Exploring signal events elicited by chemotactic

Transcription

Exploring signal events elicited by
chemotactic-receptor engagement in
migrating cells
PhD Thesis
CO-TUTELA FROM
Graduate School for Cellular and Biomedical Sciences
University of Bern
and
Graduate School in Molecular Medicine, section of Basic and
Applied Immunology Vita-Salute San Raffaele University,
Milan
Submitted by
Silvia Volpe
from Italy
UniSR matric. 002215
Ciclo di dottorato XXII
SSD MED/04 Patologia Generale
Anno accademico 2008/2009
Thesis advisor Prof. Dr. Marcus Thelen
Second Supervisors Prof. Dr. Anne-Catherine Andre
Prof. Dr. Ruggero Pardi
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Accepted by the Faculty of Medicine, the Faculty of Science and the
Vetsuisse Faculty of the University of Bern at the request of the
Graduate School for Cellular and Biomedical Sciences and by the
University Vita-Salute San Raffaele (Milano) at the request of the
Graduate School in Molecular Medicine, section of Basic and Applied
Immunology.
Bern,
Dean of the Faculty of Medicine
Bern,
Dean of the Faculty of Science
Bern,
Dean of the Vetsuisse Faculty Bern
Milano,
Supervisor Prof. Dr. Ruggero Pardi
CONSULTAZIONE TESI DI DOTTORATO DI RICERCA
La sottoscritta Silvia Volpe n° matr. 002215
nata a L’Aquila il 11/12/1980
autore della tesi di DOTTORATO dal titolo
Exploring signal events elicited by chemotactic-receptor engagement in migrating
cells
AUTORIZZA
la consultazione della tesi stessa, fatto divieto di riprodurre, in tutto
o in parte, quanto in essa contenuto.
Data
06/05/2010
Firma
Silvia Volpe
ACKNOWLEDGMENT
I would like to take this opportunity to express my gratitude to all those people who
contribute to this work.
The very sincere thanks go to my thesis advisor Prof. Dr. Marcus Thelen for giving me
the possibility to begin my scientific career by doing this Ph.D. thesis in the Signal
Transduction Laboratory at the Institute for research in Biomedicine. Was an honor to be
part of the large Thelen family together with Tiziana, Elisabetta, Sylvia and Ulrike.
Being in a sperimental cotutela setting of Ph.D. thesis between the Graduate School for
Cellular and Biomedical Sciences at the University of Bern and the Graduate School in
Molecular Medicine, section of Basic and Applied Immunology Vita-Salute San Raffaele
University (Milan), I am grateful to Prof. Dr. Anne-Catherine Andres, Prof. Dr. Erwin
Sigel and Prof. Ruggero Pardi who supervised this work. I wish to extend my gratitude to
Dr. Marlene Wolf for the effort made in organizing the cotutela.
None of this would have been possible without the strong support of my family.
4
TABLE OF CONTENTS
Table of Contents
Table of Contents
1
Abbreviations
2
Abstract
3
Introduction
4
Chemotaxis: cell’s sense of chemoattractant gradient
4
Molecular aspects
8
GPCRs: structure and mechanism of activation
8
GPCR’s transduction signal and trafficking in cell migration
11
PI(3)K
15
PI(3)K structure and classification
15
PI(3)Kγ in cell migration
18
Paper
(14 pages)
Additional Result
37
Fluorescent chemokine-dependent receptor internalization
37
Dispesable role of PI(3)K activation in cell migration
43
Discussion and Outlook
46
References
50
Curriculum Vitae and Pubblications
70
Declaration of Originality
74
1
ABBREVIATIONS
Abbreviations
7TMD
sevent transmembrane domain
DAG
diacylglycerol
ERK
extracellular-signal-regulated kinase
ERM
ezrin-radixin-moesin
fMLP
f-Met-Leu-Phe
GAP
GTPase-activating protein
GEF
guanine nucleotide exchange factor
GM-1
ganglioside GM1
GPCR
G protein-coupled receptor
GRK
G protein-coupled receptor kinase
HWT
17-hydroxy-wortmannin
ICAM
intercellular adhesion molecule 1/3
IP3
inositol 1,4,5-trisphosphate
JNK
c-Jun N-terminal kinase
LPA
lysophosphatidic acid
MAPK
mitogen-activated protein kinase
MIP
macrophage inflammatory protein
MTOC
microtubule-organizing centre
PAK1
p21-activated kinase 1
PLC
phosphoinosidite phospholipase C
PIP2
phosphatidylinositol bisphosphate
PIP3
phosphatidylinositol trisphosphate
PI(3)K
phosphatidylinositide 3-kinase
PKA
protein kinase A
PKB
protein kinase B
PKC
protein kinase C
ROCK
Rho-associated coiled-coil kinase
SHIP
Src homology2-containing inositol phosphatase]
TCR
T-cell receptor
THP-1
human acute monocytic leukemia cell line.
2
ABSTRACT
Abstract
Leukocyte migration is characterized by morphological changes which manifest in a
rapid cell polarization downstream of chemotactic receptor activation.
Controversial views of the signaling mechanisms include the localization and distribution
of chemotactic receptors within the plasma membrane, the importance of receptor
internalization for cell migration and the role of PI(3)K activity in cell polarization.
The study describes a novel monocytic cell system to investigate the cell migration
process. THP-1 cells stably transfected with the α2A-adrenergic receptor (α2AAR) show
comparable signal transduction in response to the agonist UK 14'304 as when stimulated
with CCL2/MCP-1 acting on the endogenous CCR2. Time-lapse video microscopy using
fluorescent protein tagged variants of the receptor reveals a uniform receptor distribution
in resting and polarized cells. In this experimental setting receptor internalization appears
to be dispensable for fully motile cells. By taking advantage of the CFP/YFP-based
intramolecular FRET reporter in the α2AAR (α2AAR-YFP/CFP) ligand-induced receptor
activation can be observed over the entire plasma membrane. However, PI(3)K activation
appears to occur only at the leading edge.
Freshly isolated monocytes, in contrast to neutrophils, which are known to maintain their
polarization axis, can flip the polar axis. Moving the source of chemoattractant from one
end to the other of a polarized cell reveals several differences between monocytes and
neutrophils.THP-1 cells can similarly revert their polarization axis. Interestingly, PI(3)K
activity, measured with a PIP3 specific fluorescent probe, relocates to the newly formed
leading edge. Nevertheless, reversion of the polarization axis is apparently independent of
PI(3)K activity as it occurs in the presence of wortmannin, which abrogates PIP3
production.
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INTRODUCTION
Chemotaxis: cell’s sense of chemoattractant gradient.
Throughout evolution, both prokaryotic and eukaryotic cells have developed a variety of
biochemical mechanisms to define the direction and proximity of extracellular stimuli.
Such mechanisms are essential for cells to reply properly to the environmental cues that
determine cell migration, proliferation, and differentiation.
Chemotaxis is the cellular response to chemical attractants that directs cell movement, a
process that plays a central role in many physiological situations, such as host immune
responses, angiogenesis, wound healing, embryogenesis, and neuronal patterning, among
others. In addition, aberrant cell migration takes part in pathological states, including
chronic inflammation and tumor metastasis.
To orientate its movement, theoretically a cell might use the spatial aspect of the
chemoattractant gradient or the transient intracellular signals that are generated locally
while the cell moves in the static gradient. Prokaryotes can only use the latter temporal
component as they are too small (1-2 μm) to process spatial information [1]. They
undergo a “random walk” with movement steps in all directions that are interrupted by
tumbles; when they move up the gradient the frequency of tumbling decreases and
movement in one direction is prolonged [2]. Eukaryotic cells are larger (10–20 μm in
diameter), which allows them to process both spatial and temporal information. They can
measure the difference in chemoattractant concentration between the ends of the cell, and
then move up this gradient. [3].
Eukaryotic cells can respond to differences in chemoattractant concentration as small as
2–10% between the front and the back. The ability to sense and respond to shallow
gradients of extracellular signals is remarkably similar in amoebae such as Dictyostelium
discoideum and mammalian neutrophils [4]. Both cell types use amoeboid cell migration
mechanisms which allows for greater speed due to relatively weak adhesive interactions
in contrast to mesenchymal or collective cell migration employed by tissue and cancer
cells [5].
The capability to sense and integrate signal input from the extracellular environment is a
crucial requirement for highly motile amoeboid migration. It was proposed that cells
4
INTRODUCTION
amplify the shallow external gradient of the attractant by converting it to a steeper
gradient of internal signals, resulting cell polarization with a protruding front that points
up toward the external gradient. However protrusions are periodically induced in a nearly
random direction, presumably by an internal oscillator. The directional cues stabilizes the
sefl-organizing cones pointing toward the leading edge [6]. This polarity may in turn
produce asymmetric attractant sensitivity that is greater at the cell’s protruding edge than
at the trailing one. Consequently a neutrophil confronted with a 180º reversal in the
attractant gradient does not transform a trailing edge into a leading one, instead it follows
its highly sensitive ‘nose’ and executes a U-turn [7,8].
While this phenomenon applies to a large fraction (~80%) of migrating neutrophils a
small part of the cells can completely reverse their polarity [7,9,10].This last
morphological phenotype is more evident by disrupting the cytoskeleton organization,
using specific inhibitors (Y-27632 the pharmacological inhibitor of p160-ROCK) for
actin-myosin complexes formation, rendering the cell’s posterior as sensitive to attractant
as the front; in this case reversing the gradient causes the cells to simply reverse their
polarity forming a new pseudopod from the previous tail, rather than walk around in a
circle by maintaining their polar axis [9] (Fig 1.).
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INTRODUCTION
Fig 1. Models proposed for cell directional migration toward reversed
chemoattractant source. On the left the typical U-turn observed in motile
neutrophils, while the right drawing displays a rapid reversal in polarization axis
described in different leukocytes subtypes.
Leukocyte polarization
An efficient immune reaction requires leukocytes to be at the right place at the right time.
Nearly all steps, from maturation to activation and effector function, depend upon
leukocyte migration and positioning in lymphoid and non-lymphoid tissue. After leaving
the bone marrow leukocytes reach lymphoid organs or peripheral tissues, by following a
chemoattactant cue, move toward their targets and execute effector functions. During this
route polarized leukocytes develop a small leading edge, consisting of short-lived
pseudopods, followed by the cell body that contains the nucleus, and a posterior nearcylindrical tail of 2 to 10 μm in length, termed the uropod [11].
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INTRODUCTION
Four steps mediate the amoeboid migration cycle: the leading edge protrudes one or
several pseudopds by actin flow, the membrane protrusions and thus here-contained
surface receptors interact with a subsrate, actomyosin-mediated contraction of the cell
body occurs in the mid-region, and so the rear of the cell is pulled forward. These steps
occur in a cyclic manner, generating a net forward movement [5] (Fig 2.).
Fig 2. Morphology and surface receptors of amoeboid polarized leukocytes. (A)
Round morphology of immobile or freely floating leukocyte; (B) Amoeboid shape after
polarization during random migration and chemotaxis.
After polarization, the leading edge contains rapidly forming and rebuilding networks of
filamentous actin that include abundant membrane ruffles indicative of dynamic probing
of the environment.
The leading edge is particularly sensitive to receptor engagement, including that by Fc
receptor (FcRs), T cell antigen receptors (TCRs), chemokine receptors [12] and β2
integrins. These receptors initiate contact with other cells, induce signal transduction [13]
and mediate phagocytosis after binding of opsonised bacteria [14]. However it was
demonstrated in neutrophil-like cells PBL-985 and in Dictyostelium cells that even in
polarized cells most chemoattractant receptors are homogenously expressed within the
7
INTRODUCTION
entire plasma membrane [15,16], in contrast to observations made with lymphocytes [17].
By contrast other receptors, such as β2 integrins, show discrete relocation toward the tips
of ruffles [18].
The mid-region of amoeboid cells contains the nucleus and a relatively immobile cell
region that maintains the front–rear axis. The trailing edge contains highly glycosylated
surface receptors CD43 and CD44, adhesion receptors including intercellular adhesion
molecule (ICAM)-1, ICAM-3, β1 integrins and ERM adaptor proteins, as well as GM-1type cholesterol-rich microdomains [19]. The uropod mediates cell–matrix and cell–cell
interactions during migration and has a putative anchoring function [11,20]. In addition to
regulating cell adhesion through integrins a motile cell must generate sufficient
contractile force to retract its uropod, a process mediated by the motor protein myosin II
that is highly enriched in this area [11,21].
