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(Received for publication, June 13,
1995; and in revised form, November 13, 1995) From the
In macrophages, phagosome movement is microtubule-dependent.
Microtubules are a prerequisite for phagosome maturation because they
facilitate interactions between phagosomes and organelles of the
endocytic pathway. We have established an in vitro assay that
measures the binding of purified phagosomes to microtubules. This
binding depends on the presence of membrane proteins, most likely
integral to the surface of phagosomes, and on macrophage cytosol. The
cytosolic binding factor can interact with microtubules prior to the
addition of phagosomes to the assay, suggesting that it is a
microtubule-associated protein (MAP). Consistent with this, depletion
of MAPs from the cytosol by microtubule affinity removes all binding
activity. Microtubule motor proteins show no binding activity, whereas
a crude MAP preparation is sufficient to support binding and to restore
full binding activity to MAP-depleted cytosol. We show that the
activating MAP factor is a heat-sensitive protein(s) that migrates at
around 150 kDa by gel filtration. Phagocytosis is a process whereby a cell forms a new membrane
compartment, the phagosome, to engulf particles that are too large to
be internalized by endocytosis. This organelle subsequently matures
into a phagolysosome, by a complex series of interactions with the
endocytic pathway (Pitt et al., 1992a; Pitt et al.,
1992b; Desjardins et al., 1994a; Jahraus et al.,
1994). While it is well established that the actin cytoskeleton is
important for the earliest steps of phagocytosis (Silverstein et
al., 1989), the available evidence argues that later transport
events require microtubules (D'Arcy Hart et al., 1983;
D'Arcy Hart et al., 1987; Toyohara and Inaba, 1989;
Knapp and Swanson, 1990; Desjardins et al., 1994a; Jahraus et al., 1994). The microtubule cytoskeleton is important
for the positioning and function of many organelles (Kelly, 1990),
including the endoplasmic reticulum (Terasaki et al., 1984;
Mizuno and Singer, 1994; Terasaki et al., 1986) and the Golgi
complex (Wehland and Willingham, 1983; Sandoval et al., 1984;
Ho et al., 1989; Scheel et al., 1990). Movement of
transport vesicles along microtubules is also necessary for directed
secretion (Achler et al., 1989; Kreis et al., 1989;
Lafont et al., 1994), and the transport of internalized
material from early to late endosomes (Matteoni and Kreis, 1987;
Swanson et al., 1987; Gruenberg et al., 1989; Bomsel et al., 1990; Hollenbeck and Swanson, 1990; Young et
al., 1990; Aniento et al., 1993). In the present study
we have focused on the interaction of phagolysosomes with microtubules.
For this, we used 1-µm latex beads as a convenient marker for
phagocytosis (Wetzel and Korn, 1969; Stossel et al., 1971;
Muller et al., 1980). The attraction of these beads is the
ease with which they allow the subsequent purification of
phagolysosomes on a simple flotation gradient, from J774 mouse
macrophages that have internalized them (Desjardins et al.,
1994a; Desjardins et al., 1994b). For convenience, we will
refer to all the organelles purified in this manner, irrespective of
their maturation state, as phagosomes. Previous video analysis from
our group has shown that in macrophages, endosomes and lysosomes
containing endocytosed colloidal gold and phagosomes containing latex
beads move within the cell, interacting with one another multiple times
(Desjardins et al., 1994a). Late organelles of the endocytic
pathway are known to move along microtubules (Kreis et al.,
1989; Hollenbeck and Swanson, 1990). Phagosome movements are best
observed within the first hours following bead internalization.
Movement is mainly, but not always, centripetal, leading to a gradual
accumulation of phagosomes around the nucleus, near the microtubule
organizing center. When these cells are treated with the
microtubule-depolymerizing drug nocodazole (Noc), ( The
microtubule motor proteins cytoplasmic dynein and kinesin were the
first identified molecules that could account for organelle-microtubule
interactions (Vale et al., 1985; Schroer et al.,
1989). That these motors can interact with membrane organelles is now
well established (Neighbors et al., 1988; Hollenbeck, 1989;
Pfister et al., 1989; Hirokawa et al., 1990; Hirokawa et al., 1991; Lacey and Haimo, 1992; Leopold et al.,
1992; Lin and Collins, 1992; Yu et al., 1992; Morin et
al., 1993). However, recent data argue that motors alone are
insufficient to mediate motile interactions of organelles with
microtubules (Schroer et al., 1988; Schroer and Sheetz, 1991;
Gill et al., 1991; Burkhardt et al., 1993). In
addition, evidence has been provided that motor proteins are not the
major cytosolic factors involved in the static binding of organelles to
microtubules (Scheel and Kreis, 1991). Indeed, in studies where the
static binding of membrane organelles to microtubules has been
examined, binding has been attributed to the activity of a MAP, in one
case to MAP2 (Severin et al., 1991) and in the other to a
novel MAP, CLIP-170 (Pierre et al., 1992).
Where indicated, digestion
of the phagosome membrane with proteases and mock treatment was
performed at 37 °C for 15 min. After inactivation of the enzyme
with 3,4-dichloroisocoumarin (Boehringer Mannheim), phagosomes were
repurified using a small scale version of the sucrose gradient above.
Treatment of phagosomes with 1 M NaCl was performed for 30 min
at 4 °C, and the salt was removed by flotation.
