1 Physik Depatment E22 (biophysics group), Technische Universität
München, D-85748 Garching, Germany
2 Max von Pettenkofer Institut für Medizinische Mikrobiologie,
Pettenkoferstrasse 9, Ludwig Maximilian Universität, D-80336
München, Germany
Present address: Institute of Theoretical Physics, UCSB, Santa Barbara,
USA
* Author for correspondence (e-mail: lvonna{at}ph.tum.de)
Accepted 18 October 2002
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Summary |
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Key words: Magnetic tweezers, Phagocytosis, Yersinia, Invasin, Macrophage, J774, Protrusion
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Introduction |
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Reorganizations of the actin-based cytoskeleton evoked by mechanical forces and other external stimulations (e.g. adhesion) are often difficult to distinguish. Micromechanical measurements of forces or of changes in the viscoelastic moduli associated with the reorganization of the cytoskeleton provide a valuable tool to quantify such changes.
An example of the cytoskeletal reorganization induced by adhesion is the
attraction and engulfment of pathogens by macrophages during phagocytosis.
Micromechanical studies of this process were performed by the micropipette
aspiration technique (Evans et al.,
1993; Swanson et al.,
1999
; Zhelev and Hochmuth,
1995
). In the experiments by Evans et al., for instance, the force
generation associated with the engulfment of pathogenic yeast cell by
macrophages was studied. It was found that if the neutrophils are aspired into
the micropipette with fixed lengths of protrusion, the opposite end of the
cell first spreads over the pathogen at constant tension. It then suddenly
stops and starts to build up a cortical tension, suggesting that the spreading
and the force generation are subsequent processes. To gain insight into these
sequences of steps during phagocytosis from a different perspective we studied
the response of J774 macrophages to local centripetal pulling forces applied
by magnetic tweezers (which allowed us to generate forces of up to 5 nN).
The magnetic beads were coated with invasin, a surface protein of pathogenic Yersinia species, which causes gastrointestinal infection by invasin-triggered invasion into the intestine. This protein binds to integrins containing ß1 chains and induces the recruitment of actin microfilaments and actin-associated proteins, such as filamin and talin. This cytoskeletal reorganization is also assumed to be the first step during the process of internalization of pathogens. In the present study the external force on the invasin-coated bead is applied immediately before the spreading of the phagocyte over the bead starts, and it is oriented in the same direction. Such experiments are expected to yield insight into the time evolution of the formation of cellular protrusions at the initial state of phagocytosis and into the process of generation of retraction forces resulting in the engulfment of the particles. These forces are important if phagocytes have to remove pathogens adhering strongly to surfaces, for example, to other cells or tissues. One advantage of the magnetic tweezers technique is that complex force scenarios can be applied such as sequences of pulses or staircase-like force programs. We show that it is possible to distinguish between active and passive responses of the cells to external forces by analyzing the change in deflection of the cell in response to small changes in force.
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Materials and Methods |
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Sample preparation
The experiments were performed with J774 mouse macrophages (European
Collection of Animal Cell Cultures, Salisbury, UK). Cells were cultivated in
an incubator at 37°C and 5% CO2. The cell culture medium
consisted of RPMI 1640 supplemented with 10% fetal calf serum (Life
Technologies GmbH, Eggenstein, Germany).
Tosyl-activated superparamagnetic Dynabeads (Dynal, Hamburg, Germany) with
a diameter of 4.5 µm were covered with invasin proteins according to the
procedure provided by the supplier. Expression and purification of this
protein is described in a previous paper
(Wiedemann et al., 2001). In
order to verify the binding of invasin, the beads were incubated at 4°C
with rabbit anti-invasin antibody, diluted 1:1000. Beads were then washed
three time with 1% PBS to remove unbound antibodies and then incubated with
488-labeled goat anti-rabbit antibodies, diluted 1:200 (Molecular Probes).
Beads that are effectively covered by invasin exhibit a fluorescent rim under
a confocal fluorescent microscope.
