1 Department of Geriatric and
Respiratory Medicine, To
investigate the mechanisms underlying pseudopod protrusion in
locomoting neutrophils, we measured the intracellular stiffness and
viscosity in the leading region, main body, and trailing
region from displacements of oscillating intracellular
granules driven with an optical trap. Experiments were done in control
conditions and after treatment with cytochalasin D or nocodazole. We
found 1) in the body and trailing
region, the granules divided into a "fixed" population (too stiff
to measure) and a "free" population (easily oscillated; fixed
fraction 65%, free fraction 35%). By contrast, the fixed fraction in
the leading region was <5%. 2) In
the body and trailing region, there was no difference in stiffness or
viscosity, but both were sharply lower in the leading region (respectively, 20-fold and 5-fold).
3) Neither cytochalasin D nor
nocodazole caused a decrease in stiffness, but both treatments markedly
reduced the fixed fraction in the body and trailing region to <20%
and <40%, respectively. These observations suggest a discrete lattice structure in the body and trailing region and suggest that the
developing pseudopod has a core that is more fluidlike, in the
sense of a much lower viscosity and an almost total loss of stiffness.
This is consistent with the contraction/solation hypothesis of
pseudopodial formation.
cytoskeleton; biomechanics; pseudopodia
NEUTROPHILS ARE AN important cell type in the
inflammatory response and in host defense mechanisms. Their chemotactic
response is characterized by locomotion through the formation of
pseudopods. Such pseudopods are easily visualized, but the mechanical
processes by which they form, protrude, and subsequently translate the
main body of the cell are still unknown. Because the processes of
transendothelial migration, locomotion in stroma, and transepithelial
migration require significant deformation of neutrophils, the
neutrophil cytoskeleton must be correspondingly remodeled through the
processes of polymerization and depolymerization. This response is
presumably inhomogeneously distributed between at least the locomotory
pseudopod and the main body of the cell.
There are two distinct classes of ideas about the evolving structure of
the pseudopod. One (5, 6, 12) is that the pseudopod is formed by
compression, through polymerization of cytoskeletal filaments,
especially actin, which then allows the pseudopod to "grow" at
its tip. The other (10, 19, 25) is that the pseudopod core is
essentially a passive fluid, which streams into the pseudopod as a
result of intracellular pressure; the pseudopod cortex polymerizes in
an annular fashion, but the core remains in an essentially sol or fluid state.
These two mechanisms can be distinguished in the living cell if
regional measurements of the elastic modulus, or stiffness, and
viscosity of the intracellular milieu could be made. In the pseudopodial compression model, the stiffness of the leading region would be at least as great as that in the body or trailing region; in
the pseudopodial fluid core model, the viscosity and especially the
stiffness of the leading region would be much less than those in either
the body or trailing region. (Note that throughout this paper, we use the phrase "leading region" to denote the
protruding pseudopod as a whole and not the subcortical region
immediately proximate to the pseudopodial membrane. See
DISCUSSION for the potential importance of this
distinction.)
Utilizing the recently developed laser optical trap, or "optical
tweezers," we measured both the dynamic forces on, and the displacements of, individual intracellular granules in living neutrophils, selected from each of the three regions. From these data,
we estimated the elastic modulus and viscosity in the leading region,
the body, and trailing region and assessed the regional differences in
these rheological properties.
Reagents. Krebs-Ringer phosphate with
dextrose (KRPD) was constituted by (in mM) 115 NaCl, 14 dextrose, 6 KCl, 4.6 MgSO4 · 7H2O,
3.5 NaH2PO4 · 2H2O,
and 16 Na2HPO4
in water. Normosmotic RPMI 1640 medium with L-glutamine was
purchased from GIBCO-BRL. Mono-Poly Resolving Medium, a separation
medium of blood cells into mononuclear and polymorphonuclear (PMN)
leukocytes, was purchased from Dainippon Pharmaceutical. FBS was
obtained from Cansera International. Cytochalasin D and nocodazole were
purchased from Sigma Chemical.
