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Vol. 14, Issue 5, 2016-2028, May 2003
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Department of Neurobiology, Pharmacology, and Physiology, University of Chicago, Chicago, Illinois 60637
Submitted September 26, 2002;
Revised December 16, 2002;
Accepted January 30, 2003
Monitoring Editor: Vivek Malhotra
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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amphiphysin-SH3 domain, but not a mutated form
that cannot bind to dynamin, inhibited both focal exocytosis and phagocytosis.
Immunochemical analysis of endogenous dynamin distribution in macrophages
revealed a substantial particulate pool, some of which localized to a
presumptive endosomal compartment. Expression of enhanced green fluorescent
proteindynamin-2 showed a motile dynamin pool, a fraction of which
migrated toward and within the phagosomal cup. These results suggest that
dynamin is involved in the production and/or movement of vesicles from an
intracellular organelle to the cell surface to support membrane expansion
around the engulfed particle. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Greenberg,
1999). This process has been extensively studied in macrophages
and involves complex signaling mechanisms initiated by engagement and
clustering of cell surface Fc
or complement C3 receptors
(Kwiatkowska and Sobota, 1999;
Greenberg, 2001).
Internalization after engagement of these two types of receptor is strikingly
different; in the case of Fc-mediated phagocytosis pseudopodial,
extensions reach out from the cell surface to envelop the particle, whereas in
C3-mediated phagocytosis particles seem to "sink in" to the cell
(Kaplan, 1977). One hallmark
of Fc-mediated phagocytosis is the central role played by actin
polymerization in driving the extension of the phagocytic "cup"
around the ingested particle (May and
Machesky, 2001). Coincident with this event is the postulated
insertion of new membrane at the sites of pseudopodial extension, presumably
to accommodate the growing actin filaments (for review, see
Booth et al., 2001).
This process has been named "focal exocytosis"
(Bajno et al., 2000),
a terminology we retain herein to distinguish it from other secretory
mechanisms in phagocytes (Di et
al., 2002).
Many proteins participate in phagocytosis, several of which are involved in
signaling cascades from the Fc receptor, or in the regulation of actin
assembly. Recently, the large GTPase dynamin-2 was implicated in phagocytosis
(Gold et al., 1999).
Introduction of dominant-negative dynamin into macrophages blocked particle
internalization and overexpressed dynamin was found to localize to phagocytic
cups. Dynamins constitute a family of proteins thought to be involved in
fission reactions at various membranes, including both rapid endocytosis and
receptor-mediated endocytosis at the plasma membrane, as well as transport
vesicle formation in the trans-Golgi network and possibly at
endosomal membranes (for review, see
McNiven et al.,
2000). Yet other functions have been attributed to this protein,
including a role in actin organization at podosomes as well as in the actin
"comets" that may power organelle and bacterial motion around the
cytoplasm (Orth et al.,
2002) and as an element in signal transduction cascades
(Fish et al., 2000).
A natural supposition would be that dynamin might regulate the final pinch-off
reaction that leads to internalization of the phagosome in stimulated
macrophages. However, the point of blockade with dynamin-2 dominant-negative
mutants in macrophages seems to be before the completion of the phagocytic cup
(Gold et al., 1999)
(interestingly, similar dynamin-1 dominant negatives were recently reported to
be inactive in the same assay; Tse et al., 2002). Potentially,
dynamin might modulate either focal exocytosis or the organization of actin
filaments during the early stages of particle engulfment, or it could possibly
act as a coordinating molecule regulating both aspects of pseudopodial
extension.
We recently used the electrophysiological technique of membrane capacitance
measurement to analyze exocytosis and endocytosis events in macrophages
(Holevinsky and Nelson, 1998;
Di et al., 2001,
2002). Phagocytosis can be
accurately captured in real-time by using this method, making it suitable for
kinetic analysis of processes contributing to particle uptake. Indeed, unitary
step decreases in capacitance are related to the size of the ingested
particles, and the technique can also be used to analyze the rate and
magnitude of secretion in these cells (Di et al.,
2001,
2002). An interesting feature
of these experiments was the occurrence of a rapid rise in Cm just before the
decreases characteristic of membrane internalization
(Holevinsky and Nelson, 1998).
We interpreted this increase as insertion of vesicular material into the
surface membrane, most likely by exocytosis. Such an observation of exocytosis
accompanying a phagocytic stimulus may be the electrophysiological signature
of the focal exocytosis phenomenon alluded to above. Other techniques also
reveal an increase in cell surface area that correlates with pseudopodial
extension in phagocytosing macrophages
(Cox et al., 1999;
Bajno et al., 2000).
Apart from its excellent temporal resolution, the whole-cell patch-clamp
technique has the added advantage that the intracellular milieu can be
precisely controlled via the patch pipette. For example, it is easy to set the
level of free cytoplasmic Ca2+
([Ca2+]i) by the use of buffers in the
pipette, and other small mediators can be added and deleted at will.