In order to define the initial conditions of the experiment we proceeded as follows. For each experiment the cells were detached from the culture dish and transferred onto coverglasses. After incubating with cell culture medium for 24 hours, a coverglass covered with cells was mounted on the copper block of the measuring chamber. Invasin-coated beads were then carefully added to the chamber, on top of the J774 macrophages. Immediately after this step, the motion of the beads towards the vicinity of the cells was followed by visual inspection. Occasionally, randomly distributed beads on the surface of the substrate are contacted by filopodia extending several µm into the medium and are attracted towards the cell body (L. V, unpublished). We selected beads attached to the cell, which were localized between the macrophage and the tip of the iron core. Thus, the external forces could be applied in the direction opposite to the active force exerted by the cell to engulf the bead. The distance between the cell and the tip varied between 20 and 160 µm, resulting in force amplitudes ranging from 0.1 to 9 nN. If no external forces are applied on the cell the bead is internalized after attachment to the cell within about 10 to 15 minutes.
To check the role of the actin cortex in our measurements we completely and rapidly exchanged the medium in a series of experiments by different stock solutions of medium containing Latrunculin A (Molecular probes) and dimethylsulfoxide (DMSO) (Molecular Probes), just before the engulfment of the bead. The concentration of Latrunculin A was varied from 1 µM to 10 µM; the medium:DMSO ratio was 1:150 to 1:1500.
Data acquisition
Both the position of the bead and the shape of the cell were observed with
an AXIOVERT 100 phase contrast microscope (Zeiss, Oberkochen, Germany) using a
x32/0.75 objective. Images were recorded with a CCD camera connected to
a numerical data acquisition system. The beads were tracked using homemade
software with an accuracy of 10 nm (not available on the market).
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Results |
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The following experiments and considerations show that in the linear regime
of deformation, the cell responds to the external force in an interactive way.
The initial fast and passive response, which is also clearly reflected by the
initial fast relaxation after switching off the force (see double arrow in
Fig. 1), may be caused by two
processes: firstly, an elastic deformation of the cell envelope leading to the
bulging of the cell membrane in the direction of the force while the cell body
remain fixed to the substrate; and secondly, a shear deformation of the whole
cell body, resulting in a small displacement of the contour of the cell on the
side opposite to the force direction (Lo
et al., 1998). At present we can not clearly distinguish between
these two alternatives. However, evidence for the second explanation is
provided by the frequent finding that the rim of the cell opposite to the side
of force application is displaced during the initial phase of the response.
The slow cell deformation (characterized by the deflection of the bead with
constant speed) can be described as an active growth of a cone-like
protrusion. When the applied force is switched-off (before the active
retraction sets in), the cell recovers its initial shape through the complete
retraction of the protrusion. The process of phagocytosis of the bead will
then continue until the bead is completely engulfed. At zero force the bead is
rapidly internalized by phagocytosis.
In a second series of experiments we studied the force dependence of the growth rate of the cone for the same cell. For that purpose we induced the repeated formation of protrusions by application of a sequence of force pulses of different force amplitude. Before each new pulse we waited until the protrusions were fully relaxed. The different speeds of protrusion obtained with different forces are summarized in Fig. 2 for three cells. The numbers in the figures correspond to the number of the force pulse of each sequence. No correlation between the number of the pulse and the measured speed is found, which demonstrates that the history of the cell does not influence the measured growth speed. For forces larger than about 0.5 nN, the average speed varies between v=0.020 µm second-1 and 0.175 µm second-1 but no systematic force dependence is observed. The average value of the velocity obtained by evaluating 15 cells (49 measurements) is <v>=0.065±0.020 µm second-1.
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Further evidence for the active response of the cell is provided in
Fig. 3. After application of a
force of 2.55 nN for about 150 seconds the amplitude was reduced in a
step-wise manner first to 1.7 nN, then to 0.85 nN and finally to 0 nN.
Complete relaxation is observed only after switching off the force completely.
