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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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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.
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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.

Mentions: At least three non-isogenic forms of cytoplasmic dynein have been recently identified in various systems (Tanaka et al., 1995; Vaisberg et al., 1996) and several nonkinetochore kinesin-related proteins are known to co-exist within the same cell type (reviewed in Hirokawa, 1996). We therefore wished to determine which member(s) of the kinesin and dynein families were in fact present on this organelle in vivo. Immunoblot analysis on purified phagosomes indicated that some, but not all, subunits of conventional dynein, dynactin, and kinesin could be detected (Blocker, 1995). Since all three proteins are peripherally associated with membranes, we reasoned that they might be partially lost from phagosomes during purification. We therefore decided to examine the association of these proteins with phagosomes in a more in vivo context, by performing immunoelectron microscopy on cells that had internalized latex beads. The antibody 440.4 raised against conventional chicken dynein heavy chain (CD1; however, it is not yet clear whether this antibody also recognizes the other cytoplasmic dynein heavy chains, Vaisberg, E., personal communication) labeled the cytoplasm as well as membrane organelles, in particular phagosomes, in HD11 chicken macrophages (Fig. 4 A). A subunit of the dynactin complex, p150Glued, showed a very similar distribution (Fig. 4 B and Burkhardt, J.K., A. Habermann, G. Griffiths, and T.A. Schroer, manuscript in preparation). The monoclonal antibody H1, against bovine kinesin heavy chain, was tested on NRK cells. Kinesin was found on all endocytic organelles including phagosomes (Fig. 4 C), although it was more abundant on other organelles (Blocker, A., A. Habermann, and G. Griffiths, in preparation). Although it was not possible to estimate from this data the approximate number of such molecules present per phagosome (antibody labeling efficiencies using this method are very difficult to estimate), the relative quantitative distribution of these factors on phagosomes and other organelles will be published elsewhere. The key point for the present study is that cytoplasmic dynein (mostly likely CD1), dynactin, and conventional kinesin localize to phagosomes in vivo.


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139871&req=5

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.
Mentions: At least three non-isogenic forms of cytoplasmic dynein have been recently identified in various systems (Tanaka et al., 1995; Vaisberg et al., 1996) and several nonkinetochore kinesin-related proteins are known to co-exist within the same cell type (reviewed in Hirokawa, 1996). We therefore wished to determine which member(s) of the kinesin and dynein families were in fact present on this organelle in vivo. Immunoblot analysis on purified phagosomes indicated that some, but not all, subunits of conventional dynein, dynactin, and kinesin could be detected (Blocker, 1995). Since all three proteins are peripherally associated with membranes, we reasoned that they might be partially lost from phagosomes during purification. We therefore decided to examine the association of these proteins with phagosomes in a more in vivo context, by performing immunoelectron microscopy on cells that had internalized latex beads. The antibody 440.4 raised against conventional chicken dynein heavy chain (CD1; however, it is not yet clear whether this antibody also recognizes the other cytoplasmic dynein heavy chains, Vaisberg, E., personal communication) labeled the cytoplasm as well as membrane organelles, in particular phagosomes, in HD11 chicken macrophages (Fig. 4 A). A subunit of the dynactin complex, p150Glued, showed a very similar distribution (Fig. 4 B and Burkhardt, J.K., A. Habermann, G. Griffiths, and T.A. Schroer, manuscript in preparation). The monoclonal antibody H1, against bovine kinesin heavy chain, was tested on NRK cells. Kinesin was found on all endocytic organelles including phagosomes (Fig. 4 C), although it was more abundant on other organelles (Blocker, A., A. Habermann, and G. Griffiths, in preparation). Although it was not possible to estimate from this data the approximate number of such molecules present per phagosome (antibody labeling efficiencies using this method are very difficult to estimate), the relative quantitative distribution of these factors on phagosomes and other organelles will be published elsewhere. The key point for the present study is that cytoplasmic dynein (mostly likely CD1), dynactin, and conventional kinesin localize to phagosomes in vivo.

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