Perforin Rapidly Induces Plasma Membrane
Sunil S. Metkar1, Baikun Wang1, Elena Catalan4, Gregor Anderluh2, Robert J. C. Gilbert3, Julian Pardo4,5,
Christopher J. Froelich1*
1Department of Medicine, NorthShore University HealthSystems Research Institute, Evanston, Illinois, United States of America, 2Department of Biology, Biotechnical
Faculty, University of Ljubljana, Ljubljana, Slovenia, 3Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, Oxford, United Kingdom,
4Departamento Bioquimica y Biologia Molecular y Cellular, University of Zaragoza, Zaragoza, Spain, 5Fundacio ´n Arago ´n I+D, Zaragoza, Spain
The cytotoxic cell granule secretory pathway is essential for host defense. This pathway is fundamentally a form of
intracellular protein delivery where granule proteases (granzymes) from cytotoxic lymphocytes are thought to diffuse
through barrel stave pores generated in the plasma membrane of the target cell by the pore forming protein perforin (PFN)
and mediate apoptotic as well as additional biological effects. While recent electron microscopy and structural analyses
indicate that recombinant PFN oligomerizes to form pores containing 20 monomers (20 nm) when applied to liposomal
membranes, these pores are not observed by propidium iodide uptake in target cells. Instead, concentrations of human PFN
that encourage granzyme-mediated apoptosis are associated with pore structures that unexpectedly favor phosphatidyl-
serine flip-flop measured by Annexin-V and Lactadherin. Efforts that reduce PFN mediated Ca influx in targets did not
reduce Annexin-V reactivity. Antigen specific mouse CD8 cells initiate a similar rapid flip-flop in target cells. A lipid that
augments plasma membrane curvature as well as cholesterol depletion in target cells enhance flip-flop. Annexin-V staining
highly correlated with apoptosis after Granzyme B (GzmB) treatment. We propose the structures that PFN oligomers form in
the membrane bilayer may include arcs previously observed by electron microscopy and that these unusual structures
represent an incomplete mixture of plasma membrane lipid and PFN oligomers that may act as a flexible gateway for GzmB
to translocate across the bilayer to the cytosolic leaflet of target cells.
Citation: Metkar SS, Wang B, Catalan E, Anderluh G, Gilbert RJC, et al. (2011) Perforin Rapidly Induces Plasma Membrane Phospholipid Flip-Flop. PLoS ONE 6(9):
Editor: Maria Gasset, Consejo Superior de Investigaciones Cientificas, Spain
Received June 28, 2011; Accepted August 2, 2011; Published September 12, 2011
Copyright: ? 2011 Metkar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: CF was supported through 5RO1AI04494-03 from the National Institute of Allergy and Infectious diseases, National Institutes of Health. RG is a Royal
Society University Research Fellow. GA received financial support from the Slovenian Research Agency. JP was supported by Fundacio ´n Agencia Aragonesa para
la Investigacio ´n y Desarrollo and grant SAF2008-02139 from Spanish Ministry of Science and Innovation. EC is a pre-doctoral fellow ascribed to grant SAF2007-
65144 from Spanish Ministry of Science and Innovation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation
of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
The granule secretory pathway represents an important host
defense against tumor and pathogen infected cells. This pathway is
fundamentally a form of intracellular protein delivery where the
pore forming protein perforin (PFN) contributes to the delivery of
the granule proteases (granzymes), which in turn then mediate
cytotoxic as well as additional biological effects. Although PFN
and granzymes were first discovered more than 25 years ago
[1,2,3], the mechanism through which PFN remodels the target
cell plasma membrane for granzyme passage across the bilayer
remains elusive. The original model proposed that the proteases
simply diffuse through barrel stave pores generated in the plasma
membrane of the target cell . Recent structural studies have
provided images indicating that this pore consists of a ring of about
twenty subunits with a diameter of approximately 20 nm .
Using electron microscopy or other biophysical approaches,
pores of various functional diameters are observed on membranes
when PFN is added as the isolated protein or via cytotoxic cells
[1,6,7,8,9,10,11,12]. The direct observation of the movement of
cationic proteases across the plasma membrane of target cells
through such pores remains unachieved and, perplexingly,
granzyme delivery seems to occur without detectable pore
formation [13,14,15]. A fundamental consideration in evaluating
this paradox is to ask what form PFN monomers adopt to effect
protein delivery. For example, what is the relevant number of PFN
molecules that a target cell needs to encounter to achieve this goal?
Experimentally, sufficient quantities of PFN, either in isolated
form or secreted by a cytotoxic cell, will readily induce target cell
necrosis while much lower concentrations, which leave the
membrane apparently unscathed, are necessary to deliver the
An alternative model proposes that PFN and granzymes are
autonomously internalized within endocytic vesicles from which
delivery occurs by PFN-mediated endosomolysis . Another
variant proposes that PFN pores generate sufficient calcium influx
to trigger a membrane repair response which drives internalization
of the granzyme for subsequent endocytic delivery . We have
attempted to visualize endosome lysis caused by PFN using
CLSM, without success (Metkar and Froelich, unpublished). It
remains unclear therefore how granzymes are delivered across
either the plasma or (if involved) the endosomal membrane. An
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additional problem is our inability to identify target cells prepared
by PFN for granzyme delivery.
To evaluate the effect of PFN monomers on the plasma
membrane of target cells, we have used probes that assess
permeability as well as alterations in membrane composition.
When administered alone, PFN, as determined by Annexin-V
(Ann-V) staining, causes the target cell to externalize PS from the
internal to external leaflet. The pattern of staining is distinct from
that observed for necrotic and apoptotic cells and the presence of
exposed PS is also detected by lactadherin (LA) binding. PS
externalization was shown to occur in target cells even though
such fluorophores as Propidium Iodide (PI) and Sytox Green (SG)
are completely excluded in the presence of sufficient quantities of
human perforin that deliver the granzyme. Importantly, PS
externalization was also observed after antigen specific CTLs
contacted peptide-pulsed target cells. PFN therefore appears to
induce a rapid re-organization of the plasma membrane, namely
PS flip-flop [18,19]. A possible explanation for this phenomenon is
the formation of short-lived, low-caliber pores that consist of a
matrix of lipid and PFN oligomers. Related configurations
have been described for the pro-apoptotic protein, Bax ,
for equinatoxin  and during membrane fusion [22,23]. The
formation of such structures might suggest a mechanism for
traversal of granzymes across the cell boundary, gliding over the
curved lipid surface. Furthermore, matrices of protein and lipid
have increased flexibility compared to protein channels, which
suggests an unanticipated mechanism for translocation of
granzymes across a formidable barrier- the lipid bilayer.