Antagonism of specific proteins by using antibodies and peptides can also be
readily achieved. Using this approach, we have addressed whether focal
exocytosis is a Ca2+- and GTP-dependent process and
whether it is dependent on actin polymerization in this study. To evaluate the
role of dynamin in this process, we introduced affinity-purified IgGs and
other reagents directly into macrophages. We find that dynamin antagonism
blocks exocytosis and subsequent phagocytosis, suggesting that the protein is
likely involved in the generation or insertion of exocytotic vesicles needed
for the elaboration of the phagocytic cup. In complementary studies, we show
that dynamin-2 localizes to an intracellular presumptive endosomal compartment
and becomes transiently associated with the incipient phagosome in a manner
consistent with local signaling from successively occupied Fc
receptors.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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); cells were collected and washed in Tris-buffered saline
then plated onto coverslips in DMEM/10% fetal bovine serum. After 30 min,
wells were washed twice with medium to reduce contaminating cells (largely
erythrocytes); adherent cells were used within 3 d of isolation. The mouse
J774 and mouse RAW264.7 macrophage cell lines were propagated as described
previously (Di et al.,
2001). Plasmids encoding fusion proteins of glutathione
S-transferase (GST)amphiphysin (amph)-I SH3 domain and its
mutant variant (Grabs et al.,
1997) were obtained from Dr. P. De Camilli (Yale University, New
Haven, CT), expressed in bacteria and purified by glutathione affinity
chromatography. We confirmed in binding studies that the wild-type domain
could bind dynamin-2, whereas the mutant domain was inactive in this regard
(our unpublished data). Anti-pan-dynamin antibodies were prepared in rabbits
to a conserved sequence in the G-domain by QBC (Hopkinton, MA), the IgGs were
affinity purified by sequential protein A-agarose and peptide affinity column
chromatography. All proteins were centrifugally dialyzed against internal
pipette solution before introduction into cells. Other reagents were obtained
from the following sources: wortmannin (LC Labs, Woburn, MA), herbimycin A
(Invitrogen, Carlsbad, CA), LY294002 (BIOMOL Research Laboratories, Plymouth
Meeting, PA), cytochalasin D, guanosine
5'-O-(2-thio)diphosphate (GDP
S) (Sigma-Aldrich, St.
Louis, MO), and latrunculin A (Calbiochem, San Diego, CA). A C-terminal
truncated recombinant version of Rho-GDI was obtained from Dr. Z. Derewenda
(Department of Molecular Physiology and Biological Physics, University of
Virginia, Charlottesville, VA). Human aggregated IgG (HAIGG) was prepared as
described previously (Di et al.,
2001).
Electrophysiology
Our patch-clamp capacitance methods for macrophages were as described
previously (Di et al.,
2001,
2002) and are only summarized
herein. Whole-cell capacitance recordings were obtained using an EPC-9/2
computer-controlled patch-clamp amplifier running PULSE software (HEKA
Electronik, Lambrecht, Germany). The EPC-9 includes a built-in data
acquisition interface (ITC-16; InstruTECH, Port Washington, NY). The software
package controlled stimulus delivery and data acquisition for the lock-in
amplifier in the "sine + dc" mode as described by Gillis
(2000). The temporal
resolution of the capacitance data was 40 ms/point using a 1-kHz, 20-mV sine
wave. The holding potential in the capacitance experiments was –10 mV.
Standard recording solutions were 80 mM K aspartate, 40 mM KCl, 2 mM
MgCl2, and 10 mM HEPES (pH 7.2) in the pipette with MgATP (1
mM) and MgGTP (0.3 mM) added as needed;
[Ca2+]i was buffered with EGTA to different
values as before (Di et al.,
2001). The standard bath solution contained 100 mM NaCl, 50 mM
KCl, 2 mM MgCl2, 2 mM CaCl2 and 10 NaHEPES (pH
7.4). All recordings shown herein were obtained at 37°C unless indicated
in the text. The temporal resolution of the fluorescence data was 20 ms/point.
Data was acquired using a Pentium3 PC and analyzed off-line by using the
integrated graphics package IGOR Pro (WaveMetrics, Lake Oswego, OR). All
quantitation of stimulus-induced changes in membrane capacitance were
determined as the difference between the steady-state attained after a 10-min
equilibration period and the subsequent peak of increase or decrease after
stimulation. Superoxide release was determined electrochemically using the
EPC-9/2, which allows simultaneous amperometric current and capacitance
measurements in single voltage-clamped cells
(Di et al., 2002).
Insulated polypropylene 5-µm-diameter carbon fibers (Dagan, Minneapolis,
MN) were directly connected to the headstage of the amplifier. The electrodes
were placed within 1 µm of the surface of the J774 cells used for these
assays and oxidation current was determined with 120 mV applied to the fiber.
