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. 1997 Apr 7;137(1):113-29.
doi: 10.1083/jcb.137.1.113.

Molecular requirements for bi-directional movement of phagosomes along microtubules

A Blocker  1 , F F Severin, J K Burkhardt, J B Bingham, H Yu, J C Olivo, T A Schroer, A A Hyman, G Griffiths
Affiliations

Molecular requirements for bi-directional movement of phagosomes along microtubules

A Blocker et al. J Cell Biol. .

Abstract

Microtubules facilitate the maturation of phagosomes by favoring their interactions with endocytic compartments. Here, we show that phagosomes move within cells along tracks of several microns centrifugally and centripetally in a pH- and microtubule-dependent manner. Phagosome movement was reconstituted in vitro and required energy, cytosol and membrane proteins of this organelle. The activity or presence of these phagosome proteins was regulated as the organelle matured, with "late" phagosomes moving threefold more frequently than "early" ones. The majority of moving phagosomes were minus-end directed; the remainder moved towards microtubule plus-ends and a small subset moved bi-directionally. Minus-end movement showed pharmacological characteristics expected for dyneins, was inhibited by immunodepletion of cytoplasmic dynein and could be restored by addition of cytoplasmic dynein. Plus-end movement displayed pharmacological properties of kinesin, was inhibited partially by immunodepletion of kinesin and fully by addition of an anti-kinesin IgG. Immunodepletion of dynactin, a dynein-activating complex, inhibited only minus-end directed motility. Evidence is provided for a dynactin-associated kinase required for dynein-mediated vesicle transport. Movement in both directions was inhibited by peptide fragments from kinectin (a putative kinesin membrane receptor), derived from the region to which a motility-blocking antibody binds. Polypeptide subunits from these microtubule-based motility factors were detected on phagosomes by immunoblotting or immunoelectron microscopy. This is the first study using a single in vitro system that describes the roles played by kinesin, kinectin, cytoplasmic dynein, and dynactin in the microtubule-mediated movement of a purified membrane organelle.

