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Creatine kinase-mediated ATP supply fuels actin-based events in phagocytosis.

Kuiper JW, Pluk H, Oerlemans F, van Leeuwen FN, de Lange F, Fransen J, Wieringa B - PLoS Biol. (2008)

Bottom Line: We demonstrate that endogenous CK-B in macrophages is mobilized from the cytosolic pool and coaccumulates with F-actin at nascent phagosomes.Overexpression of a catalytic dead CK-B or CK-specific cyclocreatine inhibition caused a significant reduction of actin accumulation in the phagocytic cup area, and reduced complement receptor-mediated, but not Fc-gammaR-mediated, ingestion capacity of macrophages.We propose that CK-B activity in macrophages contributes to complement-induced F-actin assembly events in early phagocytosis by providing local ATP supply.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Radboud University, Nijmegen Medical Centre, Nijmegen, The Netherlands.

ABSTRACT
Phagocytosis requires locally coordinated cytoskeletal rearrangements driven by actin polymerization and myosin motor activity. How this actomyosin dynamics is dependent upon systems that provide access to ATP at phagosome microdomains has not been determined. We analyzed the role of brain-type creatine kinase (CK-B), an enzyme involved in high-energy phosphoryl transfer. We demonstrate that endogenous CK-B in macrophages is mobilized from the cytosolic pool and coaccumulates with F-actin at nascent phagosomes. Live cell imaging with XFP-tagged CK-B and beta-actin revealed the transient and specific nature of this partitioning process. Overexpression of a catalytic dead CK-B or CK-specific cyclocreatine inhibition caused a significant reduction of actin accumulation in the phagocytic cup area, and reduced complement receptor-mediated, but not Fc-gammaR-mediated, ingestion capacity of macrophages. Finally, we found that inhibition of CK-B affected phagocytosis already at the stage of particle adhesion, most likely via effects on actin polymerization behavior. We propose that CK-B activity in macrophages contributes to complement-induced F-actin assembly events in early phagocytosis by providing local ATP supply.

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CK-B Is Involved in the Initial Steps of PhagocytosisAdhesion and uptake of FITC-labeled COZ particles in RAW 264.7 cells (A) and RAW 264.7 cells expressing CK-B(C283S) (B) or CK-B (C). Internalized particles appear green (or yellow, when colocalizing with F-actin), and external particles appear cyan. Bars indicate 10 μm.(D) Averages of total numbers of particles associated per cell (inside + outside) (±SEM). *p < 0.005; **p < 0.001.(E) Percentage particles attached to the cell, but not yet engulfed (±SEM). *p < 0.05.
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pbio-0060051-g007: CK-B Is Involved in the Initial Steps of PhagocytosisAdhesion and uptake of FITC-labeled COZ particles in RAW 264.7 cells (A) and RAW 264.7 cells expressing CK-B(C283S) (B) or CK-B (C). Internalized particles appear green (or yellow, when colocalizing with F-actin), and external particles appear cyan. Bars indicate 10 μm.(D) Averages of total numbers of particles associated per cell (inside + outside) (±SEM). *p < 0.005; **p < 0.001.(E) Percentage particles attached to the cell, but not yet engulfed (±SEM). *p < 0.05.

Mentions: Phagocytosis occurs through a series of consecutive steps that ultimately lead to the engulfment of a particle. Probing for adhesion of coat molecules, and the actual binding of the phagocytic target to specific receptors on the protruding cell surface, constitutes one of the first steps in this process. In order to specify which specific phase of the phagocytic process is linked to CK-B, we subjected the wt CK-B or CK-B(C283S) cell pools to a particle adhesion assay, using COZ particles as the phagocytic targets with most discriminative effects (Figure 7). Quantification of the total number of particles per cell (inside + outside) in images of the cell lines with adherent and already internalized particles (Figure 7A–7C; n = 3 experiments) demonstrated that an average of 1.3 ± 0.7 of COZ particles associated with RAW-CK-B(C283S) cells, significant less than with control cells, which have 2.1 ± 0.7 particles/cell (p < 0.001). Interestingly, RAW-CK-B cells bound significant higher numbers of particles than control cells (2.8 ± 0.7 particles/cell; p < 0.005). Calculation of the percentage of external COZ particles revealed that control cells have 17 ± 10% of particles attached that are not yet (fully) internalized. Overexpression of CK-B did not affect this percentage (14 ± 9% external). With RAW-CK-B(C283S) cells, a significantly higher percentage of particles remained external (27 ± 18% ; p < 0.02). Inhibition of CK-B thus apparently affects both the initial sampling of COZ particles from the added pool as well as well as the process of their internalization.