Isolated PFN is rapidly inactivated by extracellular Ca ions
Following cytotoxic cell degranulation, PFN monomers disso-
ciate from their carrier, the proteolgycan, serglycin, and face
two outcomes. Serum proteins  and, possibly Ca ions may
inactivate PFN monomers while others undergo well studied, Ca-
dependent bindingto plasma
[25,26,27]. We learned that pre-treating PFN with Ca results in
a rapid decay in permeabilizing activity with more than 80%
activity lost in approximately 5 min (Figure S1a). Furthermore,
using EM to visualize the effect of Ca ions, PFN monomers were
observed to assemble into ring-shaped oligomers in solution with
an appearance comparable to the cylinders observed on
membranes (Figure S1b). Similar to solution oligomerization that
inactivates the related cholesterol-dependent cytolysin, pneumoly-
sin , Ca induces the oligomerization of PFN, depleting the
supply of monomer that is able to interact with the target cell
Isolated PFN and GzmB initiates the lethal hit within
Cytotoxicity assays that examine the potency of PFN and
granzyme B (GzmB) in vitro involve an incubation step in which the
proteins are mixed with target cells for four hours or longer and
thereafter, a number of cell death parameters are measured. Since
PFN willbe largely inactivated within minutes by Ca, we made use of
this insight to learn whether a brief exposure to PFN and GzmB was
sufficient to induce target cell apoptosis. A 5 minute pulse with PFN
and GzmB was quite sufficient to induce target cell apoptosis as
shown by the TUNEL assay which measures DNA fragmentation
(Fig. 1a)(see Figure S1c for alternate readout after a 15 min pulse and
initiate a lethal hit, will be limited during in vitro assays.
Further efforts to identify pores induced by sublytic PFN
The study of PFN-dependent granzyme delivery in vitro
generally entails two steps. First, PFN is titrated against the target
cells of interest to identify the percentage of permeabilized cells at
various concentrations distinguishing the necrotic cells that
develop. Minimally permeabilizing (sub-lytic) concentrations of
PFN are then chosen for the delivery studies. The addition of PFN
to target cells in the presence of PI yields two subsets: 1)
undamaged target cells and 2) necrotic cells. The subset of interest,
that is, target cells prepared for granzyme delivery, resides among
the apparently normal population. As the concentration of PFN
increases, the percentage of necrotic cells rises until all the cells die
presumably due to the formation of enough pores to cause a
massive Ca influx . To our knowledge, markers that identify
the PFN-induced membrane changes that might be associated
with granzyme delivery have not been described. Indeed, we have
been unable to identify pores in target cells exposed to
concentrations of native human PFN that cause granzyme delivery
but not necrosis using fluorophores such as Propidium Iodide (PI)
 or Sytox Green (SG) (data not shown), even when the
fluorophores are added to target cells prior to PFN. This issue has
perplexed us because clearly 20 nm pores are observed when
recombinant PFN is added to liposomal membranes  and, as
determined by an osmotic protection assay, human PFN forms
structures in erythrocytes that have a hydrodynamic diameter
between 2.7 nm and 3.2 nm (Figure S1d). These values agree with
a number of studies that examine the pore diameter produced by
human and mouse PFN in erythrocytes [8,9,11].
Although fluorophores appear to be excluded from target cells
that are exposed to concentrations of human PFN that cause
granzyme delivery, calcium influx is quite evident (Fig. 1b, c) .
Thus, the measurement of Ca influx in otherwise normal-
appearing PFN-treated cells may be a useful marker to define
the subset that is rendered susceptible to granzyme translocation.
In addition, our failure to identify pores with such fluorophores as
PI may be due to the presence of the pore structures at low density
on the target cells as well as due to their rapid clearance by
membrane repair [17,30]. To address these possibilities, we
examined whether inhibition of Ca-mediated membrane repair
with the chelator, BAPTA-AM slows Ca influx and thus reduces
GzmB-induced apoptosis and reveals pores in target cells that are
identifiable with PI. Here, the slowing of membrane repair should
prolong the half-life of PFN pores in the plasma membrane of the
target cells, allowing the entry of fluorophore and shifting the PFN
concentration curve leading to more necrotic cells at lower
concentrations of PFN . The validity of this approach is
described for SLO (see Figure S1e). Unlike the technique used to
examine SLO treated cells which entails the examination of PI
entry in the presence and absence of extracellular Ca ion, BAPTA-
AM was used to effectively chelate the influx of Ca induced by a
range of non-toxic concentrations of PFN (Fig. 1d). Nevertheless,
an increase in PI uptake was not discernible (Fig. 1e) and,
surprisingly, the inhibition of Ca influx did not reduce the
induction of cell death (Fig. 1f). It thus seems that isolated human
PFN generates structures sufficient for entry of Ca ions but not low
molecular weight cationic fluorophores such PI and SG (600–700
daltons) and this signal does not contribute to granzyme delivery.
PFN alone rapidly induces PS externalization
To investigate the membrane alterations allowing Ca entry we
evaluated probes of membrane reorganization, such as the
migration of inner leaflet-specific components (e.g. phosphatidyl-
serine - PS) to the outer leaflet of the bilayer [19,31]. Two well-
known ligands of PS, the proteins, Annexin-V (Ann-V) and
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Figure 1. A brief pulse of target cells with human PFN induces calcium influx and causes PS flip-flop without evidence of membrane
pore formation as measured by the cationic fluorophore propidium iodide. a. Five min pulse with PFN and GzmB is sufficient to induce
apoptosis (TUNEL). Jurkat cells were pulsed with GzmB (1 mg/ml) and PFN at the indicated concentrations for 5 min. The reaction was then stopped by
an EGTA wash step. Cells were incubated for 90 min after which they were fixed and stained for TUNEL reactivity (mean + sd, n=3). b and c. Calcium
influx occurs in PFN-treated cells that exclude PI. Fluo-3 and Fura Red loaded Jurkat cells were treated with indicated concentrations of PFN in the
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lactadherin  were examined. We found using 5 min (data not
shown) or 15 minute pulses that human PFN rapidly induces PS
externalization as detected with Ann-V binding, without allowing
the passage of PI (Fig. 1g, see bottom right gate). Notably, Ann-V
stains the PFN-treated cells at a lower intensity compared to the
pattern observed for the discrete population of necrotic cells (top
right gate). Equinatoxin II (Eqt II), a toxin predicted to generate
pores from a matrix of protein and lipid [21,33], produced a
similar pattern of low-intensity Ann-V reactivity in nucleated cells
(Fig. 1g, see bottom right gate). Fig. 1h summarizes these data for
PFN and Eqt II. PFN induced PS externalization is also
recognized by lactadherin, a probe that binds to the anionic lipid
independent of calcium (Fig. 1i). The absence of PI influx makes it
implausible that the 40–45 kDa Ann-V and lactadherin stain cells
by entering and binding PS in the plasma membrane inner leaflet,
and we therefore conclude that the probes must be binding PS that
PFN, on a rapid timescale, has induced to migrate to the outer
PS externalization induced by PFN is not mediated by Ca
Next we asked whether Ann-V and lactadherin binding induced
by limiting concentrations of PFN could be due to calcium influx
and activation of PS translocases [34,35]. A calcium ionophore did
indeed induce measurable but minor Ann-V reactivity when
applied to the Jurkat cells (Fig. 2a). However, when the effects of
calcium influx were minimized by pre-loading targets with
BAPTA-AM, human PFN still elicited a sizable Ann-V reactive
subset (Fig. 2b–c).