Molecular aspects
GPCRs: structure and mechanism of activation
Polarization and migration of leukocytes are induced by to chemotactic gradients from
various compound classes, including chemokines and cytokines, lipid mediators,
bacterial factors and ECM degradation products including fragments of collagen,
fibronectin and elastin [22–28]. Most chemoattractants transmit signals through
heterotrimeric G-protein-coupled receptors (GPCRs). The GPCRs represent the largest
family of receptor-proteins in the human genome, encoding for more than 1000 different
molecules [29]. In addition to chemoattractants molecules different classes of messengers
can activate the receptors such as light and pain, hormones, neurotrasmitters, grow
factors, paracrine agents and odorants. Due to their typical architecture GPCRs are also
known as seven-transmembrane domain (7TMD) receptors. The general organization of
the seven-helical bundle was initially determined by biochemical studies of
Bacteriorhodopsin, an important landmark in the history of the 7TMD receptors [30–32].
The first mammalian GPCR crystal structure solved was rhodopsin, [33] followed by the
structures of the β-adrenergic receptor [34–36] and the A2A adenosine receptor [37]. All
GPCRs are predicted to share a common core topology characterized by seven
8
INTRODUCTION
membrane-spanning α-helices, an extracellular N-terminus, an intracellular C-terminus
and three interhelical loops on each side of the membrane (Fig 3.A).
Fig 3. Structure and ligand-dependent conformational changes of GPCRs. (A)
Serpentine diagram and top view for a prototypical member of the rhodopsin family of seven
transmembrane domain receptors. (B) Sequential binding and conformational stabilization
model for the molecular mechanisms of ligand action in GPCRs.
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INTRODUCTION
Based on sequence and functional similarities, GPCRs are commonly divided into five
families: rhodopsin (family A), secretin (family B), glutamate (family C), adhesion and
Frizzled/Taste2 [38,39]. The rhodopsin family is by far the largest and most diverse of
these families, and members are characterized by conserved sequence motifs, such as a
DRY motif in the second intracellular loop, and the NPXXY motif in the transmembrane
domain VII, implying shared structural features and activation mechanisms.
Despite the wide variety of agonists it has been proposed that a common molecular
activation mechanism for the 7TMD receptor family exists. According to the toggle
switch model, proposed by Schwartz et al., [40] binding of an extracellular ligand to a
GPCR induces a positional change of the transmembrane domains VI (TMVI) and
possibly VII (TMVII), stabilizing an active receptor conformation. Byond this rigid twostate equilibrium model of receptor activation more flexible models have been proposed,
postulating the existence of several receptor microstates stabilized by ligand with
modulating efficacy [41,42] (Fig 3.B).
The classical role of GPCRs is to couple the binding of the agonists to the activation of
heterotrimeric G proteins, leading to the modulation of downstream effector proteins. G
proteins are molecular switches that are activated by receptor-catalyzed GTP for GDP
exchange on the G protein alpha-subunit which triggers the dissociation of the Gα and
Gβγ subunits, the major players in intracellular signal transduction [43].
The quality of the downstream signal is in part determined by interaction with distinct
types of G proteins α subunits (Gαs Gαi Gαq Gαo Gα12 and Gα13). Gαs and Gαi stimulate
or inhibit the activity of adenylyl cyclase, respectively, regulating cytosolic levels of the
second messenger cAMP. Gαi and Gαq proteins also target phospholipase C that
generates diacylglycerol (DAG) and inositol triphosphate (IP3). Alternatively, several
GPCRs such as the type 2 beta adrenergic receptor (β2AR) can signal in a G proteinindependent manner via MAP kinases which are thought to be scaffolded by βarrestins.[44–46].
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INTRODUCTION
GPCR’s transdcuction signal and trafficking in cell migration
The GPCRs able to transduce a chemotactic signal include the fMLP (N-formyl-MetLeu-Phe) receptor, and the complement-derived anaphylotoxin receptors C5a and C3a
[47]; chemokine receptors [48]; the platelet activating factor receptor (PAFR) and the
leukotriene B4 receptor BLT1 [49]; sphingosine-1-phosphate receptors1-4 (SIP1-4)
[50,51] and lysophosphatidic acid (LPA) receprtors 1-3 [52,53].
Most GPCRs induce chemotaxis through Gi-proteins subtype. The Gi heterotrimeric
protein appears essential for chemotaxis: treatment of cells with pertussis toxin, which
catalyses the ADP-ribosylation of Gαi and thereby uncoples Gi from GPCR stinulation,
completely abolishes pseudopod formation and other leading-edge activities [54,55].
However, the GTP-loaded Gαi subunit is probably not involved in chemotaxis, but
instead is necessary to limit the action of Gβγ [56]. Indeed following receptor activation
the α subunit of the heterotrimeric G protein exchanges GDP with GTP. The classical
paradigm proposes that the GTP-loaded α-subunit then dissociates from the tightly linked
βγ complex and the receptor [57–59]. Nevertheless, others reports suggest that in
particular for Gi proteins activation occurs without dissociations of the subunits, rather is
accompanied by an intramolecular rearrangement [60,61]
Stimulation of the receptors with their specific chemotactic agonists activates a number
of characteristic signaling pathways, including PI(3)K (phosphatidylinositide-3-kinase),
phospholipase C (PLC), the MAPK cascade, Src kinases and the Rho family of small
GTPases [62].These signaling events contribute to different degree in the reorganization
of the actin cytoskeleton, polarization and directional cell migration.
Following receptor activation the Gβγ subunits stimulate two major signal transduction
enzymes, phospholipase C (PLC β2 and β3) and PI(3)Kγ. The phospholipase cleaves
phosphatidylinositol (4,5)-bisphosphate (PIP2) yielding two second messengers, inositol
(1,4,5) trisphosphate (IP3) and diacylglycerol (DAG). IP3 induces the release of Ca2+ from
intracellular stores, leading to a characteristic, transient rise of the cytosolic free calcium
concentration, and DAG activates several isoforms of protein kinase C (PKC) [63,64]. A
direct involvement of PLCβ activity in chemokine stimulated chemotaxis is still debated.
Targeted disruption of the genes encoding PLCβ isoforms, which completely abolishes
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INTRODUCTION
chemoattractant-stimulated calcium transients, had no effect on chemokine elicited
migration of neutrophils [65]. This implies a dispensable role for calcium signaling in cell
migration. On the other hand it was shown that PLCβ-dependent formation of DAG is
required for chemotaxis [66].
The PI(3)K competes with the phospholipase C for the substrate PIP2 generating PIP3.
The latter is a short lived second messenger which is selectively recognized by plecktrin
homology domains of various effector molecules, notably protein kinase B (PKB also
known as Akt) [67,68]. PKB is implicated in inducing actin polymerization and
pseudopod protrusion, such as the actin binding protein girdin [69,70]. Generally it is
assumed that PI(3)Kγ plays a critical role in cell migration [71,72]. Mice lacking PI(3)Kγ
show a strong impairment in chemokine-stimulated leukocyte recruitment [65,73].
Furthermore, it was reported that the generation of PIP3 by PI(3)Kγ regulates cell polarity
and governs the direction of migration [74,75]. However, the role of PIP3 in cell
polarization and directional migration is debated [76–78].
A central yet unanswered question in chemokine-stimulated cell migration is how
receptors are linked to actin polymerization. Current knowledge predicts that
RhoGTPases (Rac and Cdc42) act upstream of actin polymerization in cell migration.
Some studies suggest that PI(3)Kγ and RhoGTPases form a feed-back loop, which
amplifies the PIP3 formation above a critical threshold required to initiate directional
migration. Briefly, the βγ subunits activate PI(3)Kγ which then, via PIP3, activates
RhoGTPases (Cdc42 and/or Rac). The activated GTPases further stimulate PI(3)Kγ
[79,80]. The RhoGTPases activation requires functional GTP exchange factors (GEF).
About 60 different GEF have been identified so far [81]. Thus the specific RhoGTPase
activation in a signal transduction pathway depends on the GEF involved. Among the
different Rac-GEFs described member of the Vav, Sos, SWAP-70, Tiam, and P-Rex
families have been suggested to be regulated by PI(3)Ks [82–86]. By contrast PI(3)K
activity seems to be dispensable for the lymphocyte migration mediated by the Rac-GEF
DOCK2 [87] although DOCK2 contains a PIP3 binding domain [88]. In chemokine
receptor-mediated signaling it is unclear which GEF mediates Rac activation. A good
candidate could be P-Rex1, a GEF protein synergistically activated by βγ subunit and
PIP3 [86,89,90].
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INTRODUCTION
Ultimately Rac stimulates actin polymerization by binding, via intermediates, to
WAVE2, a member of the WASP family [91]. WAVE2 binds to monomeric G-Actin and
to the Arp2/3 complex, the latter catalyzing sideward branching of actin filaments
As a main downstream target/effector of PI(3)K the active RhoGTPase Rac plays an
indispensable role in leukocyte migration [92], whereas Cdc42 has been proposed to be
involved in consolidation of the leading edge, as a sensor of the chemoattractant gradient
[93,94]. Indeed several reports show that without Cdc42, leukocytes exhibit a random
walk, rather than directed migration, when placed in a chemotactic gradient [95]. [96–99].
Curiously, a target of Cdc42, PAK1, acts also upstream of it suggesting a feed-back loop
mechanism. Activated directly by Gβγ , PAK1 binds α-Pix, a GEF specific for Cdc42, to
form a complex that recruits and activates Cdc42 [94]. This signaling complex is
responsible for F-actin nucleation via activated Arp2/3 and exclusion of PI3phosphatases, the negative regulators of PIP3, from the leading edge of the cell [94,100].
Thus, promigratory signals received at the leading edge induce the local activation of
effector proteins and actin network protrusion, pushing the plasma membrane outward.
(Fig 4.).
Fig 4. Chemoattractant receptor signaling in migrating cells. Scheme for the establishment of the polar
axis: remodeling of actin cytosckeleton and presumtive gradient sensing mechanism downstream pathwayinduced signal.
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INTRODUCTION
Dissociation of G protein facilitates the phosphorylation of serine and threonine residues
at the C-terminus and possibly other intracellular domains of activated chemokine
receptors by GRKs, as well as by the second messanger protein kinases (PKC, PKA).
These processes lead to the desensitization of the receptor, as G protein coupling can no
longer occur. The two types of protein kinases differently contribute to receptor
phosphorylation, as shown for CCR5 [101]; while GRKs mediate a slow and persistent
receptor phosphorylation PKC-mediated phosphorylation is rapid and transient. It is
conceivable that the highly dynamic rearrangements of the cytoskeleton at the leading
edge, with continuous extensions and retractions of multiple protrusions are the result of
the more rapid PKC-dependent receptor phosphorylation and dephosphorylation in motile
cells [6].
Receptor phosphorylation enhances the affinity of the agonist occupied-receptor for
interaction with β-arrestins, which mediate the association of the receptor complex with
clathrin. Driven by the GTPase dynamin, the newly formed clathrin-coated pit pinches
off the membrane. This process represents the main internalization route of chemokine
receptors [102].
Alternatively, endocytosis through lipid rafts and caveolae has been discussed for a few
receptors such as CCR5 [103] and CXCR4 [104] and may take place in certain cell types.
Both process lead to receptor down-regulation at the cell membrane.
While GRKs can mediate β-arrestin-dependent receptor downregulation [105–107], the
role of the second-messanger dependent protein kinase phosphorylation in β-arrestin
recruitment is less clear. In addition to their function in endocytosis, arrestins have been
implicated in crosstalk with other signaling pathways through the interation with
components of the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal
kinase (JNK) MAPK cascades [108–110]. Furthermore β-arrestins may also contribute to
the spatial control over actin assembly proteins. There is evidence for a role of β-arrestins
as scaffold that regulate cofillin activity in PAR-2-mediated chemotaxis. [111].
Once internalized the chemokine receptors are targeted to recycling or degradation
pathways. Sorting of the receptor to distinct endocytic pathways is a tightly regulated
process with a stong impact on the cell’s responsiveness to further stimulation [112].
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INTRODUCTION
After short-term incubation and subsequent removal of the agonist, some internalized
chemokine receptors can recycle back to the membrane. This type of re-sensitization has
been shown for CCR5 [113], CCR7 [114] and CXCR2 [115]. At prolonged chemokine
exposure however, delivery to the late endosomes and lysosomes typically leads to downregulation of cell surface receptor [115], and long-term desensitization of the cell to
extracellular chemokine.
The requirement of receptor internalization for chemotaxis is still controversial. Several
reports indicate that internalization of chemokine receptors is necessary for chemotaxis
[107,116,117] while others provide evidence that internalization is not required [54], or
even can enhance chemokine-mediated cell migration [118].
PI(3)K.