Preparation of cells. Human
neutrophils were isolated from whole blood by a density gradient
technique using Mono-Poly Resolving Medium according to the
manufacturer's directions. Briefly, 24 ml of peripheral blood were
drawn from normal subjects with a heparinized syringe. The sample was
put in a 50-ml sterile polypropylene tube and centrifuged at 175 g for 20 min at room temperature. The
upper platelet-rich plasma layer was carefully removed using a Pasteur
pipette and was replaced by the same amount of KRPD. After being mixed
gently, it was equally divided into four sterile polypropylene tubes.
Mono-Poly Resolving Medium (4 ml) was gently added so as to underlay
the blood without significant mixing in each tube. The samples were
then centrifuged at 330 g for 25 min at room temperature. This procedure resulted in the following four
layers, in order from the top: KRPD solution, monocyte/lymphocyte layer, PMN cell layer, and red blood cell layer. The PMN cell layer was
collected and rinsed with KRPD solution. It was then centrifuged at 250 g for 10 min. KRPD was removed, and
the PMN cells were resuspended with 10 ml of medium (RPMI Medium 1640 + 5% FBS).
Chamber preparation. A chamber was
prepared with two clean uncoated coverslips as the top and the bottom
surfaces, separated by ~600 µm using sheet paraffin wax spacers.
Edges of the coverslips were sealed with valap
(beeswax-lanolin-petrolatum, 1:1:1 by wt). Two 23-gauge needles had
been introduced into the space before sealing to be used as entrance
and exit ports. Gentle suction on the exit needle or gravity drainage
was used for filling. The sample was placed on a heated microscope
stage maintained at 37°C. Many of the cells remained floating or
adhered to the glass surface only loosely, appearing round and
inactivated. A modest fraction adhered to the glass strongly and began
to spread and locomote. The chamber was then rinsed with 2 ml of medium
to remove floating or loosely adherent cells. The remaining
neutrophils, in the process of lamellipodial protrusion and locomotion,
were used for this study.
Inhibition of F-actin formation and microtubule
assembly. To disrupt F-actin or microtubules, we
introduced cytochalasin D or nocodazole into the chamber, diluted,
respectively, to 2 and 10 µM in medium containing 0.1% DMSO. The
sample was then incubated at 37°C for 5 or 10 min, respectively.
Video-enhanced differential interference contrast
microscopy with optical tweezers. Samples were observed
under a differential interference contrast microscope (Diaphot TMD300;
Nikon) equipped with a Plan Apochromat ×100 oil-immersion
objective lens (numerical aperture 1.4), an oil-immersion condenser
lens for high-magnification objectives (numerical aperture 1.4), and a
100-W halogen lamp. Images were detected with a Newvicon tube video
camera (C2400-07; Hamamatsu), enhanced with an image processor
(DVS-3000; Hamamatsu), and recorded at 30 frames/s with a super-VHS
videocassette recorder (SVO-9650; Sony). A video printer (UP-860; Sony)
was used for video prints of taped images. A linearly polarized laser
beam from a Nd:YAG laser (SL902T; Spectron Laser Systems) emitting at
1,064 nm was introduced into the epifluorescence port of the microscope
with the aid of galvano mirrors and collimating lenses. The laser beam
was manipulated in two dimensions over the field of camera view using
the galvano scanner controller (CX-660; General Scanning), which was
operated by an external voltage signal from a function generator
(Iwatsu). The position of the trap center was monitored on the video
image by a superposed digital recording of the voltage level. Rotation
of a Glan-laser polarizer or half-wave plate inserted between the laser
and galvano scanner allowed attenuation of the laser beam and hence
different trap forces in different experiments. The laser power was
monitored by a thermal detector (model 835; Newport). The
force/displacement characteristics of the trap were determined by
oscillating isolated granules in medium, as described below.
Displacement data collection. Each
frame to be analyzed on the videotape was captured by a frame
grabber card (CinemaGear; Interware, Tokyo, Japan). Clock
time, laser power, and the relative displacement of the trap were
printed on each image, so that the position of the center of the trap
was recorded on each image. There was approximately one frame delay in
writing the image; this time delay of 30 ms was subtracted from all
clock times. The amplitude and phase of the oscillating granule were
determined by locating the granule centroid at its positive
and negative extremes and at the time of the zero crossing of
the trap. This was performed on a Macintosh computer using the public
domain National Institutes of Health (NIH) Image program (developed at the United States NIH and available on the Internet at
http://rsb.info.nih.gov/nih-image/). A grid with 10-µm squares was
used for calibration, and one pixel on the image was determined to be
equal to 50 nm.