PS externalization induced by PFN is enhanced by
phospholipids that increase membrane curvature and by
cholesterol depletion of the plasma membrane
Since PFN appears to be directly inducing reconfiguration of
the plasma membrane lipids, we then sought to investigate the
lipid dependence of this phenomenon. To do this we made use of
oleoyl lysophosphatidylcholine (oLPC), a phospholipid that
induces positive membrane curvature [31,36,37] and by reducing
the cholesterol content of the plasma membrane. We first
demonstrated the suitability of this approach by showing that
the oLPC increased the percentage of Ann-V reactive cells among
Eqt II treated target cells (Fig. 3a). This technique however was
problematic for the study of PFN because the assay ordinarily is
performed in the presence of fluid-phase oLPC, a condition that
inhibits the capacity of the PFN monomers to bind to the plasma
membrane of the target cells due to their interaction with the LPC
headgroups . Therefore, Jurkat cells were preincubated with
oLPC, exposed to PFN and, then monitored for PS flip-flop.
Despite the limited exposure, the data show that the pre-
incubation of the target cells with the oLPC increased the
percentage of Ann-V positive cells after the target cells were
exposed to PFN (Fig. 3b).
Depleting the relatively rigid cholesterol molecule from the
target cell plasma membrane mediated by non-toxic concentra-
tions of methyl-b-cyclodextrin (MBCD) was also observed to
increase PS flip-flop induced by both Eqt II and PFN (Fig. 3c–d).
Among the many effects that cholesterol has on the organization of
the plasma membrane, its removal is predicted to increase the
disorder of the phospholipid acyl chains as well as the size of
ordered and disordered domains [39,40,41]. These observations
further strengthen the likelihood that PS flip-flop originates from
reconfigurations among the lipids themselves by PFN.
PFN-induced PS flip-flop is the first marker that correlates
with a cell’s susceptibility to GzmB-induced apoptosis
Since the measurement of Ca influx was not a reliable marker
for granzyme delivery, we examined whether PFN-induced PS
flip-flop correlated with cellular susceptibility to GzmB. Targets
were split into two aliquots with one exposed to PFN alone to
measure Ann-V and PI reactivity (Fig. 4a) while the other was
treated with both PFN and GzmB followed by measurement of cell
death by mitochondrial depolarization (Fig. 4b). The results show
a statistically significant correlation between the identified Ann-V
reactivity and the percentage of cells that undergo apoptosis
(r2=0.89, p=0.0047; Fig. 4b inset). These data therefore suggest
that the low intensity Ann-V positive subset induced by PFN
identifies a portion of the cells that undergo apoptosis if exposed to
GzmB. For comparison, the pattern of Ann-V staining is presented
after a 15 min PFN-GzmB pulse (Fig. 4c) as well as after a 2 hr
incubation period to verify that the pulse resulted in apoptosis
(Figure S1f). GzmB, despite the short exposure time, modified the
pattern generated by PFN producing a low intensity PI population
as well as the low intensity Ann-V cells, cells.
PFN-induced PS flip-flop is induced by antigen specific
To determine physiological relevance, we then asked whether
target cells exposed to CTLs develop rapid PS flip-flop. For these
studies, ex vivo CD8 T cells from LCMV-immune (day 7) WT,
GzmA/B2/2and PFN/GzmA/GzmB2/2mice were added to
target cells in the presence and absence of the immunizing peptide,
gp 33. PI was added prior to formation of the conjugates to
evaluate membrane damage and Ann-V staining of PS flip-flop
presence of PI and the percent events in PI negative subset enumerated over 6 minutes (b). Intracellular Calcium flux was measured concomitantly
using the ratio of Fluo-3/Fura Red MFI in the PI negative subset (c). Cells were followed to 6 minutes. Data is representative of one of three
independent experiments. d. PFN mediated Ca influx can be blocked by BAPTA-AM. Fluo-4 and BAPTA-AM/DMSO loaded cells were treated with the
indicated concentrations of PFN in presence of PI and intracellular Calcium flux measured in PI negative cells. Data is presented as background
subtracted Fluo-4 MFI over the indicated times and is representative of 1 of 2 independent experiments. e. Intracellular Calcium chelation does not
increase PFN induced necrosis. BAPTA-AM/DMSO loaded cells were treated with the indicated concentrations of PFN in presence of PI for 6 min and
analyzed by flow. Data is representative of 1 of 2 independent experiments. Numbers represent percent events in the various gates. f. Inhibition of Ca
influx does not interfere with PFN-mediated granzyme delivery. Media, DMSO or BAPTA-AM loaded cells were treated with GzmB and PFN at the
indicated concentrations for 1 hour at 37uC in presence of the Cell Event Caspase 3/7 reagent and PI. Cells demonstrating activated Caspase-3/7 were
enumerated using flow cytometry (mean + sd, n=2). g. PS flip-flop occurs in PFN-treated cells that exclude PI – annexin-V binding. Jurkat cells were
treated with PFN for 15 min, washed and stained with Ann-V-FITC. PI was present throughout the assay. Eqt II was used as a positive control. Data
from one representative experiment is shown. Numbers represent percent events in the various gates. h. PFN induces PS flip-flop in cells that exclude
PI. Jurkat cells were treated as described above and events in the Ann-V positive, PI negative gate induced after PFN and Eqt II treatment enumerated
for three experiments (mean + sd). i. PS flip-flop occurs in PFN-treated cells that exclude PI –Lactadherin binding. Jurkat cells were pulsed with indicated
concentrations of PFN for 15 min in presence of PI, washed and stained with either Ann-FITC or Lactadherin-FITC in calcium containing buffer.