Fractionation of cytosol was performed on a Superose 12
column on a SMART system; 50-µl fractions were collected. Heat
treatment of MAPs was performed according to Kuznetsov et
al.(1978) and Kim et al.(1979). MAPs were adjusted to 1 M NaCl and heated to 95 °C for 5 min. A mock sample was
adjusted to 1 M NaCl and left on ice. Samples were cooled on
ice and centrifuged at 300,000
Figure 1:
Transfer of horseradish peroxidase
from late endosomes/lysosomes to phagosomes in intact cells requires
microtubules. J774A.1 cells were fed horseradish peroxidase for 20 min
and, after thorough washing, chased for 1 h. At this point latex beads
were pulsed for 20 min, and the cells were chased in the presence or
absence of nocodazole for up to an additional 120 min. At this time the
drug was washed out, and the cells were chased for a subsequent 120
min. At each time point phagosomes were purified, and horseradish
peroxidase was quantified. The data are expressed as arbitrary units of
horseradish peroxidase activity, corrected for bead content of each
time point. This experiment was repeated 3 times and gave similar
results, though the final values could not be merged to produce error
bars.
Figure 2:
Visualization of the binding assay and of
the phagosome-microtubule complex. Typical fields of binding assays
incubated in the absence of phagosomes to allow visualization of the
microtubule lawn (A), in the absence of cytosol but with
phagosomes (bar = 10 µm) (B), and in the presence
of 2 mg/ml cytosol and phagosomes (C) are shown. D,
electron micrograph of the floated phagosome-microtubule complex (bar = 0.2 µm). Phagosomes were incubated with
taxol-stabilized microtubules in the presence of 2 mg/ml macrophage
cytosol. Phagosomes were then repurified by flotation and and processed
for thin section electron microscopy. Only in the presence of cytosol
do microtubules bind to the phagosomes and become copurified. Multiple
microtubules are seen in association with the phagosome membrane (arrowheads). In addition, a network of microtubules is
floated with the organelles (small
arrows).
Figure 3:
Binding requires microtubules, cytosol,
and proteins on the phagosome membrane. A, phagosome binding
was tested in the presence of the indicated final cytosol
concentrations. Optimal binding reproducibly occurred at 2 mg/ml
cytosol. At higher concentrations inhibition was observed. B,
binding to the microtubule lawn of uninternalized fish skin gelatin
beads (beads) and phagosomes purified after fish skin gelatin
bead internalization (1-h pulse, 1-h chase, phags) was tested
in the absence (- cyt) or presence (+ cyt)
of 2 mg/ml cytosol. Only phagosomes displayed cytosol-dependent
binding, suggesting that both cytosolic and phagosome membrane factors
are required. Background binding of phagosomes to the chamber in the
absence of microtubules (no MTs, phags + cyt) was
minimal. Treatment of phagosomes with 100 µg/ml trypsin as
described under ``Materials and Methods'' completely
abolished their cytosol-dependent microtubule binding (trypsin
phags + cyt), suggesting that membrane proteins are required.
In each case, samples were adjusted for difference in bead content. C, phagosome binding activity is modulated by maturation of
the organelle. Phagosomes were prepared after a 20-min pulse or after a
1-h pulse followed by 4, 12, or 24 h of chase. Phagosomes adjusted to
the same bead concentration were tested for their ability to bind to
microtubules in the presence or absence of 2 mg/ml
cytosol.
The ability of the phagosomes to bind to microtubules is a
property of their surrounding membranes, since uninternalized latex
beads coupled to fish skin gelatin showed very little tendency to bind
to microtubules, even in the presence of cytosol (Fig. 3B). Moreover, phagosome-microtubule interaction
was abolished by heating the phagosomes to 80 °C for 10 min (data
not shown) and by digestion of the phagosome membrane with trypsin (Fig. 3B). Phagosome-microtubule interaction was also
inhibited by digestion of the phagosomes with chymotrypsin or
proteinase K but not by V8 protease. ( Previous studies have shown that the protein
composition of phagosomes changes with time, as these organelles fuse
with endocytic compartments to become progressively more late
endosome/lysosome-like (Pitt et al., 1992a; Pitt et
al., 1992b; Desjardins et al., 1994a; Desjardins et
al., 1994b). We therefore asked whether the protein machinery
responsible for microtubule binding competence also changes with time.
Phagosomes were prepared at various times after bead internalization
and tested for their ability to bind microtubules. As shown in Fig. 3C, the ability of phagosomes to bind microtubules
was highest at the earliest times after internalization, decreasing
steadily with time to about of its original value. The ability to bind
microtubules in the presence of cytosol was never lost but reached a
stable low level after 12 h that persisted for 24 h after
internalization.
To examine
whether the binding factor could independently interact with
microtubules, the microtubule lawn was preincubated with cytosol at 2
mg/ml in the absence of phagosomes. Cytosol was then washed out of the
chamber using assay buffer, and phagosomes were added in the presence
or absence of 2 mg/ml additional cytosol. As shown in Fig. 4,
when microtubules were preincubated with cytosol, phagosomes bound in
the absence of additional cytosol. The number of phagosomes bound was
not as high as under standard assay conditions (i.e. simultaneous addition of phagosomes, microtubules, and cytosol),
presumably because of the intervening wash step, but it was
significantly higher than in the control reaction lacking cytosol
altogether. This indicates that the binding activity can interact first
with microtubules and then with the phagosome membrane. The fact that
the binding factor can interact independently with microtubules
suggests that it could be a MAP.
Figure 4:
The
phagosome-microtubule binding factor can interact with microtubules
independently of phagosomes. Assay chambers containing microtubules
were preincubated for 20 min at room temperature with assay buffer or
cytosol at 2 mg/ml (without phagosomes). The buffer or cytosol was
washed away. Phagosomes were then perfused in the presence or absence
of fresh cytosol at 2 mg/ml, incubated, washed, and counted as
usual.