By contrast, the step-wise reduction of the force to 1.7 nN and to 0.85 nN,
respectively, leads first to a transient viscoelastic relaxation (indicated by
arrows in the figure), but this process is then followed by a renewed growth
of the cone-like tip with about the same velocity as before the reduction of
the force. This type of behavior has been observed many times. It is
interesting to note that the relaxation time of the transient retraction of
the protrusion after the reduction of the force is of the same order (20
seconds) as the response time characterizing the transition from the elastic
deflection to the linear flow regime when the force is switched on.
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After prolonged application of the external force, the cells retract the protrusions again against the largest external forces available (5 nN). An example is show in Fig. 4 for an applied force of 3 nN. It is seen that after application of the force for about 60 seconds the growth rate of the protrusion first decreases and the protrusion is finally reversed. Until now we had not found a systematic correlation between the onset of the retraction force and the length of the protrusion or the time over which the force is applied.
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To clarify the role of the actin network in the active growth of the cone
we studied the response of cells treated with Latrunculin A. This agent
strongly binds to monomeric actin and thus prevents the generation of a new
actin cortex (Spector et al.,
1983). We found two scenarios. In many cases (such as shown in
Fig. 5) a viscoelastic response
is found, consisting of an elastic deflection, a relaxation process and a flow
regime (A to B in Fig. 5),
which is followed by the pulling of a tether (B to C in
Fig. 5). In some cases the
viscoelastic response is also observed but is preceded by the formation of a
short tether. In the first scenario the viscoelastic response and flow regime
(A to B in Fig. 5) is also
associated with the formation of a cone-like protrusion. However, this is a
passive bending deformation since the flow rate is 1.6 µm
second-1 and is thus much faster than the active process
(<v>=0.065±0.020 µm second-1).
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Discussion |
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The mechanical response of the macrophage to local force pulses applied on invasin functionalized beads, which are bound to integrins exhibiting ß1 chains, is characterized by the following features:
The threshold behavior of the cone growth process is similar to that of the
tension-induced elongation of axons by forces applied through microglass
needles to the tip of the growth cones of neurons
(Zheng et al., 1991). The
threshold force in this case varied between 0.5 and 1.5 nN and is thus similar
to the present value of
0.5 nN. Threshold behavior has also been reported
for the formation of tube-like protrusions by aspiration of the cell envelope
by micropipettes (Merkel et al.,
2000
; Zhelev and Hochmuth,
1995
). However, in these experiments the threshold tension is an
order of magnitude higher (typically 2 nN) and is determined by the fracture
of the bilayer membrane from the actin-based cytoskeleton.
The force-induced elongation of the protrusion of macrophages and of the
axon of neurons (Zheng et al.,
1991) differ in one aspect. The growth rate of neurites is
proportional to the applied stress, whereas no correlation between rate of
advancement and the applied stress was observed in our case. The average speed
of advancements of the neurite at 2 nN is a factor of three smaller than the
average value of <v>=0.065 µm second-1 observed by us.
There are some interesting similarities between the force-induced growth of
the cone-like protrusion and the spreading of the envelope of macrophages over
pathogenic cells (Evans et al.,
1993), although the driving force in this case is cell-cell
adhesion. First, the speed of advancement of the cell lobe over the pathogen
(v
0.1 µm second-1) is similar to the cone growth rate
(<v>
0.065 µm second-1). Secondly, the advancement of
the cell lobe over the pathogenic cell stops abruptly (after about 30 seconds)
and a retraction force builds up, which is similar to the behavior of the
macrophage shown in Fig. 4. The
retraction force exhibits a value of about 10 nN and is thus beyond the force
range of our technique.
Several observations of our study can be explained by assuming that the
force-induced cone formation is associated with the formation of an actin
cortex. One source of evidence comes from the experiment with Latrunculin A.
The rather fast viscoelastic response associated with the formation of a
conical deformation (Fig. 6A)
and the subsequent tether formation is attributed to the partial decoupling of
the actin cortex from the plasma membrane and the impediment of actin
repolymerization by this agent. The decoupling facilitates tether formation
owing to the fracture of bonds between the actin cortex and the intracellular
domains of membrane receptors such as integrins
(Hochmuth et al., 1996).