Percent events in the respective gates are depicted.
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was measured after the conjugates had been incubated for the
times designated. Unlike our studies with isolated human PFN, a
discrete subset of targets display low intensity Ann-V staining and
subtle PI uptake (Fig. 4d). Presumably the PS flip-flop associated
pores that are induced by isolated human PFN are somewhat
smaller in size than those induced by the CTLs (see below). As
described above for the isolated proteins, adding the combination
of PFN and GzmB prevents us from reliably detecting PS
flip-flop due to PFN alone (Fig. 4d – right panel). However, the
GzmAxB2/2 CD8 cells which contain PFN but lack the pro-
apoptotic GzmB as well as the non-cytotoxic GzmA  rapidly
induce low intensity Ann-V reactivity suggesting that ex vivo
antigen specific CD8 cells are able to stimulate rapid PS flip-flop in
recognized target cells through the action of PFN. Taken together,
mouse PFN secreted by antigen specific CD8 cells induces PS flip-
flop as well as allows PI to enter target cells where the fluorophore
remains minimally excitable in the cytosol compared to the much
greater excitation that occurs after the probe binds to nuclear
DNA of necrotic cells (see Fig. 5b–c, see below). This pattern is
only observable when the probe is added prior to PFN challenge.
Human PFN can be manipulated to produce low
intensity PI cells
Since isolated mouse PFN was unavailable, we explored the
possibility that human PFN could also induce simultaneously low
intensity PI and Ann-V staining if conditions were modified to
increase monomer binding. Reducing the pH of the buffer will
increase the density of monomers that bind to target cells. This
strategy generates low intensity PI cells  and results in the
development of low intensity Ann-V+cells (See Figure S1g).
However, the relatively harsh incubation at acidic pH and the
complex experimental design could introduce systematic errors
Figure 2. PFN-mediated Calcium influx does not induce Ann-V reactivity in Jurkat cells. a. Calcimycin treated Jurkat cells demonstrate PS
flip-flop. Cells were exposed to Calcimycin at the indicated concentrations for 15 min in presence of PI, followed by Ann-V-FITC staining. Numbers
represent percent events in the respective gates. Data described for one of two experiments. b. Ann-V reactivity in PFN treated cells is not inhibited by
calcium chelation. Cells were loaded with BAPTA-AM, treated with human PFN in presence of PI and stained with AnnV-FITC. Density plots from 1 of 2
independent experiments is shown. c. Intracellular Calcium chelator BAPTA-AM prevents Calcium flux in cells treated with PFN. Jurkat cells were loaded
with Fluo-4 with/without BAPTA-AM, washed and treated with PFN in presence of PI. Calcium flux was measured over 6 min. Data is presented as
background subtracted Fluo-4 MFI over the indicated times and is representative of 1 of 2 independent experiments.
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that might lead to the measurable low intensity Ann-V and PI
staining. We, therefore, corroborated this work with a simpler
design that involved the incubation of the target cells with PFN in
the presence of 0.2 mM Ca. The rationale for this approach was
our previous observation that cell associated PFN was more
readily detectable by anti-DG9 mAb staining and flow cytometry
after incubation of the targets with PFN and 0.2 mM Ca as
opposed 1.25 mM Ca. This finding suggested that a greater
number of monomers bound to the target cells in the reduced Ca
buffer , perhaps because fewer were inactivated in the solution
phase. Treatment of the Jurkat cells with human PFN in the
presence of 0.2 mM Ca produced cells that were both low
intensity Ann-V and PI positive (Fig. 5a). To clarify the
localization of the PI staining, Jurkat cells that contained low
levels of PI by flow cytometry were evaluated by CLSM. The
observed low intensity PI staining could be attributed to cytosolic
localization of the fluorophore compared to the more intense
nuclear fluorescence observed for necrotic cells (Fig. 5b). Imaging
of HeLa cells confirmed the cytosolic localization of the low
intensity PI (Fig. 5c). These results suggest that structures formed
in target cells by mouse and human PFN are similar with the low
intensity PI uptake induced by the CD8 cells attributable to a
higher net level of PFN binding to the target cells.
How PFN delivers GzmB into cells without causing detectable
damage to the cell membrane is a matter of intense interest
especially since an understanding of this phenomenon will allow
the development of more rational strategies for intra-cellular
protein delivery. The evidence presented here suggests that the
mechanism involves lipid components of the membrane them-
selves, as well as PFN monomers inserted into the membrane. For
example, single channel conductance measurements show that the
physical characteristics of the pore including its size, its stability
and its mechanism of formation are affected by the degree of
ordering in the membrane lipids [45,46]. Pore diameters formed
by PFN appear to vary depending on membrane composition
ranging from homogenous phospholipids of a single headgroup to
erythrocyte membrane and the plasma membrane of nucleated
cells [1,5,8,9,11,12,47]. Despite stringent efforts to uncover the
20 nm pores, when PFN is applied to target cells at concentrations
Figure 3. Effect of long chain positive curvature lipid, oLPC and Cholesterol depletion on Eqt II and PFN-induced PS flip-flop.
a. LysoPC augments Eqt II induced PS flip-flop. Cells were treated with Eqt II in the presence and absence of oLPC for 15 min, washed and then stained
with Ann-V-FITC. Data describe percent events in the Ann-V positive, PI negative gate (mean 6 sd, n=3). b. LysoPC augments PFN induced PS flip-flop.
Cells were treated with oleoyl LysoPC for 15 min to allow for plasma membrane insertion, washed and then treated with PFN at the indicated
concentrations for 15 min. Cells were washed and stained with Ann-V-FITC. PI was present throughout the assay. Data (mean 6 sd) represent percent
events in the Ann-V positive, PI negative gate for two independent experiments. c. Cholesterol depletion augments Eqt II induced PS flip-flop. Cells pre-
treated with MBCD were treated with Eqt II for 15 min, washed and then stained with Ann-V-FITC. Data describe percent events in the Ann-V positive,
PI negative gate (mean 6 sd, n=2). d Cholesterol depletion augments PFN induced PS flip-flop. Cells pre-treated with MBCD were exposed to PFN at
the indicated concentrations for 15 min. Cells were washed and stained with Ann-V-FITC. PI was present throughout the assay. Data (mean 6 sd,
n=2) represent percent events in the Ann-V positive, PI negative gate for two independent experiments. A two-tailed paired t-test was used to
determine statistical significance.
