Limits...
The zipper mechanism in phagocytosis: energetic requirements and variability in phagocytic cup shape.

Tollis S, Dart AE, Tzircotis G, Endres RG - BMC Syst Biol (2010)

Bottom Line: Highly curved shapes are not taken up, in line with recent experimental results.This suggests that biochemical pathways render the evolutionary ancient process of phagocytic highly robust, allowing cells to engulf even very large particles.The particle-shape dependence of phagocytosis makes a systematic investigation of host-pathogen interactions and an efficient design of a vehicle for drug delivery possible.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular Biosciences, South Kensington Campus, Imperial College London, SW72AZ London, UK.

ABSTRACT

Background: Phagocytosis is the fundamental cellular process by which eukaryotic cells bind and engulf particles by their cell membrane. Particle engulfment involves particle recognition by cell-surface receptors, signaling and remodeling of the actin cytoskeleton to guide the membrane around the particle in a zipper-like fashion. Despite the signaling complexity, phagocytosis also depends strongly on biophysical parameters, such as particle shape, and the need for actin-driven force generation remains poorly understood.

Results: Here, we propose a novel, three-dimensional and stochastic biophysical model of phagocytosis, and study the engulfment of particles of various sizes and shapes, including spiral and rod-shaped particles reminiscent of bacteria. Highly curved shapes are not taken up, in line with recent experimental results. Furthermore, we surprisingly find that even without actin-driven force generation, engulfment proceeds in a large regime of parameter values, albeit more slowly and with highly variable phagocytic cups. We experimentally confirm these predictions using fibroblasts, transfected with immunoreceptor FcγRIIa for engulfment of immunoglobulin G-opsonized particles. Specifically, we compare the wild-type receptor with a mutant receptor, unable to signal to the actin cytoskeleton. Based on the reconstruction of phagocytic cups from imaging data, we indeed show that cells are able to engulf small particles even without support from biological actin-driven processes.

Conclusions: This suggests that biochemical pathways render the evolutionary ancient process of phagocytic highly robust, allowing cells to engulf even very large particles. The particle-shape dependence of phagocytosis makes a systematic investigation of host-pathogen interactions and an efficient design of a vehicle for drug delivery possible.

Show MeSH

Related in: MedlinePlus

Statistical analysis of phagocytic cup shapes. (A) Experimental cup variability, defined in Figure 4, for 20-40% engulfed particles, for COS-7 cells transfected with wild-type FcγR (WT-FcγR) or signaling-dead mutant (Y282F/Y298F-FcγR) in DMEM medium, as well as WT-FcγR cells treated with 0.2 μM cytochalasin D (WT-FcγR+CytoD). The data points represent the variability averaged over all the phagocytic cups measured for given condition (receptor type and treatment) and range of engulfment. Error bars represent the statistical standard error of the cup variability. Student's t-test: p-values are (*) 0.0002 and (**) 0.0003. (Inset) Theoretical cup variability calculated from corresponding model simulations. Data points and error bars represent respectively the statistical average and standard error of the cup variability over several simulations. Student's t-test (*): p-value is less than 0.0001. (B) Experimental cup variability for 40-60% engulfed particles. (Inset) Theoretical cup variability calculated from corresponding model simulations. Student's t-test (*): p-value is given less than 0.0001. (C) Repartitioning of regular and variable cups for different experimental conditions, i.e. wild-type receptor (WT-FcγR), signaling-dead mutant (Y282F/Y298F-FcγR) and cytochalasin-D treated cells (WT-FcγR+CytoD), for engulfment between 20 and 40%. The numbers indicate the contribution (in %) of the three conditions in the overall sample of regular (left) and variable (right) cups.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2991294&req=5