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Figures

Figure 1
Figure 1
Phagosome movement is microtubule-dependent and sensitive to cytosolic pH. (A) NRK cells were pulsed with latex beads for 30 min and chased for 3 h. At this time all the beads are observed to be in the perinuclear region of the cells. (B) Indirect immunofluorescence labeling of microtubules in the same cells. (C) NRK cells were pulsed with beads as above but subsequently treated and chased in the presence of nocodazole. The phagosome distribution in these cells appears random. (D) The same cells as in D stained using an anti α-tubulin antibody. (E) NRK cells pulsed and chased as above but then treated with acetate Ringers for 20 min. About 30– 50% of the phagosomes were seen to move out to the periphery of the cell (arrows indicate cell boundary). This is observed in ∼70% of the cells. (F) NRK cells loaded with beads, incubated in acetate Ringers for 20 min, and then physiological Ringers for 15 min. Rebound movement of phagosomes to restore tight perinuclear localization of these organelles is observed. Only ∼50% of the cells survive this treatment. (G) NRK cells loaded with beads as above but treated with nocodazole before treatment with acetate Ringers in the presence of nocodazole. Phagosomes do not move to the periphery of the cells but instead appear to be “released” from the perinuclear region (arrows point to the increased space between phagosomes and the nuclear membrane). When cells are treated with nocodazole after the phagosomes have reached the perinuclear region rather than during the chase period, their distribution is very similar (not shown). (H) NRK cells treated with acetate Ringers and then nocodazole before treatment with physiological Ringers containing nocodazole. Phagosomes do not return to a tight perinuclear localization. Bar, 10 μm.
Figure 2
Figure 2
In vitro motility of phagosomes along polarity-marked microtubules. (A) The first (left) and last (right) recorded frames of a typical motility assay field, as analyzed by the tracking program (numbers given by the program to each phagosome have been omitted to allow better visualization of the tracks). Arrows indicate the starting position of three phagosomes that have moved significant distances. By following the tracks starting at these arrows in the left panel, one sees that phagosomes can move relatively long distances and often switch microtubules in the process. The star and square, respectively, indicate phagosomes demonstrating Brownian movement and remaining stationary throughout, for comparison. (B) A close-up of a minus-end directed movement. The arrowhead indicates the brightly labeled rhodamine tubulin “seed” marking the minus end of the microtubule along which the phagosome is moving. The short arrows indicate the original starting point of each phagosome. Long arrows are drawn parallel to the microtubule along which the phagosome is moving to allow better visualization; the arrow points towards the plus end of the microtubule. Frames are shown at 3-s intervals. (C) A plus-end directed movement. (D) A bidirectional movement.
Figure 3
Figure 3
Movement requires energy, cytosol, and proteins of the phagosome membrane, and is regulated during phagosome maturation. (A) The motility assay was performed with 2 M NaCl- or mock-stripped phagosomes in the presence of 15 mg/ml cytosol and an ATP regenerating system (salt and mock salt). Saltstripped phagosomes did not move in the presence of 1 mg/ml casein (salt + casein), nor in the presence of cytosol but in the absence of the ATP regenerating system (−ATP). Uninternalized fish skin gelatin-coated beads (FSG-beads) did not move in the presence of cytosol and ATP. Treatment of phagosomes with 30 μg/ml chymotrypsin for 30 min at 4°C (protease; the protease was then inhibited by treatment with 3,4-dichloroiscoumarin and the phagosomes were separated from residual active protease by flotation into a small sucrose gradient as described in Blocker et al., 1996) reduced their ability to move by eightfold over control (mock protease). (B) Latex bead containing phagosomes were purified from J774 macrophages after various times of internalization: a 20-min pulse, a 1-h pulse followed by a 1-, 4-, 12-, or 24-h chase. The different phagosome preparations were adjusted for bead content in the assay by optical density measurement (see Materials and Methods). This figure was generated using non–saltstripped phagosomes. The dotted line represents an optimized curve fit generated by the computer program KaleidoGraph. Each value represents the mean of the average movements/field/ min of at least two, but often many more, identical motility chambers; errors are population standard deviations. Each experiment was independently repeated at least twice, but often many more times. For each point at least two different preparations of cytosol and phagosomes was tested.
Figure 4
Figure 4
Subunits of cytoplasmic dynein, the dynactin complex, and kinesin are found on phagosomes. HD-11 chicken macrophages (A and B) or NRK cells (C) were pulsed and chased with 1 μm diameter latex beads for 1 h and then processed for cryosectioning and immunoelectron microscopy. Immunolocalization on phagosomes of (A) cytoplasmic dynein heavy chain, using the 440.4 monoclonal antibody; (B) the dynactin subunit p150Glued, using the 150.1 monoclonal antibody; and (C) kinesin heavy chain using the H1 monoclonal antibody. Note the labeling (arrowheads) on the limiting membrane of latex-bead containing phagosomes. Bar, 0.25 μm.
Figure 5
Figure 5
(A) (Left) J774 cytosol was immunodepleted of cytoplasmic dynein with the dynein intermediate chain monoclonal antibody 70.1 (70.1), and mock depleted with an anti-chondritin sulphate antibody (control); the blot was probed with a polyclonal anti-cytoplasmic dynein heavy chain (DHC). The blot was also probed with 150B, an antibody to p150Glued of dynactin (below). (Right) Macrophage cytosol was depleted of dynactin complex with the Arp1 monoclonal antibody 45A (45A), and mock depleted with P5D4 (control); the blot was probed with 150B. The blot was reprobed with anti-DHC (below). (B) Pure bovine brain cytoplasmic dynein (dynein), dynactin (dynactin), and J774 macrophage ATP release (ATP release) were run on a 6–15% gradient gel and stained with Coomassie blue. (C) Phagosomes were purified from HD11 chicken monocyte after different chase periods within cells: 20 min pulse, no chase and 1 h pulse followed by a 4- or 12-h chase. The fractions were normalized for bead content, blotted along side cytosol (Cyt) and a crude membrane fraction (Memb) from HD11 monocytes, at the same protein concentration, and probed with 160.9.1, a monoclonal antibody against chicken kinectin.