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that contribute to granzyme delivery, even low molecular
fluorophores such as PI do not enter the target cells. Instead, we
present evidence that PFN reconfigures membrane lipids on live
cells that results in unanticipated phospholipid flip-flop. We show
by Ann-V and lactadherin staining that PS appears in the outer
leaflet of the bilayer, and that this marks cells that are prepared for
GzmB delivery. PS flip-flop is not caused by PFN-induced calcium
influx and seems to increase when cells are exposed to an oLPC
that enhances positive membrane curvature and by cholesterol
PFN appears to induce flip-flop of PS from the inner to the
outer membrane leaflet that does not rely on the induction of
intracellular translocases and flippases. The externalization of PS
we observe can most easily be explained by the fusion of the inner
and outer leaflets of the bilayer and the lipid’s migration over the
resulting lip, a process that does not occur during the formation of
Figure 4. Cells demonstrating PS flip-flop are susceptible to GzmB induced apoptosis. a. PFN induces PS flip-flop in PI negative cells. Cells
were treated with PFN, washed, and stained with Ann-V-FITC. GzmB (1 mg/ml) was added to paired samples to assess delivery (see Fig. 4b). An auto-
fluorescence control is also shown. b. The presence of Annexin V reactivity (PS flip-flop) correlates with susceptibility of target cells to GzmB-mediated
apoptosis. Paired sample (PFN alone and PFN plus GzmB) were incubated for 45 min in presence of CMX-ROS. PFN concentrations were chosen for
their ability to induce PS flip-flop in PI negative cells from (a). Cells undergoing mitochondrial depolarization were enumerated and results are
presented as mean + sd of 2 experiments. Inset: A significant correlation (r2=0.89; p=0.0047) was observed between PI negative events exhibiting
flip-flop in presence of PFN and events describing mitochondrial depolarization in presence of PFN and GzmB. c. A comparison of PS flip-flop in PFN
versus PFN-GzmB pulsed Jurkat cells. To compare the pattern of Ann-V-FITC staining produced by PFN versus PFN plus GzmB, cells were pulsed with
PFN in the presence and absence of GzmB (1 mg/ml) for 15 min at 37uC followed by Ann-V-FITC staining (data for one of two experiments). d. gp33
specific murine CTL that contain PFN induce Ann-V + subset in gp33 loaded targets. Ex vivo CD8 cells from LCMV mice were isolated, labeled with
CellTracker Green and added to EL4 cells 6 gp33 at E:T of 2:1. PI (10 mg/ml) was added at onset with distribution of PS flip-flop and PI reactivity within
targets determined by FACS at 10, 20 and 120 min in the CellTracker negative population (EL4 cells). Data representative of 1 of 2 independent
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transmembrane proteinaceous channels. A related toroidal
structure is known to arise during electroporation [48,49] as well
as during membrane fusion events that occur upon viral cell entry
and transfer of intracellular cargo between vesicular compartments
PFN monomers have been reported to clearly oligomerize into a
cylindrical pore-forming proteinaceous channel which span the
membrane bilayer to form a pore and this mechanism must
contribute to certain biological effects mediated by the toxin
[5,45]. Many other proteins are known to form pores using a
similar mechanism of oligomerization and insertion. Nevertheless,
biophysical data have suggested that bacterial peptides such as
melittin [19,50], toxins including Equinatoxin II  and small
proteins like Bax , all may induce pores consisting of a mixture
of incompletely-oligomerized protein and the membrane phos-
pholipids themselves. Whether larger toxins such as bacterial
CDCs and the related MACPF domain proteins such as PFN
form these structures is highly controversial  but must be
considered possible given the apparent effects of the membrane
lipid components on pore structure, stability and lifetime [45,52].
Pores formed by such matrices of protein and lipid would have the
necessary characteristics to allow PS migration between PM leaflets
and would be similar to pore structures induced during membrane
fusion [22,23]. Using a variety of biophysical approaches including
electron microscopy , atomic force microscopy  and X-ray
diffraction , the existence of these unusual pores in model
membranes has been argued to exist. However, even if evidence for
their formation in vitro is accepted, their appearance in biological
membranes has remained unproven, due to their presumed
evanescence as shown by transient Ca influx and the lack of
technologies capable of imaging complex membranes at the
One of the major concerns in understanding how a membrane
is altered by an applied protein is whether its insertion achieves the
biological effect observed in vivo. PFN may be added to target
cells at concentrations that induce widespread necrosis but such
toxic levels neither mirror the conditions necessary for granzyme
delivery nor reflect the levels encountered by a target cell after
CTL engagement (see Fig. 4). Our data establish for the first time
guidelines that allow the identification of physiologically relevant
PFN concentrations for cellular and biophysical studies. We
hypothesized previously that PFN oligomers might disrupt the
plasma membrane allowing granzymes to cross the bilayer in the
absence of discrete pores . The phenomena we see evidenced
in the movement of PS to the upper leaflet of the membrane would
correlate with such an event.
Based on recent structural data, PFN is predicted to oligomerize
to a single-sized pore (20 nm) regardless of extracellular conditions
or membrane composition that serves as the platform for insertion.
However, data reported here and elsewhere suggest that the
structures formed by PFN are variable in size  depending on at
least two factors that influence binding density and oligomerization
rates. These include 1) The concentration of Ca ions and inhibitory
proteins  that PFN encounters and 2) the composition and
and then insert within. Together these conditions appear to
influence the characteristics of pores with cylinders predominating
above a concentration threshold while membrane structures that
give rise to PS flip-flop are more common when the level of
monomers is limiting (Fig. 6). These latter structures may be
incomplete ring oligomers, or arcs, that have been observed
While PFN is observed to form cylinders and arcs on liposomes
and sheep erythrocyte ghosts when evaluated by EM, a systematic
description of membrane alterations that might be induced by the
toxin has not been undertaken. In this regard, we have learned that
PFN induces membrane invaginations in both LUVs and giant
unilamellar vesicles (manuscript submitted). Although additional
work is necessary to understand how PFN induces such structures, it
is intriguing to speculate that PFN arcs may generate positive
membrane curvature that partly contributes to the observed
invaginations and the counterpart to these morphological alter-
ationsis transient membranefusion and phospholipidflip-flop when
PFNis appliedto nucleatedcells inlimiting amounts associated with
granzyme delivery. Inasmuch as cylindrical pore structures have not
been identified in target cells that would allow passage of the
granzymes, the observed flip-flop becomes the first viable pathway
that could offer a route for GzmB translocation. However,
translocation would depend on the availability of GzmB to interact
with membrane phospholipids of the outer leaflet. Since GzmB
would preferentially bind the glycosaminoglycan (GAG) side chains
of cell surface proteolgycans, a GzmB-phospholipid interaction
would require a radical movement of the proteoglycans and their
associated Glycosaminoglycan chains outward from the phospho-
lipid binding sites on the leaflet. Such a possibility may occur
efficiently in the CTL synapse where major alteration in the
topological distribution of integral membrane proteins has been
PFN induced Ca influx is considered to be mechanistically
linked to granzyme delivery . In this model, PFN triggers a
plasma membrane-repair response that depends on inward
movement of Ca ions. The response then drives endocytic uptake
of GzmB and its delivery, an outcome inhibited by BAPTA-AM.