PI(3)K and PIP3 formation is an hallmark of Gi-GPCR, however the role of PIP3 in cell
migration is far from being clear. Currently PI(3)K is the first detectable asymmetrical
event, as visualized by translocation of GFP-tagged probes for the lipid products of
PI(3)K activity to the cell membrane facing the chemoattractant source [119].
Considering a uniform distribution of chemokine receptors at the plasma membrane of
motile cells, the mechanisms of preferential receptor sensitivity at the leading edge may
include local signal amplification mechanisms [120] and the exclusion of counterregulatory proteins. With this strategy chemoattractant-responsive cells are able to
translate a shallow extracellular chemical gradient into a steep intracellular gradient of
signaling molecules.
PI(3)K structure and classification
PI(3)Ks consitute a family of lipid kinases that catalyze the phosphorylation of the 3-OH
position of
the inositol head groups of phosphoinositide (PI) lipids, namely
Phosphatidylinositol(PtdIns),
Phosphatidylinositol(4)phosphate
(PtdIns(4)P),
Phosphatidylinositol(5)phosphate (PtdIns(5)P) and Phosphatidylinositol(4,5)bisphosphate
(PtdIns(4,5)P2) resulting in the formation of PtdIns(3)P, PtdIns(3,4)P2, PtdIns(3,5)P2 and
PtdIns(3,4,5)P3 respectively, collettively termed 3’-phosphoinositide lipids [121,122]
(Fig.5.A).
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INTRODUCTION
Fig 5. Key steps of 3-phosphoinositides synthesis and graphical view of
PI(3)K domains. Class IA regulatory subunits have several modular domains
that can regulate function of the heterodimer. Each isoform has two SH2 domains
selective for binding pTyr-X-X-Met sequences, an interaction that appears
critical for enzyme activation. The Rac-binding domain in p85α and p85β is
homologous to RacGAPs for Rho family small G proteins but lacks GAP
activity. The SH3 and proline-rich motifs can also participate in intramolecular
and intermolecular interactions.
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INTRODUCTION
PtdIns(3)P is costitutively present in eukaryotic cells, its levels are largely unaltered upon
cell stimulation and it is thought to be involved in the regulation of membrane trafficking
[121]. In contrast PI(3,4)P2 and PI(3,4,5)P3 are generally absent from resting cells, but
their membrane concentration rises markedly upon stimulation via a variety of receptors.
PI(3)Ks are evolutionary conserved from yeast eukaryotes and are divided into three
main classes I, II and III PI(3)Ks, according to their molecular structure, cellular
regulation and sustrate specificities [123] (Fig 5.B). The class I PI(3)Ks can
phosphorylate PI, PI(4)P and PI(4,5)P2.
The prototypical class IA PI(3)Ks are heterodimers consisting of a catalytic subunit of
110-kDa and a tightly associated regulatory/adaptor subunit of 85-kDa that link them to
different upstream signaling events. The existence of multiple isoforms of both the
regulatory (e.g. p85α/β, p55γ) and catalytic (e.g. p110 α/β/δ) components indicates the
possibility of considerable variation of the hetrodimers between tissues as well as for
coupling to different receptors. The class I PI(3)Ks interact with the active GTP-bound
Ras [124]. Besides PI3Kδ, which is mainly expressed in leukocytes the expression of the
class IA isoforms is ubiquitous [121,125].
The class IA PI(3)Ks are activated by tyrosine-kinase associated receptors, including
antigen, co-stimulatory and cytokine receptors. Briefly the tyrosine kinase phosphorylates
membrane proteins in specific YXXM motifs. The phosphotyrosine sequence provides
the docking sites for the Src-homology2 (SH2) domains of the regulatory subunits of
PI(3)K, brings the p110 catalytic subunit to the membrane, where it catalyses the
conversion op PIP2 in PIP3 . The direct interaction between p110and activated Ras
represent an alternative mechanism of PI(3)K activation [126,127]. P85α and p85β also
contain proline-rich regions and SH3 domanis that can facilitate additional proteinprotein interactions, in addition and p85 has been reported to bind Cdc42 and Rac [128–
130]. Specific isoforms of class IA PI3Ks have been implicated in chemotaxis. For
example p110α and p110β have been implicated in macrophages chemotaxis to colonystimulated factor 1 [131] and p110δ deficiency strongly affected CXCL13/BCA-1–
mediated B cell homing and homeostasis [132,133]. The ability of p110δ to function
downstream of chemokine receptors in lymphocyte chemotactic responses is consistent
17
INTRODUCTION
with the finding that dominant-negative p85 reduces chemotaxis to CXCL12/SDF1[134,135]. Furthermore a critical requirement of PI3K δ in PIP3 synthesis during
neutrophil migration has been reported showing a unique role in directional movement
[136].
The 110-kDa catalytic subunit of PI(3)Kγ, the only class IB PI(3)K isoform described so
far, associates with one of two regulatory subunits, p101 and p84 that are distinct form
any of the p85 subunits [137]; [138]. Typically PI(3)Kγ (class IB) activation is mediated
by the βγ subunits of heterotrimeric G proteins and signal downstream the Gi subtype of
GPCRs. In addition PI(3)Kγ binds and becomes activated by GTP-bound Ras.
The class II and class III PI(3)K are less relevant in sell migration, despite some data
suggesting that class II PI(3)Ks may be involved in migration of some cell lines
[139,140].
PI(3)Kγ in cell migration
The first evidence for the involvement of PI(3)K in chemokine-stimulated cell migration
was the demonstration that chemotaxis and polarization of T-cells induced by CCL5/
RANTES could be inhibited by PI(3)K inhibitors such as wortmannin and LY294002
[141] in contrast to the fMLP-stimulated neutrophil migration [77]. Subsequent studies by
several groups have shown that other CC chemokines (e.g. CCL20/MIP-3α, CCL2) as
well as CXC chemokines (e.g. CXCL8/IL-8 and CXCL12) stimulate wortmanninsensitive chemotaxis of eosinophils, THP-1 cells, as well as neutrophils and T
lymphocytes [142–145]. PI(3)K signals at the plasma membrane are interpreted by PIP3binding proteins that traslocate and/or become activated in response to PIP3 binding.
Translocation of PH domain-containing proteins to the inner face of the plasma
membrane is an early event that marks the directional response of migrating cells, as in
Dictyostelium discoideum [68] and neutrophil-like HL-60 cells [146].
From these studies it was concluded that the production and degradation of 3’phosphoinositide lipids is crucial in maintaining asymmetrical signaling gradients. The
asymmetric distribution of this lipid seems to be maintained through spatially and
temporally controlled positive feedback loops and negative regulation. The assumption
18
INTRODUCTION
was supported by experimental evidence that extracellular addiction of PIP3 analogs were
shown to be sufficient to cause polarization and subsequent migration in neutrophils and
HL-60 cells [147,148].
Furthermore evidence indicated that polarized neutrophil morphology induced by
exogenous PIP3 is blocked by wortmannin and LY294002 [80]. A likely mechanisms for
this positive feedback involves polymerized actin [75,149] and Rho GTPases (which can
act both up and downstream of PI(3)K [150]. Inactivation of the Rho-GTPases Rac, Rho
and/or Cdc42 with different Clostridium toxins and dominant-negative mutants, both
result in the inhibition of membrane traslocation of GFP-tagged probes for the lipid
products of PI(3)Ks. [93].
The second leg of the positive feedback loop, the generation of more PIP3 downstream of
Rac activation, is less well defined and controversial. There are reports showing that
receptor stimulation enhances subsequent Rac-induced PIP3 production in PC12 cells
[151]; while others reports found no significant Rac generated PIP3 [152]. In addition to a
positive feedback loop on PI(3)K activity, asymmetric PIP3 accumulation is maintained
by actively excluding it from the uropod through regulation by phosphoinositide
phosphatases.
The phosphoinositide phosphatases PTEN and SHIP catalyse the conversion of PIP3 into
PI(4,5)P2 and PI(3,4)P2, respectively, thus acting as negative regulators of PI3K activity
[153–155]. During polarization the lipid phosphatase PTEN is proposed to be
downstream of activated RhoA and Cdc42. Cdc42 is activated by α-PIX in a complex
containing Gβγ subunits the protein kinase PAK [94]. Active Cdc42 is required to localize
RhoA; and active RhoA at the back of the cells activates Rock, which then
phosphorylates and activates PTEN [156,157]. After chemotactic stimulation, the tumor
suppressor PTEN rapidly localizes to the cell membrane [158]. In Dictyostelium it has
been shown that the complementary localization of PI(3)Ks and PTEN amplifies the
distribution of PIP3 with respect to the gradient [159]. Indeed PTEN does not localize to
the leading edge, where PI(3)Ks are recruited, but instead to the sides and the rear of
polarized cells. A similar distribution pattern of PTEN has also been noted in mammalian
cells [156,160] (Fig 6.).
19
INTRODUCTION
Fig 6. Spatial restriction for the phosphoinositide metabolizing enzymes in polarized cells.
Unstimulated resting cells have a spherical shape with a uniform distribution of phosphoinositide lipids.
When cells are placed in a gradient of chemoattractant the delicate balance between the activation of
PI(3)K and PTEN coordinates the local enrichment of PIP3 at the leading edge. Members of the
RhoGTPase family of protein are involved in this process.
The importance of PTEN as a regulator of chemotaxis of Dictyostelium is unequivocal; in
contrast the evidence that neutrophils exposed to fMLP require PTEN as a regulator of
the chemotactic response is far less convincing [161,162]. Selective deletion of PTEN in
myeloid cells has demonstrated only a minor inhibitory function for PTEN during
migration to fMLP [162,163].
These results prompted to investigate the other regulator of PIP3 degradation, the inositol
5-phosphatase SHIP. SHIPs role in cell migration was demonstrated by overexpression of
constutively active SHIP resulting in the inhibition of chemotaxis of Jurkat T-cells [164].
Nishio et al. clearly demonstrated that loss of the gene leds to comparable defects
observed when Dictyostelium PTEN was knocked out. The ship1-/- neutrophils had a flat,
unpolarized phenotype due to an increase in the number of membrane extensions labelled
by AKTPH-GFP [163,165].
20
INTRODUCTION
Thus increased mislocalized levels of phosphoinositides have severe effects suggesting
that excess amounts of PIP3 can disrupt polarity and directed migration by promoting the
extension of lateral pseudopodia (Fig 7.).
Fig 7. The effect of PtdIns(3,4,5)P3 on chemotaxis. The image emphasizes the marginal effect of
PI(3)Kγ downregulation in contrast to SHIP1 deletion on efficient migration and polarization.
21
INTRODUCTION
The idea that chemotacting cells orientate by means of PIP3 gradients in their plasma
membrane is deduced from genetic and ablation inhibitor studies in Dictyostelium [119],
neutrophils [146]and fibroblast [166].
However other reports clearly show a dispensable role for PIP3 gradient and PI(3)Ks
activity in directional cell migration [76,167–170]. In particular Ferguson et al. showed
that PI(3)Kγ-/- neutrophils have a strong adhesion defect inferring a role for PI(3)Ks in
cytoskeletal organization required for cell polarization and movement instead of in
direction sensing [170].
In conclusion, a signaling pathway from the receptor to the cytoskeleton, which is able to
guide cells independently of polarized PIP3 must exist [171].
22
Polarization of Migrating Monocytic Cells Is Independent
of PI 3-Kinase Activity
Silvia Volpe1, Sylvia Thelen1, Thomas Pertel2, Martin J. Lohse3, Marcus Thelen1*
1 Institute for Research in Biomedicine, Bellinzona, Switzerland, 2 Department of Microbiology and Molecular Medicine, University of Geneva, Geneva, Switzerland,
3 Rudolf Virchow Center and Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany
Abstract
Background: Migration of mammalian cells is a complex cell type and environment specific process. Migrating
hematopoietic cells assume a rapid amoeboid like movement when exposed to gradients of chemoattractants. The
underlying signaling mechanisms remain controversial with respect to localization and distribution of chemotactic
receptors within the plasma membrane and the role of PI 3-kinase activity in cell polarization.
Methodology/Principal Findings: We present a novel model for the investigation of human leukocyte migration. Monocytic
THP-1 cells transfected with the a2A-adrenoceptor (a2AAR) display comparable signal transduction responses, such as
calcium mobilization, MAP-kinase activation and chemotaxis, to the noradrenaline homlogue UK 14’304 as when stimulated
with CCL2, which binds to the endogenous chemokine receptor CCR2. Time-lapse video microcopy reveals that chemotactic
receptors remain evenly distributed over the plasma membrane and that their internalization is not required for migration.