Oscillation protocol. For each series
of experiments, we measured the trap amplitude, the granule amplitude,
and the phase of the oscillating granule relative to the trap. The trap
amplitude, a0, was measured by trapping an
extracellular granule or plastic bead and oscillating it at full laser
power in the medium to ensure that the displacement of the granule
faithfully represented the displacement of the center of the trap. The
a0 was taken as
half the peak-to-peak displacement. This calibration of
a0 was done before every sequence of intracellular granule measurement.
The intracellular granule amplitude
x0 was estimated,
as above, by half the peak-to-peak displacement. To determine the phase
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
of the granular motion relative to the trap, we note that the displacement of a granule when the trap crosses zero, denoted xz,
is given by
xz = x0 sin
(note
that for the granule lagging behind the trap motion,
< 0). The
xz
was estimated as one-half the difference of the displacement of the
granule at the two zero crossings of the trap per cycle. The phase was
then taken to be
We selected intracellular granules (or occasional aggregates) whose
diameters were ~0.6 µm (range 0.5-0.7 µm) for the study. These granules were trapped and oscillated by the optical tweezers with
an amplitude a0
of 0.5 µm and at frequencies of 0.3, 1, and 3 Hz. The experimental
runs were performed at the leading region where granules flowed in, the
main body not proximate to the nucleus, or the trailing region.
(1)
Estimating the elastic modulus G and viscosity
. The primary data, together with the force/length
characteristics of the optical trap described below, were then used to
compute the elastic modulus (or stiffness)
G and the viscosity
of the
cytoplasmic milieu, characterized as a simple viscoelastic material,
with parallel stiffness and viscous elements. The displacement of the granule lags behind the trap displacement, and therefore the
computation of the corresponding force is indirect, through the
combined granule plus trap system.
To this end, we first derive the equation of motion for the driven
granule. Let x and
a be, respectively, the time-dependent displacement of the granule and the trap center, both with respect to a
stationary laboratory coordinate system, the zeros of both being chosen
as the point about which the oscillations take place. The displacement
of the trap relative to the granule is thus
(a x); let the spring constant of the
trap (i.e., the force per unit length of displacement of granule
relative to the trap center, determined by calibration runs described
below) be denoted k. Let
G1 and
1 be, respectively, the uniaxial stiffness (force per
unit length of granule displacement) and damping (force per unit
granule velocity) of the cytoplasmic medium. There is a simple relationship between G1
and
1 and the desired material descriptors G and
described below. Because the
granule and the trap are mechanically in series, the force
(F) applied to the granule, given by the sum of the
elastic and viscous forces, is equal to the force applied by the trap
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(2) |
In the complex Fourier domain, the trap and granule displacements can be written as
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(3) |
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(4) |
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(5) |
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(6) |
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(7) |
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(8) |
To translate these uniaxial moduli into the more conventional material
moduli, where the elastic moduli and viscosity have units of
force/area and force × time/area,
respectively, we first note the simple relationship between force and
velocity in Stokes flow of a sphere of radius
r moving with velocity
u in a medium of viscosity : F = 6
ru. This is the same as writing
the damping force as F =
1
,
from which we make the immediate identifications
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(9) |
Calibrating the stiffness of the trap (i.e., the value of k90) can be easily accomplished using the same methodology as described above. This is important, since the stiffness of the trap depends on the optical properties of the trapped object, especially its index of refraction. This is not known for intracellular granules, but the force/length characteristic of the trap for granules actually used in the experiments can be measured directly. The calibration procedure is as follows and is similar to the method of Simmons et al. (15). If an extracellular granule (obtained from spontaneously lysed cell debris) is oscillated in RPMI 1640 medium, the same governing equations as above still apply. However, in this case, it is known a priori that the stiffness of the medium is zero and that the viscosity is essentially that of water, here taken to be 0.01 poise. Thirty-one measurements of amplitude decrement and phase delay were performed, with laser power ranging from 0.4 to 1.3 mW and frequencies from 0.1 to 1.5 Hz. Equations 7 and 8 can then be solved for the trap stiffness k90. The result of these measurements showed that, for 0.3-µm radius granules, k90 was given by 0.030 pN/nm.
