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A mechanical bottleneck explains the variation in cup growth during FcgammaR phagocytosis.

van Zon JS, Tzircotis G, Caron E, Howard M - Mol. Syst. Biol. (2009)

Bottom Line: Here, we study the internalization of immunoglobulin G-coated particles in cells transfected with Fcgamma receptors (FcgammaRs) through the formation of an enveloping phagocytic cup.We explain these observations in terms of a mechanical bottleneck using a simple mathematical model of the overall process of cup growth.Our analysis gives a coherent explanation for the importance of geometry in phagocytic uptake and provides a unifying framework for integrating the key processes, both biochemical and mechanical, occurring during cup growth.

View Article: PubMed Central - PubMed

Affiliation: Centre for Integrative Systems Biology Imperial College (CISBIC), South Kensington Campus, Imperial College London, London, UK.

ABSTRACT
Phagocytosis is the process by which cells internalize particulate material, and is of central importance to immunity, homeostasis and development. Here, we study the internalization of immunoglobulin G-coated particles in cells transfected with Fcgamma receptors (FcgammaRs) through the formation of an enveloping phagocytic cup. Using confocal microscopy, we precisely track the location of fluorescently tagged FcgammaRs during cup growth. Surprisingly, we found that phagocytic cups growing around identical spherical particles showed great variability even within a single cell and exhibited two eventual fates: a cup either stalled before forming a half-cup or it proceeded until the particle was fully enveloped. We explain these observations in terms of a mechanical bottleneck using a simple mathematical model of the overall process of cup growth. The model predicts that reducing F-actin concentration levels, and hence the deforming force, does not necessarily lead to stalled cups, a prediction we verify experimentally. Our analysis gives a coherent explanation for the importance of geometry in phagocytic uptake and provides a unifying framework for integrating the key processes, both biochemical and mechanical, occurring during cup growth.

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Typical cup shape and distribution of FcγR–GFP (green) and F-actin (red) in the phagocytic cup at 0, 4 and 10 min after the start of phagocytosis. COS-7 cells were transfected with (A, B) WT FcγR, (C, D) Y282F/Y298F FcγR, and (E, F) WT FcγR treated with 0.2 μM cytochalasin D. (A), (C) and (E) Representative images, showing the projection of fluorescence along the vertical axis. (B), (D) and (F) Corresponding averaged phagocytic cup cross-section (see Materials and methods for averaging methodology). For each image, the position of the particle is indicated by the dotted white lines. (G) Definition of the phagocytic cup size S, where S is the distance along the phagocytic cup from the cup center to the cup rim measured along the particle surface. (H) Shape and localization of FcγRs (green) as a function of time as predicted by our model for parameters corresponding to WT FcγRs.
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f2: Typical cup shape and distribution of FcγR–GFP (green) and F-actin (red) in the phagocytic cup at 0, 4 and 10 min after the start of phagocytosis. COS-7 cells were transfected with (A, B) WT FcγR, (C, D) Y282F/Y298F FcγR, and (E, F) WT FcγR treated with 0.2 μM cytochalasin D. (A), (C) and (E) Representative images, showing the projection of fluorescence along the vertical axis. (B), (D) and (F) Corresponding averaged phagocytic cup cross-section (see Materials and methods for averaging methodology). For each image, the position of the particle is indicated by the dotted white lines. (G) Definition of the phagocytic cup size S, where S is the distance along the phagocytic cup from the cup center to the cup rim measured along the particle surface. (H) Shape and localization of FcγRs (green) as a function of time as predicted by our model for parameters corresponding to WT FcγRs.

Mentions: Using confocal microscopy we systematically studied the distribution of F-actin, using fluorescent phalloidin, and also WT FcγR tagged with green fluorescent protein (GFP) (see Materials and methods). First, we confirmed by phagocytosis assays that COS-7 cells expressing GFP-tagged WT FcγR properly ingested IgG-opsonized spherical latex particles 3 μm in diameter (Figure 1). As expected, cells left untransfected or transfected with GFP alone could not bind IgG-opsonized particles; neither could FcγR-transfected cells bind non-opsonized particles (data not shown). GFP-tagged, FcγR-transfected cells were presented with IgG-opsonized particles, fixed at 2-min intervals and imaged by confocal microscopy to obtain a complete picture of the cortical deformation as well as the distribution of FcγR and F-actin at various times following synchronizing cold treatment (see Materials and methods). Figure 2A shows typical examples of WT FcγR cup morphology at various time points. We find that for cells transfected with WT FcγR, phagocytic cups progress over the particle in a radially symmetric manner, leading to cup closure in 4–10 min for a significant fraction of particles. In addition, we find that F-actin and FcγR are strongly co-localized, as can be seen in Figure 2A and B, in agreement with previous observations (Greenberg et al, 1990; Caron and Hall, 1998).