Measurements of intramolecular fluorescence resonance energy transfer (FRET) of a2AAR-YFP/CFP suggest a uniform
activation of the receptors over the entire plasma membrane. Nevertheless, PI 3-kinse activation is confined to the leading
edge. When reverting the gradient of chemoattractant by moving the dispensing micropipette, polarized monocytes – in
contrast to neutrophils – rapidly flip their polarization axis by developing a new leading edge at the previous posterior side.
Flipping of the polarization axis is accompanied by re-localization of PI-3-kinase activity to the new leading edge. However,
reversal of the polarization axis occurs in the absence of PI 3-kinase activation.
Conclusions/Significance: Accumulation and internalization of chemotactic receptors at the leading edge is dispensable for
cell migration. Furthermore, uniformly distributed receptors allow the cells to rapidly reorient and adapt to changes in the
attractant cue. Polarized monocytes, which display typical amoeboid like motility, can rapidly develop a new leading edge
facing the highest chemoattractant concentration at any site of the plasma membrane, including the uropod. The process
appears to be independent of PI 3-kinase activity.
Citation: Volpe S, Thelen S, Pertel T, Lohse MJ, Thelen M (2010) Polarization of Migrating Monocytic Cells Is Independent of PI 3-Kinase Activity. PLoS ONE 5(4):
e10159. doi:10.1371/journal.pone.0010159
Editor: Jean Kanellopoulos, University Paris Sud, France
Received December 28, 2009; Accepted March 23, 2010; Published April 15, 2010
Copyright: ß 2010 Volpe 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: The study was co-financed by the European Community through a Marie Curie Action (INTEGRAMM) and the 6th Framework Program, Network of
Excellence Grant (MAIN) LSHG-CT-2003-502935 and supported by the Swiss National Science Foundation program R’Equip 316000-117395. The Institute for
Research in Biomedicine is supported by the Helmut Horten Foundation. 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: [email protected]
A central downstream regulatory element in receptor-mediated
cell migration is the activation of phosphatidylinositide 3-kinase (PI
3-kinase) [3–5]. The kinase contributes, but is not mandatory to
convey extracellular gradients to the intracellular organization of
the responses. Furthermore, redundant pathways in chemotaxis
exist for which PI 3-kinase activity is dispensable [6–10].
Model systems for the analysis of the signal transduction events
in cells undergoing chemotaxis have provided much insight to our
current knowledge on leukocyte migration. Monitoring the spatiotemporal activation of pathways has allowed refining different
signaling events to the leading and trailing edge, respectively [11].
Currently few suitable systems are available that can easily be
interrogated for the specific function of signal transduction
components in amoeboid-like migration. Many studies were
performed in Dictyostelium where protein expression levels can
easily be altered [12,13]. The chemotaxis of primary neutrophils
Introduction
Cell migration is an essential process for the functional
positioning of cells in higher organisms. In most cases cells follow
a guidance cue formed by chemoattractants that bind to specific
cell surface receptors to promote chemotaxis. While the migration
of tissue cells is slow and characterized by strong adhesions,
leukocytes have adapted a highly motile amoeboid mechanism of
migration which is in many aspects reminiscent of the amoeba
Dictyostelium discoideum [1]. Leukocyte trafficking is a central
regulatory mechanism for immune homeostasis and immune
responses [2]. Upon injury neutrophils and monocytes are among
the first cells leaving the blood stream to approach the site of
lesion. The cells are attracted by a variety of stimuli, such as
chemokines, bioactive lipids, anaphylatoxins and bacterial derived
peptides, which all bind to Gi-protein-coupled receptors (GiPCR).
PLoS ONE | www.plosone.org
1
April 2010 | Volume 5 | Issue 4 | e10159
THP-1 Cell Migration
medium (Invitrogen). After 24 h, the transfection medium was
replaced with fresh complete RPMI medium. Forty-eight hours
posttransfection, the LVs containing medium was collected and
filtered through a 0.45 mm filter (Millipore). THP-1 cells cultured
in 6-well plates were treated with 2 ml of VLP. Positive clones
were selected by FACS.
and monocytes can be monitored by time laps video-microscopy,
but manipulation of the expression levels of proteins in these cells
is not straightforward. Most commonly neutrophil-like HL-60 cells
are used to study molecular events during leukocyte migration
[3,14–17]. However, the cells must be differentiated to assume a
functional neutrophil-like phenotype and to respond to typical
agonists, such as f-Met-Leu-Phe. As a rule, however, differentiation leads to heterogeneous cell populations.
In this study we introduce the monocytic THP-1 cells to study
chemotaxis. We show that cells stably transfected with the a2Aadrenoceptor (a2AAR) migrate towards the a2AAR agonist UK
14’304 (brimonidine). The efficacy of the chemotactic response is
comparable to the stimulation with the chemokine CCL2 which
binds to the endogenous CCR2. Measurements of PIP3 formation
indicate that the cells promote the local activation of PI 3-kinase at
the leading edge in response to an extracellular agonist gradient.
In contrast to neutrophils and Dictyostelium, where the cells
predominantly maintain their polarization axis and perform a
U-turn in response to inversion of the chemotactic gradient, we
observed that monocytes and THP-1 cells rapidly switch their
polarization axis. PI 3-kinase activity rapidly relocates to what was
before the uropod and after reversal of the gradient acts as leading
edge. However, PIP3 production appears dispensable for cell
polarization. Consistent with the capability of a rapid relocation of
the polarization axis, receptors remain evenly distributed over the
plasma membrane during cell migration.
FACS analysis of receptor expression
THP-1 cells (200 ml containing 105cells) were incubated with
200 nM CCL2 [20] or 1mM UK 14’304 at 37uC for the indicated
times. Incubations were terminated by the addition of ice cold PBS
and washing of the cells. The cells were stained for CCR2 with
5 mg/ml anti CCR2 (MAB150 R&D System) or 5 mg/ml anti HA
(12CA5, Roche) for 30 min on ice, washed and incubated with
10 mg/ml goat anti-mouse IgG-RPE (Southern Biotech). Isotype
control was performed with 5 mg/ml mouse IgG22b (Southern
Biotech).
Calcium and Chemotaxis
THP-1 cells (ATCC) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS, HyClone), 1% glutamax,
50 U/ml of penicillin and 50 mg/ml of streptomycin (Pen/Strep,
all Invitrogen).
Intracellular free calcium was measured as previously described
[25]. Chemotaxis assays were performed in triplicate in 48-well
Boyden chambers (NeuroProbe, Gaithersburg, MD), using 5mm
pore-sized polyvinylpyrrolidone-free polycarbonate membranes.
Chemotaxis medium (RPMI-1640 supplemented with 1% FBS,
1% glutamax, Pen/Strep) containing chemoattractants was added
to the lower wells. Cells (105 per well) in chemotaxis medium were
added to the upper wells and incubated for 40 min at 37uC in 5%
CO2 atmosphere. Cells were removed from the upper part of the
membrane with a rubber policeman. Cells attached to the lower
side of the membrane were fixed and stained. Migrated cells were
counted in five randomly selected fields of 100-fold magnification.
Where indicated, cells were pretreated at 37uC for 2 h with 2 mg/
ml Bordetella pertussis toxin (Sigma).
Transfection
Western blot analysis
Materials and Methods
Cells and cell culture
The cells were starved for 6 h in serum-free RPMI 1640
medium supplemented with 2% bovine serum albumin (BSA,
Sigma). When indicated pertussis toxin 2 mg/ml (Sigma) was
added to the medium. Cells were washed once in phosphate
buffered saline (PBS) and stimulated at 37uC for 2 min with UK
14’304 300 nM and CCL2 100 nM. Incubations were terminated
by the addition of ice-cold trichloro-acetic acid. Processing of cell
lysates and ERK phosphorylation was performed as described
[20].
Plasmids (pcDNA3) encoding for wild type a2AAR, a2AAR-YFP
and a2AAR-YFP/CFP were as previously described [18,19]. All
receptor sequences included an HA epitope at their N-terminus.
THP-1 cells were transfected by NucleofectionH (Amaxa, Lonza)
according to the manufacturer’s instructions. Transfected cells
were grown in selection medium containing 0.7 mg/ml G418
(Invitrogen) and subcloned for highest receptor expression by
FACS (FACS Aria, BD Biosciences).
Transduction
Microscopy
The PH-PKB sequence [20] was subcloned in frame with a
nonapeptide linker sequence (GSGGSGGSG) into pEGFP
(Invitrogen) where the GFP was replaced with the sequence of
the red fluorescent protein mCherry [21]. The PH-PKB-mCherry
sequence was amplified by PCR using primers to incorporate Xba1
and Sma1 sites at the 59 and 39 ends (forward: 59-AAAATCTAGAATGAACGACGTAGCCATTGTGAA; reverse 59-TGAATTCCCGGGTTACTTGTAGAGCTCGTCCATGC). The PCR
product was cloned into the pAIP-WPRE-IRES-Puro lentiviral
vector [22]. Vesicular stomatitis virus-G (VSV-G)-pseudotype
lentiviral vectors (LVs) were generated by co-transfecting pAIPPH-PKB-mCherry, the HIV-1 packaging plasmid psPAX2 and
the pMD2.G plasmid encoding the VSV-G envelope glycoprotein
into 293T cells as described [23,24]. Briefly, nearly confluent
293T cells in 6-well-plates were co-transfected with pALPS-PHPKB-mCherry, psPAX2, and pMD2.G at a ratio of 4:3:1 (3.3 mg
of total plasmid DNA) using 7.5 ml of polyethylenimine 1 mg/ml
stock (MW 25000, Polysciences) 250 mL in serum-free OptiMEM
Receptor internalization was determined on fixed permeabilized specimens as follows. THP-1 cells were plated on poly-Dlysine coated coverslips in medium. Following adherence the cells
were stimulated for 30 min at 37uC. Cells were washed with warm
PBS and then fixed with 4% paraformaldehyde on PBS for 20 min
at room temperature. Cells were washed again with PBS, the
reaction quenched with 100 mM glycine in PBS (20 min), and
permeabilized for 5 min with PBS containing 0.1% (w/v)
TritonX-100 (Sigma). Cells were washed with PBS containing
0.02% Tween 20 (w/v) (PBST) and incubated with primary rabbit
anti-CCR2 (Sigma) for one hour. Following washing with PBST
the cells were incubated with secondary anti-mouse- or anti-goatIgG conjugated with Alexa488 (Invitrogen), DAPI and phalloidinAlexa594 (Invitrogen). Coverslips were mounted as previously
described [20].
Laser scanning confocal microscopy was performed with a Leica
DI6000 microscope stand connected to a SP5 scan head equipped
2
with a temperature controlled chamber (Cube, LIS, Basel). Live
cell cultures were placed in a humidified and CO2-controlled
incubator which was mounted on the microscope stage (Brick,
LIS, Basel).
For time-lapse video microscopy cells (0.56106/ml) were
resuspended in D-PBS containing calcium and magnesium
(Invitrogen) supplemented with 1% FBS, Pen/Strep, 0.04 mM
sodium pyruvate, 1 mg/ml fatty acid free BSA (Sigma), 1 mg/ml
glucose (Fluka). Cells were plated on glass bottom petri-dishes
(MatTek cultureware) which were coated before with D-polylysine (5 mg/ml) and subsequently overlaid with a 1:80 diluted
MatrigelH (BD Biosciences) solution at 4uC for 30 min.
Chemoattractants were dispersed with a micropipette (Femtotip
II, Eppendorf) controlled by micromanipulator (Eppendorf) at a
constant backpressure of 15 hPa (Femtojet, Eppendorf).
For CFP/YFP FRET measurements cells were excited with a
UV laser (405 nm) at low power setting (,15%), and the emission
of CFP (FCFP = 465 nm-505 nm) and the FRET signal (YFP
emission, FYFP = 525 nm–600 nm) were measured contemporaneously. Ratio FRET (rFRET) was calculated as FRET/FCFP
(FYFP/FCFP) as described [26] using the Metamorph software
package (Visitron). Due to the low laser intensity used for
excitation fading of CFP and YFP was usually ,5% over 1 min.
Results
Non-chemokine pertussis toxin-sensitive Gi-protein coupled
receptors can mediate chemotaxis [27]. Therefore we reasoned
that the Gi-protein-coupled a2A-adrenergic receptor (a2AAR),
when expressed in the migration competent THP-1 cells, could
induce cell migration. The a2AAR belongs to the group of
adrenoceptors that transduce responses to catecholamines during
neurotransmission and is known to mediate hypotension, sedation
and analgesia [28], but it is not known to stimulate cell migration.