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The sequestering of G actin by Latrunculin is expected to prevent the
repairing of the fractures. The speed of the cone growth agrees reasonably
well with biochemically stimulated growth rates of actin networks, such as the
comet-like tails of listeria bacteria (v0.1 µm second-1)
(Noireaux et al., 2000
). These
tails grow by assembly of a new actin gel at the surface of the bacteria
through a mechanism similar to the formation of lamellipodia; that is by
polymerization of actin between the advancing membrane lobe and the actin
cortex by new synthesis of an actin network beneath the advancing membrane
lobe (Borisy and Svitkina,
2000
).
The protrusion assumes a conical shape, exhibiting a smooth transition into
the main cell body (Fig. 6).
The situation is very similar to the generation of a trumpet-like protrusion
generated by a local force in the center of a circular disk of radius R.
Following Bruinsma (Bruinsma,
1996), the contour of such protrusions is approximately given by
X(r)=X(a).ln(R/r)/ln(R/a), where a is the radius of the contact area between
the bead and the cell membrane and R is the radius of the disk, which is about
equal to the radius of the cell. For our case (a
2 µm and R
10 µm)
the above equation holds with an accuracy of 20%. The extension
x is
proportional to the force f0
. The force constant
is
. This shows that, to a first
approximation, the cortical tension does not depend on the length of the
protrusion. It provides the driving force for the retraction of the protrusion
after switching off the force before the active contraction sets in. The close
analogy between the observed cellular deformation and the trumpet-like
protrusions generated in a circular plate through a local force strongly
suggest that the constant tension is built up within a thin layer close to the
cell envelope.
The constant speed of advancement <v> of the tip could be explained by two mechanisms:
First it could be determined by the rate of formation of free volume at the
advancing tip generated by the force-induced disruption of the lipid/protein
bilayer from the cytoskeleton. If this disruption is the rate-limiting step,
the process corresponds to the Brownian ratchet model of cell locomotion
(Mogilner and Oster, 1996).
This model contradicts our finding of a much lower threshold force (
0.5
nN) than the fracture force required for the disruption of the actin-membrane
coupling (Merkel et al.,
2000
). A second possibility is that the point force mediates the
activation of a cell signaling pathway that controls the growth of cellular
protrusions such as the formation of lamellipodia. The link between the
mechanical force and the cell signaling pathway could be the stress
sensitivity of the integrin receptors containing ß1 chains, which are
known to coupled to invasin. Actin would in this case act as a
mechanotransducer. Evidence for such a role for integrin has been provided by
various groups (Choquet et al.,
1997
; Ingber,
1991
; Nebe et al.,
1995
; Pommerenke et al.,
1996
; Wang et al.,
1993
). In particular, Schmidt et al.
(Schmidt et al., 1998
) induced
local deformation of osteoblastic cells through magnetic beads functionalized
with antibodies that bind specifically to integrins exhibiting either ß1
or ß2 units (or both). They showed that mechanical excitation induces
tyrosine phosphorylation of cytoskeletal anchoring proteins and most probably
activates MAP kinases and thus couples these proteins to genetic expression
pathways.
One likely pathway mediating the growth of an actin cortex is one involving
an increase in the Ca2+ level, which is known to be accompanied by
an increase in the concentration of F-actin
(Zhao and Davis, 1999).
Another pathway may be mediated by the small GTP-binding protein of the Rho
family (Cho and Klemke, 2002
),
which is known to stimulate pseudopodium formation in the absence of external
forces. Our previous results also suggest activation of the Rho GTPases Rho,
Rac and CDC42Hs upon binding of invasin-coated beads to ß1 integrins
(Wiedemann et al., 2001
),
although no pseudopodium formation, but invagination of the cell envelope
followed by the engulfment of the bead, occurs in this case. By contrast, the
activation of the Rho GTPase may well promote the growth of the protrusion
directed towards the outside of the cell after stimulation by an external
force. Consistent with this idea, it was shown recently that application of
shear stress to endothelial cells caused Rho activation via
Vß3
integrins (Tzima et al.,
2001
).
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Acknowledgments |
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