Figure 5:
A,
the binding is highly sensitive to increasing salt concentrations. KCl
was titrated into the usual assay using 2 mg/ml cytosol. B,
binding is insensitive to nucleotides or nonhydrolyzable nucleotide
analogues. The ATP-depleting agents apyrase and glucose/hexokinase and
the ATP-regenerating system ATP/creatine phosphate/creatine
phosphokinase were added exactly as in Bomsel et al.(1990).
Phagosomes were mixed in the presence or absence of 2 mg/ml cytosol
with the indicated nucleotides or analogs at 2 mM and perfused
into chambers coated with poly-L-ornithine prior to the
addition of the microtubules. This method was used to minimize the loss
of microtubules from the lawn due to force exerted by cytosolic
microtubule motor proteins in the presence of hydrolyzable nucleotides.
Poly-L-ornithine slightly increased background binding without
affecting the stimulation normally observed in the presence of
cytosol.
Figure 6:
A,
cytosols depleted of MAPs or of MAPs and motor proteins as described
under ``Materials and Methods'' were blotted with IF5.2.2, an
anti-MAP4 antibody, or KMTBX, an antibody that recognizes the motor
domain of multiple kinesin-like proteins. B, silver-stained 6%
SDS-polyacrylamide gel of ATP-eluted motors and NaCl-eluted MAPs. C, kinesin was depleted with SUK4 anti-kinesin heavy chain and
blotted with KMTBX; the mock sample was incubated with P5D4, an
isotype-matched control antibody. Note that the 116-kDa kinesin heavy
chain is removed while other kinesin-related proteins remain. D, cytoplasmic dynein was immunodepleted with 70.1 anti-dynein
intermediate chain and blotted with rabbit anti-dynein heavy chain; the
mock sample was treated with matched control antibody
F13.
Figure 7:
MAPs,
but not known microtubule motors, are necessary and sufficient for
binding. A, phagosomes were tested for their ability to bind
to microtubules in the presence of 2 mg/ml cytosol depleted of MAPs or
of MAPs and motors by microtubule affinity (A). The cytosol
solely depleted of MAPs but where motors remained had lost all binding
activity. B, binding was tested in the presence of 2 mg/ml
cytosol immunodepleted of cytoplasmic dynein with 70.1 or kinesin with
SUK4; these depletions had no effect on the binding. Mock
immunodepletion with isotype-matched control antibodies F13 and P5D4
(blots shown in Fig. 6, C and D) also had no
effect. C, macrophage MAPs (20 µg/ml in the assay) are
able to restore activity to MAP-depleted cytosol (
Motor proteins
and MAPs can be eluted from the microtubule pellets using ATP or high
salt, respectively, as shown in Fig. 6B. Readdition of
the MAP preparation to MAP-depleted cytosol restored binding activity (Fig. 7C), while readdition of motor proteins had
little effect (data not shown). These results indicate that motor
proteins do not represent the predominant cytosolic microtubule binding
activity measured in our assay. Instead, one or more MAP proteins are
absolutely required for phagosome-microtubule binding. To determine
whether the proteins removed by microtubule affinity are sufficient to
mediate the binding of phagosomes to microtubules in the absence of
other cytosolic activities, the eluted MAP and motor preparations were
tested alone in the assay. Fig. 7D shows that only the
MAP preparation was able to support any significant binding. The low
level of binding supported by the motor fraction was not ATP-sensitive
(data not shown); hence we attribute it to contaminating MAPs. To
determine whether the MAP factor could interact directly with the
phagosome membrane, we tested the ability of the MAP preparation (50
µg/ml) to mediate the interaction of 1 M NaCl-stripped
phagosomes with microtubules. The MAPs mediated this interaction to the
same level as 2 mg/ml cytosol (67.2 ± 4.9 for mock-treated
phagosomes versus 52.8 ± 8.9 for salt-washed
phagosomes), suggesting that all the required soluble components are
present in this preparation.
Figure 8:
The cytosolic binding factor behaves as a
150-kDa globular protein upon fractionation on Superose 12. Cytosol was
fractionated on Superose 12 (V
The finding that a number of MAPs, including MAP4, are
heat-stable facilitated their purification. We therefore tested the
thermoresistance of the phagosome-binding factor. All binding activity
was lost following brief heating of the MAP fraction to 95 °C (41.6
± 1 for the mock-treated fraction versus 15.5 ±
5 for the heat-treated fraction). Thus, the binding factor is
heat-sensitive. Although considerable progress has been made toward
understanding the molecular mechanisms of microtubule motors, our
knowledge of motor-membrane interactions remains rudimentary. Still
less is known about the possible role in membrane traffic of the
heterogeneous family of proteins operationally classified as MAPs. Slow
progress in this field is due largely to technical difficulties in
purifying a suitable membrane organelle in biologically active form.