THP-1 cells were stably transfected with the a2AAR and tested in
chemotaxis assays using modified Boyden chambers. Figure 1
shows the migratory behavior of wild type (top right) and
transfected (top left) THP-1 cells in response to the chemokine
CCL2, an agonist of the endogenous CCR2, and to UK 14’304,
an agonist with high affinity and specificity for a2AAR [29]. In
THP-1 cells expressing a2AAR (top left) CCL2 and UK 14’304
induced chemotaxis with similar efficacy, albeit with different
Figure 1. Chemotaxis of THP-1 cells mediated by endogenous and transfected receptors. Either wild type (upper right) or transfected
cells (panel labeling denotes expressed receptor) were subjected to chemotaxis assays in modified Boyden chambers for 40 min. Chemotaxis was
stimulated with UK 14’304 (circles) or CCL2 (triangles) without (open symbols) or with pretreatment with pertussis toxin (closed symbols).
doi:10.1371/journal.pone.0010159.g001
3
intracellular loop, appears to be preserved, because the response
was abolished by pertussis toxin pretreatment.
To continuously monitor migration of THP-1 cells, time-lapse
video microscopy was established. Glass bottom culture dishes
coated with a monolayer of poly-Lys were incubated with a diluted
MatrigelH solution at 4uC for 30 min. Excess of MatrigelH was
removed and the dishes rinsed with PBS. Because MatrigelH does
not polymerize at low temperatures, the procedure leads to a
monolayer like coating of the dishes allowing observation of cell
migration at high resolution in a single optical plane. Among
different coatings tested, such as fibrinogen, fibronectin and
collagen, coating with MatrigelH appeared to provide the optimal
substratum to support THP-1 migration. The chemoattractants
CCL2 or UK 14’304 were released from a micropipette driven
by constant backpressure. Movie S1 shows typical migration
sequences of wild type and THP-1 cells expressing the double
tagged a2AAR-YFP/CFP in response to UK 14’304 and CCL2.
Both agents attracted the cells towards the tip of the dispensing
pipette and moving the pipette reoriented the migration of the
cells.
Analysis of tracks recorded by time-lapse video microscopy
from multiple cells stimulated with 10 nM CCL2 or 1 mM
UK14’304 indicated that migration towards the chemokine was
somewhat more efficient (Figure 2A and B), despite of similar
velocity of the movement (Figure 2C). The migratory index (MI),
i.e. the distance between the starting point and the end point
divided by the total migrated distance, was 0.70460.15 (SD) for
CCL2 and 0.61460.17 (SD) for UK 14’304. The mean velocity
of THP-1 cell migration on matrigel coated glass appears to be
slightly slower than that of mouse neutrophils migrating in EZTaxiscan chambers on fibrinogen coated glass, 0.08 mm/sec vs.
0.11 mm/sec, respectively [10]. Overall the velocity of migration
and the MI of THP-1 cells are only moderately less efficient
efficiencies. The differences in efficiency was unexpected because
both agonist possess comparable affinities for their respective
receptors (Kd ,2–3 nM CCL2/CCR2; Kd ,2 nM UK 14’304/
a2AAR) [29–31]. A maximum of the typical bell shape migratory
response for CCL2 was obtained between 1–3 nM, whereas the
UK 14’304 stimulated response peaked at 100 nM. Wild type
THP-1 cells did not respond to UK 14’304 (top right), indicating
that chemotaxis observed in the transfected cells was entirely
dependent on the newly introduced a2AAR. Pertussis toxin
pretreatment fully abrogated cell migration in response to CCL2
and UK 14’304, confirming that both receptors couple to Giproteins.
For the study of receptor activity and localization in polarized
migrating cells we took advantage of a2AAR variants which were
tagged with yellow fluorescent protein (YFP) at the C-terminus
(THP-1 a2AAR-YFP) or with yellow fluorescent protein (YFP) at
the third intracellular loop and CFP at the C-terminus (THP-1
a2AAR-YFP/CFP). Both constructs were previously characterized
for ligand binding and response properties [18,19,32]. After stably
transfecting the constructs in THP-1 cells, we tested if the tagged
receptors, which bear a considerable increase in molecular mass,
were competent in mediating chemotaxis. Figure 1 (bottom right)
shows that fusion of YFP to the C-terminus of a2AAR had no
major effect on UK 14’304 stimulated chemotaxis. Similarly, the
response to CCL2 was also not affected (not shown). Even more
critical for receptor function could be the incorporation of two
fluorescent proteins. However, THP-1 cells expressing a2AARYFP/CFP showed almost identical chemotactic responses to UK
14’304 and CCL2 as THP-1 a2AAR cells (Figure 1 left panels).
Thus, the inclusion of the tags does not appear to compromise
coupling of the receptor to downstream elements responsible for
mediating cell migration. Moreover signaling via Gia, which
requires the interaction of the G-protein with the second and third
Figure 2. Velocity of THP-1 cells migrating towards different agonists. THP-1 a2AAR-YFP/CFP cells were plated on glass bottom coverslips
coated with poly-D-lysine and MatrigelH. (A) CCL2 (10 nM) and (B) UK 14’304 (1 mM) were dispensed from a micropipette with constant backpressure.
Time-lapse videos for each conditions were recorded at 5 sec interval. Tracks were analyzed and plotted aligning their average directional vector with
the y-axis (distance in mm). (C) Mean velocity of tracks shown in (A) and (B) were calculated using Metamorph. Data from two independent
observations, n = 17 (A) and n = 20 (B).
4
The localization of chemotaxis-mediating receptors on the
plasma membrane of leukocytes is still contentious. While studies
on lymphocytes conclude that receptors are recruited to the
leading edge and accumulate at the immunological synapse
[43–48], others have shown that in amoebae and neutrophil like
cells (PBL-985, HL-60) the receptors remain evenly distributed
over the plasma membrane of polarized cells [49–52]. We used the
fluorescently tagged receptors to address the question whether the
GPCRs distribute uniformly at the plasma membrane in polarized
monocytic cells. THP-1 cells expressing a2AAR-YFP/CFP were
labeled with the Vybrant DiD cell-labeling solution (DiD) and then
subjected to time-lapse video microscopy. Figure 5A shows
representative confocal frames at time 0 and after 5 min from
movie S2 of cells migrating in response to UK 14’304 taken with
high optical resolution (63x) and a narrow pinhole at the confocal
unit. Over the total observation period, the (red) dye appeared
evenly associated with the plasma membrane and accumulated in
intracellular organelles. Similarly, the fluorescence of YFP seemed
uniformly distributed over the entire plasma membrane in the
confocal images. Ratiometric analysis of DiD/YFP fluorescence
intensity shows a homogeneous color marking over the entire
plasma membrane in resting and migrating cells. The observation
is consistent with the concentration of membrane marker and
receptors being constant and suggests that the ratio does not alter
when cells polarize and migrate. The quantitative analysis from
multiple cells of the DiD/YFP fluorescence intensity ratio of the
leading edge and the rear confirms the conclusion (Figure 5B).
Thus, the constant ratio over the entire membrane illustrates that
the receptors do not re-localize to the membrane of the leading
edge in THP-1 cells.
Despite the even distribution of chemoattractant receptors in
polarized cells in amoebae and leukocytes, local activation of PI
3-kinase occurs solely at the leading edge [4,53,54]. We tested
the activation of PI 3-kinase in THP-1 cells stably transformed
with a reporter construct specific for the kinase product
phosphatidylinositol (3,4,5) triphosphate (PIP3) [53]. The construct consisted of the PH-domain of protein kinase B (PKB)
fused to a short linker sequence and cloned in frame to the Nterminus of the red fluorescent protein mCherry (PH-PKBmCherry). THP-1 cells expressing the a2AAR-YFP/CFP and the
PH-PKB construct were analyzed by time-lapse video microscopy. Unstimulated cells revealed a moderate red fluorescence in
the cytoplasm and at the plasma membrane. When the cells
started to polarize and migrated towards the micropipette
dispensing UK 14’304 a pronounced accumulation of the
reporter construct at the leading edge was observed (Figure 6A
and movie S3). Given that the a2AAR-YFP/CFP does not alter
its distribution during migration (Figure 5B), the YFP fluorescence was used to normalize the PH-PKB-mCherry fluorescence
intensity per voxel. Figure 6A and movie S3 shows a marked
increase of the mCherry fluorescence intensity with respect to
the YFP signal of the receptor consistent with a pronounced PIP3
formation at the leading edge. The asymmetric distribution of
the PKB-PH domain further indicates that PI 3-kinase is almost
exclusively activated at the leading edge and that the PIP3
formation occurs concomitantly with the cell becoming polarized
(movie S3). When a2AAR-YFP/CFP and PH-PKB-mCherry
expressing THP-1 cells were stimulated with CCL2 a similar
strong activation of PI 3-kinase at the leading edge was observed
(not shown). Figure 6B reports the mean fluorescence intensity in
resting non-polarized cells and in UK 14’304-induced polarized
cells at the front and the rear. The analysis indicates an average
two-fold increase in PIP3 after 30-40 sec at the leading edge
compared to the rear (Figure 6B).
compared with parameters reported for primary mouse neutrophils migrating in EZ-Taxiscan chambers on fibrinogen coated
glass [10].
Next we investigated the ability of a2AAR transfected THP-1
cells to trigger changes in intracellular free calcium [Ca2+]i.
Figure 3A (lower left) shows a typical rise in [Ca2+]i upon
stimulation of wild type THP-1 cells with CCL2, but as expected
from the chemotaxis experiments no response was obtained with
UK 14’304 (upper left). In THP-1 a2AAR cells, CCL2 stimulated a
similar response, but in addition these cells also displayed a
marked rise in [Ca2+]i following stimulation with UK 14’304. Also
cells expressing the doubly tagged receptor THP-1 a2AAR-YFP/
CFP exhibited typical calcium elevations in response to CCL2 and
UK 14’304 (right panels). When cells were exposed sequentially to
either agonist, an almost complete desensitization to a second
bolus of the same agonist was observed, but the response to the
other stimulus remained normal. Thus, neither CCL2 nor UK
14’304 cross-desensitized each other indicating that the two
receptors function autonomously. Following treatment of the cells
with pertussis toxin, calcium elevations with either stimulus were
abolished (not shown).
Rapid phosphorylation of the MAPK kinases ERK1/2
following stimulation with chemokines is amply reported as a
measure of receptor-mediated cell activation. We used Western
blot analysis to detect dual phosphorylated ERK1/2 (dpERK) and
show that in THP-1 cells CCL2 induced a marked activation of
ERK1/2 (Figure 3B, lane 2). The addition of UK 14’304 to wild
type THP-1 had no effect, further confirming that the cells do not
express endogenous functional receptors for this agonist. However,
THP-1 a2AAR displayed a marked activation of ERK2 in
response to CCL2 and to UK 14’304 (Figure 3B lane 8 and 9).
Similarly, THP-1 a2AAR-YFP/CFP responded to both agonists
with a marked activation of ERK1/2 (right panel). In all instances
phosphorylation of ERK1/2 was prevented if the cells were
pretreated with pertussis toxin, confirming that the receptors
couple to heterotrimeric Gi-proteins. Taken together these
findings indicate that a2AAR and its tagged variants when
expressed in THP-1 cells induce similar responses as endogenous
chemokine receptors.
In general GPCR internalize following agonist stimulation via
clathrin-mediated endocytosis [33]. However, the necessity of
receptor internalization during cell migration remains controversial. Several reports indicate that internalization of chemokine
receptors is required for chemotaxis [34–37], while others provide
evidence that internalization is not required [27] or show that lack
of internalization can enhance chemokine-mediated cell migration
[38]. Stimulation of THP-1 cells with CCL2 resulted in a timedependent down regulation of CCR2 (Figure 4A). By contrast, the
a2AAR, either wild type or tagged with fluorescent proteins, was
not down regulated from the cell surface even after extended
treatment with UK 14’304. Receptor internalization was measured by FACS analysis using the mAb 12CA5 which detects the
HA epitope at the N-terminus of the a2AAR constructs. Similarly,
confocal microscopy revealed refractoriness of a2AAR-YFP/CFP
to become internalized following stimulation, whereas upon
activation with CCL2 CCR2 was readily detected on endosomal
structures (Figure 4B). Our observation that UK 14’304 does not
induce major internalization of a2AAR in the THP-1 cells mirrors
findings obtained with the non-hematopoietic HEK293 and COS1 cells transfected with a2AAR [39–42]. Together these data
indicate that a2AAR-YFP/CFP-mediated chemotaxis, calcium
mobilization and ERK2 phosphorylation, which are comparable
with the responses elicited by the endogenous CCR2, do not
depend on receptor internalization.
