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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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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.
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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.

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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139871&req=5

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.
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