Statistical analysis. Regional differences (body, leading region, and trailing region) and treatment differences (normal, cytochalasin D, and nocodazole) were assessed by two-way ANOVA. Statistical significance was assessed a priori at P < 0.05, but posterior significances were found to be much stronger.
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RESULTS |
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In all, successful measurements were made on >1,000
granules in >200 cells. The distribution of these measurements over
the three cellular regions (body, leading region, and trailing region) and the three treatment groups (control, cytochalasin D, and
nocodazole) is displayed in matrix form in Table
1.
|
Three frames from the video taken during the oscillation of a typical
granule are shown in Fig. 1. They show the
granule at its maximum positive displacement from the origin,
x0, its
displacement when the trap center crosses zero,
xz,
and its maximum negative displacement from the origin,
x0. Figure
1, top, shows the sinusoidally varying
trap displacement, the granule displacement, and the times at which the
images were taken. The granule amplitude is systematically less than
the trap amplitude, and the granular motion lags behind the trap.
|
In the body and trailing regions of the control group of cells, many
granules were so rigid that no observable oscillation took place;
searching was required to find granules that could be oscillated.
Granules whose amplitudes were so low that they could not be reliably
measured
(x0/a0 < 0.4 at maximum laser power) or were being dragged by intracellular
motions beyond the trap's strength were not included in the data
analysis (and are not part of the counts displayed in Table 1).
Inspection of the data revealed that very few granules had relative
displacements in the range of 0.4-0.6; they were either much too
stiff to be measured (the "fixed" population) or had
displacements typically in the range 0.6 < x0/a0 < 1.0 (the "free" population). The distribution of the percentage of
free granules over the three cellular regions and over the three
treatment groups is shown in Fig. 2. In the main body or trailing region of control neutrophils, the fractions of
granules displaying fixed behavior and free behavior were ~65 and
35% respectively. By contrast, the fixed fraction in the leading region regardless of treatment was <5%, implying an apparent absence of the fixed population in this region. Both cytochalasin D and nocodazole induced a reversal in the free and fixed populations; the
fixed fraction in the body and trailing region of cells treated with
cytochalasin D fell to <20% and with nocodazole fell to <40%. By
contrast to control conditions, in both treatment cases the population
of fixed granules was outweighed by the population of free granules.
|
As described in MATERIALS AND METHODS, the transfer
function can be used to estimate the uniaxial elastic modulus
G1 and viscosity 1. In Fig.
3A, we
show
Re(T
1
1) versus 1/k for all
measurements (means ± SE) on free granules in the separate regions
of the cell under control conditions. The constant of proportionality
between
Re(T
1
1) and 1/k, or slope of this
graph, is an estimate of the uniaxial elastic modulus
G1 (this comes
from the real part of Eq. 8). In Fig. 3B, we show the corresponding
data for
ImT
1
versus
/k, where the slope is an
estimate of the uniaxial viscosity
1 (from the imaginary part of
Eq. 8).
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Figure 4 shows a bar graph representation
(means ± SE) of the values of the material properties
G (Fig.
4A) and (Fig.
4B) for the three regions of the
cell and for the three conditions of control, treatment with
cytochalasin D, and treatment with nocodazole. These values were
obtained from the corresponding uniaxial values of Eq. 9. For both G and
,
there was no significant difference between the body and trailing
regions. Surprisingly, neither cytochalasin D nor nocodazole had any
effect on either G or
in these
regions. By contrast, both G and
are significantly lower in the leading region under all conditions when
compared with either the body or trailing region. Interestingly,
G and
in the leading region were
not significantly different between control and nocodazole treatment
conditions, whereas both G and
in
the cytochalasin D group (while less than the corresponding values in
the body or trailing region) were significantly higher than
G and
in the normal and nocodazole
groups. Morphologically, the neutrophils treated with cytochalasin D
also showed a marked drop in the speed of pseudopodial protrusion,
including occasional stoppages.