5
Figure 3. THP-1 cells-mediated signal transduction by endogenous and transfected receptors. (A) Intracellular calcium mobilization. Wild
type and transfected THP-1 cells were loaded with Fura-2 and stimulated with 300 nM UK 14’304 (U) or 100 nM CCL2 (C). Fluorescence signals were
normalized to maximum calcium influx elicited by the addition of ionomycin (I) as described in Methods. (B) Activation of ERK1/2 in THP-1 cells. Wild
type and transfected THP-1 cells non-treated or pretreated with pertussis toxin (PTX) were stimulated for 2 min with 100 nM CCL2 or 300 nM
UK14’304. Upper images are Western blots of dual phosphorylated ERK1 and 2, below total ERK2 (loading reference).
6
7
Figure 4. Receptor internalization. (A) FACS analysis. THP-1 cells were incubated with 200 nM CCL2 and CCR2 expression was measured after the
indicated times (upper left). Similarly, THP-1 cells stably transfected with a2AAR (upper right), a2AAR-YFP and a2AAR-YFP/CFP (lower panels) were
incubated with 1mM UK 14’304. Red lines represent isotype controls (IgG2b). (B) Confocal microscopy images of wild type THP-1 cells (upper panels)
and THP-1 transfected with a2AAR-YFP/CFP (lower panels). Cells were untreated (control) or stimulated with 200 nM CCL2 or 1mM UK14’304 (UK) for
30 min fixed and processed as described in Methods. Receptor expression in wild type (CCR2) was revealed with specific anti-CCR2 (left panel, green).
For localization of a2AAR-YFP/CFP the fluorescence of receptor associated YFP was measured (left panel, yellow). The middle panels depict F-actin
revealed with phalloidin-Alexa594. Right panels are merged images including nuclear staining (DAPI, blue).
reversal of the gradient reorient their leading edge to the source of
the gradient by performing U-turns [4,54,57]. However, a small
fraction (up to 20%) can revert their polarization [60]. Movie S5
illustrates the typical behavior of most neutrophils stimulated with
10 mM f-Met-Leu-Phe. By contrast, almost all freshly isolated
human monocytes fully reverse their polarization axis in less than
one minute upon reversal of the gradient produced by 100 nM
CCL2. Moreover, movie S5 demonstrates that the polarization
axis could be switched several times. Similar results were obtained
with THP-1 cells (movie S6 and S7). Figure 8A depicts selected
frames from the Movie S6 which demonstrate that PI 3-kinase
activity upon the reversal of the polarization axis re-localized to
the new leading edge (right panels). Interestingly, reversal of the
polarization axis observed in the phase images appears to precede
the activation of PI 3-kinase at the new leading edge. When the
experiment was performed in the presence of 100 nM Wortmannin, the cells were able to migrate toward the dispensing pipette
and to flip their polarization axis following displacement of the
source of attractant (movie S7). Moreover, neither polarization nor
reversal of the axis was associated with PI3-kinase activation. The
frames from the movie shown in Figure 8B illustrate that the PKBPH domain probe in the presence of Wortmannin does not
localize at the leading edge, but rather remains diffuse in the
cytoplasm. These observations suggest that receptor-mediated
activation of PI 3-kinase is not required for monocyte polarization.
The observation is consistent with a number of publications that
have contested a general requirement of PI 3-kinase activation in
cell migration [6,9,10,61–64].
The above observations suggest that receptor-mediated signal
transduction leading to PI 3-kinase activation occurs by and large
at the leading edge. However, the gradient which is formed by the
dispensing pipette should also be sensed at the back of the cells
albeit at up to 30% lower concentration. The exact chemoattractant concentration over the cell is difficult to determine in this
experimental set up. The flow rate from the micropipette cannot
accurately be determined due to the non-linear flow conditions
imposed by the narrow orifice of the pipette and the free diffusion
of the ligand. However, cell migration can be induced with a wide
range of concentrations of the filling solution, such as 30 nM–
3 mM UK 14’304 or CCL2 with similar efficiencies. Given the
dissociation constant of UK 14’304 for a2AAR-YFP/CFP being 3–
4 nM [19] and an estimated concentration difference between the
front and the rear of a migrating cell of ,30%, this indicates that
at any of the above conditions the concentration of the attractant
at the rear of the cells is sufficiently high to occupy the receptors.
Taking these reflections into account it is unclear why PIP3
production occurs only at the leading edge. It has been proposed
that at the rear of a polarized cell the signal transduction from
GPCRs is offset at the Gi-protein level through a global inhibitory
process [55]. To test receptor activity at the front and the rear we
took advantage of the intramolecular FRET of the a2AAR-YFP/
CFP as an indicator. The stoichiometry of the donor (CFP) and
acceptor (YFP) of the construct is constant, therefore the FRET
signal is independent of the local concentration of the receptor
[26,56]. Ligand-induced a2AAR-YFP/CFP activation causes a
conformational change leading to the reduction of the FRET
efficiency between CFP and YFP [19]. The maximum change in
FRET efficiency measured as normalized ratio FRET (FYFP/FCFP)
of the a2AAR-YFP/CFP expressed in THP-1 exposed to 1 mM
UK 14’304 was ,10%, similar to the changes reported in
HEK293 cells [19]. Figure 7A depicts frames from a time lapsevideo at time 0 and 152 sec of THP-1 a2AAR-YFP/CFP cells
stimulated with 300 nM UK 14’304 from a pipette. At both times
the FRET efficiency is continuous over the entire plasma
membrane. However, markedly different ratios were observed,
showing a high FRET efficiency at time 0 and a reduced efficiency
after 152 sec (false color scale) indicating homogeneous receptor
activation over the entire membrane. Movie S4 displays the
complete sequence of the gradual change in FRET efficiency over
time. Importantly, although a clear net change in FRET was
obtained, we observed at no time a difference in FRET efficiency
between the leading edge and the rear of the cells. A quantitative
analysis of the FRET efficiency measured at the front and the rear
of polarized cells exposed for 2 min to 30 nM, 300 nM and 3 mM
UK 14’304 is shown in Figure 7B. The data reveal within the
limits of the accuracy of the measurements that there was no
significant difference in FRET efficiency between the leading edge
and the posterior side.
Amoeboid migrating cells, such as neutrophils and Dictyostelium,
once polarized in a gradient, largely maintain their polarization
axis [54,57]. Dictyostelium when stimulated with a relatively large
concentration of cAMP (100 mM) or upon rapid reversal of flowinduced hydrodynamic shear stress can, however, reverse its
polarization axis [58,59]. The vast majority of neutrophils upon
Discussion
Only few model systems for the study of leukocyte migration are
currently available. The here described monocytic THP-1 cells
bear some advantage over the classical neutrophil-like HL-60 cells.
THP-1 can easily be transfected or virally transduced and do not
need to be differentiated to reach a chemotaxis-competent
phenotype. The relatively homogenous cell populations of wild
type or transformed cells uniformly respond to ligands of
endogenous chemotactic receptors such as CCL2 (CCR2) or
CXCL12 (CXCR4, not shown). In addition the cells are capable
to migrate in response to stimulation of ectopically expressed
receptors such as the a2AAR. Interestingly the highly conserved
DRY(L/I)AI(V/I) motif of chemokine receptors that is considered
critical for chemoattractant-induced and Gi-protein-mediated cell
migration is only partially conserved in the DRYWSIT sequence of
the a2AAR. Nevertheless the present data demonstrate that the
structural properties of the a2AAR suffice to mediate efficient
chemokine receptor-like signal transduction and cellular responses
in THP-1 cells stimulated with the a2AAR agonist UK 14’304.
Following global stimulation most chemokine receptors internalize and become down regulated by an b-arrestin depended
pathway [65,65,66]. In THP-1 cells CCR2 follows this paradigm;
upon stimulation its appearance at the plasma membrane becomes
punctuated, consistent with ligand-induced lateral segregation of
the receptor, as initial step in clathrin-mediated endocytosis
leading to the localization of the receptor in large endosomal
8
Figure 5. Receptor localization in resting and polarized cells. (A) Selected frames from Movies S2 just after insertion of the micropipette
dispensing 300 nM UK 14’304 (0 sec, upper images) and after 5 min (300 sec, lower images). Red fluorescence derives from the membrane marker
DiD (594 nm excitation/620–680 nm emission), yellow fluorescence derives from the YFP tag of a2AAR CFP/YFP (514 nm excitation/525–590 nm
emission). Both fluorescences were recorded contemporaneously. The false color shows the ratio of red/yellow. Gray, corresponding phase image.
The bar in the phase images represents 5 mm. (B) Average ratio-intensity analysis of regions of interest (ROI) measured in resting cells (white bar, non
polarized) and polarized cells stimulated with 300 nM UK 14’304 at the front (black) or rear (grey). Data represent the mean values from multiple
frames from 3 different cells each from 3 independent experiments. The threshold for data inclusion was set equal in all experiments.
9
Figure 6. PIP3 formation occurs exclusively at the leading edge. (A) Selected frames from Movies S3 just after insertion of the micropipette
dispensing 300 nM UK 14’304 (0 sec, upper images) and after 5 min (250 sec, lower images). The emission of THP-1 cells transduced with PH-PKBmCherry (red fluorescence, 594 nm excitation/610–680 nm emission) and expressing a2AAR-YFP/CFP (yellow fluorescence YFP, 514 nm excitation/
525–580 nm emission) was recorded contemporaneously (right panels). The false color shows the ratio of red/yellow. Gray, corresponding phase
image. The bar in the phase images represents 5 mm. (B) Average ratio-intensity analysis of regions of interest (ROI) measured in resting cells (white
bar, non polarized) and polarized cells stimulated with 300 nM UK 14’304 at the front (black) or rear (grey). Data represent the mean values from
multiple frames from 3 different cells each from 3 independent experiments. The threshold for data inclusion was set equal in all experiments.
structures [67]. By contrast, neither wild type nor fluorescent
protein tagged a2AAR expressed on THP-1 cells appear to
segregate or internalize. Previous studies in COS-1 cells indicated
that a2AAR requires b-arrestin2 for internalization [41]. Our
observation indicates that for efficient migration receptor internalization might be dispensable. However, we can not exclude that
during migration at the leading edge, rapid recycling of the
receptors may occur, which does not go beyond the formation of
very early endosomes and does not lead to fusion with large
endosomes [68]. Such early endosomes must remain close to the
plasma membrane and can therefore not be visualized by confocal
microscopy.
Our data provide strong evidence that chemotactic receptors in
THP-1 cells remain uniformly distributed in the plasma
membranes and do not accumulate in the membrane forming
the leading edge. In polarized cells we observed a net increase in
plasma membrane towards the front of the cells which is caused by
ruffling and lamellipodia formation. Macroscopically this would
indicate a relative increase in receptor density at the front versus
the sides and the rear of the cells where the membrane appears
to be more smooth. As a consequence, through a feedback
mechanism more signal transduction events could be engaged
at the front leading the stabilization of the polarization axis.
However, the fact that the polarization axis of monocytic cells can
10
Figure 7. FRET efficiency of a2AAR-YFP/CFP in migrating cells. (A) Selected frames from Movies S4 just after insertion of the micropipette
dispensing 300 nM UK 14’304 (0 sec, upper images) and after 152 sec (2:32, lower images). Ratio FRET of THP-1 cells expressing a2AAR-YFP/CFP was
measured as described in Methods. The false color shows FRET efficiencies. Gray, corresponding phase image. (B) Quantitative analysis of FRET
efficiencies at the beginning (Cont., white bar) and 2–3 min after stimulation measured at the front (black bar) and rear (grey bar). Mean values of 3
cells from 3 independent experiments (30 nM and 300 nM UK 14’304) and 3 cells from 2 experiments for 3 mM UK 14’304. Stars indicate statistical
relevance of p,0.0001 (T-test).
11
12
Figure 8. Role of PIP3 formation during reversal of polarization. Selected frames from Movies S6 just after insertion of the micropipette
dispensing 300 nM UK 14’304 (0 sec, upper images) and after 5:40 min (lower images). The emission of THP-1 cells transduced with PH-PKB-mCherry
(red fluorescence, 594 nm excitation/610–680 nm emission) and expressing a2AAR-YFP/CFP (yellow fluorescence YFP, 514 nm excitation/525–
580 nm emission) was recorded contemporaneously. The false color shows the ratio of red/yellow (right panels). Gray, corresponding phase image.
(A) control cells, (B) cell pretreated for 10 min with 100 nM Wortmannin. (514 nm excitation/525–590 nm emission), (Phase) phase images taken with
DIC settings, and (Ratio) False color shows the ratio of red/yellow.
was recorded at 6 sec interval with DIC optics at 206magnification.
In the second part the cells were stimulated with 1 mM UK 14’304.
