conditions, both
cellular F-actin and F-actin in cytosolic extracts is highly
upregulated (Jahraus et al., 2001). With J774 phagosomes, a low
[ATP] stimulates actin assembly whereas a high [ATP] inhibits actin
assembly. S1P stimulates the system at high [ATP], but inhibits at
low [ATP] (Anes et al., 2006) (Fig. 1C).
With BMMs containing the P2X7R, the pattern was the same as
in J774 cells; low [ATP] stimulated and high [ATP] inhibited,
whereas S1P stimulated actin assembly at high [ATP] but inhibited
at low [ATP] (Fig. 3A). With BMMs lacking P2X7R there was no
difference from the results in wild-type phagosomes at low [ATP],
both with and without S1P (Fig. 3A); both stimulated actin assembly
in a similar manner. The inability of these phagosomes to be
inhibited in actin assembly by S1P at low [ATP] is not readily
explained at present, but the results argue that the P2X7R is not
necessary for actin assembly under ischemic conditions (Anes et
al., 2003). More interesting for this study were the effects seen at
high [ATP]; here, S1P was unable to significantly stimulate the
assembly of actin, in contrast to wild-type phagosomes (Fig. 3A).
Thus, the P2X7 receptor is important to allow S1P to enhance the
ability of phagosomes to assemble actin filaments under
physiological ATP conditions.
Immunofluorescence labeling of isolated phagosomes using an
antibody against the cytoplasmic domain of P2X7R showed a
punctate labeling of 22±4% of phagosomes from wild-type cells,
but no significant labeling of phagosomes from P2X7R-knockout
macrophages (Fig. 3B). Similar levels of P2X7R labeling were seen
with J774-LBP (Fig. 3B, inset). In both J774 and wild-type BMMs,
the P2X7R labeling was often seen in distinct patches on the
phagosome surface (Fig. 3B). Western blotting of spleen tissue from
the different mice confirmed the presence of P2X7R as a band of
Lipids regulate actin
5
approximately 80 kDa in the wild type, but not in the knockout
tissues (Fig. 3C).
Discussion
The data we present are consistent with the model shown in Fig.
4. The lipids that stimulate LBP actin assembly, the (+) lipids, such
as S1P and PIP, are incorporated into the phagosomal membrane,
presumably into the cytoplasmic leaflet. Under physiological
conditions, high levels of these lipids are more likely to be the result
of their synthesis by the lipid kinases, such as sphingosine kinase
or phosphoinositide 4-kinase, which can be activated in response
to receptor activation (Hait et al., 2006; Taha et al., 2006). The
presence of high concentrations of the (+) lipids in the membrane
is proposed to open a channel or transporter that selectively
transports ADP (but not ATP) across the membrane. The precise
mechanism by which ADP selectively crosses the membrane
remains to be elucidated. However, our data show that for efficient
ADP transport into the phagosome lumen, micromolar levels of ATP
must also be added to the bathing solution around phagosomes. It
has been shown that this ATP can activate a large number of
phagosomal membrane kinases (Emans et al., 1996; Defacque et
al., 2002), some of which might be needed for ADP transport. The
ATP might also be needed to maintain a proton or electrochemical
gradient across the phagosomal membrane (Huynh and Grinstein,
2007).
Within the lumen, an adenylate kinase converts ADP to ATP and
AMP. Although the identity of this adenylate kinase is unknown,
the isoform AK2 is detected in the proteome of J774 LBP (M.
Desjardins, pers. commun.) and in the proteomic analysis of human
neutrophil LBPs (Burlak et al., 2006). Our attempts to test for AK1
and AK2 were hampered by the poor quality of the available
antibodies.
Our data are consistent with the hypothesis that the ATP that is
made by the adenylate kinase can bind and activate the P2X7R,
presumably via its high affinity ATP-binding site (Kuehnel et al.,
2009). This receptor, which we could detect in LBPs by western
blotting, could then transmit a signal from its luminal domain to
its cytoplasmic domain, with which it interacts physically, and
thereby switches on the machinery that assembles actin on the
cytoplasmic surface of phagosome. This scenario is consistent with
a number of publications linking the P2X7R with actin (Kim et al.,
2000; Li et al., 2003; Pfeiffer et al., 2004; Chen et al., 2006)
Further evidence consistent with a role for the P2X7R in
phagosome actin assembly came from the analysis of LBPs isolated
from BMMs. With physiological (5 mM) [ATP] and in the presence
of S1P, the levels of phagosomal actin assembly was significantly
reduced in BMMs from P2X7R-knockout mice relative to wildtype BMMs.
The model we present (Fig. 4) is strongly supported by our
parallel analysis of receptor-mediated actin assembly at the plasma
membrane of intact macrophages (Kuehnel et al, 2009). In that
system, the outer luminal surface of the membrane is easily
accessible to reagents of interest, whereas the cytoplasmic surface
is inaccessible under physiological conditions. This is the opposite
situation to that in phagosomes, where the cytoplasmic surface is
accessible to experimentation, but the lumen can only be accessed
by physical disruption or by using detergent. In both systems, when
S1P is added to the cytoplasmic surface of LBPs or to the
extracellular surface of macrophages (where it can activate S1P
receptors), it induces ADP to cross the membrane into the lumen.
An adenylate kinase activity then converts ADP to ATP, which is
Fig. 4. Model showing the role of P2X7R in phagosome actin assembly. AK,
adenylate kinase.
hypothesized to bind and activate the P2X7 receptor to stimulate
actin assembly on the cytoplasmic surface of the membrane.