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DISCUSSION |
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There are three main issues addressed by these experiments on locomoting neutrophils: 1) the presence of fixed and free populations of granules; 2) regional differences (body, leading region, and trailing region) in intracellular stiffness and viscosity; and 3) differences in these rheological properties when the neutrophils are treated with either of the cytoskeletal disrupters cytochalasin D or nocodazole.
Fixed and free granules. In the body and trailing region of normal cells, there appeared to be a discontinuous distribution of granule properties; the granules behaved in effect as if there were two distinct populations. In the fixed granule population, trapped granules exhibited little if any oscillatory displacement, even at maximum laser power. In the free granule population, trapped granules could easily be oscillated. These observations are consistent with a discrete lattice structure to the cytoskeleton, at least in the body and trailing regions, with fixed granules mechanically or chemically bound to it and with free granules in the lattice interstices sampling the cytosolic component of the intracellular milieu. Our data suggest that the lattice spacing, or size of such granular "cages," would be at least about the magnitude of our oscillation amplitudes (1 µm peak to peak), since a smaller size would have prevented virtually any successful observations of oscillatory displacements. We cannot speculate on an upper bound for the cage size, since the population of fixed to free granules could be determined by biochemical factors independent of mechanical constraints. On the other hand, a cursory inspection of the Brownian motion of the granules (magnitude and mixing) suggests that the cages are probably not much larger than the above estimate. Future studies with variable oscillation amplitudes may shed light on this question.
The fraction of granules in the fixed population fell to essentially zero in the leading region. This is consistent with the idea that there is a marked depolymerization of the cytoskeletal structure at the site of and during the course of pseudopod protrusion.
It is known that there are at least four different types of granules in neutrophils (1) that in turn can be classified by whether they remain within the cell, performing intracellular lysosomal functions, or whether they are exocytotic or secretory. This observation invites two competing hypotheses. First, the fixed granule population may consist of the nonsecretory type, whereas the free granule population is of the secretory type. In this case, we speculate that the transport of the free secretory granules may be effected by directed Brownian motion while the nonsecretory granules remain fixed. This notion of directed Brownian transport would be a consequence of directed cytoskeletal remodeling that allows for granular transport without an active transport mechanism. By contrast, the converse hypothesis may be true, wherein the fixed granules are secretory in nature and are transported by molecular motors, and the nonsecretory granules remain free but caged.
Regional differences. First, we note
that, in all experiments, neither the stiffness nor the viscosity was
significantly different between the main body and the trailing region
of the cell. This is perhaps not surprising, to the extent that the
evolving dynamic activity is preferentially restricted to the region
near the protruding pseudopod. On the other hand, our observations of
significantly lower viscosity, and especially stiffness, in the leading
region have direct implications to the rheological nature of the
protruding pseudopod during locomotion. The fact that both
G and are lower in the leading
region implies that the pseudopodial core is more fluidlike. This is
inconsistent with the hypothesis that the core is a continually growing
body of polymerizing actin in compression, which would lead to a
relatively larger stiffness compared with the cell body. Rather, it is
more suggestive of simple fluid flow of the sol state of the cytoplasm,
driven presumably by intracellular pressure secondary to cortical
contraction. (See below for remarks on the rheological properties of
the core versus the pseudopodial tip.)
This interpretation is further strengthened by inspection of the
relaxation time constants. For the simple parallel viscoelastic description used in this work, the time constant , defined by
=
/G, is a convenient parameter that
describes the extent to which fluidlike behavior can be quantified.
Thus a purely elastic medium is characterized by
= 0, whereas a
purely fluid medium is characterized by
=
. We find, for the
body, trailing region, and the leading region, that
(body) = 0.34 s,
(trailing region) = 0.48 s, and
(leading region) = 1.71 s. The
observation that the rheological time constant is longer in the leading
region compared with the body and trailing region is then a
quantification of the statement that the leading region is more
fluidlike. Note that the rheological time constant is an independent
descriptor of the material. For example, honey is more fluidlike than
Jello, in this sense of time constant, despite having a larger
viscosity. Our experiments thus show that the leading edge of
locomoting neutrophils is more fluidlike in both the sense of a lower
viscosity and stiffness as well as in the shift of time constant along
the solid/fluid continuum.