For these studies, an arbitrary sub-lytic dose of PFN was employed
without distinguishing the subset of cells that PFN renders
susceptible to GzmB. By adding PI before PFN, we could identify
and recognize those cells that succumb to GzmB versus targets
that became rapidly necrotic. We were thus surprised to observe
Figure 5. PFN induces both low intensity PI and Ann-V reactivity in target cells. a. Target cells treated with human PFN under low calcium
develop both low intensity PI and Ann-V reactivity. Jurkat cells were treated with human PFN in the presence of 0.2 mM calcium, washed with 0.2 mM
calcium containing buffer and then stained with Ann-V-FITC in presence of 1.25 mM calcium. Density plots from 1 of 2 independent experiments is
shown. b. A PI low intensity subset is induced by PFN as visualized by flow cytometry and CLSM: Jurkat cells were treated with rat PFN in the presence of
PI for 15 min, washed and either run on a flow cytometer (i) or imaged by CLSM (ii) as describe in methods. Histograms show PI staining where
normal, low intensity and high intensity PI subsets are marked along with the percentages in the respective regions. Confocal images visualizing the
PI hi and low cells along with Media control are shown in the lower panel. Data is representative of 1 of 3 experiments. c. Imaging of low intensity PI
cells induced by PFN: HeLa cells were treated with PFN (150 ng/ml) in the presence of FM1-43 and PI and imaged by CLSM. The upper left panel shows
unenhanced image of three Hela cells, two with low intensity PI uptake and one unaffected (normal). The upper right panel show the same field with
green channel (FM1-43) enhancement. Enlarged lower left panel show enhancement of red channel to visualize PI. Lower right panel depicts PFN at a
lytic concentration (250 ng/ml), note the intensity of PI staining in the nucleus in this un-enhanced image. Data are representative of 1 of 2
independent experiments; the most in-frame section is shown. Enhancement of select panels was performed with Adobe Photoshop where highlight
value for select color channel was in all instances changed from 255 to 100.
Perforin Induces Phospholipid Flip-Flop
PLoS ONE | www.plosone.org9 September 2011 | Volume 6 | Issue 9 | e24286
that intracellular chelation of PFN-induced Ca influx did not
reduce granzyme delivery and apoptosis induction when detected
by a highly sensitive probe for caspase-3/-7 activation. This issue
notwithstanding, Ca influx and PS flip-flop appear to represent
entirely separate biological processes where Ca influx indeed may
serve to protect cells as the intracellular levels of Ca rise with
increasing deposition of cylindrical pores.
PFN-induced phospholipid flip-flop appears to correlate highly
with target cells that undergo GzmB-induced apoptosis, but PS
translocation may serve other functions. PFN is considered to act in
host defense primarily as the delivery agent for the granzymes.
However, PFN has been shown to be both necessary and sufficient
in the absence of the granzyme A and B to eliminate tumor burden
. PFN alone would be predicted to act by inducing tumor cell
necrosis, which is viewed as deleterious to the host by inducing
autoimmune responsiveness. Perhaps, PFN-induced phospholipid
flip-flop is instead sufficient to stimulate phagocytic clearance of
tumor cells though mechanisms described for apoptotic cells.
In summary, the observation that a 5 min pulse of PFN and
GzmB is sufficient for apoptosis suggests that the granzyme may
simply translocate through structures that reflect PS flip-flop at the
plasma membrane. The validity of these concepts await our ability
to image target cells treated with the physiologically relevant
concentrations of the pore forming protein, to determine whether
mutations that alter the oligomerization of membrane bound PFN
encourages formation of the proposed arc-like structures, and to
learn whether GzmB exploits these structures to translocate to the
Materials and Methods
All experimental work involving mice were performed accord-
ing to FELASA guidelines under the supervision and approval of
Comite ´ E´tico para la Experimentacio ´n Animal (Ethics Committee
for Animal Experimentation) from CITA (Agrifood Research and
Figure 6. Factors that influence the outcome of PFN interaction with cells. Once secreted from effector cells, PFN monomers are rapidly
inactivated by Calcium thereby limiting their availability to interact with the plasma membrane of the target cell. The complexity of the plasma
membrane further influences the density of monomer binding and rate of oligomerization. Together these variables may influence the tendency of
PFN to form barrel stave pores of varying diameters or, possibly, arcs.
Perforin Induces Phospholipid Flip-Flop
PLoS ONE | www.plosone.org10 September 2011 | Volume 6 | Issue 9 | e24286
Technology Centre from Aragon), Approval # CEEA-01, 27/01/
2011, Nu 2011/01.
Cell lines and reagents
Jurkat and HeLa cells were purchased from ATCC and cultured
in RPMI – 10% fetal bovine serum (FBS) media. All reagents and
tissue culture supplies were purchased from either Sigma-Aldrich
(St. Louis, MO) or Invitrogen (Carlsbad, CA). Granzyme B (GzmB)
and human PFN (PFN) were isolated as described [16,55]. PFN was
then stabilized to tolerate freeze thaw cycles by the addition of 1%
fatty acid-free BSA. Eqt II was isolated as described  and
Streptolysin O (SLO) was a kind gift from S. Bhakdi. Oleoyl
Lysophosphatidylcholine (18:1 Lyso PC; 1-oleoly-2-hydroxy-sn-
glycero-3-phosphocholine) was purchased from Avanti Polar Lipids
(Alabaster, AL). Sheep red blood cells (SRBC) were purchased from
of varying Mr (2,000 to 10,000 daltons) and Propidium Iodide (PI)
were from Sigma-Aldrich (St. Louis, MO). The terminal deox-
ynucleotidyl transferase dUTP nick end labeling (TUNEL) and
Annexin-V-FITC kits were from BD Biosciences (San Jose, CA).