Total amperometric charge was calculated using the automated event detection
program events written for IGOR by Segura et al.
(2000).
Immunocytochemistry
Alveolar macrophages, J774 or RAW 267.4 cells were plated onto coverslips
and cultured overnight before fixation in 4% paraformaldehyde in Hanks'
balanced salt solution for 20 min at room temperature. Cells were blocked and
permeabilized in PBS containing 0.25% fish skin gelatin plus 0.01% saponin
with 0.2% NaN3. To visualize dynamin-2 and various markers, fixed
cells were exposed to primary IgGs as follows: dynamin-2 polyclonal antibody
(pAb) (1:1000; Oncogene Research, Cambridge, MA) or MC-63 anti-pandynamin pAb
(0.5 µg/ml; kind gift of Mark McNiven, Mayo Institute); TGN-38 monoclonal
antibody (mAb) (1:100; Transduction Laboratories, Lexington, KY); giantin mAb
(1:100; kind gift of Adam Linstedt, Carnegie Mellon University, Pittsburgh,
PA); EEA-1 mAb (1:100; Transduction Laboratories); and clathrin heavy chain
mAb X-22 (1:100; Affinity Bioreagents, Golden, CO). In general, overnight
incubation in primary antibody at 4°C was used; Alexa 488-coupled goat
anti-rabbit secondary antibodies (Molecular Probes, Eugene, OR) were used to
visualize dynamin, whereas rhodamine- or Alexa 633-coupled secondaries were
used to visualize other antigens (1-h incubation at room temperature). To
label the endosomal system, we incubated cells at 37°C for various times
in either Cy5-transferrin (50 µg/ml; Jackson Immunoresearch Laboratories,
West Grove, PA) or lysine-fixable rhodamine-dextran (1 mg/ml;
Mr 70,000; Molecular Probes), taken up by
receptor-mediated and fluid phase endocytosis, respectively. In some
experiments, fixed and permeabilized cells were treated with
rhodamine-phalloidin (165 nM; Molecular Probes) to visualize polymerized
actin. For bead uptake experiments, cells were exposed to either unlabeled or
rhodamine-labeled latex beads (Spherotech, Libertyville, IL) that were
IgG-coated, or opsonized zymosan particles (Molecular Probes). Cells were
examined in an IX70 Fluoview confocal microscope (Olympus, Tokyo, Japan);
images were captured and digitized with Fluoview software then processed using
Image J software (National Institutes of Health) and Adobe Photoshop.
Preparation and Expression of GFPDynamin-2
A plasmid encoding rat dynamin-2 (bb form) was obtained from Drs. P.
Okamoto and R.B. Vallee (University of Massachusetts Medical School,
Worcester, MA; Okamoto et al.,
1997). The coding sequence was excised and inserted in-frame with
the sequence for enhanced green fluorescent protein (EGFP) at the N terminus
by using the pEGFP-C2 vector (BD Biosciences Clontech, Palo Alto, CA). The
sequence of the resultant plasmid pEGFP-N-dynamin-2 was verified and
functional expression was tested by transient transfection into human
embryonic kidney 293 cells. A protein of the appropriate size (
140 kDa)
was detected by immunoblotting (our unpublished data). RAW 264.7 cells were
transiently transfected with the same vector by using Superfect (QIAGEN, Santa
Clara, CA; 5-h incubation), resulting in 25% transfection efficiency as
judged by counting fluorescent cells 24–48 h later. At this time,
coverslips containing transfected cells were exposed to unlabeled opsonized
latex beads (0.8 or 3 µm in diameter) or zymosan particles and allowed to
internalize for periods of 10 min to 1 h. Coverslips were fixed and
counterstained with rhodamine-phalloidin then analyzed by confocal microscopy
as described above.
EGFP-dynamin-2 K44A was prepared by point mutation of the appropriate base in pEGFP-N-dynamin-2 by using the Quick-Change system (Stratagene, La Jolla, CA). The product was verified by sequencing and expressed in RAW 264.7 cells exactly as described above for the wild-type vector. Cells were incubated 24 h for expression and then subjected to patch-clamp capacitance recording as described above, after location of expressing cells by fluorescence, or to bead uptake followed by microscopic analysis.
