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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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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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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.

Mentions: Although phagosomes moved predominantly in the minus-end direction, we observed a significant number of movements (∼30%) toward the plus-end of microtubules. Our immunolocalization data (Fig. 4 C) indicated that conventional kinesin was present on the phagosome surface, suggesting that it was serving as the plus-end motor. To explore this question further, we used the mouse monoclonal antibody against sea urchin kinesin heavy chain, SUK4, which is known to inhibit the kinesin ATPase (Ingold et al., 1988). When SUK4 IgG was added at 100 μg/ml to the assay, bidirectional motility was nearly completely inhibited whereas addition of control IgG had no effect; 10 μg/ml SUK4 had little effect (Fig. 8 A). However, when we depleted 95% of kinesin heavy chain using the SUK4 antibody, we saw no significant decrease in minus-end directed motility but a 51% decrease in plus-end directed motility (n = 21, statistically significant at P = 0.01). These results strongly suggest that plus end–directed phagosome movement is driven by kinesin.


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)

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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Related In: Results  -  Collection

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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.
Mentions: Although phagosomes moved predominantly in the minus-end direction, we observed a significant number of movements (∼30%) toward the plus-end of microtubules. Our immunolocalization data (Fig. 4 C) indicated that conventional kinesin was present on the phagosome surface, suggesting that it was serving as the plus-end motor. To explore this question further, we used the mouse monoclonal antibody against sea urchin kinesin heavy chain, SUK4, which is known to inhibit the kinesin ATPase (Ingold et al., 1988). When SUK4 IgG was added at 100 μg/ml to the assay, bidirectional motility was nearly completely inhibited whereas addition of control IgG had no effect; 10 μg/ml SUK4 had little effect (Fig. 8 A). However, when we depleted 95% of kinesin heavy chain using the SUK4 antibody, we saw no significant decrease in minus-end directed motility but a 51% decrease in plus-end directed motility (n = 21, statistically significant at P = 0.01). These results strongly suggest that plus end–directed phagosome movement is driven by kinesin.

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