Figure 5: Statistical analysis of phagocytic cup shapes. (A) Experimental cup variability, defined in Figure 4, for 20-40% engulfed particles, for COS-7 cells transfected with wild-type FcγR (WT-FcγR) or signaling-dead mutant (Y282F/Y298F-FcγR) in DMEM medium, as well as WT-FcγR cells treated with 0.2 μM cytochalasin D (WT-FcγR+CytoD). The data points represent the variability averaged over all the phagocytic cups measured for given condition (receptor type and treatment) and range of engulfment. Error bars represent the statistical standard error of the cup variability. Student's t-test: p-values are (*) 0.0002 and (**) 0.0003. (Inset) Theoretical cup variability calculated from corresponding model simulations. Data points and error bars represent respectively the statistical average and standard error of the cup variability over several simulations. Student's t-test (*): p-value is less than 0.0001. (B) Experimental cup variability for 40-60% engulfed particles. (Inset) Theoretical cup variability calculated from corresponding model simulations. Student's t-test (*): p-value is given less than 0.0001. (C) Repartitioning of regular and variable cups for different experimental conditions, i.e. wild-type receptor (WT-FcγR), signaling-dead mutant (Y282F/Y298F-FcγR) and cytochalasin-D treated cells (WT-FcγR+CytoD), for engulfment between 20 and 40%. The numbers indicate the contribution (in %) of the three conditions in the overall sample of regular (left) and variable (right) cups.

Mentions: To test whether passive engulfment leads to more variable cups than active engulfment, we developed an image-analysis method illustrated in Figure 4B-F. The cup shape varibility was quantified by the standard deviation of the distribution of cell-membrane (FcγR-GFP fluorescence) height around the particle, divided by the square root of the average membrane height. The unit of membrane height is given by the distance (0.4 μm) between consecutive confocal image planes (see Methods). Figure 5A shows that for small particles engulfed between 20 and 40% of their surfaces, cup variability increases from cells transfected with wild-type receptor to cells transfected with signaling-dead mutant receptor to WT-Fcγ+CytoD cells. The lowest variability, found for cells expressing wild-type receptor, is statistically significant against both passive zipper types (Student's t-test, p-value < 0.001). This result is consistent with model predictions: Figure 5A, inset shows the cup variability from simulations, revealing that the active zipper leads to significantly less variable cups. In contrast, for the ranges of engulfment between 40 and 60% (Figure 5B) and between 60 and 100% (see Additional file 1, Figure S7) we observed no noticeable difference in cup variability between the three experimental conditions, while our model consistently predicts more variable cups for the passive zipper (Figure 5B, inset). This discrepancy may indicate that active processes such as contraction by myosin motor proteins become important at later stages of engulfment, limiting our model's full validity to the early events in phagocytosis (see Conclusion section).


The zipper mechanism in phagocytosis: energetic requirements and variability in phagocytic cup shape.

Tollis S, Dart AE, Tzircotis G, Endres RG - BMC Syst Biol (2010)

Statistical analysis of phagocytic cup shapes. (A) Experimental cup variability, defined in Figure 4, for 20-40% engulfed particles, for COS-7 cells transfected with wild-type FcγR (WT-FcγR) or signaling-dead mutant (Y282F/Y298F-FcγR) in DMEM medium, as well as WT-FcγR cells treated with 0.2 μM cytochalasin D (WT-FcγR+CytoD). The data points represent the variability averaged over all the phagocytic cups measured for given condition (receptor type and treatment) and range of engulfment. Error bars represent the statistical standard error of the cup variability. Student's t-test: p-values are (*) 0.0002 and (**) 0.0003. (Inset) Theoretical cup variability calculated from corresponding model simulations. Data points and error bars represent respectively the statistical average and standard error of the cup variability over several simulations. Student's t-test (*): p-value is less than 0.0001. (B) Experimental cup variability for 40-60% engulfed particles. (Inset) Theoretical cup variability calculated from corresponding model simulations. Student's t-test (*): p-value is given less than 0.0001. (C) Repartitioning of regular and variable cups for different experimental conditions, i.e. wild-type receptor (WT-FcγR), signaling-dead mutant (Y282F/Y298F-FcγR) and cytochalasin-D treated cells (WT-FcγR+CytoD), for engulfment between 20 and 40%. The numbers indicate the contribution (in %) of the three conditions in the overall sample of regular (left) and variable (right) cups.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2991294&req=5