This is a prerequisite for any attempts to identify the molecules
essential both for stable binding and for motility of membrane
organelles along microtubules. Latex bead-enclosing phagosomes are a
powerful model system to study the interactions of defined membrane
organelles with microtubules. Phagosomes move along microtubules in
vivo, We describe here a novel in
vitro light microscopy assay to measure the binding of phagosomes
to microtubules. This assay requires minimal amounts of biological
material and it is simple, fast, and reproducible in quantitative
terms. Using this assay, we show that the binding of phagosomes to
microtubules depends on both membrane proteins, probably integral to
the surface of phagosomes, and exogenous cytosol. Sensitivity of
phagosome-microtubule binding to proteases exhibiting different
specificities suggests that a phagosome protein(s) functions as a
receptor on the phagosome surface for the cytosolic microtubule binding
factor. The fact that significantly more phagosomes bound, in the
presence of the same cytosol preparation, when they were isolated at
earlier rather than later times after their internalization indicates
that the presence or activity of the membrane receptor is regulated. We
are now pursuing the identification of this receptor activity using its
protease sensitivity profile and established two-dimensional gel maps
of the phagosome preparation (Desjardins et al., 1994b;
Burkhardt et al., in press). The cytosolic factor
responsible for phagosome-microtubule binding is a heat-sensitive
MAP(s) with a molecular weight in the range of 150 kDa. Classification
of this factor as a MAP is based on several criteria. First, it is able
to bind to microtubules in the absence of phagosomes. Second, its
binding to microtubules is sensitive to moderate salt concentration but
not to nucleotides. Third, it is removed from cytosol under conditions
where MAPs are removed. Finally, it is present in a crude MAP
preparation. Our data lead us to conclude that two proteins previously
recognized as mediators of membrane organelle-microtubule interactions
are probably not involved in the interaction we observe; the gel
filtration profiles of CLIP-170 and MAP4 (some properties of which
resemble neuronal MAP2; Olmsted(1993) and Walden(1993)) are different
from, albeit overlapping, that of the activity we measure. Our factor
is heat-sensitive, which provides evidence that it is not MAP2/MAP4
which are known to belong to the family of heat-stable MAPs (Herzog and
Weber, 1978; Parysek et al., 1984). CLIP-170 demonstrates
nucleotide- and phosphorylation-sensitive binding to microtubules,
whereas the phagosome-microtubule binding factor does not (Rickard and
Kreis, 1991; Scheel and Kreis, 1991). It therefore appears that the
activating MAP factor is most likely a novel MAP or a previously
identified MAP for which no such role has yet been demonstrated. At
first glance, the MAP activity we describe seems redundant to the
activity of a motor, which must itself mediate some form of
organelle-microtubule binding. Although there is substantial evidence
that purified motors can interact with organelles, there is so far no
evidence that organelles carrying only bound motors can interact with
microtubules. In fact, there is evidence that motors alone are
insufficient to mediate motile interactions of organelles with
microtubules (Schroer et al., 1988; Gill et al.,
1991; Schroer and Sheetz, 1991; Burkhardt et al., 1993). As
was found previously by Scheel and Kreis(1991) for endocytic vesicles,
we find that immunodepletion of conventional kinesin and cytoplasmic
dynein from cytosol has no effect on the binding of phagosomes to
microtubules. Moreover, we tested the entire subset of proteins that
bind to microtubules in an ATP-sensitive manner and found that this
microtubule motor preparation, which contains cytoplasmic dynein,
kinesin, and at least several kinesin-like proteins (see blot of
microtubule pellet with a pan-kinesin antibody in Fig. 7A), was also unable to support binding. We
therefore conclude either that microtubule motors are unable to support
organelle-microtubule binding on their own or that the binding they
support is too weak to be detected in our assay. Together with the work
of Scheel and Kreis (1991), Severin et al.(1991) and Pierre et al. (1992), our results show that interaction of three
different kinds of organelles with microtubules requires a type of MAP. What functional role might these MAPs play? It has been previously
suggested that the position of organelles such as the Golgi complex
reflects an equilibrium between plus and minus end-directed motors.
This is consistent with the work of others (Heuser, 1989; Parton et
al., 1991; Lin and Collins, 1992; Feiguin et al., 1994).
Yet the weight of recent evidence suggests that MAPs must also play a
role in this process. We propose that the function of the MAP factor is
to create a high affinity static link between the organelle and the
microtubule, a function that a motor may not be able to perform.
Specific MAP linker type molecules could function to define the
position of each organelle relative to microtubules within cells,
perhaps by antagonizing the action of a motor. Alternatively, or
additionally, these proteins could facilitate motor-driven movements by
creating a high affinity microtubule-organelle link, which may be
required to initiate lower affinity motor interactions. The MAP may be
lost from the organelle-motor complex when the organelle begins to
move, or motors could just loosen this static link and perhaps use it
to stabilize their transient interactions with microtubules while
moving. Any of these options requires a form of regulation of the
linker MAP, to allow the switch between tethering and movement. We have
already seen that the activity we describe is regulated on the membrane
side as the phagosome matures. Rickard and Kreis(1991) have shown that
CLIP-170 is released from microtubules by phosphorylation. We have been
unable to modulate the activity of the binding factor by manipulating
the nucleotide or phosphorylation state of the cytosol. However, a
possibility for regulation in our system comes from our observation
that at above 2 mg/ml cytosol, the MAP binding activity is inhibited.
Such an inhibitory activity was not observed in other binding studies
(van der Sluijs et al., 1990; Scheel and Kreis, 1991; Severin et al., 1991) partly, we think, because cytosol above 5 mg/ml
was not tested. We have evidence that the binding and inhibitory
activities are distinct and that the inhibitor is also a MAP. ( Clearly, the most
direct approach to understanding the nature of the binding/motility
switch is to study tethering and movement in parallel. Toward this goal
we have established a second in vitro assay, very similar to
the one described here, which reconstitutes the movement of purified
phagosomes along polarity-marked microtubules in macrophage
cytosol.
Volume 271,
Number 7,
Issue of February 16, 1996 pp. 3803-3811
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)phagosome movement ceases. (
)These results
suggest that phagosomes move within the cell along microtubules.