A mechanical bottleneck explains the variation in cup growth during FcgammaR phagocytosis.

van Zon JS, Tzircotis G, Caron E, Howard M - Mol. Syst. Biol. (2009)

Typical cup shape and distribution of FcγR–GFP (green) and F-actin (red) in the phagocytic cup at 0, 4 and 10 min after the start of phagocytosis. COS-7 cells were transfected with (A, B) WT FcγR, (C, D) Y282F/Y298F FcγR, and (E, F) WT FcγR treated with 0.2 μM cytochalasin D. (A), (C) and (E) Representative images, showing the projection of fluorescence along the vertical axis. (B), (D) and (F) Corresponding averaged phagocytic cup cross-section (see Materials and methods for averaging methodology). For each image, the position of the particle is indicated by the dotted white lines. (G) Definition of the phagocytic cup size S, where S is the distance along the phagocytic cup from the cup center to the cup rim measured along the particle surface. (H) Shape and localization of FcγRs (green) as a function of time as predicted by our model for parameters corresponding to WT FcγRs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2736656&req=5

f2: Typical cup shape and distribution of FcγR–GFP (green) and F-actin (red) in the phagocytic cup at 0, 4 and 10 min after the start of phagocytosis. COS-7 cells were transfected with (A, B) WT FcγR, (C, D) Y282F/Y298F FcγR, and (E, F) WT FcγR treated with 0.2 μM cytochalasin D. (A), (C) and (E) Representative images, showing the projection of fluorescence along the vertical axis. (B), (D) and (F) Corresponding averaged phagocytic cup cross-section (see Materials and methods for averaging methodology). For each image, the position of the particle is indicated by the dotted white lines. (G) Definition of the phagocytic cup size S, where S is the distance along the phagocytic cup from the cup center to the cup rim measured along the particle surface. (H) Shape and localization of FcγRs (green) as a function of time as predicted by our model for parameters corresponding to WT FcγRs.
Mentions: Using confocal microscopy we systematically studied the distribution of F-actin, using fluorescent phalloidin, and also WT FcγR tagged with green fluorescent protein (GFP) (see Materials and methods). First, we confirmed by phagocytosis assays that COS-7 cells expressing GFP-tagged WT FcγR properly ingested IgG-opsonized spherical latex particles 3 μm in diameter (Figure 1). As expected, cells left untransfected or transfected with GFP alone could not bind IgG-opsonized particles; neither could FcγR-transfected cells bind non-opsonized particles (data not shown). GFP-tagged, FcγR-transfected cells were presented with IgG-opsonized particles, fixed at 2-min intervals and imaged by confocal microscopy to obtain a complete picture of the cortical deformation as well as the distribution of FcγR and F-actin at various times following synchronizing cold treatment (see Materials and methods). Figure 2A shows typical examples of WT FcγR cup morphology at various time points. We find that for cells transfected with WT FcγR, phagocytic cups progress over the particle in a radially symmetric manner, leading to cup closure in 4–10 min for a significant fraction of particles. In addition, we find that F-actin and FcγR are strongly co-localized, as can be seen in Figure 2A and B, in agreement with previous observations (Greenberg et al, 1990; Caron and Hall, 1998).

Bottom Line: Here, we study the internalization of immunoglobulin G-coated particles in cells transfected with Fcgamma receptors (FcgammaRs) through the formation of an enveloping phagocytic cup.We explain these observations in terms of a mechanical bottleneck using a simple mathematical model of the overall process of cup growth.Our analysis gives a coherent explanation for the importance of geometry in phagocytic uptake and provides a unifying framework for integrating the key processes, both biochemical and mechanical, occurring during cup growth.

View Article: PubMed Central - PubMed

Affiliation: Centre for Integrative Systems Biology Imperial College (CISBIC), South Kensington Campus, Imperial College London, London, UK.

ABSTRACT
Phagocytosis is the process by which cells internalize particulate material, and is of central importance to immunity, homeostasis and development. Here, we study the internalization of immunoglobulin G-coated particles in cells transfected with Fcgamma receptors (FcgammaRs) through the formation of an enveloping phagocytic cup. Using confocal microscopy, we precisely track the location of fluorescently tagged FcgammaRs during cup growth. Surprisingly, we found that phagocytic cups growing around identical spherical particles showed great variability even within a single cell and exhibited two eventual fates: a cup either stalled before forming a half-cup or it proceeded until the particle was fully enveloped. We explain these observations in terms of a mechanical bottleneck using a simple mathematical model of the overall process of cup growth. The model predicts that reducing F-actin concentration levels, and hence the deforming force, does not necessarily lead to stalled cups, a prediction we verify experimentally. Our analysis gives a coherent explanation for the importance of geometry in phagocytic uptake and provides a unifying framework for integrating the key processes, both biochemical and mechanical, occurring during cup growth.

Show MeSH
Related in: MedlinePlus