MitoTracker red (CMXRos), Fluo-3, Fluo-4, Fura Red, the
intracellular Calcium chelator, BAPTA-AM, and the Cell Event
Caspase 3/7 detection reagent were from Invitrogen (Carlsbad,
CA). Bovine Lactadherin conjugated to FITC was from Hemato-
logical Technologies (Essex Junction, VT).
Production of the lethal hit
Target cells were suspended at 16107per ml in the high Ca
buffer (HCB) that consisted of 150 mM NaCl, 20 mM Hepes and
2.5 mM Ca (pH 7.4). PFN was diluted in a no calcium buffer
(NCB) that consisted of 150 mM NaCl, 20 mM Hepes, and 1%
BSA (pH 7.4). PFN was added to microwells of a 96 well plate.
GzmB, at desired concentration, was then added (50 ml) followed
by target cells (50 ml) and incubated for 5 min at 37uC. PI was
present at final concentration of 10 mg/ml throughout. The cells
were then washed twice with PBS containing 5 mM EGTA
followed by single wash with PBS and resuspended in 1 ml of
RPMI, 10% FBS. The cells were separated into two aliquots
where the first was exposed to MitoTracker red CMXRos (50 nM)
while the second received additional PI (10 mg/ml). After 45 min,
the cells were analyzed by flow cytometry.
Cell death assays
potential loss using CMXRos was measured as reported . Cell
viability was measured using PI (10 mg/ml) and flow cytometry.
Target cells were pulsed with PFN in
presence of GzmB for 5 min as described above, washed with
EGTA and incubated for 90 min. Cells were then fixed and death
measured by TUNEL .
Activation of Caspase3/7.
PFN and GzmB as described above in presence of the Cell Event
Caspase 3/7 detection reagent (5 mM, Invitrogen, Carlsbad, CA),
and PI for 5 min (pulse) or continuously for 1 hour incubation at
37uC. PI was present at final concentration of 10 mg/ml
throughout. In case of a pulse, after an EGTA wash, the cells
were resuspended in buffer containing PI and Cell Event Caspase
3/7 reagent, allowed to incubate for 55 min at 37uC and analyzed
by flow cytometry.
Target cells were treated with
Calcium influx in PFN treated targets
Jurkat cells preloaded with Fluo-3 and Fura Red (1 mM each)
were treated with PFN in presence of PI (10 mg/ml) and flow
cytometry data acquired in real-time (up to 360 seconds). The
final extracellular calcium concentration was 1.25 mM. Cells in
the PI negative gate (Fluorescence intensity ,10) were analyzed
for Fluo-3 and Fura Red fluorescence and data expressed as the
ratio of MFI for Fluo-3 over Fura Red. Ionomycin treated cells
served as positive controls.
In experiments utilizing the intracellular calcium chelator
BAPTA-AM, calcium flux was measured using Fluo-4. Cells were
loaded with 100 mM BAPTA-AM and Fluo-4 (5 mM) for 40 min
at room temperature in the dark. Cells were then washed and
treated with PFN in presence of PI (10 mg/ml) and changes in
Fluo-4 fluorescence measured in real time over 360 sec. Cells in
the PI negative gate were analyzed for Fluo-4 fluorescence and
data was expressed as MFI for Fluo-4 in treated samples minus
MFI in control sample (background subtracted).
The effect of Calcium chelation on PFN mediated GzmB
delivery was studied using the Caspase 3/7 reagent. Here cells
were first loaded with DMSO (vehicle control) or BAPTA as
described above, followed by washes and then treatment with
GzmB and PFN for 1 hour at 37uC in presence of PI and Cell
Event Caspase 3/7 detection reagent (5 mM).
PFN induced PS flip-flop in targets measured by Annexin
Cells were treated with designated concentrations of PFN for 5
to 15 min in presence of PI (10 mg/ml) at a final extracellular
calcium concentration of 1.25 mM. Cells were then washed and
stained with Ann-V-FITC per the manufacturer’s instructions.
Data were acquired as Ann-V-FITC versus PI density plots.
Equinatoxin II (Eqt II) and Streptolysin O (SLO) were used in
select experiments as controls to monitor formation of proteo-lipid
and barrel stave pores respectively.
PFN induced PS flip-flop in targets undergoing
intracellular calcium chelation
To test whether PFN induced PS flip-flop was dependant on
calcium activated PS translocase activity, intracellular calcium
chelation was performed using BAPTA-AM. Here cells were
loaded with BAPTA-AM (100 and 50 mM) or DMSO (vehicle
control) in RPMI, 1% BSA for 40 min at RT. Cells were then
washed and treated with either human or rat PFN for 15 min at
37uC with extracellular calcium concentration of 1.25 mM. Ann-
V-FITC staining was then performed. PI (10 mg/ml) was present
through out the assay.
PFN induced PS flip-flop in targets undergoing GzmB
To distinguish the pattern of Ann-V-FITC staining in presence
of PFN alone versus PFN-mediated GzmB delivery (apoptosis),
target cells were pulsed with PFN in the presence or absence of
GzmB (1 mg/ml) for 15 min at 37uC. Ann-V-FITC staining was
performed immediately with PI (10 mg/ml) present throughout the
assay. To ascertain the pattern of Ann-V-FITC staining in a
typical apoptosis assay, cells were treated with PFN in the presence
or absence of GzmB (1 mg/ml) for 120 min at 37uC followed by
PFN induced PS flip-flop measured by Lactadherin
Cells were treated with human PFN for 15 min in presence of
10 mg/ml PI at a final extracellular calcium concentration of
1.25 mM. Cells were then washed and stained with lactadherin-
FITC per manufacturer’s instructions. PI was present throughout
Perforin Induces Phospholipid Flip-Flop
PLoS ONE | www.plosone.org11September 2011 | Volume 6 | Issue 9 | e24286
the assay. Data was acquired as lactadherin-FITC versus PI
Effect of oleoyl Lysophosphatidylcholine on PFN induced
To test whether the positive curvature lipid, oleoyl Lysophos-
phatidylcholine (oLPC) could augment PFN induced PS flip-flop,
cells were incubated with oLPC (8 and 4 mM) for 15 min at 37uC
in absence of BSA. Cells were then washed once in 1.25 mM
CaCl2containing 150 mM NaCl, 20 mM Hepes, pH 7.4 and
resuspended in the same buffer supplemented with 0.125% BSA.
Cells were incubated with PFN for 15 min at 37uC, washed and
stained with Ann-V-FITC. PI (10 mg/ml) was present throughout
To test the effect of oleoyl Lysophosphatidylcholine on Eqt II
induced PS flip flop, cells were incubated concomitantly with
oLPC (4 mM) and Eqt II at the indicated concentrations for
15 min at 37uC in absence of BSA. Cells were then washed once
in 1.25 mM CaCl2containing 150 mM NaCl, 20 mM Hepes,
pH 7.4, 0.5% BSA and stained with Ann-V-FITC. PI (10 mg/ml)
was present throughout the assay.