Cell Culture
J774A.1 macrophages were obtained
from American Type Tissue Collection and maintained as described
previously (Desjardins et al., 1994a), except with 10% newborn
calf serum (Seromed). Cells were grown adherent in a 5% CO atmosphere and passaged by vigorous pipetting. For cytosol
preparations, cells were grown as spinner cultures to 5 
10
/ml in Joklik's modified Eagle's medium (Life
Technologies, Inc.) containing 2 g/liter NaHC0
and
supplemented with 100 units/ml penicillin and 100 µg/ml
streptomycin (both from Life Technologies, Inc.).Preparation of Latex Beads
Naked polystyrene latex
beads, either untreated or carboxylate-modified, were found to have
high intrinsic nonspecific binding to microtubules. Therefore, to block
the bead surface for binding studies, carboxylate-modified, orange
fluorescent latex beads, 1 µm in diameter (Molecular Probes) were
incubated with 1 mg/ml fish skin gelatin (Sigma) in 50 mM MES
buffer, pH 6.7, and covalently coupled using
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, according to the
manufacturer's recommendations.Phagosome Purification
Phagosomes were isolated
using the high buoyant density of latex, as described previously
(Desjardins et al., 1994a), except that cells were broken
using several passes through a 22-gauge syringe needle, and the sucrose
gradient was simplified to three steps: the postnuclear supernatant
adjusted to 40% sucrose, and steps of 25 and 10% sucrose. Protease
inhibitors (PIs) in the form of 1 µg/ml pepstatin A, 0.5 µg/ml N-tosyl-L-phenylalanine, 0.5 µg/ml leupeptin, and
4 µg/ml aprotinin (all from Sigma), were included throughout the
isolation procedure. Phagosomes were collected from the 10%/25% sucrose
interface and frozen in liquid nitrogen.Measurement of Fusion between Phagosomes and Late
Compartments of the Endocytic Pathway
Macrophages grown on 15-cm
dishes were allowed to internalize horseradish peroxidase (Sigma) at 2
mg/ml for 20 min at 37 °C in internalization medium (MEM; 10 mM Hepes, 5 mMD-glucose, pH 7.4). Cells were
washed twice at 4 °C with PBS, twice with PBS containing 5 mg/ml
bovine serum albumin, and chased for 1 h at 37 °C in growth medium.
Cells were then pulsed for 20 min at 37 °C with latex beads at a
0.05% solids suspension in growth medium, returned to 4 °C, and
washed several times with cold PBS. Noc (Sigma) was then added to 10
µM, and cells were returned to 37 °C for the indicated
times of chase. At the end of 120 min of chase, Noc was washed out with
PBS, and the cells were returned to normal medium for an additional
120-min chase period. At the end of each chase time, cells were
transferred to ice, phagosomes were isolated as described above, and
horseradish peroxidase activity in each sample was determined as
described by Gruenberg et al.(1989). To normalize samples for
varying recovery of phagosomes, the optical density of the latex beads
at 600 nm was determined. The data are expressed as arbitrary units of
horseradish peroxidase activity, corrected for bead content of each
time point.Preparation of Cytosol
Cells were collected by
centrifugation and washed twice in cold PBS, resuspended in
PMEE/sucrose (35 mM Pipes buffered with KOH to pH 7.4, 5
mM MgSO
, 1 mM EGTA, 0.5 mM EDTA,
0.25 M sucrose), and pelleted at 2,500 g.
Cells were resuspended using 0.9 volume of PMEE/sucrose with PIs and 1
mM dithiothreitol (DTT, Sigma) and homogenized using several
passages through a 22-gauge syringe needle. A postnuclear supernatant
was generated by centrifugation at 6,000 g for 15 min
at 4 °C and centrifuged at 200,000 g for 30 min at
4 °C to remove particulates. The top lipid layer was discarded, and
the underlying clear cytosol fraction was collected and frozen in
liquid nitrogen. For larger scale preparations, cells were first
concentrated 10-fold using a Millipore cell concentrator and then
collected by centrifugation, washed three times in PBS, and broken
using several strokes in a Dounce homogenizer fitted with a B pestle.
Both methods reproducibly produced cytosol at 30-50 mg/ml
protein.Depletion of MAPs and Motors by Microtubule
Affinity
This procedure is derived from modifications of methods
published by Vallee (1982), Vale et al.(1985), Paschal et
al. (1987), Schroer et al.(1989), and Scheel and
Kreis(1991). Microtubules were polymerized from
phosphocellulose-purified tubulin obtained from bovine brain (Mitchison
and Kirschner, 1984) in PMEE containing 1 mM GTP (Boehringer
Mannheim) and 20 µM taxol (Sigma) for 20 min at 37 °C
and collected by centrifugation either in an airfuge at 25 p.s.i. or in
a conventional ultracentrifuge at 70,000 g for 5 min
at 20 °C. To those cytosol samples that were to be depleted of
either MAPs (sample 1) or MAPs and motors (sample 2), protein
concentration of polymerized microtubules resuspended in PMEE, 20
mM GTP, and 200 µM taxol was added; to the mock
sample (sample 3) only PMEE was added. To sample 1 volume of an
ATP-regenerating system, freshly prepared by mixing a 1:1:1 volume of
stock solutions of 100 mM ATP, pH 7, with HCl, 800 mM creatine phosphate, and 4 mg/ml creatine phosphokinase at 800
units/ml in 50% glycerol (both from Boehringer Mannheim), was added;
sample 2 was brought to 2 mM AMP-PNP (Sigma) and received
volume of an ATP depletion system, freshly prepared from a 1:1 mix of
stock solutions of hexokinase (Boehringer Mannheim; 1500 units/ml in
50% glycerol) and 1 MD-glucose; to sample 3 an
equivalent amount of PMEE was added. All samples were incubated at 37
°C for 15 min and then centrifuged at 70,000 g for
20 min at 20 °C to pellet microtubules and associated proteins. The