Creatine kinase-mediated ATP supply fuels actin-based events in phagocytosis.

Kuiper JW, Pluk H, Oerlemans F, van Leeuwen FN, de Lange F, Fransen J, Wieringa B - PLoS Biol. (2008)

CK-B Is Involved in the Initial Steps of PhagocytosisAdhesion and uptake of FITC-labeled COZ particles in RAW 264.7 cells (A) and RAW 264.7 cells expressing CK-B(C283S) (B) or CK-B (C). Internalized particles appear green (or yellow, when colocalizing with F-actin), and external particles appear cyan. Bars indicate 10 μm.(D) Averages of total numbers of particles associated per cell (inside + outside) (±SEM). *p < 0.005; **p < 0.001.(E) Percentage particles attached to the cell, but not yet engulfed (±SEM). *p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2265766&req=5

pbio-0060051-g007: CK-B Is Involved in the Initial Steps of PhagocytosisAdhesion and uptake of FITC-labeled COZ particles in RAW 264.7 cells (A) and RAW 264.7 cells expressing CK-B(C283S) (B) or CK-B (C). Internalized particles appear green (or yellow, when colocalizing with F-actin), and external particles appear cyan. Bars indicate 10 μm.(D) Averages of total numbers of particles associated per cell (inside + outside) (±SEM). *p < 0.005; **p < 0.001.(E) Percentage particles attached to the cell, but not yet engulfed (±SEM). *p < 0.05.
Mentions: Phagocytosis occurs through a series of consecutive steps that ultimately lead to the engulfment of a particle. Probing for adhesion of coat molecules, and the actual binding of the phagocytic target to specific receptors on the protruding cell surface, constitutes one of the first steps in this process. In order to specify which specific phase of the phagocytic process is linked to CK-B, we subjected the wt CK-B or CK-B(C283S) cell pools to a particle adhesion assay, using COZ particles as the phagocytic targets with most discriminative effects (Figure 7). Quantification of the total number of particles per cell (inside + outside) in images of the cell lines with adherent and already internalized particles (Figure 7A–7C; n = 3 experiments) demonstrated that an average of 1.3 ± 0.7 of COZ particles associated with RAW-CK-B(C283S) cells, significant less than with control cells, which have 2.1 ± 0.7 particles/cell (p < 0.001). Interestingly, RAW-CK-B cells bound significant higher numbers of particles than control cells (2.8 ± 0.7 particles/cell; p < 0.005). Calculation of the percentage of external COZ particles revealed that control cells have 17 ± 10% of particles attached that are not yet (fully) internalized. Overexpression of CK-B did not affect this percentage (14 ± 9% external). With RAW-CK-B(C283S) cells, a significantly higher percentage of particles remained external (27 ± 18% ; p < 0.02). Inhibition of CK-B thus apparently affects both the initial sampling of COZ particles from the added pool as well as well as the process of their internalization.

Bottom Line: We demonstrate that endogenous CK-B in macrophages is mobilized from the cytosolic pool and coaccumulates with F-actin at nascent phagosomes.Overexpression of a catalytic dead CK-B or CK-specific cyclocreatine inhibition caused a significant reduction of actin accumulation in the phagocytic cup area, and reduced complement receptor-mediated, but not Fc-gammaR-mediated, ingestion capacity of macrophages.We propose that CK-B activity in macrophages contributes to complement-induced F-actin assembly events in early phagocytosis by providing local ATP supply.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Radboud University, Nijmegen Medical Centre, Nijmegen, The Netherlands.

ABSTRACT
Phagocytosis requires locally coordinated cytoskeletal rearrangements driven by actin polymerization and myosin motor activity. How this actomyosin dynamics is dependent upon systems that provide access to ATP at phagosome microdomains has not been determined. We analyzed the role of brain-type creatine kinase (CK-B), an enzyme involved in high-energy phosphoryl transfer. We demonstrate that endogenous CK-B in macrophages is mobilized from the cytosolic pool and coaccumulates with F-actin at nascent phagosomes. Live cell imaging with XFP-tagged CK-B and beta-actin revealed the transient and specific nature of this partitioning process. Overexpression of a catalytic dead CK-B or CK-specific cyclocreatine inhibition caused a significant reduction of actin accumulation in the phagocytic cup area, and reduced complement receptor-mediated, but not Fc-gammaR-mediated, ingestion capacity of macrophages. Finally, we found that inhibition of CK-B affected phagocytosis already at the stage of particle adhesion, most likely via effects on actin polymerization behavior. We propose that CK-B activity in macrophages contributes to complement-induced F-actin assembly events in early phagocytosis by providing local ATP supply.

Show MeSH
Related in: MedlinePlus