Effect of Methyl-b-cyclodextrin (MBCD) on PFN and Eqt II
induced PS flip-flop
To test the effect of cholesterol depletion on PFN and Eqt II
induced PS flip-flop, cells were washed twice in 2.5 mM CaCl2
containing 150 mM NaCl, 20 mM Hepes, pH 7.4 buffer. They
were resuspended in the same buffer and treated with 0 or 1.5 mM
MBCD for 1 hr at 37uC. After 1 hr, the cells were plated in wells
containing equal volume of PFN or Eqt II diluted in 150 mM
NaCl, 20 mM Hepes, pH 7.4 buffer containing 0.5% BSA. After a
15 min treatment at 37uC, cells were washed and stained with
Ann-V-FITC. PI (10 mg/ml) was present throughout the assay.
PFN induced PS flip-flop in targets under low Calcium
Cells were treated with designated concentrations of PFN for
15 min in presence of PI (10 mg/ml) at a final extracellular
calcium concentration of 0.2 mM. Cells were then washed with
0.2 mM calcium containing 150 mM NaCl, 20 mM Hepes,
pH 7.4 buffer and stained with Ann-V-FITC in presence of
1.25 mM calcium containing 150 mM NaCl, 20 mM Hepes,
pH 7.4 buffer, 0.5% BSA. Data were acquired as Ann-V-FITC
versus PI density plots.
Imaging of targets that exhibit low intensity PI staining
after PFN treatment
Jurkat cells were incubated with PFN in the presence of PI
(10 mg/ml) for 30 min at 37uC in 1.25 mM Calcium containing
Hepes-NaCl buffer, pH 7.4. Cells were washed and an aliquot
analyzed by flow cytometry. PFN concentrations yielding a
substantial PI low population (fluorescence intensity ranging
between 10 and 600) were selected for imaging. Cells were
layered on to poly-L-lysine coated slides, allowed to adhere and
imaged on a Leica Confocal Laser Scanning Microscope System
with a DMIRE2 inverted microscope (Leica Microsystems, Exton
PA). Twelve-bit fluorescence images of the fluorophores were
acquired with 100 X oil-immersion objectives (N.A. 1.25 and 1.4
respectively) using the Leica Confocal software. In experiments
with HeLa, cells were plated on coverslips overnight. Cells were
then washed and treated with PFN in presence of FM1-43 (5 mM)
Preparation and use of ex vivo CTLs
Mice (WT, gzmAxB2/2, and pfnxgzmAxB2/2) were inoculat-
ed with 105pfu LCMV followed by splenectomy 8 days later.
CD8+ cells were positively selected (MACS), stained with Cell
tracker green (1 mM) and added to EL4 cells in the presence and
absence of the immunizing peptide, gp33, at an effector: target
(E:T) ratio of 2:1 . PI (10 mg/ml) was added at onset with PI
and Ann-V reactivity of targets determined after 10, 20 and
120 min. Under this immunizing protocol, similar numbers of
LCMV-specific CTLs (analyzed by tetramer staining) were
recovered from the three mouse strains.
Methodology used to generate supporting information is
included under Methods S1.
was incubated in 1.25 mM Ca buffer – 0.5% BSA buffer at 37uC
for the indicated times and then incubated with cells for an
additional 20 min in presence of PI (10 mg/ml). Samples were
then run on the cytometer to enumerate the PI Hi cells. b: Fluid
phase PFN structures observed by cryo-EM: Electron micrograph of PFN
oligomers in fluid phase in presence of Calcium. PFN was
incubated with 2 mM Ca and the resulting oligomers stained with
uranyl acetate. Images were grouped by multivariate statistical
analysis using the program IMAGIC and the classes of similar
images averaged to produce these class sums. The numbers given
indicate the size of each class (number of individual images it
comprises) and the associated percentages. Scale bar, 20 nm. c:
Five min pulse with PFN and GzmB is sufficient to induce Caspase 3/7
activation. Jurkat cells were incubated with PFN and GzmB (1 mg/
ml) at the indicated concentrations for 1 hr in a continuous
incubation (left) or pulsed with GzmB and PFN for 5 min (right).
For the pulse set, the reaction was stopped by an EGTA wash step
and cells incubated for another 55 min in presence of PI and Cell
Event Caspase3/7 reagent after which they were analysed by flow
cytometry (data for one of two experiments). d: Sizing PFN pores in
SRBCs using the PEG osmotic protection assay. SRBCs were exposed to
increasing concentrations of human PFN in the presence and
absence of PEGs ranging from 2,000 through 6,000 dalton.
Hemoglobin release was determined as described in methods; data
represents one of two experiments. Background Hemoglobin
release ranged from 8 to 9.1%. e: Membrane repair after SLO requires
extracellular Calcium. Jurkat cells were treated with the indicated
concentrations of SLO for 15 min at 37uC in presence or absence
of Calcium and acquired on the cytometer. Data for one
representative experiments of two is shown. f: Typical pattern of
Ann-V and PI during GzmB induced apoptosis. To compare the pattern
of Ann-V-FITC staining produced by PFN versus PFN plus
GzmB, cells were treated with PFN in the presence and absence of
GzmB (1 mg/ml) for 120 min at 37uC followed by Ann-V-FITC
staining (data for one of two experiments). g: Target cells coated with
human PFN at pH 6.0 develop both low intensity PI and Ann-V reactivity.
Jurkat cells were treated with human PFN (pH 6) in the presence
of calcium and Na Azide at 4uC, washed (pH 7.4) at 4uC and then
incubated with 1.25 mM calcium at 37uC for 15 min. Thereafter,
cells were stained with Ann-V-FITC as described (one of two
Methodology used to generate supporting
a: Calcium inactivates PFN in fluid phase. Human PFN
Perforin Induces Phospholipid Flip-Flop
PLoS ONE | www.plosone.org12 September 2011 | Volume 6 | Issue 9 | e24286
Conceived and designed the experiments: SM GA RG JP CF. Performed
the experiments: SM BW EC JP. Analyzed the data: SM GA RG JP CF.
Contributed reagents/materials/analysis tools: GA RG JP. Wrote the
paper: SM GA RG JP CF.
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Perforin Induces Phospholipid Flip-Flop
PLoS ONE | www.plosone.org 13 September 2011 | Volume 6 | Issue 9 | e24286