supernatants were collected and were desalted into PMEE containing PIs
and 1 mM DTT on PD-10 columns (Pharmacia Biotech Inc.), which
were quenched for protein binding by pretreament in 5 mg/ml casein in
PMEE, PIs, 1 mM DTT. These samples were frozen in liquid
nitrogen.Immunodepletions of Motor Proteins
Cytosol was
depleted of kinesin with the SUK4 monoclonal IgG bound to protein
A-Sepharose beads as described by Ingold et al.(1988). Another
monoclonal IgG (P5D4), raised against a viral antigen (Kreis, 1986),
was used as a control antibody. The same procedure (Bomsel et
al., 1990; Aniento et al., 1993b) was used to deplete
cytoplasmic dynein with the 70.1 monoclonal IgM (Sigma) coupled to goat
anti-mouse agarose beads. Monoclonal IgM F13, raised as an
anti-idiotype antibody to a viral antigen (Vaux et al., 1988)
served as a matched control.Preparation of J774 MAPs and Motors
To generate a
MAP preparation, macrophage cytosol was treated initially as above for
MAP depletion (sample 1). To generate a motor preparation, macrophage
cytosol was treated as for MAP and motor depletion (sample 2). In each
case the microtubules were pelleted for 20 min at 20 °C and 75,000
g through a sucrose gradient containing steps of 15
and 25% sucrose, in PMEE, PIs, 1 mM DTT, 10 µM taxol. Microtubule pellets were resuspended in a large volume of
PMEE, PIs, 1 mM DTT with 10 µM taxol and pelleted
again over the same sucrose gradient. The microtubule pellets were then
resuspended in PMEE, PIs, 1 mM DTT, 10 mM taxol, 10
mM ATP and incubated at room temperature for 30 min to remove
any contaminating motor proteins in the case of sample 1 and to elute
the motor proteins in the case of sample 2. The microtubules were
pelleted at 22,000 g for 15 min at 20 °C, and the
supernatant of the pellet from sample 1 was discarded, whereas the
supernatant of sample 2, the motor preparation, was collected. The
microtubule pellet of sample 1 was then resuspended in PMEE, PIs, 1
mM DTT, 10 µM taxol containing 0.5 M NaCl and incubated at room temperature for 30 min to elute the
MAPs. The microtubules were removed by centrifugation as above, and the
supernatant, the MAP fraction, was collected. Both fractions were
desalted into PMEE, PIs, 1 mM DTT using a fast desalting PC
3.2/10 column on a SMART system (Pharmacia) and frozen in liquid
nitrogen. g for 20 min at 4
°C. The supernatant was desalted as directly above.Binding Assay
Microscope chambers were built from
a glass microscope slide and two pieces of double sided tape (Scotch)
onto which an 11-mm circular glass coverslip (Menzel) was sealed,
forming a 3-µl chamber. Chambers were perfused with rhodamine
polarity-marked microtubules, prepared as in Howard and Hyman(1993), in
PMEE containing 10 mM taxol. A dense lawn of these
microtubules adhered to the glass coverslip within a short time. The
excess microtubules were washed away by perfusion of 2 volumes of assay
buffer (PMEE, PIs, 1 mM DTT, 10 µM taxol, 250
µg/ml casein) containing antifade in the form of 0.1 mg/ml glucose
oxidase, 0.1 mg/ml catalase (both from Sigma), and 10 mM glucose. Where indicated, coverslips were precoated with
poly-L-ornithine prior to addition of the microtubules as
described by Urrutia et al.(1993). This results in a lawn of
strongly bound microtubules, which are not dislodged by the activity of
microtubule motor proteins. Any free poly-L-ornithine was
neutralized by a wash with 10 mg/ml aspartic acid at pH 7 followed by a
wash in assay buffer. The chambers were then perfused with 2 volumes of
the phagosome-binding reaction containing 1-2 µl of purified
phagosomes diluted into assay buffer and cytosol where indicated, and
the organelles were allowed to bind to the microtubules in humidified
chambers for 20 min at room temperature. Unbound phagosomes were washed
away with 2 volumes of assay buffer with antifade. Chambers were
observed using a Zeiss photomicroscope with a 10 ocular and a
Zeiss Planapo 63 lens (field surface area of 35,000
µm). The bound phagosomes were counted by eye, and in
each experiment values from at least 10 fields were averaged. The
errors reported are the population standard deviations from at least
two separate but identical reactions.Electron Microscopy
Phagosomes were mixed in assay
buffer with taxol-stabilized microtubules in the presence or absence of
2 mg/ml macrophage cytosol and allowed to bind in solution for 30 min
at room temperature. Samples were then mixed with 1 volume of 62%
sucrose, 3 mM imidazole, pH 7.4, and repurified in the
presence of taxol using a small version of the sucrose gradient used
for phagosome purification. Floated phagosomes and any bound
microtubules were collected, mixed with an excess of 1% glutaraldehyde,
0.2 M sodium cacodylate, pH 7.2, and pelleted at 15,000
g. Pellets were postfixed in osmium tetroxide,
dehydrated, and embedded in epon according to standard procedures. Thin
sections were contrasted with lead citrate and uranyl acetate.Antibodies and Electrophoresis
SUK4 (Ingold et
al., 1988) was obtained from Developmental Studies Hybridoma Bank,
and 70.1 (Steuer et al., 1990) was purchased from Sigma.
KMTBX, a rabbit affinity-purified polyclonal antibody raised against a
peptide of the motor domain of the Xenopus kinesin-like
protein Eg5, was a gift of Drs. I. Vernos and E. Karsenti (Vernos et al., 1995). The Db1 rabbit affinity-purified polyclonal
antibody was a gift from Dr. E. A. Vaisberg (Vaisberg et al.,
1993). Professor T. E. Kreis donated 
55, an affinity-purified
rabbit polyclonal to CLIP-170 (Rickard and Kreis, 1990) and P5D4. Dr.