Figure 6
Figure 6
Phagosome motility requires cytoplasmic dynein and dynactin complex. (A) Phagosome motility driven by cytosol subjected to two rounds of immunodepletion with the anti-cytoplasmic dynein intermediate chain monoclonal antibody 70.1 (70.1) or mock adsorbed with an isotype-matched monoclonal antibody (anti-chondroitin sulfate; control). Bar 70.1+DE shows the activity of dynein-depleted cytosol to which 25 nM bovine brain dynein was added. (B) Cytosol was immunodepleted of dynactin by two rounds of adsorption with the anti-Arp1 of dynactin monoclonal antibody 45A (45A) or mock depleted with an isotypematched control monoclonal antibody (P5D4; control) and then assayed for motility. The bar labeled 45A+DA shows the activity of dynactin-depleted cytosol to which 25 nM dynactin was added. n is the total number of movements of clear polarity scored for each condition. The percents of plus end–directed movements observed in dynein and dynactin depleted cytosols are significantly different from those seen in the control depleted cytosols at P = 0.01 and P = 0.10, respectively. Black bars correspond to the percent of minus end–directed movements; white bars correspond to the percent of plus end–directed movements; and gray bars represent total motility. Each value represents the mean of the average movements/field/min of at least two, but often many more identical motility chambers; errors are population standard deviations. Each experiment was independently repeated at least twice, but often many more times. For each point at least two different preparations of cytosol, phagosomes, and purified protein or ATP release were tested.
Figure 7
Figure 7
Dynactin activity requires at least Activator X, which may be a dynactinassociated kinase. All assay samples, with the exception of bar 17 (mock-depleted cytosol; control), contained dynactin-depleted cytosol prepared as in Fig. 6. (A) Effect of different concentrations of ATP release on dynactindependent motility. Dynactin-depleted cytosol was reconstituted with different concentrations of macrophage ATP release (0.4 mg, 4 μg, or 0.8 μg/ml) and assayed for motility. The sample containing 0.8 μg/ml ATP release also contained 25 nM bovine brain dynactin. (B) Removal of the “dynactin-activating” activity from ATP release by immunoadsorption of dynactin. ATP release samples were immunoadsorbed with monoclonal antibody 45A (dynactin depleted ATP release) or mock-adsorbed with a control monoclonal antibody (control depleted ATP release), then tested for ability to restore activity to dynactin-depleted cytosol in combination with exogenous bovine brain dynactin (± dynactin). A high salt eluate of the 45A immunoadsorbent (ATP release dynactin eluate; 1.5 μg/ml), control immunoadsorbent (ATP release control eluate; 1.5 μg/ml), or the eluate of 45A immunoadsorbent from the original depletion of cytosol (cytosol dynactin eluate; 20 μg/ml) were also tested for activity. Bar 5 represents the same data as in bar 1 for comparison. Values for bars 9 and 10 are significantly different at P = 0.05. (C) Effect of the general protein kinase inhibitor, staurosporine, on the dynactin-activating activity of the ATP release. 50 nM staurosporine was added to dynactin-depleted cytosol immediately before addition of bovine dynactin and ATP release or to mock-depleted cytosol (control; motility is similar to control cytosol alone, compare with Fig. 6 B; and was also unaffected by addition of DMSO alone, not shown). Bar 13 illustrates the same data as in bars 1 and 5; bar 14 illustrates the same data as bar 3; and bar 15 illustrates the same data as bar 6; these three values are included for comparison. Each value in this figure represents the mean of the average movements/field/min of at least two, but often many more, identical motility chambers; errors are population standard deviations. Each experiment was independently repeated at least twice, but often many more times. For each point at least two different preparations of cytosol, phagosomes, and purified protein or ATP release were tested.
Figure 8
Figure 8
Phagosome motility requires kinesin and kinectin. (A) Effects of kinesin antibodies on phagosome motility. The function-blocking kinesin antibody, SUK4, was tested for its effects on phagosome motility when added directly to the assay at two different concentrations (100 μg/ml, SUK4 IgG 100 and 10 μg/ml, SUK4 IgG 10). Bar control IgG 100 shows the effect on motility of 100 μg/ml control isotype-matched IgG (P5D4). (B) Effects of kinectin peptides on phagosomes motility. 10 μM chicken kinectin fragments corresponding to amino acids 295-612 (aa 295-612), 568-943 (aa 568-943), and 924-1321 (aa 924-1321) were added directly to the phagosome motility assay. For both A and B, each value represents the mean of the average movements/field/min of at least two, but often many more, identical motility chambers; errors are population standard deviations. Each experiment was independently repeated at least twice, but often many more times. For each experiment at least two different preparations of cytosol and phagosomes were tested. Only one preparation of purified IgGs or protein fragments were tested.
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References

    1. Allan VJ. Protein phosphatase 1 regulates the cytoplasmic dynein-driven formation of endoplasmic reticulum networks in vitro. J Cell Biol. 1995;128:879–891. - PMC - PubMed
    1. Allan VJ. Role of motor proteins in organizing the endoplasmic reticulum and Golgi apparatus. Semin Cell Biol. 1996;7:335–342.
    1. Allan VJ, Vale RD. Cell cycle control of microtuble-based membrane transport and tubule formation in vitro. J Cell Biol. 1991;113:347–359. - PMC - PubMed
    1. Aniento F, Emans N, Griffiths G, Gruenberg J. Cytoplasmic dynein-dependent vesicular transport from early to late endosomes. J Cell Biol. 1993;123:1373–1387. - PMC - PubMed
    1. Ashkin A, Schütze K, Dziedzic JM, Euteneuer U, Schliwa M. Force generation of organelle transport measured in vivo by an infrared laser trap. Nature (Lond) 1990;348:346–348. - PubMed

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