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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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Transient Recruitment of EYFP-Tagged CK-BTime-lapse microscopy of zymosan uptake in RAW 264.7 cells transiently transfected with EYFP-tagged CK-B. Eight subsequent images captured at 6-s intervals over a 48-s recording period are shown. Asterisks indicate zymosan particles binding at the cellular membrane, during the process of engulfment, and after internalization. Bar indicates 5 μm.
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pbio-0060051-g002: Transient Recruitment of EYFP-Tagged CK-BTime-lapse microscopy of zymosan uptake in RAW 264.7 cells transiently transfected with EYFP-tagged CK-B. Eight subsequent images captured at 6-s intervals over a 48-s recording period are shown. Asterisks indicate zymosan particles binding at the cellular membrane, during the process of engulfment, and after internalization. Bar indicates 5 μm.

Mentions: As CK-B's role might involve the delicate interplay between compartmentalized energy supply and local molecular dynamics in the cell cortex area, we monitored the profile and timing of CK-B recruitment at the phagosome in more detail. To obtain dynamic information, we transiently expressed enhanced yellow fluorescent protein (EYFP)-tagged CK-B (via N-terminal fusion) in RAW 264.7 cells, and applied live cell microscopy imaging after induction of phagocytosis. Earlier work showed that N-terminal tagging of CK-B does not affect its enzymatic or structural properties [33]; (unpublished data). In the first (Figure 2A) of eight sequential frames of a movie (see Video S1) of typical CK-B behavior in an active macrophage, one particle is already being internalized (indicated by an asterisk), but at that point in time, EYFP-CK-B appears nonpartitioned and is still diffusely distributed throughout the cytoplasm. In subsequent frames (Figure 2B–2F), a clear CK-B accumulation in the phagocytic cup is observed, dissipation of which occurs when the particle is fully internalized (Figure 2G). A second phagocytic event with recruitment is initiated in the same cell at a later time point (Figure 2G and 2H). In control cells expressing nonfused EYFP, no significant accumulation at the site of zymosan ingestion was ever seen. A relatively straightforward interpretation of these observations would be that the spatially confined recruitment of CK-B serves phagocytic cup formation and/or closure, presumably via local delivery of ATP.


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)

Transient Recruitment of EYFP-Tagged CK-BTime-lapse microscopy of zymosan uptake in RAW 264.7 cells transiently transfected with EYFP-tagged CK-B. Eight subsequent images captured at 6-s intervals over a 48-s recording period are shown. Asterisks indicate zymosan particles binding at the cellular membrane, during the process of engulfment, and after internalization. Bar indicates 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pbio-0060051-g002: Transient Recruitment of EYFP-Tagged CK-BTime-lapse microscopy of zymosan uptake in RAW 264.7 cells transiently transfected with EYFP-tagged CK-B. Eight subsequent images captured at 6-s intervals over a 48-s recording period are shown. Asterisks indicate zymosan particles binding at the cellular membrane, during the process of engulfment, and after internalization. Bar indicates 5 μm.
Mentions: As CK-B's role might involve the delicate interplay between compartmentalized energy supply and local molecular dynamics in the cell cortex area, we monitored the profile and timing of CK-B recruitment at the phagosome in more detail. To obtain dynamic information, we transiently expressed enhanced yellow fluorescent protein (EYFP)-tagged CK-B (via N-terminal fusion) in RAW 264.7 cells, and applied live cell microscopy imaging after induction of phagocytosis. Earlier work showed that N-terminal tagging of CK-B does not affect its enzymatic or structural properties [33]; (unpublished data). In the first (Figure 2A) of eight sequential frames of a movie (see Video S1) of typical CK-B behavior in an active macrophage, one particle is already being internalized (indicated by an asterisk), but at that point in time, EYFP-CK-B appears nonpartitioned and is still diffusely distributed throughout the cytoplasm. In subsequent frames (Figure 2B–2F), a clear CK-B accumulation in the phagocytic cup is observed, dissipation of which occurs when the particle is fully internalized (Figure 2G). A second phagocytic event with recruitment is initiated in the same cell at a later time point (Figure 2G and 2H). In control cells expressing nonfused EYFP, no significant accumulation at the site of zymosan ingestion was ever seen. A relatively straightforward interpretation of these observations would be that the spatially confined recruitment of CK-B serves phagocytic cup formation and/or closure, presumably via local delivery of ATP.

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