Online Supplemental Material
Several videos and confocal image stacks showing details of dynamin
localization in macrophages are included in supplemental material accessible
via the MBC Web site.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Artalejo et al.,
1995). Our previous experiments showed that macrophages exhibit a
rapid rise in Cm before the fall in surface area concomitant with particle
ingestion (Holevinsky and Nelson,
1998). This can be interpreted as a cycle of exocytosis followed
by pinch-off of the phagosome, as illustrated in
Figure 1. To evaluate the
generality of this pattern, we examined the phenomenon in three types of
macrophage in response to IgG-bead challenge. As shown in
Figure 2, rat primary alveolar
macrophages as well as the murine J774.1 and RAW 264.7 cell lines all
exhibited rapid rises in Cm (peaking in <2 min) that preceded bead
internalization, reflected as a stepwise decrease in Cm. It is likely that
some overlap between focal exocytosis and phagocytosis occurs in such
recordings, as also exists in neuroendocrine secretion
(Artalejo et al.,
1995), resulting in some attenuation of the Cm rise. The time
course of this event is in keeping with the kinetics of phagocytosis
determined morphologically. For example, in the synchronous phagocytosis of
prebound erythrocytes investigated by Diakanova et al. (2002) in bone
marrow macrophages, formation of the phagocytic cup occurred within
10–20 s after stimulation. The increase in cell surface area represents
a significant enlargement of up to 30% in total cell surface at peak (for
baseline values, see legend to Figure
2). These results suggest that exocytosis generally precedes
phagocytosis in macrophages and may contribute to the formation of the
phagocytic cup.
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Exocytosis before Phagocytosis Is Ca2+ Independent but Is
Blocked by GDPS
Exocytosis in several systems is regulated by
[Ca2+]i and in some hematopoietic cells also
by GTP (sometimes in concert with [Ca2+]i).
We, therefore, tested whether focal exocytosis was dependent on either of
these two effectors. As shown in Figure 3,
A and B, a rapid increase in Cm was observed when cells contained
[Ca2+]i close to "normal" values
(40 nM in the patch pipette, rising to 300 nM on IgG stimulation) or when
[Ca2+]i is reduced to vanishingly low levels
by using the chelator BAPTA (10 mM; [Ca2+]i
<1 nM) (Di et al.,
2001). A striking difference between the two conditions was that
in normal [Ca2+]i phagocytosis occurred, as
reflected in the decrease in Cm, whereas in low
[Ca2+]i phagocytosis was blocked. Global
secretion in macrophages has been shown to require GTP and to be stimulated by
guanosine 5'-O-(3-thio)triphosphate and inhibited by GDPS
(Di et al., 2002). To
determine whether focal exocytosis is dependent on G proteins, we introduced
GDPS intracellularly before stimulation with IgG-coated beads.
GDPS completely blocked the initial Cm rise in normal
[Ca2+]i, suggesting that G proteins are
essential to focal exocytosis (Figure
3C). We did not use GTPS in this study because it induces
secretion of other intracellular vesicles that would contaminate measurements
of focal exocytosis (Di et al.,
2001).
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Focal Exocytosis Is Blocked by Compromising Tyrosine Phosphorylation
or Actin Polymerization but Not by Phosphatidylinositol 3 (PI3) Kinase
Inhibition
Signaling from Fc receptors requires the participation of various
cytoplasmic tyrosine kinases including Src-family members and Syk (Greenberg,
1999,
2001). Phagocytic cups
accumulate phosphotyrosine immunoreactivity and blockade of tyrosine
phosphorylation inhibits phagocytosis, but it is not known whether tyrosine
phosphorylation is required for focal exocytosis. In one study, it was shown
that pseudopod extension in fibroblasts transfected with various Fc
receptors did not require the presence of tyrosine activation motifs in the
cytoplasmic tail (Lowry et al.,
1998). However, we found that preincubation of J774 cells with the
inhibitor herbimycin A, a potent blocker of several tyrosine kinases and
phagocytosis, inhibited both the initial rise and subsequent fall in Cm,
suggesting that tyrosine phosphorylation is required for focal exocytosis (our
unpublished data). Phagocytosis is crucially dependent on actin
polymerization, initiated by the activation of Rho-family proteins, mediated
in part through Fc-mediated tyrosine phosphorylation of the Rho-GEF Vav
(Caron and Hall, 1998).
Although actin assembly has been thought to provide structural integrity to
the growing pseudopod, it has not been determined whether it is also involved
in focal exocytosis. We blocked actin polymerization by using three different
approaches: cytochalasin D (caps barbed ends), latrunculin A (binds actin
monomers), and introduction of the Rho-family inhibitor Rho-GDI (blocks all
Rho protein actions) via the patch pipette into the cell interior. All three
strategies totally prevented the typical Cm changes associated with IgG-bead
addition (Figure 4).
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Another prominent response to the occupation of Fc receptors is the
activation of PI3 kinase (Ninomiya et
al., 1994). Agreement exists that interference with PI3
kinase activity blocks phagocytosis, but there is debate about the phase of
phagocytosis that is affected, with some claiming an effect on an early stage,
whereas others posit an effect at a stage close to phagosome closure
(Araki et al., 1996;
Cox et al., 1999). To
investigate this question in our paradigm of focal exocytosis, we treated
cells with the PI3 kinase antagonists wortmannin and LY294002. Both were found
to leave increases in Cm untouched but to block the subsequent decline
(Figure 4). These results
suggest that PI3 kinase is not involved in the early stage of vesicular
production and fusion, but may well be required for later steps that result in
scission of the phagosome from the cell surface.