Mechanisms responsible for pseudopod formation have been extensively investigated; some are still controversial, but all lean toward one or the other of two distinct hypotheses. One is that cortical contraction generates an increase in intracellular hydrostatic pressure or in gel osmotic pressure and associated solation, which causes a protrusion of the anterior region to form a pseudopod (9, 10, 11, 26). The other is that polymerization and cross-linking of actin at the anterior tip push the cell membrane forward as the pseudopod grows (5, 6, 12, 17). In support of the first hypothesis, contractility of the cortical layer in motile cells has been widely demonstrated. In vitro studies show that contraction coupled to solation occurs with changes in Ca2+ concentration or pH in a gel from amoebae extracts (9) or in an actin gel mixed with myosin and gelsolin (10). Histochemical colocalization of actin and myosin at the pseudopodial base in Dictyostelium amoebae (13) has been observed. Finally, cortical contraction is consistent with direct measurements of intracellular pressure in Amoeba proteus (25).
Support for the second hypothesis has largely come from histochemical studies showing that heavy condensation of F-actin exists in the lamellipodia of D. amoebae (6), fibroblasts (18), fish keratocytes (20), or neutrophils especially chemotactically stimulated (2). Both of these modes of pseudopod protrusion may exist in amoeboid cells; which mechanism is involved in any given circumstance may depend on the cell type or conditions of chemotactic induction.
The leading region itself may also be regionally heterogeneous, particularly the streaming cytoplasmic core versus the pseudopodial tip. All of our stiffness and viscosity measurements in the leading region were done at sites where intracellular granules flowed into the lamellipod. The absence of granules at the very tip of the pseudopod prevented any rheological measurements there, and thus we cannot compare the tip with the core. At least two possibilities may be suggested. First, continued pseudopodial growth may be associated with simple pressure forces, even at the pseudopodial tip, which is supported by evidence of decreased F-actin in the protruding pseudopod (4, 11, 14). Second, even with a fluid core, the pseudopod may grow through actin polymerization at its tip, being further anchored by the cytoskeleton to the substrate. This is supported by observations of increased pseudopodial F-actin (16, 17, 23). Which of these mechanisms underlies pseudopodial protrusion is no doubt cell type dependent and remains open.
Finally, there are a number of other experimental techniques by which to assess rheological properties of cells. We cannot make any direct comparisons at this time, however, because, unlike previous methods, the work reported here estimates intracellular stiffness and viscosity in different regions within individual living and locomoting cells. By contrast, rheological measurements made with cell aspiration into a micropipette (8) involve large-scale distortions of the entire cell and do not distinguish among different cell regions. Estimates of stiffness (21, 22) by magnetic twisting cytometry are restricted to large populations of cells and represent a weighted average of any regional differences in how ligand-coated beads bind to the cell membrane. Cell poking experiments are done on single cells (7, 24), but the separate assessment of stiffness and viscosity between the body or trailing region on the one hand, and the leading region on the other, appears to be technically difficult.
Differences in rheological properties with cytochalasin D or nocodazole treatment. Cytochalasin D and nocodazole are known to disrupt the cytoskeleton through inhibition of polymerization of the filamentous actin and microtubules, respectively. These compounds have been used extensively in investigations of the role played by the cytoskeleton in cell mechanics (3, 24) and in the identification of receptor binding to the cytoskeleton (21, 22). In all such experiments, the cell stiffness decreases with treatment with either drug. In sharp contrast to these observations, we found no differences in either stiffness or viscosity in the body or trailing regions of neutrophils with or without treatment with either cytochalasin D or nocodazole.
How might these apparently contradictory observations be reconciled? Recall that, in the body and trailing region of control cells, there appeared to be in effect two distinct populations of granules, one fixed and the other free. Despite the lack of difference seen between the rheological properties shown by free granules in the control and in the cytochalasin D or nocodazole treatment group, one striking difference did emerge. Specifically, the fraction of fixed granules was substantial (65%) in the body and trailing regions of the control group and fell sharply in the two treatment groups to <20% and <40%, respectively.
