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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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Cytosolic CK-B Accumulates in the Phagocytic Cup Area of MacrophagesUptake of zymosan in primary microglia (A–D), primary peritoneal macrophages (E and F), and RAW 264.7 macrophages (G–J). Fixation followed by (immuno)staining with CK-B antibodies (A, C, E, G, I, and K) or phalloidin (B, D, F, H, J, and L) reveals the co-accumulation of CK-B and F-actin at the phagocytic cup (arrows).(C and D) Saponin (sap) extraction of phagocytosing microglia prior to fixation and CK-B or actin staining (I–L) RAW 264.7 macrophages overexpressing mouse CK-B. Note that CK-B shows additional pronounced spot-like accumulation at the distal tips of filopodia (arrows, [K and L]). Bar represents 10 μm.
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pbio-0060051-g001: Cytosolic CK-B Accumulates in the Phagocytic Cup Area of MacrophagesUptake of zymosan in primary microglia (A–D), primary peritoneal macrophages (E and F), and RAW 264.7 macrophages (G–J). Fixation followed by (immuno)staining with CK-B antibodies (A, C, E, G, I, and K) or phalloidin (B, D, F, H, J, and L) reveals the co-accumulation of CK-B and F-actin at the phagocytic cup (arrows).(C and D) Saponin (sap) extraction of phagocytosing microglia prior to fixation and CK-B or actin staining (I–L) RAW 264.7 macrophages overexpressing mouse CK-B. Note that CK-B shows additional pronounced spot-like accumulation at the distal tips of filopodia (arrows, [K and L]). Bar represents 10 μm.

Mentions: Phagocytic cup formation is characterized by a localized expansion of the plasmalemmal membrane, coupled to highly active remodeling and myosin-based contraction of the actin cytoskeleton. We studied the possible fate and role of endogenous CK-B in this process, in primary microglia and peritoneal macrophages after induction of phagocytosis with nonopsonized zymosan. Macrophages and microglia [2,30] are cells of the immune system that are very active in ruffle extension and uptake of extracellular particles. Although it has been reported that primary macrophages express CK-B [31], no data are available on the enzyme's behavior under conditions of active phagocytosis. Figure 1A and 1E shows that a fraction of CK-B always remained diffusely distributed throughout the cytosol, as in nonphagocytosing cells, but that a substantial portion of CK-B accumulated around the engulfed zymosan particles at nascent phagosomes. This accumulation did not occur exactly simultaneously in all cells because phagocytosis was not initiated fully synchronously throughout the culture, but at later time points, the concentrated staining dissipated (unpublished data), indicating that CK-B associated only transiently with phagosome structures.


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)

Cytosolic CK-B Accumulates in the Phagocytic Cup Area of MacrophagesUptake of zymosan in primary microglia (A–D), primary peritoneal macrophages (E and F), and RAW 264.7 macrophages (G–J). Fixation followed by (immuno)staining with CK-B antibodies (A, C, E, G, I, and K) or phalloidin (B, D, F, H, J, and L) reveals the co-accumulation of CK-B and F-actin at the phagocytic cup (arrows).(C and D) Saponin (sap) extraction of phagocytosing microglia prior to fixation and CK-B or actin staining (I–L) RAW 264.7 macrophages overexpressing mouse CK-B. Note that CK-B shows additional pronounced spot-like accumulation at the distal tips of filopodia (arrows, [K and L]). Bar represents 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pbio-0060051-g001: Cytosolic CK-B Accumulates in the Phagocytic Cup Area of MacrophagesUptake of zymosan in primary microglia (A–D), primary peritoneal macrophages (E and F), and RAW 264.7 macrophages (G–J). Fixation followed by (immuno)staining with CK-B antibodies (A, C, E, G, I, and K) or phalloidin (B, D, F, H, J, and L) reveals the co-accumulation of CK-B and F-actin at the phagocytic cup (arrows).(C and D) Saponin (sap) extraction of phagocytosing microglia prior to fixation and CK-B or actin staining (I–L) RAW 264.7 macrophages overexpressing mouse CK-B. Note that CK-B shows additional pronounced spot-like accumulation at the distal tips of filopodia (arrows, [K and L]). Bar represents 10 μm.
Mentions: Phagocytic cup formation is characterized by a localized expansion of the plasmalemmal membrane, coupled to highly active remodeling and myosin-based contraction of the actin cytoskeleton. We studied the possible fate and role of endogenous CK-B in this process, in primary microglia and peritoneal macrophages after induction of phagocytosis with nonopsonized zymosan. Macrophages and microglia [2,30] are cells of the immune system that are very active in ruffle extension and uptake of extracellular particles. Although it has been reported that primary macrophages express CK-B [31], no data are available on the enzyme's behavior under conditions of active phagocytosis. Figure 1A and 1E shows that a fraction of CK-B always remained diffusely distributed throughout the cytosol, as in nonphagocytosing cells, but that a substantial portion of CK-B accumulated around the engulfed zymosan particles at nascent phagosomes. This accumulation did not occur exactly simultaneously in all cells because phagocytosis was not initiated fully synchronously throughout the culture, but at later time points, the concentrated staining dissipated (unpublished data), indicating that CK-B associated only transiently with phagosome structures.

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