Focal Exocytosis Is Blocked by Anti-Dynamin-2 IgGs and
Amphiphysin-Src Homology 3 (SH3) Domain, but Not by a Mutant Amphiphysin-SH3
Domain
Having demonstrated that the capacitance technique provides considerable
insight into the initial stages of phagocytosis, we proceeded to investigate
the role of the large GTPase dynamin. To determine whether the protein is
involved in focal exocytosis, we introduced affinity-purified anti-pandynamin
IgGs into J774 macrophages and allowed 10 min for equilibration before
stimulation with IgG beads (Figure
5B). We have shown previously that a similar strategy abolishes
rapid endocytosis in chromaffin cells
(Artalejo et al.,
1995). Both the early increase in Cm, as well as the later
decrease representing phagocytosis, were blocked by this procedure; preimmune
IgG was inactive, suggesting that the effect was specific (our unpublished
data). The antibody itself had no discernible influence on baseline
capacitance in cells not exposed to beads, over periods comparable with those
used in the present study. This suggests that dynamin inhibition in the short
term does not affect some ongoing constitutive endocytotic process that might
contribute to subsequent phagocytosis in macrophages. Dynamin has been
reported to interact with the protein amphiphysin in receptor-mediated
endocytosis, and an isoform of amphiphysin-II is present in macrophages and is
involved in phagocytosis (Gold et
al., 2000). Amphiphysin links to the proline-rich C terminus
of dynamin via its SH3 domain, and GSTamph-SH3 domain constructs can
block receptor-mediated endocytosis in fibroblasts and neurons
(Shupliakov et al.,
1997; Wigge et al.,
1997). Introduction of GSTamph-SH3 protein into macrophages
blocked focal exocytosis and phagocytosis in a manner similar to anti-dynamin
IgG (Figure 5, C and E).
Interaction with dynamin has been localized to a key sequence of six amino
acids in amph-SH3; mutation of two of these residues ablates binding to
dynamin and destroys the dominant negative behavior of the domain in vivo
(Grabs et al., 1997).
Introduction of this mutated domain into macrophages had no effect on
IgG-bead–induced exocytosis or phagocytosis
(Figure 5, D and E), indicating
that the effects of the wild-type domain are specific. Finally, we tested the
effects of expressing a mutant dynamin that acts as a dominant-negative in
other systems (van der Bliek et
al., 1993). Previous studies showed that ectopic expression
of dominant-negative dynamin-2 in phagocytes inhibited particle
internalization, probably at a stage before final closure of the phagocytic
cup (Gold et al.,
1999). In agreement with this study, we found that overexpression
of EGFP-dynamin-2 K44A in RAW 264.7 macrophages blocked Cm changes
characteristic of focal exocytosis and phagocytosis
(Figure 5F) and internalization
of fluorescent latex beads (our unpublished data). These studies place the
effects of dynamin antagonism clearly at an early stage in the phagocytic
process, rather than simply in final pinch-off of the internalized
particle.
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To further assure the specificity of these effects on focal exocytosis, we
studied the influence of anti-dynamin IgGs on other types of exocytosis in
macrophages. Recently, we have shown that phagosomes in cells loaded with
opsonized latex beads could be induced to undergo secretion upon a second
stimulation by HAIGG (Di et al.,
2002). This type of secretion can be observed
electrophysiologically as a stepwise increase in Cm after HAIGG challenge of
cells preloaded with IgG beads, as well as a quantal release of superoxide
anions into the extracellular space that can be detected by amperometry
(Di et al., 2002)
(Figure 6A). Introduction of
anti-dynamin IgG (Figure 6B) or
GST-amphiphysin-SH3 (our unpublished data) into bead-loaded cells
significantly reduced, but did not abolish, exocytosis under these conditions.
In this situation, exocytosis is likely a mixture of rapid focal exocytosis
(<2 min) and slower release of granular material, including phagosomes
(>2 min). Anti-dynamin IgG seemed to block the first phase while leaving
the second phase intact as revealed by time-to-peak analysis
(). That
this interpretation is likely correct was supported by the fact that the IgG
had no effect on superoxide release, which we previously showed is due to
phagosomal secretion (Figure 6, A and
B, bottom; D).
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Intracellular Localization of Endogenous Dynamin-2 in
Macrophages
If dynamin is involved in focal exocytosis then it may well localize to and
be a marker for those intracellular membranes that provide vesicular material
for pseudopodial extension in macrophages. Immunoblotting studies confirmed
that macrophages express only dynamin-2 (our unpublished data; cf.
Gold et al., 1999),
and subcellular fractionation showed that dynamin is localized for the most
part to a postnuclear particulate fraction in J774 macrophages
(Figure 7A). Confocal
microscopy revealed that dynamin-2 staining in J774 or primary alveolar
macrophages is punctate and throughout the cell
(Figure 7, B and C). These
results resemble the distribution of dynamin-2 seen by others in epithelial
cells (Cao et al.,
1998; Nicoziani et
al., 2000). Only a minor pool of dynamin seemed to colocalize
with peripheral polymerized actin ruffles in resting cells
(Figure 1, supplemental data).