S. Fuller gave us F13, and Dr. J. Olmsted gave us IF5.2.2, a rat
monoclonal antibody against mouse MAP4. ()SDS-polyacrylamide
gel electrophoresis was performed as by Laemmli(1970), and blots were
performed using the ECL detection system (Amersham Corp.).
Microtubules Facilitate Exchanges of Material between
Phagosomes and Late Endocytic Compartments
Desjardins et al. (1994a) showed that treatment of macrophages having internalized
latex beads with microtubule-depolymerizing drugs inhibits the
acquisition by the phagosomes of two lysosomal glycoproteins, Lamp1 and
Lamp2, membrane markers of late endocytic/lysosomal compartments
(Kornfeld and Mellman, 1989). To further assess the role of
microtubule-based movement in phagosome-lysosome interactions, J774
cells were fed the fluid phase marker horseradish peroxidase under
conditions that selectively load late endocytic compartments
(Desjardins et al., 1994a). The cells were then pulsed with
latex beads and chased in the presence or absence of Noc. At each chase
time, the phagosomes were purified, and the quantity of horseradish
peroxidase transferred to the phagosome compartment was determined. As
shown in Fig. 1, Noc treatment inhibited the transfer of
horseradish peroxidase into the phagosomes by nearly 70% over a 2-h
chase period of the latex beads. The effects of nocodazole were
reversible. When the treated cells were returned to nocodazole-free
medium and the microtubule cytoskeleton was allowed to reform,
horseradish peroxidase was transferred to the phagosomes at a rate that
was comparable with, if slightly slower than, the rate found in control
cells. This result shows that late endocytic organelles and phagosomes
interact with microtubules in intact cells and that this interaction is
important for facilitating the exchange of material between them.
Reconstitution of Phagosome-microtubule Binding in
Vitro
To probe the molecular details of phagosome-microtubule
interactions, we established an in vitro phagosome-microtubule
binding assay. Latex bead-containing phagosomes were isolated by
flotation in a discontinuous sucrose gradient, a procedure that yields
phagosomes of high purity, with a defined protein composition
(Desjardins et al., 1994a; Desjardins et al., 1994b;
Burkhardt et al., in press). Unless otherwise stated,
macrophages were allowed to internalize beads for 1 h and were chased
for 1 h prior to phagosome purification. The ability of the phagosomes
to bind to microtubules was tested using a modification of the assay
developed by Sorger et al.(1994) to measure
kinetochore-microtubule interactions. For this, dimly labeled rhodamine
microtubules, stabilized with taxol, were perfused into a small
microscopy chamber and allowed to adsorb to the coverslip (Fig. 2A). After washing away excess microtubules,
phagosomes containing rhodamine labeled latex beads were added in the
presence or absence of macrophage cytosol and allowed to bind. The
phagosomes that remained bound after a wash and were in the focal plane
of the microtubule lawn were counted by direct observation. Binding of
phagosomes to microtubules was minimal in the absence of cytosol, but
when macrophage cytosol was added, numerous phagosomes bound (Fig. 2, B and C).
Visualization of the Phagosome-Microtubule
Complex
To visualize the phagosome-microtubule interaction at
higher resolution, the complex was analyzed by electron microscopy. For
this, phagosomes were incubated in solution with microtubules in the
presence or absence of cytosol under standard binding assay conditions.
The phagosomes and bound microtubules were separated from unbound
material by flotation, collected, and processed for electron
microscopy. When cytosol was omitted from the reaction, no microtubules
were floated with the organelles (data not shown). When cytosol was
included in the reaction, microtubules were observed tangentially
associated with the phagosomes and in close apposition (2-10 nm)
to their enclosing membranes (Fig. 2D). In some cases
dense material was observed in the intervening space.Binding Is Dependent on Cytosol and Phagosome Membrane
Proteins
The absolute number of phagosomes bound per field in
our light microscopy assay varied essentially linearly with the number
of input phagosomes (data not shown), but the stimulation by cytosol
remained a constant, typically 5-10-fold (Fig. 3, A and B). Binding was stimulated by cytosol concentrations
up to approximately 2 mg/ml in the assay (Fig. 3A). At
higher concentrations of cytosol, however, binding was inhibited by 50%
or more. Therefore, all subsequent assays were performed at 2 mg/ml
cytosol. Phagosome binding absolutely required the presence of
microtubules on the coverslip (Fig. 3B), was
time-dependent, saturating at 20-30 min of incubation, and showed
little difference whether performed at 25 or 37 °C (data not
shown).
)Stripping of
phagosomes with 1 M NaCl diminished subsequent binding in the
presence of cytosol only slightly (78.0 ± 7.1 for mock-treated
phagosomes, versus 52.3 ± 1.8 for salt-washed
phagosomes). This indicates that if peripheral membrane proteins are
required for binding, these can be supplied by the added cytosol. Taken
together, these results indicate that the binding requires the activity
of one or more specific, and probably integral, membrane proteins on
the phagosome surface.The Binding Factor Can Interact with Microtubules in the
Absence of Phagosomes
To probe the mode of interaction among
phagosomes, microtubules, and the cytosolic binding factor,
order-of-addition experiments were performed. In the absence of
microtubules, phagosomes were incubated with cytosol for 20 min,
floated, and then tested in the binding assay in the presence or
absence of additional cytosol. A 2-fold stimulation of binding was
sometimes apparent after preincubation with cytosol, but this was not
reproducible (n = 6, with a stimulation of binding seen
in about 50% of trials; data not shown). We conclude from this that
under the conditions tested, the cytosolic binding factor interacts
only weakly with the phagosome membrane or that, in the absence of
microtubules, it binds in a partially inactive form.