Figure 5: Statistical analysis of phagocytic cup shapes. (A) Experimental cup variability, defined in Figure 4, for 20-40% engulfed particles, for COS-7 cells transfected with wild-type FcγR (WT-FcγR) or signaling-dead mutant (Y282F/Y298F-FcγR) in DMEM medium, as well as WT-FcγR cells treated with 0.2 μM cytochalasin D (WT-FcγR+CytoD). The data points represent the variability averaged over all the phagocytic cups measured for given condition (receptor type and treatment) and range of engulfment. Error bars represent the statistical standard error of the cup variability. Student's t-test: p-values are (*) 0.0002 and (**) 0.0003. (Inset) Theoretical cup variability calculated from corresponding model simulations. Data points and error bars represent respectively the statistical average and standard error of the cup variability over several simulations. Student's t-test (*): p-value is less than 0.0001. (B) Experimental cup variability for 40-60% engulfed particles. (Inset) Theoretical cup variability calculated from corresponding model simulations. Student's t-test (*): p-value is given less than 0.0001. (C) Repartitioning of regular and variable cups for different experimental conditions, i.e. wild-type receptor (WT-FcγR), signaling-dead mutant (Y282F/Y298F-FcγR) and cytochalasin-D treated cells (WT-FcγR+CytoD), for engulfment between 20 and 40%. The numbers indicate the contribution (in %) of the three conditions in the overall sample of regular (left) and variable (right) cups.
Mentions: To test whether passive engulfment leads to more variable cups than active engulfment, we developed an image-analysis method illustrated in Figure 4B-F. The cup shape varibility was quantified by the standard deviation of the distribution of cell-membrane (FcγR-GFP fluorescence) height around the particle, divided by the square root of the average membrane height. The unit of membrane height is given by the distance (0.4 μm) between consecutive confocal image planes (see Methods). Figure 5A shows that for small particles engulfed between 20 and 40% of their surfaces, cup variability increases from cells transfected with wild-type receptor to cells transfected with signaling-dead mutant receptor to WT-Fcγ+CytoD cells. The lowest variability, found for cells expressing wild-type receptor, is statistically significant against both passive zipper types (Student's t-test, p-value < 0.001). This result is consistent with model predictions: Figure 5A, inset shows the cup variability from simulations, revealing that the active zipper leads to significantly less variable cups. In contrast, for the ranges of engulfment between 40 and 60% (Figure 5B) and between 60 and 100% (see Additional file 1, Figure S7) we observed no noticeable difference in cup variability between the three experimental conditions, while our model consistently predicts more variable cups for the passive zipper (Figure 5B, inset). This discrepancy may indicate that active processes such as contraction by myosin motor proteins become important at later stages of engulfment, limiting our model's full validity to the early events in phagocytosis (see Conclusion section).

Bottom Line: Highly curved shapes are not taken up, in line with recent experimental results.This suggests that biochemical pathways render the evolutionary ancient process of phagocytic highly robust, allowing cells to engulf even very large particles.The particle-shape dependence of phagocytosis makes a systematic investigation of host-pathogen interactions and an efficient design of a vehicle for drug delivery possible.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular Biosciences, South Kensington Campus, Imperial College London, SW72AZ London, UK.

ABSTRACT

Background: Phagocytosis is the fundamental cellular process by which eukaryotic cells bind and engulf particles by their cell membrane. Particle engulfment involves particle recognition by cell-surface receptors, signaling and remodeling of the actin cytoskeleton to guide the membrane around the particle in a zipper-like fashion. Despite the signaling complexity, phagocytosis also depends strongly on biophysical parameters, such as particle shape, and the need for actin-driven force generation remains poorly understood.

Results: Here, we propose a novel, three-dimensional and stochastic biophysical model of phagocytosis, and study the engulfment of particles of various sizes and shapes, including spiral and rod-shaped particles reminiscent of bacteria. Highly curved shapes are not taken up, in line with recent experimental results. Furthermore, we surprisingly find that even without actin-driven force generation, engulfment proceeds in a large regime of parameter values, albeit more slowly and with highly variable phagocytic cups. We experimentally confirm these predictions using fibroblasts, transfected with immunoreceptor FcγRIIa for engulfment of immunoglobulin G-opsonized particles. Specifically, we compare the wild-type receptor with a mutant receptor, unable to signal to the actin cytoskeleton. Based on the reconstruction of phagocytic cups from imaging data, we indeed show that cells are able to engulf small particles even without support from biological actin-driven processes.

Conclusions: This suggests that biochemical pathways render the evolutionary ancient process of phagocytic highly robust, allowing cells to engulf even very large particles. The particle-shape dependence of phagocytosis makes a systematic investigation of host-pathogen interactions and an efficient design of a vehicle for drug delivery possible.

Show MeSH
Related in: MedlinePlus