Some dynamin was found in the trans-Golgi network, prominently
labeled in some other cell types (Cao
et al., 1998), as evidenced by colocalization with TGN-38
(Figure 7D), but dynamin did
not overlap with the cis-Golgi marker giantin
(Figure 2, supplemental data).
Dynamin-2 also showed minimal overlap with clathrin at the plasma membrane
(our unpublished data) or with the early endosome marker EEA1
(Figure 7E). To ascertain
whether dynamin was located in part to an endosomal compartment, we allowed
macrophages to internalize dye-labeled transferrin or dextran. Significant
overlap was found between the dextran and dynamin-2 signals (Figures
3, supplemental data; and
7F). Little dynamin-2 was found
associated with transferrin at early times (1 min) after internalization
(Figure 7G), but at later times
(10 min) when transferrin should have permeated the entire endosomal system,
we found significant overlap of the two signals, especially evident toward the
middle of the cell in confocal sections (Figures
4, supplemental data; and
7H). These results suggest that
dynamin-2 associates with a part of the endosomal network in macrophages, as
is true in other cells (Nicoziani et
al., 2000; van Dam and
Stoorvogel, 2002). We then localized dynamin-2 in cells ingesting
fluorescent IgG-coated beads. In favorable circumstances, we were able to
observe proximity between the dynamin-2 signal and the periphery of the bead,
marked by an accumulation of polymerized actin. Nevertheless, most of the
dynamin remained associated with punctate cytoplasmic structures under these
conditions (Figure 7I).
|
Dynamin-2 Migrates toward Sites of Particle Internalization and
Transiently Associates with the Phagocytic Cup
To visualize the kinetic behavior of dynamin-2 during phagocytosis, we
expressed an EGFP-tagged version of dynamin-2 in RAW macrophages and studied
the disposition of the protein after IgG-bead challenge. Overexpression of
this construct, even to high levels, did not seem to compromise particle
internalization in these cells. As shown in selected fixed images
(Figure 8A) accumulation of
dynamin-2 around the bead periphery could be seen, colocalizing to some extent
with polymerized actin, visualized with rhodamine-phalloidin. Comparison of
this result with that found in untransfected cells
(Figure 7I) suggested that the
ectopic version of the protein behaved quite similarly to endogenous
dynamin-2. Time-lapse studies showed that the punctate
dynamin-2–containing structures were motile and transiently associated
with large membrane ruffles (supplemental data, Movie 1). Moreover, when cells
were challenged with opsonized beads or zymosan, EGFP-dynamin-2 transiently
accumulated in the phagocytic cup (Figure
8B; supplemental data, Movie 2); sequential rounds of dynamin
recruitment could be seen in cells ingesting more than one particle
(supplemental data, Movie 4 and corresponding intensity profiles in Movie 5).
These results suggest that a pool of dynamin-2 is mobilized near internalizing
beads. That this is likely a transitory event during the formation of the
phagosome is illustrated in Figure
8C, where two internalized zymosan particles are seen to lack
significant peripheral EGFP-dynamin-2 accumulation (see also three-dimensional
reconstruction in supplemental data, Movie 6). Close examination of dynamin
behavior during the internalization process revealed an intensity pattern that
seemed to progress around the captured particle (supplemental data, Movies 2,
4, and 5). Moreover, strands of fluorescent material apparently
"peel" away from the cup after internalization (supplemental data,
Movie 3), suggesting that dynamin is dissociating from the base of the
phagosome while still being recruited to the tip. These results are consistent
with an active role for dynamin throughout particle internalization and
support the "zipper" model of signaling during Fc-mediated
phagocytosis (see DISCUSSION).
