(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), ()phagosome movement ceases. (
)These results
suggest that phagosomes move within the cell along microtubules.
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 g for 20 min at 4
°C. The supernatant was desalted as directly above.
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. ()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.
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 () 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.
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 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.
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, 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.
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. ()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.
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. 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.