The Cytosolic Binding Activity Behaves Like a
MAP
As expected of a MAP or a motor, binding was linearly
sensitive to an increasing addition of salt (Fig. 5A), with
a complete abolishtion of binding occurring at 150 mM KCl. In
order to determine whether the activity is that of a motor or a true
MAP, we examined the ability of phagosomes to bind to microtubules in
the presence of a variety of nucleotides and nucleotide analogs. As
shown in Fig. 5B, phagosome-microtubule binding was not
greatly affected by depletion of endogenous cytosolic ATP, by the
addition of ATP, by the nonhydrolyzable ATP analogs AMP-PNP and
ATPS, or by GTP or its nonhydrolyzable analog GTP
S. These
results suggest that the majority of phagosome binding in this assay is
not mediated by motor proteins, as motors are expected to bind more
strongly (in the rigor state) in the absence of ATP or in the presence
of nonhydrolyzable ATP analogs.
MAPs Are Necessary and Sufficient for Binding
To
assess more directly the role of MAPs and motors, microtubule affinity
depletion was used to remove these proteins from the macrophage
cytosol. Excess exogenous taxol-stabilized microtubules were added to
the macrophage cytosol, and the proteins that bound to these
microtubules were removed by pelleting. If this is performed in the
presence of ATP, MAPs are depleted but motors remain. If an ATP
depletion system and AMP-PNP are added, both MAPs and motor proteins
are depleted. Western blotting of both types of depleted cytosols
confirms the specific depletion of representative MAP and motor
proteins (Fig. 6A). As shown in Fig. 7A, the
MAP-depleted cytosol failed to support phagosome binding, whether or
not the motor proteins were removed. Since by microtubule affinity it
is not possible to selectively deplete only the motor proteins, these
were removed by immunodepletion (Fig. 6, C and D).
As shown in Fig. 7B, immunodepletion of either
cytoplasmic dynein or kinesin (the only motors for which adequate
reagents were available) had no affect on binding.
) to the same
levels as mock-depleted cytosol (*). D, the cytosolic binding
activity is recovered in the MAP but not in the motor fraction. Crude
fractions of MAPs and motors eluted sequentially from the same
microtubule pellet (shown in Fig. 6B) were titrated by
sequential 2-fold dilution for their ability to support phagosome
binding in the absence of cytosol. A dilution factor of 1 represents
the maximal amount of MAPs or motors that can be added to the assay,
representing 140 µg/ml protein for MAPs and 40 µg/ml protein
for motors.
The Cytosolic Binding Factor Is a Heat-sensitive MAP in
the Range of 150 kDa
As shown in Fig. 8, fractionation of
macrophage cytosol on a Superose 12 column yields a single peak of
binding activity. As with the activity in whole cytosol (Fig. 3C), the active fractions preferentially
supported the binding of newly formed versus late phagosomes
(data not shown). Though it is still unclear whether the activity
represents a single protein, the active fraction corresponds to a
globular protein around 150 kDa. Close to this size fall two previously
identified MAPs, which were candidate proteins to mediate this binding.
These are CLIP-170, identified as a MAP that mediates binding of
endocytic vesicles to microtubules (Pierre et al., 1992), and
MAP4, which shares structural features with neuronal MAP2, itself
identified as the mediator of chromaffin granule-microtubule binding
(Severin et al., 1991). Western blotting of column fractions
from whole cytosol shows that despite some fractions containing these
two proteins showing binding activity, the peak of activity does not
exactly co-migrate with either protein; both MAP4 and CLIP-170 run
slightly faster than the peak of activity (Fig. 8). This
suggests that our factor is likely to represent either a new MAP or a
known MAP to which no such organelle-binding function has yet been
assigned.
is found at
fraction 3, V
at fraction 36). Fractions were
pooled sequentially two-by-two and tested for binding activity.
Fractionation of the cytosol yielded a single activity peak around 150
kDa. Note that when active fractions were assayed individually the
150-kDa peak was found to be contained mainly in fraction 12. Blotting
of pooled cytosol fractions with 55 anti-CLIP-170 and IF5.2.2
anti-MAP4 shows that the profiles of these proteins do not correspond
with the binding activity.
and this movement is required for their
interaction with compartments of the endocytic pathway (Desjardins et al., 1994a; Jahraus et al., 1994). As phagosomes
are generated by engulfment of individual latex beads, they are large,
discrete, and labeled organelles. Moreover, the buoyancy
characteristics of latex make phagosomes remarkably easy to purify.
These properties facilitate in vivo and in vitro analysis. Finally, since phagosomes are formed de novo,
one can study their biogenesis and how this relates to their binding
and motility along microtubules.)Since we have been unable to modulate the activity of the
binding factor, we are investigating the possibility that it is the
activity of the inhibitor which is regulated. In this assay phagosomes move bidirectionally
along microtubules, although mainly toward the minus end. Used in
tandem, the two assays provide an excellent model to identify the
molecules that mediate anchoring and movement of organelles along
microtubules and to study how they function coordinately.
)S, adenosine
5`-3-O-(thio)triphosphate; GTP
S, guanosine
5`-3-O-(thio)triphosphate; CLIP, cytoplasmic linker protein;
MAP, microtubule-associated protein; PI, protease inhibitor; MES,
4-morpholineethanesulfonic acid.
))))
-We thank Drs S. Fuller, E. Karsenti, T. E.
Kreis, J. Olmsted, and E. Vaisberg for generous gifts of antibodies. We
are grateful to Drs. M. Way and R. G. Parton for critical comments on
the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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