These observations, taken together, suggest the following interpretation. In control cells, there is a free granular population that interacts only weakly with the cytoskeleton, perhaps only through restriction of large-scale displacements. Similarly, there is the complementary fixed population that interacts strongly with the cytoskeleton, through chemical or mechanical interactions. If the only effects of cytochalasin D and nocodazole are on the extent of polymerization of the F-actin and microtubule components of the cytoskeleton, then one might expect that the sole effect of drug treatment would be a sharp decrease in the fraction of fixed versus free granules and that, furthermore, the free granules in the control case would exhibit the same rheological properties as in the drug treatment groups. This is precisely what we observed. By contrast, other techniques, such as cell aspiration, involve large-scale deformation of the entire cell. It is not surprising, therefore, that they should show sharp decreases in stiffness when the actin filaments or microtubules are disrupted; these observations are thus consistent with our own, following the above interpretation.
In contrast to our observations in the body or trailing regions, we observed no change in the stiffness or viscosity in the leading region with nocodazole treatment. This suggests few microtubules in the flowing core of the pseudopod, or at least a much diminished biochemical and mechanical interaction with pseudopodial granules. This is consistent with a flowing fluid core and is supported by histochemical studies (13). On the other hand, with cytochalasin D treatment, both stiffness and viscosity (while less than in the body or trailing region) in fact increased over values seen common to both control and nocodazole groups. The origin of this surprising result is less clear, but some speculative ideas deserve mention. The network of F-actin is almost certainly necessary for any development of intracellular pressure. To the extent that an increased intracellular pressure is necessary to drive pseudopodial protrusion, any disruption of the network, such as an assembly inhibition by cytochalasin D, would imply a corresponding drop in intracellular pressure. This in turn would decrease the normal separation of fluid from the cytoskeleton as the protrusion progresses, and F-actin fragments [similar to the phenomena reported by Safiejko-Mroczka and Bell (14)] could be dragged into the much more slowly developing pseudopod seen in cytochalasin D-treated cells. Indeed, even if intracellular pressure were unchanged, the presence of short F-actin polymers flowing into the pseudopod would increase the apparent stiffness and viscosity compared with control or nocodazole treatment conditions. We must emphasize that these ideas remain speculative, insofar as we did not measure the F-actin concentrations in any of these experiments and cannot yet conclude that cortical tension and increased intracellular pressure are the causal agencies effecting pseudopodial protrusion.
In conclusion, we have found that, first, in normal locomoting neutrophils, the intracellular granules appear to behave as two distinct populations. In the body and trailing region of the cell, those in the majority fixed population were stiffer than can be measured by displacements with a 90-mW optical trap, whereas those in the minority free population were easily oscillated. This was in striking contrast to the virtual absence of any fixed granules in the leading region of the neutrophils. Second, the free granules in the pseudopodial core showed a substantial drop in both stiffness and viscosity when compared with the body or trailing regions (the body and trailing region were not significantly different). Third, neither cytochalasin D nor nocodazole caused a significant drop in stiffness or viscosity in the body or trailing region of the cell. Taken together, these results suggest the presence of a discrete filament lattice to which the fixed granules are bound, within which the free granules sample the cytosolic phase and which is essentially absent in the flowing core of a protruding pseudopod. Furthermore, the uniform drop in stiffness and viscosity measured with free granules in the pseudopod strongly supports the hypothesis that the pseudopodial core is more fluidlike than the cytosolic component of the body or trailing region of the neutrophil and that the pseudopod does not protrude secondary to a continuously polymerizing actin assembly throughout the entire pseudopod.
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ACKNOWLEDGEMENTS |
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We are grateful to C. M. Doerschuk for very helpful advice, suggestions, and critique of this work.
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FOOTNOTES |
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This study was partly supported by grants provided by the Ministry of Education, Japan (nos. 08559015 and 10670529), and by National Heart, Lung, and Blood Institute Grant HL-33009.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Yanai, Dept. of Geriatric and Respiratory Medicine, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan (E-mail: myan{at}geriat.med.tohuku.ac.jp).
Received 8 February 1999; accepted in final form 21 May 1999.
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