Materials and Methods
Cells and mice
J774 A.1 mouse macrophages were grown as described (Anes et al., 2003). The
primary P2X7R-knockout and WT bone marrow macrophages were isolated as
described (Al-Haddad et al., 2001) from wild-type and P2X7R–/– C57 BL/6 mice
(Solle et al., 2001)
Isolation of latex bead phagosomes
LBPs were isolated from J774 macrophages following a 1 hour pulse and 1 hour
chase was carried out as described (Kuehnel, 2004; Anes et al., 2003). Apyrase was
conjugated to carboxylated latex beads using EDAC crosslinking. These were
internalized for 1 hour before LBP isolation. For isolation of LBP from BMM, 3⫻106
BMMs per Petri dish were used at 7-10 days of age and incubated for 1 hour with
OD600 0.1 latex beads. After 1 hour, non-internalized beads were removed by washing
with PBS and subsequently, cells were incubated for another hour in complete medium.
On average over 95% of cells phagocytosed beads at this point. Cells were scraped
on ice and phagosome isolation was carried out as described (Kuehnel, 2004; Anes
et al., 2003).
Actin assembly
This assay was carried out as described (Defacque et al., 2000a).
Application of lipids
For all lipid treatments, the stock lipids were dissolved in ethanol and used as follows:
arachidonic acid (AA) (1.6 M; used at 125 μM), ceramide (Cer) (1 mg/ml used at 1
μM), ceramide-1-P (C1P) (1 mM; used at 1 μM), phosphatidic acid (PA) (1 mM;
used at 50 μM), phosphatidylcholine (PC) (1 mM used at 100 nM), sphingosine (Sph)
(5 mM; used at 100 nM), S1P (5 mM; used at 100nM), PIP2 (5 mM; used at 50 μM)
phosphatidyl-inositol-4-phosphate (PIP) (5 mM; used at 50 μM). Diacylglycerol
(DAG) was dissolved in chloroform:methanol (2:1) (1 mM; used at 10 μM). In all
experiments, optimal lipid concentrations were based on previously determined
empirical values (Anes et al., 2003). Solvent without lipids was routinely tested and
is referred to in the figures as ‘control’. Additionally, a subset of inhibitors was used
in the assays as follows: adenosine (inhibits phosphoinositide 4-kinase) at 50 μM,
dihydrosphingosine (DHS) (inhibits sphingosine kinase) at 1 μM, di-adenosinepentaphosphate (Ad2P5) (inhibits adenylate kinase) 1 mM.
HPLC
The HPLC detection of ATP, ADP and AMP was done as described (Murphy et al.,
1996).
ATP-synthesis assay for phagosomes
Phagosomes were diluted to an OD600 of 1 in a buffer system containing 10 mM
Tris-HCl pH 7.4, 0.1 mM DTT, 2 mM MgCl2, 0.2 mM CaCl2, 0.2 mM KCl, 1 mM
ADP and 1 mM ATP and incubated either alone, or with single lipids or combinations
of lipids on ice. The reaction was allowed to proceed for 20 minutes at room
temperature. Subsequently, 100 μl of phagosomes were mixed with 100 μl ice-cold
63% sucrose and overlaid with 1 ml of 25% sucrose and then 200 μl of 8% sucrose
before centrifugation at 24,000 r.p.m. in a SW60 rotor for 15 minutes at 4°C.
Phagosomes were recovered and remaining extraphagosomal nucleotides were
removed by adding 1 U apyrase (for 5 minutes). Apyrase was then inactivated by
boiling for 30 seconds Subsequently samples were cooled on ice and vortexed in the
presence of 0.1% Triton X-100 and, after pelleting lysed phagosomes by centrifugation
6
Journal of Cell Science 122 ()
for 10 minutes at 14,000 g, the ATP concentrations in supernatants (the phagosomal
lumen ATP) were determined using the BioOrbit ATP detection kit and the BioOrbit
1250 luminometer, following the manufacturer’s instructions.
Nucleotide translocation assay
Phagosomes were treated as described in the ATP-synthesis assay (above) with 1
mM ADP and 1 mM ATP but in addition, the ATP or ADP pools were labeled with
tritiated ATP or ADP (1 μCi) respectively. After release of the content of the
phagosome lumen (as above, the radioactivity was measured using a Beckmann
Coulter counter.
Immunolabelling of P2X7R in western blot and fluorescence
microscopy
Western blots were carried out on isolated spleen tissue for P2X7R using a rabbit
polyclonal antibody against the anti-P2X7R provided by Blanche Schwappach
(ZMBH, Heidelberg) that was made against the highly conserved sequence
CRWRIRKEFPKSEGQYSGFKSPY at the C-terminus of the human P2X7 receptor,
which also recognizes the mouse P2X7R. For immunofluorescence microscopy,
isolated phagosomes were attached to coverslips and labeled for P2X7R using the
above-mentioned antibody at a 1:50 dilution combined with a Alexa Fluor 488 antirabbit antibody. The LBPs were visualized by phase-contrast microscopy. In Fig. 3B,
LBP labelling was converted to a red signal by false coloration.
We would like to thank Sabrina Marion and Simi Antony for their
technical support. We greatly appreciate the suggestions by Jens Reich
and Thomas Dandekar. Thanks also to Christopher Bleck for preparing
Fig. 4 and to Luis Mayorga, Sabrina Marion and Maximiliano Gutierrez
for their comments on the manuscript. Blanche Schwappach
generousely provided the anti-P2X7R antibody. Sergei Kuznetsov
provided labeled actin. We thank Michel Desjardins for sharing data
with us. M.K. was supported by a DFG SPP grant. P.K.A. was supported
by the Alexander von Humboldt foundation, Germany. E.A. was
supported by Fundação para a Ciência e a tecnologia (FCT) grant with
co-participation of the EU fund FEDER Programme.
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