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Dynamin 2 is required for phagocytosis in macrophages.

Gold ES, Underhill DM, Morrissette NS, Guo J, McNiven MA, Aderem A - J. Exp. Med. (1999)

Bottom Line: Cells internalize soluble ligands through endocytosis and large particles through actin-based phagocytosis.Dynamin 2 is enriched on early phagosomes, and expression of a dominant-negative mutant of dynamin 2 significantly inhibits particle internalization at the stage of membrane extension around the particle.This arrest in phagocytosis resembles that seen with inhibitors of phosphoinositide 3-kinase (PI3K), and inhibition of PI3K prevents the recruitment of dynamin to the site of particle binding.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Washington, Seattle, Washington 98195, USA.

ABSTRACT
Cells internalize soluble ligands through endocytosis and large particles through actin-based phagocytosis. The dynamin family of GTPases mediates the scission of endocytic vesicles from the plasma membrane. We report here that dynamin 2, a ubiquitously expressed dynamin isoform, has a role in phagocytosis in macrophages. Dynamin 2 is enriched on early phagosomes, and expression of a dominant-negative mutant of dynamin 2 significantly inhibits particle internalization at the stage of membrane extension around the particle. This arrest in phagocytosis resembles that seen with inhibitors of phosphoinositide 3-kinase (PI3K), and inhibition of PI3K prevents the recruitment of dynamin to the site of particle binding. Although expression of mutant dynamin in macrophages inhibited particle internalization, it had no effect on the production of inflammatory mediators elicited by particle binding.

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Related in: MedlinePlus

DynK44A inhibits membrane extension around the forming phagosome. Transiently transfected cells were sorted by FACS®, and cells expressing the highest levels of GFP were studied by scanning electron microscopy. Sorted cells were incubated with IgG-coated SRBCs for 10 min (A and B) or for 1 h (C and D). After a 10-min internalization, control pTIGZ2–transfected cells demonstrated various stages of particle internalization (A), and after 1 h, >90% of the particles were internalized (C). By contrast, cells expressing dynK44A could only extend membrane partially around the bound particles at both time points (B and D). The size bar shown in D applies to A–D.
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Figure 5: DynK44A inhibits membrane extension around the forming phagosome. Transiently transfected cells were sorted by FACS®, and cells expressing the highest levels of GFP were studied by scanning electron microscopy. Sorted cells were incubated with IgG-coated SRBCs for 10 min (A and B) or for 1 h (C and D). After a 10-min internalization, control pTIGZ2–transfected cells demonstrated various stages of particle internalization (A), and after 1 h, >90% of the particles were internalized (C). By contrast, cells expressing dynK44A could only extend membrane partially around the bound particles at both time points (B and D). The size bar shown in D applies to A–D.

Mentions: The defect in phagocytosis was not due to an effect of dynK44A on the level of phagocytic receptors, since transfected cells expressed normal cell surface levels of FcRs (CD16 and CD32) and CRs (Mac-1) (data not shown), and particle binding was unimpaired (Table ; and see Fig. 4 and Fig. 5). Cell viability and other actin-based processes were unaffected by the expression of dynK44A as demonstrated by the observations that RAW-TT10 cells expressing dynK44A were able to migrate, polarize, extend and retract ruffles, and spread in response to phorbol esters (data not shown). Further, engagement of phagocytic receptors stimulated actin polymerization at the site of particle binding (Fig. 4). In addition, mutant dynamin had no effect on particle-induced TNF-α production (see Fig. 7, below), demonstrating that one arm (internalization) of a bifurcating signaling pathway was selectively inhibited.


Dynamin 2 is required for phagocytosis in macrophages.

Gold ES, Underhill DM, Morrissette NS, Guo J, McNiven MA, Aderem A - J. Exp. Med. (1999)

DynK44A inhibits membrane extension around the forming phagosome. Transiently transfected cells were sorted by FACS®, and cells expressing the highest levels of GFP were studied by scanning electron microscopy. Sorted cells were incubated with IgG-coated SRBCs for 10 min (A and B) or for 1 h (C and D). After a 10-min internalization, control pTIGZ2–transfected cells demonstrated various stages of particle internalization (A), and after 1 h, >90% of the particles were internalized (C). By contrast, cells expressing dynK44A could only extend membrane partially around the bound particles at both time points (B and D). The size bar shown in D applies to A–D.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195719&req=5

Figure 5: DynK44A inhibits membrane extension around the forming phagosome. Transiently transfected cells were sorted by FACS®, and cells expressing the highest levels of GFP were studied by scanning electron microscopy. Sorted cells were incubated with IgG-coated SRBCs for 10 min (A and B) or for 1 h (C and D). After a 10-min internalization, control pTIGZ2–transfected cells demonstrated various stages of particle internalization (A), and after 1 h, >90% of the particles were internalized (C). By contrast, cells expressing dynK44A could only extend membrane partially around the bound particles at both time points (B and D). The size bar shown in D applies to A–D.
Mentions: The defect in phagocytosis was not due to an effect of dynK44A on the level of phagocytic receptors, since transfected cells expressed normal cell surface levels of FcRs (CD16 and CD32) and CRs (Mac-1) (data not shown), and particle binding was unimpaired (Table ; and see Fig. 4 and Fig. 5). Cell viability and other actin-based processes were unaffected by the expression of dynK44A as demonstrated by the observations that RAW-TT10 cells expressing dynK44A were able to migrate, polarize, extend and retract ruffles, and spread in response to phorbol esters (data not shown). Further, engagement of phagocytic receptors stimulated actin polymerization at the site of particle binding (Fig. 4). In addition, mutant dynamin had no effect on particle-induced TNF-α production (see Fig. 7, below), demonstrating that one arm (internalization) of a bifurcating signaling pathway was selectively inhibited.

Bottom Line: Cells internalize soluble ligands through endocytosis and large particles through actin-based phagocytosis.Dynamin 2 is enriched on early phagosomes, and expression of a dominant-negative mutant of dynamin 2 significantly inhibits particle internalization at the stage of membrane extension around the particle.This arrest in phagocytosis resembles that seen with inhibitors of phosphoinositide 3-kinase (PI3K), and inhibition of PI3K prevents the recruitment of dynamin to the site of particle binding.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Washington, Seattle, Washington 98195, USA.

ABSTRACT
Cells internalize soluble ligands through endocytosis and large particles through actin-based phagocytosis. The dynamin family of GTPases mediates the scission of endocytic vesicles from the plasma membrane. We report here that dynamin 2, a ubiquitously expressed dynamin isoform, has a role in phagocytosis in macrophages. Dynamin 2 is enriched on early phagosomes, and expression of a dominant-negative mutant of dynamin 2 significantly inhibits particle internalization at the stage of membrane extension around the particle. This arrest in phagocytosis resembles that seen with inhibitors of phosphoinositide 3-kinase (PI3K), and inhibition of PI3K prevents the recruitment of dynamin to the site of particle binding. Although expression of mutant dynamin in macrophages inhibited particle internalization, it had no effect on the production of inflammatory mediators elicited by particle binding.

Show MeSH
Related in: MedlinePlus