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Molecular requirements for bi-directional movement of phagosomes along microtubules.

Blocker A, Severin FF, Burkhardt JK, Bingham JB, Yu H, Olivo JC, Schroer TA, Hyman AA, Griffiths G - J. Cell Biol. (1997)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Programme, European Molecular Biology Laboratory, Heidelberg, Germany. ablocker@pasteur.fr

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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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.
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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.

Mentions: To test this hypothesis, we determined whether dynactin-enriched fractions taken from different steps of the purification could restore phagosome motility to dynactindepleted cytosol. A partially purified mixture of dynactin and cytoplasmic dynein did not restore motility (the 20S sucrose gradient pool, see Schroer and Sheetz, 1991; data not shown). However, phagosome motility could be fully restored by macrophage microtubule “ATP release” (prepared as in Blocker et al., 1996; Fig. 7 A). This fraction is enriched in microtubule-binding proteins that dissociate from microtubules in the presence of ATP and contains a number of abundant, defined polypeptides (including dynein and dynactin), as well as many other minor, unidentified proteins (its composition is shown in Fig. 5 B). Dynactin represents ∼5% of the total protein in this fraction, as estimated by quantitative immunoblotting. That a crude, ATP release fraction, but not partially or highly purified dynactin could restore activity to dynactin-depleted cytosol suggested that ATP release contained an additional “dynactin-activating” component necessary for dyneinmediated vesicle motility.


Molecular requirements for bi-directional movement of phagosomes along microtubules.

Blocker A, Severin FF, Burkhardt JK, Bingham JB, Yu H, Olivo JC, Schroer TA, Hyman AA, Griffiths G - J. Cell Biol. (1997)

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.
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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.
Mentions: To test this hypothesis, we determined whether dynactin-enriched fractions taken from different steps of the purification could restore phagosome motility to dynactindepleted cytosol. A partially purified mixture of dynactin and cytoplasmic dynein did not restore motility (the 20S sucrose gradient pool, see Schroer and Sheetz, 1991; data not shown). However, phagosome motility could be fully restored by macrophage microtubule “ATP release” (prepared as in Blocker et al., 1996; Fig. 7 A). This fraction is enriched in microtubule-binding proteins that dissociate from microtubules in the presence of ATP and contains a number of abundant, defined polypeptides (including dynein and dynactin), as well as many other minor, unidentified proteins (its composition is shown in Fig. 5 B). Dynactin represents ∼5% of the total protein in this fraction, as estimated by quantitative immunoblotting. That a crude, ATP release fraction, but not partially or highly purified dynactin could restore activity to dynactin-depleted cytosol suggested that ATP release contained an additional “dynactin-activating” component necessary for dyneinmediated vesicle motility.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Programme, European Molecular Biology Laboratory, Heidelberg, Germany. ablocker@pasteur.fr

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.

Show MeSH
Related in: MedlinePlus