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Although the membrane trafficking events contributing to pseudopodial
extension in Fc-mediated phagocytosis have come under increased
scrutiny, no definitive conclusions have been reached. Local insertion of
internally derived vesicles might be required for surface membrane expansion
to accommodate the developing phagocytic cup. In the present work, we show
that such addition of membrane to the macrophage cell surface can be appraised
in real time by using capacitance measurements. This technique has several
advantages over other methods, having particularly high resolution in the time
domain along with the ability to follow the entire cycle of
exocytosis-endocytosis with a single recording. Other methods, like
dye-labeling with FM1-43, for example (Cox
et al., 1999; Bajno
et al., 2000), do not reveal the complex kinetics of both
focal exocytosis and phagocytosis. Our results support the contention that
rapid plasma membrane expansion, seen as a rise in Cm, is an essential prelude
to particle internalization. All three macrophage types surveyed displayed the
increase, and maneuvers that blocked the Cm rise also ablated the subsequent
Cm fall characteristic of particle internalization. At present, it is not
possible to state with certainty whether the increase in Cm represents the
fusion of many small vesicles or lesser numbers of larger structures with the
plasma membrane. In most cases, we observed a fairly smooth increase in Cm
with little indication of step-like events, whereas in some instances
spike-like increases in Cm were seen that might be indicative of sudden
merging of larger cytoplasmic structures with the cell surface. A recent study
showed that elements of the endoplasmic reticulum could fuse with macrophage
plasma membrane at the site of enveloping phagosomes, resulting in deposition
of endoplasmic reticulum markers in the phagosomal membrane
(Gagnon et al.,
2002). The occasional recording of sudden large jumps in Cm (see
Figure 1B in particular) might
reflect such an event and is worthy of further analysis. It is interesting to
speculate on how many vesicles would be needed to give rise to the magnitude
of signals we commonly see (1–4 pF). If vesicular dimensions were
comparable with synaptic vesicles (50 nm; 70 aF) from 30,000 to
120,000 would be needed, but only 250-1000 vesicles of a size similar to
chromaffin granules (300 nm; 2.5 fF) would suffice. Evidently, the former
number is prodigious, whereas the latter seems manageable. It is likely that
the truth lies somewhere in between. Cm measurements also do not indicate the
location of vesicular insertion in the plasma membrane. It will be necessary
to use various membrane- or compartment-specific labeling techniques to
approach this question at the morphological level.
Using the Cm assay, we have analyzed the requirements for focal exocytosis
in macrophages and come to some novel conclusions that cast light on the
regulation of this phenomenon. Unlike many other types of regulated
exocytosis, elevated cytoplasmic Ca2+ is apparently not
required for focal exocytosis. However, cells did not phagocytose when
[Ca2+]i was strongly buffered, suggesting
that some aspect of particle internalization is
[Ca2+]i dependent. A
[Ca2+]i requirement for phagocytosis has been
controversial and may be cell and stimulus dependent
(Greenberg, 1999). Although
there is little doubt that ligation of Fc receptors leads to a rise in
[Ca2+]i flux, some have reported that
phagocytosis can continue even when [Ca2+]i
is buffered to extremely low levels
(McNeil et al., 1986;
but see Edberg et al.,
1995). Our results suggest that particle pinch-off is blocked (or
delayed) when [Ca2+]i is buffered to
<10–9 M. Thus, our data delimit the
participation of Ca2+ to a late stage in phagocytosis.
These results also clearly differentiate macrophages from neutrophils, where
elevated [Ca2+]i is apparently required for
fusion of intracellular granules with the invaginating phagosome
(Tapper et al.,
2002). In contrast to the results with Ca2+
chelators, blockade of G proteins with the antagonist GDPS eliminated
both focal exocytosis and subsequent phagocytosis. Because vesicular
trafficking requires participation of diverse G proteins, this is not
inherently surprising; indeed, several G proteins could be involved in focal
exocytosis, including Rab or Rho-family GTPases and dynamin-2.
Two other characteristics of focal exocytosis were less expected:
insensitivity to PI3 kinase blockade and necessity for actin polymerization.
PI3 kinases are activated after Fc receptor stimulation, probably by
recruitment to the tyrosine kinase Syk
(Greenberg, 2001). Activation
is essential for phagocytosis because antagonism of these enzymes inhibits
particle uptake. Type I PI3 kinase, which generates primarily
phosphatidylinositol-3,4,5-trisphosphate is activated early in the phagocytic
cascade and the lipid product of this enzyme has been localized to phagocytic
cups before and including their closure
(Vieira et al.,
2001). In contrast, elevated type III PI3 kinase activity,
generating primarily phosphatidylinositol 3-phosphate, occurs later and may be
involved in phagosome maturation (Ellson
et al., 2001; Vieira
et al., 2001). The precise role of type I PI3 kinase and
phosphatidylinosiotl-3,4,5-trisphosphate before phagosome pinch-off is not
clear. Some claim the lipid may only be essential for final engulfment
(Araki et al., 1996;
Gagnon et al., 2002),
whereas others postulate an earlier role, possibly in focal exocytosis
(Cox et al., 1999).
Our results suggest that PI3 kinase inhibition does not affect focal
exocytosis per se, but does impair particle internalization. The fact that PI3
kinase inhibition did not affect focal exocytosis while dynamin-2 antagonism
did, suggests that PI3 kinase and its lipid products act downstream rather
than upstream of dynamin-2 in phagocytosis.
Actin polymerization is also essential for phagocytosis. There is
convincing evidence that components of the actin assembly network (e.g.,
N-WASP and Arp2/3) are localized to developing phagosomes and that
interference with these proteins compromises particle uptake
(May and Machesky, 2001).