Found at: doi:10.1371/journal.pone.0010159.s001 (9.18 MB
WMV)
rapidly reverse impinges on the model. In movie S5 we show the
repeated rapid reversal of the polarization axis in monocytes which
were attracted with 10 mM CCL2. Thus, the reorientation initiates
at a site with markedly lower membrane density suggesting that
the relative receptor density may not be sufficient to maintain the
polarization axis in monocytes. Furthermore, flipping of the
polarization was observed with different agonist concentrations
(30 nM–10 mM). In Movie S5 we applied 10 mM CCL2, a
concentration sufficiently high to saturate CCR2 at the front and
presumably also at the rear. Considering the slow dissociation rate
of the CCL2 from the CCR2 (half life .200 min) [69], it is
conceivable that the receptors remain ligated during the entire
recording period. Taken together, the observations suggest that
chemotactic receptors presumably sense small differences in the
relative agonist concentration rather than the absolute amount
[52]. The conclusion is consistent with our FRET-based receptor
activation measurements and would predict that differences in
receptor activity are minimal and below the detection limit of the
probe.
Extracellular gradients are assumed to translate into steep
intracellular ramps of enzymatic reactions. Local activation of PI
3-kinase at the leading edge has been proposed as hallmark of
polarized cells [54,62]. Several downstream effectors which are
directly or indirectly regulated by 3-phosphoinositides and are
involved in chemotaxis have been identified [3,4]. In agreement
with these observations we show that PI 3-kinase is rapidly
activated during THP-1 cell polarization where PIP3 accumulation is restricted to the leading edge. The two-fold increase is
probably a cautious estimate considering the basal membrane
association of the reporter construct in non-polarized cells
(Figure 6B). The absence of PIP3 at the posterior end of the cell
may reflect several points of interference with agonist-stimulated
PI 3-kinase activation. Besides the potential direct inhibition of any
signal transduction step leading to PIP3 formation local activation
of phosphatases such as PTEN or SHIP-1 could account for the
absence. Both phosphatases have been implicated in restricting
PIP3 to the leading edge and preventing the lateral diffusion
[70,71]. It was also proposed that in polarized cells feedback loops
enhance PIP3 formation at the leading edge even in the absence of
receptor activation [3,62]. On the other hand time-lapse video
microscopy showed that efficient chemotaxis can occur in the
absence of PI 3-kinase activation [9]. It was proposed that PI 3kinase activity is involved in integrin mediated neutrophil adhesion
and F-action accumulation at the leading edge [10]. Here we
provide evidence (Movie S6) that cells can migrate towards the
dispensing pipette and that polarization occurs in the absence of
the activation of PI 3-kinase. Moreover, in the presence of
wortmannin the polarization axis can be reversed further
underlining that polarization per se is independent of PI 3-kinase
activity.
Movie S2 Receptor localization in resting and polarized cells.
Frames were taken at 4 sec interval with a 636magnification. (DiD)
Red fluorescence derives from the membrane marker DiD (594 nm
excitation/620–680 nm emission), (YFP) yellow fluorescence derives from the YFP tag of a2AAR-YFP/CFP (514 nm excitation/
525–590 nm emission), (Phase) phase images taken with DIC
settings, and (Ratio) False color shows the ratio of red/yellow.
WMV)
Movie S3 PIP3 formation occurs exclusively at the leading edge.
Frames were taken at 5 sec interval with a 636 magnification.
(PH) Red fluorescence derives from PH-PKB-mCherrry probe
which binds to PIP3, (YFP) yellow fluorescence derives from the
YFP tag of a2AAR-YFP/CFP (514 nm excitation/525–590 nm
emission), (Phase) phase images taken with DIC settings, and
(Ratio) False color shows the ratio of red/yellow.
WMV)
Movie S4 FRET efficiency of a2AAR-YFP/CFP in migrating
cells. Frames were taken from THP-1 cells expressing a2AAR-YFP/
CFP and stimulated with 1 mM UK 14’304 at 4 sec interval with a
636 magnification. (FRET YFP) FRET signal (YFP emission,
FYFP = 525 nm–600 nm), (CFP) CFP emission (FCFP = 465 nm–
505 nm), (PHASE) phase images taken with DIC settings, and
(Ratio FRET) Ratio FRET (rFRET) was calculated as FRET/
FCFP (FYFP/FCFP). The false color shows FRET efficiencies.
WMV)
Movie S5 Migration of freshly isolated human neutrophils and
monocytes. Neutrophils were prepared as previously described
[64], frames were taken at 3 sec interval with DIC optics. Cells
were stimulated with 10 mM f-Met-Leu-Phe. Monocytes were
prepared as described [65] were taken at 3 sec interval with DIC
optics. Cells were stimulated with 100 nM CCL2.
WMV)
Supporting Information
Movie S6 Role of PIP3 formation during reversal of polarization.
Frames were taken at 5 sec interval with a 636magnification from
control THP-1 a2AAR-YFP/CFP cells stimulated with 300 nM UK
14’304. (PH) Red fluorescence derives from PH-PKB-mCherrry
probe which binds to PIP3, (YFP) yellow fluorescence derives from
the YFP tag of a2AAR-YFP/CFP (514 nm excitation/525–590 nm
emission), (Phase) phase images taken with DIC settings, and (Ratio)
False color shows the ratio of red/yellow.
WMV)
Movie S1 CCR2-mediated migration of THP-1 a2AAR-YFP/
CFP cells. Cells were plated on glass bottom coverslips coated with
poly-D-lysine and MatrigelH. CCL2 (100nM) was dispensed from a
micropipette (center) with constant backpressure. Time-lapse video
Movie S7 Role of PIP3 formation during reversal of polarization. Frames were taken at 5 sec interval with a 636magnification
from THP-1 a2AAAR-YFP/CFP cells pretreated for 10 min with
100 nM Wortmannin and stimulated with 300 nM UK 14’304.
13
(PH) Red fluorescence derives from PH-PKB-mCherrry probe
which binds to PIP3, (YFP) yellow fluorescence derives from the
YFP tag of a2AAR-YFP/CFP (514 nm excitation/525–590 nm
emission), (Phase) phase images taken with DIC settings, and
(Ratio) False color shows the ratio of red/yellow.
WMV)
Acknowledgments
We would like to thank Drs. E. Cameroni and J. Luban for helpful
discussions.
Author Contributions
Conceived and designed the experiments: SV MT. Performed the
experiments: SV ST. Analyzed the data: SV ST. Contributed reagents/
materials/analysis tools: TP MJL. Wrote the paper: MT.
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15
ADDITIONAL RESULTS
Additional results
The following section contains data which are not shown in the included paper.
Fluorescent chemokine-dependent receptor internalization.
To further investigate chemokine receptors during migration experiments were performed
using fluorescent labeled CC-chemokines engineered in the laboratory. Briefly, human
chemokines were tagged at the C-terminus with different fluorescent proteins and
expressed in insect cells under the control of the insect-specific PH promoter. Secretion
of the recombinant proteins was efficienty mediated by the human leader sequences,
allowing recovery of the recombinant proteins from the cell supernatants (14417;
Cameroni E., and Thelen M., unpublished).
Peripheral blood monocytes were isolated to undergo CCL2-mediated cell migration
acting on the endogenous receptor. For time-lapse video microscopy of moving cells
recombinant CCL2 fused to the red fluorescent protein mCherry was employed [217] (Fig
1.).
37
ADDITIONAL RESULTS
F
38
ADDITIONAL RESULTS
Fig 1. Uptake of fluorescently labeled chemokine by moving cells. Selected frames
from time-lapse video microscopy of migrating monocytes at different intervals (min)
after insertion of the micropipette dispensing the agonist CCL2-mCherry 100nM.
Gray (left column) corresponding phase image. Red fluorescence (middle column)
derives from the recombinant chemokine mCherry-tagged (ex 594 nm excitation/620680 nm emission). The overlay color (right column) corresponding merge image.
The data recapitulates the traditional paradigm of chemokine receptor internalization
following agonist-induced activation [112,194]. The rapid (after 85 sec) initial ligandreceptor interaction is visualized by the red rim surrounding the cell body.
The appearance of red-stained intracellular vesicles (after 180 sec) might be the
consequence of the clathrin-mediated cargo endocytosis flowing the ligand-induced
lateral segregation of the receptor [212]. Fusion of small endosomal vesicles, probably
starting from the leading edge, leads to the formation of large intracellular compartments
(starting at 355 sec and subsequent) which appear to be stored at the trailing edge of
migranting cells. Moreover, cells remain fully motile suggesting that internalized
receptors are efficiently resorted to the plasma membrane through a rapid recycling
pathway [112].
In order to prove that the observed phenomenon was receptor-mediated the same
experiment was performed in the presence of an irrelevant recombinant chemokine
CCL20 tagged with GFP (Fig 2.).
39
ADDITIONAL RESULTS
40
ADDITIONAL RESULTS
Fig 2. CCR2-specific CCL2 uptake in moving monocytes. Selected frames from
time-lapse video microscopy of migrating monocytes at different intervals (min) after
insertion of the micropipette dispensing a mix solution of CCL2-mCherry agonist
1μM together with CCL20-GFP 1μM. The emission of mCherry-CCL2 (594 nm
excitation/620-680 nm emission) and CCL20-GFP (488 nm exitation 500-520nm
emission) was recorded contemporaneously. Only the effective red fluorescence was
reported (middle column). Gray (left column) corresponding phase image. The
overlay color (right column) corresponding merge image including the GFP emission
signal for the irrelevant chemokine (CCL20-GFP).
CCL20 (also known as LARC/MIP3α/Exodus) is a unique ligand for CCR6 whose
expression is restricted to lymphocytes and dendritic cells (DC) [218,219].
When cells were exposed to a mixture of CCL2-mCherry/CCL20-GFP only the red
emission signal from CCL2-mCherry was detected at the surfaces (after 25 sec) and
intracellularly at the level of the large endosomal compartments (starting at 195 sec and
subsequent). The observation is consistent with a CCR2-specific CCL2 uptake in
directionally migrating monocytes and makes pinocytosis less probable.
The contribution of the chemokine decoy receptors in this process was also assessed. It is
known that the chemokine scavenging receptors bind, internalize and target their ligands
for lysosomal degradation [220–222]. In particular the receptor D6 was reported to be
expressed by primary monocytes [223] and its engagement by inflammatory CC
chemokine resulted in efficient agonist internalization [220,224].
The role of D6 in CCL2 endocytosis during monocyte migration was investigated using a
combination of two D6 ligands CCL2-mCherry (10nM) and CCL22/MDC in 100 fold
excess (10μM). If CCL2 uptake was mediated by the decoy receptor D6 the addition of
unlabelled ligand in large excess would compete with CCL2-mCherry for D6 receptor
occupancy. A marked decrease in the intracellular fluorescent signal would be expected.
The result shown in Fig 3. clearly demonstrates that the CCL2 uptake is not affected by
the presence of CCL22, strongly indicating that CCL2 uptake is independent of D6
activity.
41
ADDITIONAL RESULTS
42
ADDITIONAL RESULTS
Fig 3. CCR2-specific CCL2 uptake is D6-independent. Selected frames from timelapse video microscopy of migrating monocytes at different intervals (min) after insertion
of the micropipette dispensing the agonist CCL2-mCherry 100nM in combination with
two fold higher CCL22 concentration (10μM). Gray (left column) corresponding phase
image. Red fluorescence (middle column) derives from the recombinant chemokine
mCherry-tagged (ex 594 nm excitation/620-680 nm emission). The overlay color (right
column) corresponding merge image
Dispensable role of PI(3)K activation in cell migration.
To further support the findings that regulation of migration is independent of the PI(3)K
activation chemotaxis was recorded by time-lapse video microscopy using freshly
isolated monocytes from peripheral blood. Cells were treated with wortmannin (100nM),
a concentration that fully blocks PI(3)K [225,226]. Fig 4. clearly shows that the migration
of monocytes is not perturbed by the PI(3)K inhibitor, neither their polarization nor the
CCR2-meiated CCL2 endocytosis.
43
ADDITIONAL RESULTS
44
ADDITIONAL RESULTS
Fig 4. Irrelevant effect of wortmannin to the migratory response of monocytes. Selected
frames from time-lapse video microscopy of migrating monocytes at different intervals (min)
after insertion of the micropipette dispensing the agonist CCL2-mCherry 100nM. Cells were
under HWT treatment (100nM) during the migratory assay. Gray (left column) corresponding
phase image. Red fluorescence (middle column) derives from the recombinant chemokine
mCherry-tagged (ex 594 nm excitation/620-680 nm emission). The overlay color (right
column) corresponding merge image.
