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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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(A) (Left) J774 cytosol was immunodepleted of cytoplasmic dynein with the dynein intermediate chain monoclonal  antibody 70.1 (70.1), and mock depleted with an anti-chondritin  sulphate antibody (control); the blot was probed with a polyclonal anti-cytoplasmic dynein heavy chain (DHC). The blot was  also probed with 150B, an antibody to p150Glued of dynactin (below). (Right) Macrophage cytosol was depleted of dynactin complex with the Arp1 monoclonal antibody 45A (45A), and mock  depleted with P5D4 (control); the blot was probed with 150B.  The blot was reprobed with anti-DHC (below). (B) Pure bovine  brain cytoplasmic dynein (dynein), dynactin (dynactin), and J774  macrophage ATP release (ATP release) were run on a 6–15%  gradient gel and stained with Coomassie blue. (C) Phagosomes  were purified from HD11 chicken monocyte after different chase  periods within cells: 20 min pulse, no chase and 1 h pulse followed  by a 4- or 12-h chase. The fractions were normalized for bead  content, blotted along side cytosol (Cyt) and a crude membrane  fraction (Memb) from HD11 monocytes, at the same protein concentration, and probed with 160.9.1, a monoclonal antibody  against chicken kinectin.
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Figure 5: (A) (Left) J774 cytosol was immunodepleted of cytoplasmic dynein with the dynein intermediate chain monoclonal antibody 70.1 (70.1), and mock depleted with an anti-chondritin sulphate antibody (control); the blot was probed with a polyclonal anti-cytoplasmic dynein heavy chain (DHC). The blot was also probed with 150B, an antibody to p150Glued of dynactin (below). (Right) Macrophage cytosol was depleted of dynactin complex with the Arp1 monoclonal antibody 45A (45A), and mock depleted with P5D4 (control); the blot was probed with 150B. The blot was reprobed with anti-DHC (below). (B) Pure bovine brain cytoplasmic dynein (dynein), dynactin (dynactin), and J774 macrophage ATP release (ATP release) were run on a 6–15% gradient gel and stained with Coomassie blue. (C) Phagosomes were purified from HD11 chicken monocyte after different chase periods within cells: 20 min pulse, no chase and 1 h pulse followed by a 4- or 12-h chase. The fractions were normalized for bead content, blotted along side cytosol (Cyt) and a crude membrane fraction (Memb) from HD11 monocytes, at the same protein concentration, and probed with 160.9.1, a monoclonal antibody against chicken kinectin.

Mentions: The previous results suggested that the motility of phagosomes is driven by cytoplasmic dynein (CD1; see Vaisberg et al., 1996), dynactin, and conventional kinesin. To verify this hypothesis, we used immunological reagents specific for these different proteins to determine whether each was functionally involved in mediating phagosome motility in our in vitro assay. Immunodepletion of cytoplasmic dynein was performed using the monoclonal antibody 70.1 against the conventional cytoplasmic dynein 74-kD intermediate chain (however, it is still unclear whether cytoplasmic dynein heavy chains share associated subunits, Vaisberg, E., personal communication). Performing two rounds of immunodepletion removed ∼80% of the original dynein heavy chain (Fig. 5 A). Removal of dynein reduced phagosome motility significantly as compared to a mock-depleted control. Minus end–directed motility was reduced from 70– 25% while plus-end movement was not affected (Fig. 6 A). We estimated the amount of dynein present in cytosol to be 2 nM by quantitative immunoblotting using purified bovine dynein as a standard. Motility of dynein depletedcytosol could be fully restored by addition of 25 nM purified bovine brain dynein (Figs. 5 B and 6 A; this was the lowest concentration that supported microtubule gliding, see Materials and Methods). Thus, cytoplasmic dynein (most likely CD1) is the motor for minus end–directed movement of phagosomes along microtubules.


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)

(A) (Left) J774 cytosol was immunodepleted of cytoplasmic dynein with the dynein intermediate chain monoclonal  antibody 70.1 (70.1), and mock depleted with an anti-chondritin  sulphate antibody (control); the blot was probed with a polyclonal anti-cytoplasmic dynein heavy chain (DHC). The blot was  also probed with 150B, an antibody to p150Glued of dynactin (below). (Right) Macrophage cytosol was depleted of dynactin complex with the Arp1 monoclonal antibody 45A (45A), and mock  depleted with P5D4 (control); the blot was probed with 150B.  The blot was reprobed with anti-DHC (below). (B) Pure bovine  brain cytoplasmic dynein (dynein), dynactin (dynactin), and J774  macrophage ATP release (ATP release) were run on a 6–15%  gradient gel and stained with Coomassie blue. (C) Phagosomes  were purified from HD11 chicken monocyte after different chase  periods within cells: 20 min pulse, no chase and 1 h pulse followed  by a 4- or 12-h chase. The fractions were normalized for bead  content, blotted along side cytosol (Cyt) and a crude membrane  fraction (Memb) from HD11 monocytes, at the same protein concentration, and probed with 160.9.1, a monoclonal antibody  against chicken kinectin.
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Figure 5: (A) (Left) J774 cytosol was immunodepleted of cytoplasmic dynein with the dynein intermediate chain monoclonal antibody 70.1 (70.1), and mock depleted with an anti-chondritin sulphate antibody (control); the blot was probed with a polyclonal anti-cytoplasmic dynein heavy chain (DHC). The blot was also probed with 150B, an antibody to p150Glued of dynactin (below). (Right) Macrophage cytosol was depleted of dynactin complex with the Arp1 monoclonal antibody 45A (45A), and mock depleted with P5D4 (control); the blot was probed with 150B. The blot was reprobed with anti-DHC (below). (B) Pure bovine brain cytoplasmic dynein (dynein), dynactin (dynactin), and J774 macrophage ATP release (ATP release) were run on a 6–15% gradient gel and stained with Coomassie blue. (C) Phagosomes were purified from HD11 chicken monocyte after different chase periods within cells: 20 min pulse, no chase and 1 h pulse followed by a 4- or 12-h chase. The fractions were normalized for bead content, blotted along side cytosol (Cyt) and a crude membrane fraction (Memb) from HD11 monocytes, at the same protein concentration, and probed with 160.9.1, a monoclonal antibody against chicken kinectin.
Mentions: The previous results suggested that the motility of phagosomes is driven by cytoplasmic dynein (CD1; see Vaisberg et al., 1996), dynactin, and conventional kinesin. To verify this hypothesis, we used immunological reagents specific for these different proteins to determine whether each was functionally involved in mediating phagosome motility in our in vitro assay. Immunodepletion of cytoplasmic dynein was performed using the monoclonal antibody 70.1 against the conventional cytoplasmic dynein 74-kD intermediate chain (however, it is still unclear whether cytoplasmic dynein heavy chains share associated subunits, Vaisberg, E., personal communication). Performing two rounds of immunodepletion removed ∼80% of the original dynein heavy chain (Fig. 5 A). Removal of dynein reduced phagosome motility significantly as compared to a mock-depleted control. Minus end–directed motility was reduced from 70– 25% while plus-end movement was not affected (Fig. 6 A). We estimated the amount of dynein present in cytosol to be 2 nM by quantitative immunoblotting using purified bovine dynein as a standard. Motility of dynein depletedcytosol could be fully restored by addition of 25 nM purified bovine brain dynein (Figs. 5 B and 6 A; this was the lowest concentration that supported microtubule gliding, see Materials and Methods). Thus, cytoplasmic dynein (most likely CD1) is the motor for minus end–directed movement of phagosomes along microtubules.

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