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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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Related in: MedlinePlus

Phagocytosis assays in FcγR-transfected COS-7 cells. Cells were transfected with wild-type (WT) or Y282F/Y298F FcγR and subsequently challenged with IgG-opsonized particles for 10 min with or without 0.2 μM cytochalasin D. Fifty FcγR-expressing cells were scored for the number of particles attached but not internalized versus the number internalized. The fraction of successful internalizing events was then calculated.
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f1: Phagocytosis assays in FcγR-transfected COS-7 cells. Cells were transfected with wild-type (WT) or Y282F/Y298F FcγR and subsequently challenged with IgG-opsonized particles for 10 min with or without 0.2 μM cytochalasin D. Fifty FcγR-expressing cells were scored for the number of particles attached but not internalized versus the number internalized. The fraction of successful internalizing events was then calculated.

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)

Phagocytosis assays in FcγR-transfected COS-7 cells. Cells were transfected with wild-type (WT) or Y282F/Y298F FcγR and subsequently challenged with IgG-opsonized particles for 10 min with or without 0.2 μM cytochalasin D. Fifty FcγR-expressing cells were scored for the number of particles attached but not internalized versus the number internalized. The fraction of successful internalizing events was then calculated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Phagocytosis assays in FcγR-transfected COS-7 cells. Cells were transfected with wild-type (WT) or Y282F/Y298F FcγR and subsequently challenged with IgG-opsonized particles for 10 min with or without 0.2 μM cytochalasin D. Fifty FcγR-expressing cells were scored for the number of particles attached but not internalized versus the number internalized. The fraction of successful internalizing events was then calculated.
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