Similar results were obtained by measuring the migratory response of THP-1 using
modified Boyden chambers. The Fig 5. shows the effect of wortmannin (100 nM), on the
migratory behavior of THP-1 cells elicited by the optimum concentration of CCL2
(3nM).
Fig 5. Chemotactic response of THP-1 cells to CCL2 is unaffected by HWT
treatment. THP-1 cells were subjected to chemotaxis assays in modified Boyden
chambers for 40 min. Chemotaxis was stimulated at the CCL2 peak value of 3nM
without (black bars) or with (grey bars) pretreatment with wortmannin (100 nM
for 30 min). The data are representative of three independent observations
determined in triplicate. Two independent experiments are shown.
The data demonstrate that, at least for monocytic cells, the chemoattractant-stimulated
migration is completely insensitive to PI(3)K inhibitor, as already published for other cell
types [77,170,227].
45
DISCUSSION AND OUTLOOK
Discussion and outlook
The present study introduces THP-1 cells as a suitable model system to investigate the
complex machinery underlying leukocyte migration. THP-1 cells offer several
advantages over currently available model systems such as the neutrophilic-like HL60
cells. The cells are more permissive to transfection and transduction protocols,
furthermore their inherent chemotaxis-competent phenotype eliminates the requirement
to induce in differentiation, a process resulting in inhomogeneous cell populations. THP1 cells show fast and prominent chemotactic responses which can be elicited by different
ligands such as CCL2 or CXCL12 acting on the endogenous receptors CCR2 and
CXCR4, respectively. Moreover, exogenously expressed Gi-coupling α2A adrenoreceptor
(α2AAR) mediates efficient chemokine receptor-like signal transduction and cellular
responses upon stimulation with the α2AAR agonist UK 14’304, indicating that THP-I
cells are suitable to the chemotactic potential of diverse receptors.
The fluorescent protein labeled variants of the α2AAR (α2AAR-YFP and α2AARYFP/CFP) are suitable tools to investigate the controversial issue about the localization
and distribution of chemoattractant receptors in responding cells [15–17]. By normalizing
the fluorescence emission of the receptor and an evenly distributing plasma membrane
marker we observed an unequivocally homogenous distribution of the receptor in
polarized cells. In other words functional receptors are not specifically recruited at the
leading edge of migrating cells.
However our data do not rule out a local receptor enrichment due to the net increase of
plasma membrane at front of the cells which is caused by ruffling and lamellipodia
formation. This macroscopically polarized distribution of chemosensory receptors might
contribute to an asymmetric amplification of signal transduction events through a positive
feedback mechanism leading to the stabilization of the polarization axis. However the
observation that the polarization axis of monocytic cells can be rapidly (~90 sec) reversed
argues against the rigid polarization axis implied by this model mechanism. Indeed timelapse video microscopy data of migrating monocytes and THP-1 cells towards the CCL2
gradient show high flexibility in the polar axis organization in response to a change in the
relative position of the chemokine source. The rapid rebuilding of a new leading edge on
46
the preexistent uropod, which has a markedly lower membrane density, suggests that an
increased relative receptor density may not be sufficient to maintain the polarization axis
in monocytic cells.
The homogenous membrane receptor distribution is consistent with the uniform receptor
activation observed by monitoring agonist-induced intramolecular FRET variations,
which reflect the receptor conformational states. Taken together, these observations
suggest that the driving force for cell polarization might reside in the intrinsic ability of
the chemotactic receptor to respond to and to transduce small differences in the relative
agonist concentration rather than in the absolute amount of receptors being activated by
the chemoattractant. Subtle differences in receptor activity along the front-rear axis could
be below the detection limit of our experimental setting.
In general most chemokine receptors following prolonged agonist stimulation are
internalized by a β-arrestin dependent pathway [48,211]. Briefly, ligand binding induces
GRK-mediated receptor phosphorylation and the phosphorylated motifs operate as
docking sites for β-arrestin, which subsequently coordinates clathrin-coated pit mediated
endocytosis. As expected in THP-1 cells CCR2 was efficiently removed from the plasma
membrane upon CCL2 stimulation. Conversely neither WT nor the fluorescently tagged
protein constructs of the α2AAR appeared to follow this paradigm, as already
demonstrated for COS-1 cells which require cotransfection of α2AAR and β-arrestin2 to
sustain a modest agonist-depend α2AAR internalization [198].
Given that cells readily polarized and migrated in response to the agonist UK 14’304
suggests a dispensable role of receptor internalization for directional cell migration.
However our data do not rule out rapid recycling of the internalized α2AAR. Such rapid
receptor traffic route might exploit the formation of very early endosomes which are
smaller in size and may not proceed in the fusion with large endosomes, but may rather
remain in the immediate vicinity of the plasma membrane and therefore can not be
visualized by confocal microscopy. Theoretically by using selective inhibitors for this
critical step in the maturation of the endosome by applying electron microscopy it might
be possible to investigate the hypothesis.
47
A transient and rapid process of receptor desensitization not involving internalization
may be postulated [6]. It was elegantly demonstrated for CCR5 that protein kinases
differently contribute to receptor phosphorylation, while GRKs mediate a slow and
persistent receptor phosphorylation, the second messenger-dependent protein kinases
(PKA, PKC) mediate a rapid and transient phosphorylation of the receptor [101]. Thus it
is conceivable that the highly dynamic rearrangements of the cytoskeleton at the leading
edge of motile cells, with continuous extensions and retractions of multiple protrusions
are the result of the more rapid phosphorylation events mediated by second messengerdependent protein kinase.
Assuming a uniform distribution of chemokine receptors at the plasma membrane of
motile cells, preferential agonist responsiveness at the leading edge may be supported by
local signal amplification mechanisms [120] as well as by the exclusion of counterregulatory proteins. With this strategy chemoattractant-responsive cells are able to
translate a shallow extracellular chemical gradient into a steep intracellular gradient of
signaling molecules.
A great deal of evidence suggests that PI(3)K has an important role in establishing and
maintaining cell polarity by regulating the subcellular localization and activation of
downstream effectors that are essential for proper chemotaxis [79,228–230]. The catalytic
activity of PI(3)K has been assayed by using probes detecting its phosphorylated product
PIP3 which is found to be highly enriched at the inner surface of the plasma membrane at
the front of migrating cells.
Our data using a fluorescent sensor for PIP3 support this observation showing that PI(3)K
is rapidly activated during THP-1 cell polarization inducing a PIP3 accumulation
restricted to the leading edge. The asymmetric distribution of phosphoinositide lipids is
achieved by spatially segregated antagonistic reactions: positive feedback loops sustains
the PI(3)K activity while counter-regulatory proteins confines the PIP3 production
[72,80]. Negative regulators of PIP3 accumulation include the 3’ lipid phosphatase PTEN
and the 5’ lipid phosphatase SHIP which have been implicated in restricting PIP3 to the
leading edge and preventing lateral diffusion [161,163]. It has been also demonstrated the
existence of feedback loops which enhance the PIP3 formation even in the absence of
48
receptor activation [75,152]. However our data clearly show that the impaired
accumulation of PIP3 at the up-gradient front of migrating THP-1 cells does not affect the
chemotactic response, neither at the level of gradient sensing nor at the polarization stage,
being the cells able to reverse the polar axis. This result is in agreement with observations
proposed in other reports postulating a dispensable role for PIP3 gradient formation and
PI(3)K activity in directional cell migration [76,77,167–170].
Indeed alternative pathways which cooperate with the canonical PI(3)K-mediated signal
have been shown to exist for polarization and migration [163,171,173]. However it would
be worth investigate new role of other potential factors indirectly involved in the PI(3)K
regulation such as the small Rho-GTPase Rac, known to be part of the positive feedback
loop which modulate the PI3K activity [79,80].
49
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69
CURRICULUM VITAE AND PUBBLICATIONS
• Contact
Silvia Volpe
IRB (Institute For Research in Biomedicine)
Via Vincenzo Vela 6, 6500, Bellinzona Switzerland
Phone: +41 91 820 0337
Mobile: +41 77 431 5568 (CH); +39 349 763 5365 (IT)
E-mail: [email protected]
• Education
March 2006 to April 2010
PhD supervised by Prof. Marcus Thelen at the Institute for research in Biomedicine,
Bellinzona, Switzeland (Phd fellowship within the Europian commission “Marie Curie
Programme” for early stage researchers training)
Second Supervisors:
Prof. Dr. Anne-Catherine Andres, Graduate School for Cellular and Biomedical Sciences,
University of Bern, Switzerland;
Prof. Dr. Ruggero Pardi at the Graduate School in Molecular Medicine Section of Basic
and Applied Immunology of Vita-Salute San Raffaele University in Milan, Italy
October 2005
MSc in Biology at the University of L’ Aquila (Italy)
1999-2005
Biology studies at the University of L’Aquila (Italy)
1986-1999
Primary and Secondary education, graduation from the Liceo Classico “D.Cotugno”,
L’Aquila, Italy
70
• Appointments and Experience
Institute for Research in Biomedicine since 2006
PhD student project entitled “GPCR activation during cell migration determined by
FRET” in the Signal Transduction Lab headed by Prof. Marcus Thelen
University of L’Aquila 2004-2006
Graduate assinstant in the Biochemestry lab University of L’Aquila, Italy headed by Prof.
Giuseppina Pitari. The lab also hosted the master thesis project entitled “The Effect of
Cysteamine on Activity of Detoxifying and Antioxidative Enzymes in Vanin-1 NullMice”
• Experimental Skills
Biochemical
techniques:
protein
biochemistry,
electrophoretichal
analysis,
chromatography, spectroscopical UV-Vis and spettrofluorimetric techniques
Molecular Biology.
Cell biology methods: cell culture, primary human blood cells and FACS.
Imaging: confocal microscopy (FRET, FRAP analysis, Metamorph and IMARIS
softwares for image processing)
• Training and courses
November 2006
Imaging Platform Course on “Dynamic Imaging of DCs and Associated Molecules”,
sponsored by Dendritic Cells and New Immunotherapies (DC THERA), Centre
d’Immunologie de Marseille-Luminy
November 2007
International Workshop “Inflammation and Cancer”, Humanitas, Rozzano, Milan, Italy
September 2008
“Innovative chemochine-based therapeutic-strategies for autoimmunity and chronic
inflammation (INNOCHEM) Practical Course on “Pharmacology of 7-transmembrane
71
domain receptors”, sponsored by the Europian commission sixth framework programme,
Copenhagen, Denmark
August 2009
EMBO meeting, Amsterdam, Holland
• Conferences and presentations
November 2006
The second “MAINNOE Targeting cell Migration in Chronic inflammation” Annual
Conference”, Castelnuovo del Garda Verona (Italy)
Poster “Potential mechanisms of G-protein coupled-receptor mediated cell migration”
INNOCHEM Annual meeting in Bellinzona, Switzerland.
Poster “Potential mechanisms of G-protein coupled-receptor mediated cell migration”
May 2007
MAINNOE meeting, Villars-sur-Ollon, Switzerland.
Oral presentation “Characterization of a potential tool to study the activity of GPCRs
during cell migration”
September 2007
INNOCHEM meeting in San Lorenzo De El Escorial, Spain
Poster “GPCR activity during cell migration determined by FRET”
May 2008
MAINNOE meeting, Certosa di Pontignano, Siena, Italy.
Oral presentation “GPCR activity during cell migration determined by FRET”
January 2009
USGEB meeting in Iterlaken, Switzerland
Poster “GPCR activity during cell migration determined by FRET”
February 2010
USGEB meeting in Lugano, Switzerland
72
Poster “Study of GPCR activation in polarized cells: while receptors remain evenly
distributed cellular responses are localized in migrating cells”
• Publications
Silvia Volpe, Sylvia Thelen, Thomas Pertel, Martin J. Lohse and Marcus Thelen
“Polarization of migrating monocytic cells is independent of PI 3-kinase activity” PLoS
One. 2010 Apr 15;5(4):e10159
73
DECLARATION OF ORIGINALITY
Declaration of Originality
Last name, first name:
Volpe, Silvia
Matriculation number:
06-128-995
I hereby declare that this thesis represents my original work and that I have used no other
sources except as noted by citations.
All data, tables, figures and text citations which have been reproduced from any other
source, including the internet, have been explicitly acknowledged as such.
I am aware that in case of non-compliance, the Senate is entitled to divest me of the
doctorate degree awarded to me on the basis of the present thesis, in accordance with the
“Statut der Universität Bern (Universitätsstatut; UniSt)”, Art. 20, of 17 December 1997.
Place, date
Signature
……………………………………………
……………………………………………
74

Exploring signal events elicited by chemotactic

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