Members of the Rho family of small GTPases likely control actin polymerization
via N-WASP and Arp 2/3. Both Rac and Cdc42 are activated during Fc
signaling, probably due to the enhancement of various guanine nucleotide
exchange factors (e.g., the Rac1 guanine nucleotide exchange factor Vav is
stimulated by tyrosine phosphorylation;
Caron and Hall, 1998). We found
that blockade of Rho family members by using intracellular application of
Rho-GDI, or antagonism of actin polymerization by using drugs, unexpectedly
ablated focal exocytosis. Whether the effect occurs at the level of vesicle
production, transport, or insertion is not known, but the results suggest that
there must be some coordination between membrane insertion and actin
polymerization during pseudopodial extension.
Antagonism of dynamin-2 function by using three independent approaches also
abolished the Cm rise after opsonized particle challenge. This result
complements previous findings that dominant-negative dynamin-2 overexpression
in macrophages interfered with particle uptake
(Gold et al., 1999)
and intimates that the locus of the effect is at an early stage in the
process. Immunocytochemical analysis of endogenous dynamin-2 in macrophages
revealed punctate structures, possibly of an endosomal nature. These
compartments could be labeled in part by rhodamine-dextran, which enters by
fluid-phase endocytosis and is thought to mark the entire endosomal system,
and to a lesser extent by labeled transferrin. Current evidence supports a
model in which focal exocytosis involves an endosomal compartment, and there
is ample evidence that fusion of endosomes with internalized phagosomes plays
an important role in phagosomal maturation
(Vieira et al.,
2002). Phagocytosis seems to involve soluble
N-ethylmaleimide-sensitive factor attachment protein receptor
(SNARE)-dependent fusion processes, as demonstrated by its partial inhibition
on microinjection of v-SNARE degrading protease toxins
(Hackam et al.,
1998), and appearance of the v-SNARE vesicle-associated membrane
protein-3 on early phagosomal membranes
(Bajno et al., 2000).
Endosomes whose normal function may be to recycle cell surface components such
as the transferrin receptor have been implicated in focal exocytosis, and
components such as Rab11, that regulate recycling from this compartment, may
be involved (Cox et al.,
2000). In some cells dynamin-2 is involved in the production of
vesicles from this pool and has been localized at the electron microscope
level to tubular recycling endosomes (van
Dam and Stoorvogel, 2002). Thus, an attractive explanation for the
effects of antagonizing dynamin-2 on focal exocytosis is prevention of vesicle
budding from such a compartment.
It proved difficult to find association between endogenous dynamin-2 and
particle uptake sites in fixed cells, thus the possibility of a transient
association was investigated using overexpressed EGFP-dynamin-2. In
transfected cells, mobilization of dynamin-2 toward the incipient phagocytic
cup was apparent and time-lapse studies revealed that the dynamin-2 signal
completely encircled the internalizing particle during engulfment. During this
process, the intensity of the dynamin signal followed the production of the
phagocytic cup, with the highest intensity ultimately occurring at the distal
end or tip, whereas the proximal end or base became fainter. On completion of
phagocytosis, EGFP-dynamin-2 rapidly fell away from the phagosomal surface and
totally internalized particles showed no evidence of dynamin enrichment. Such
a cycle is compatible with the zipper model of Fc-mediated phagocytosis
(for review, see Swanson and Baer,
1995), in which signaling from successively occupied receptors
induces local recruitment of relevant proteins and structures in a progressive
manner as the particle becomes surrounded. An intriguing possibility is that
dynamin-2 serves to coordinate vesicle production with actin assembly
processes, possibly using its fission and actin-binding properties in a
sequential manner, as has been proposed for receptor-mediated endocytosis
(Qualmann et al.,
2000). Recent studies with total internal reflectance microscopy
revealed a close association between coated vesicle fission by dynamin at the
plasma membrane and assembly of actin, possibly as part of a force-generating
system powering movement of coated vesicles into the cytoplasm
(Merrifield et al.,
2002). To understand the role of dynamin in macrophages, it will
be important to identify dynamin-2 interacting species. Several proteins,
including amphiphysin, have been postulated to be dynamin partners in
regulating receptor-mediated endocytosis, in addition to the lipid messenger
phosphatidylinositol bisphosphate (PIP2). An amphiphysin-II isoform
has been found in macrophages and overexpression of a truncated version,
acting as a dominant negative, led to a phenotype much like mutant dynamin
expression in these cells (Gold et
al., 2000). Localized changes in PIP2 have been
reported to occur in the vicinity of the phagocytic cup
(Botelho et al.,
2000). We have recently found that, aside from amphiphysin,
macrophages express syndapin II and cortactin (our unpublished data), both
putative dynamin partners in regulating the actin cytoskeleton. It will be
interesting to determine whether either of these proteins or PIP2
interacts with macrophage dynamin-2 in vivo.
|
Footnotes |
|---|
Online version of this article contains video material for some figures.
Online version available at
www.molbiolcell.org. 
* Corresponding author. E-mail address: hpalfrey{at}midway.uchicago.edu.
|
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