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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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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.
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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.

Mentions: To dissect the mechanism of phagosome transport in molecular detail, we have reconstituted the movement of purified phagosomes along polarity-marked microtubules in vitro using a fluorescence video microscopy assay. A uniform lawn of dimly fluorescent microtubules marked at their minus ends by brightly labeled “seeds” (Howard and Hyman, 1993) was laid down on the coverglass of a perfusion chamber. A mixture of purified, salt-stripped phagosomes (containing weakly fluorescent latex beads coupled to fish skin gelatin, FSG; see Materials and Methods), J774 macrophage cytosol, and an ATP regenerating system were then added. In the presence of both ATP and cytosol, phagosomes displayed movements, sometimes many microns in length, along microtubules (Figs. 2 A and 3 A). A small number of microtubules in each field displayed gliding which was essentially plus-end directed (not shown).


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)

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.
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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.
Mentions: To dissect the mechanism of phagosome transport in molecular detail, we have reconstituted the movement of purified phagosomes along polarity-marked microtubules in vitro using a fluorescence video microscopy assay. A uniform lawn of dimly fluorescent microtubules marked at their minus ends by brightly labeled “seeds” (Howard and Hyman, 1993) was laid down on the coverglass of a perfusion chamber. A mixture of purified, salt-stripped phagosomes (containing weakly fluorescent latex beads coupled to fish skin gelatin, FSG; see Materials and Methods), J774 macrophage cytosol, and an ATP regenerating system were then added. In the presence of both ATP and cytosol, phagosomes displayed movements, sometimes many microns in length, along microtubules (Figs. 2 A and 3 A). A small number of microtubules in each field displayed gliding which was essentially plus-end directed (not shown).

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