TI - Discussion . AB - Phagocytosis requires a rapid and spatially confined reorganization of the actin cytoskeleton . The underlying molecular processes , such as actin polymerization and actomyosin force generation , generate a sudden and localized demand for cellular ATP [22,23] . To reciprocate this challenge , sites of ATP production should be coupled tightly to sites of ATP consumption . From studies in cell and animal models , we know that CK isozymes are particularly well equipped for this role , as they provide the cell with a fast ATP regeneration and delivery system that can adequately provide high-energy phosphoryl groups to cellular locales with high energy turnover . Here , we established a tight functional and spatial link between the CK system and the actin-based cytoskeletal machinery in macrophages during phagocytosis . A similar relationship was found for the muscle isoform of CK , CK-M , which associates with the M - and I-bands of skeletal muscle and fuels local ATP-consuming processes , including actomyosin contraction and calcium pumping [26,43] . Importantly , CK-M's role in these physiological processes is supportive , not absolutely vital [27,43] , just as we report here for CK-B's role in phagocytosis . For CK-M , the molecular nature of events that support its role has been unraveled to some extent . For example , we know that interaction of CK-M with the sarcomeric M-band is mediated via conserved lysine residues . In addition , particular amino acid segments in CK-M enable the protein to bind indirectly to the I-band via the glycolytic enzymes phosphofructokinase ( PFK ) and aldolase , which have actin-binding properties [44] . Interestingly , glycolytic enzymes are also known to be recruited to phagosomes [45,46] , so there may be parallel mechanisms . Unfortunately , to explain CK-B dynamics , it is not possible to use simple analogy since the lysine residues involved in CK-M M-band interaction are not conserved in CK-B [47] , and direct sequence comparison is not yielding clear clues for other binding modes--for example to glycolytic enzymes . Several mechanisms could therefore be involved in the recruitment of CK-B . One model would be the transient availability of CK-B binding sites at the nascent phagosome by modification of local proteins or presence of CK-B interacting proteins . Based on its colocalization with CK-B , we tested whether actin could be a candidate for such scaffolding , but pull-down assays and FRAP experiments did not reveal a tight interaction . Also yeast two hybrid assays did not disclose any CK-B to actin binding opportunities ( unpublished data ) . As another possibility , CK-B binding characteristics could also be transiently modified at the enzyme itself , possibly by ( enzymatic ) events located at the forming cup . Indeed , CK-B is prone to covalent modifications such as PHOSphorylation [48] , oxidation [49] , methylation [50] or ubiquination [51] . Simple presence of SUBstrate may also determine binding ability , as recently shown for CK-M [52] . Further studies are necessary to discriminate between all these possibilities . Because phagocytosis is a metabolically demanding process , CK-B recruitment to phagocytic cups could serve to promote or safeguard local events or--reciprocally--shield the rest of the cell from excessive local energy demand . To distinguish between these mechanistic models and elucidate CK-B global and local physiological roles in macrophages more precisely will be technically challenging because of the confined character of the events . This , therefore , also remains a topic for future study . Of particular importance was our finding that displacement of endogenous CK-B by ECFP-CK-B (C283S) during phagocytosis reduced the local accumulation of EGFP-actin in the phagocytic cup area . The observation that inhibition of CK-B activity impairs global F-actin polymerization in RAW 264.7 cells was equally revealing . Normally , actin polymerization requires the incorporation of ATP-actin at the barbed end of actin filaments . During filament elongation , ATP is hydrolyzed and ADP-actin is being released at the pointed end . Thus , constant reloading of actin with ATP is required for the continuation of the polymerization cycle [12,41,42] . We propose here , that CK-B could specifically enhance this process by regenerating ATP at sites of active actin remodeling . In addition to the polymerization reaction , actin nucleation and branching play an important role . The Arp2/3 complex , together with the Wiskott-Aldrich syndrome ( adapter ) protein , WASP [8] , and the motor proteins myosin-I [53 , 54] and myosin-II [19] , helps to guide these processes during shaping of the cup of nascent phagosomes . Recently , the involvement of the formin mDia has also been implicated in phagocytosis of COZ . Inhibition of mDia results in decreased F-actin recruitment at the phagocytic cup and induces a concomitant decrease in efficiency of CR3 -mediated , -mediated , phagocytosis [55] . Formins promote actin polymerization by increasing polymerization-associated ATP hydrolysis up to 15 times via profilin [56] . Finally , regulation of cell structure via RhoA activation or AMP -activated kinase involvement is also directly energy dependent [57,58] . Thus , different types of actin regulatory processes form local and temporal energy drains , which may need compensation by CK-B -mediated ATP regeneration . Various associated myosin motor mechanisms involved in formation of the specialized structures at the phagosome may also be CK-B dependent , because motor activity of myosins is controlled by ATP/ADP ratio . In Dictyostelium , myosin-VII was found to be important for initial adhesion in phagocytosis [21] . In addition , another closely related myosin , myosin-X , was implicated in adhesion and phagocytosis [20 , 59] . However , at this point , we need more mechanistic information about how ATP fuelling separately serves actin and myosin activities in the phagocytic cup before we can analyze CK-B's presumed roles in detail further . Intriguingly , Olazabal and coworkers [19] have reported that inhibition of myosin-II decreased actin recruitment in phagocytic cups during CR3 -mediated phagocytosis , . Our results regarding differences in ingestion capacity for COZ or IgG-coated zymosan and beads after genetic blocking or pharmacological inhibition of CK activity fit with a model in which ATP-driven actomyosin activities differentially contribute to discrete phagocytic processes . Only phagocytosis of COZ and zymosan was significantly affected under conditions in which CK activity was lowered , Fc-gammaR -mediated activity was not significantly affected by cyclocreatine or dominant-negative CK inhibition . Of note , phagocytosis of COZ is mainly , but not exclusively , mediated by complement molecules present on COZ . Also , sugar residues that are present on ( non ) opsonized zymosan facilitate recognition by lectin domains in CR3 [60-62] or alternative phagocytic receptors such as dectin-1 [63] . Based on our findings and these background notions regarding differences in pathways involved in cup formation in complement or IgG modes of phagocytosis [7,19,55,64] , it is tempting to speculate that CK-B enhances phagocytosis by modulating specific processes up - or downstream of CR3 . It may be important to note that differences in the actin levels in the phagocytic cup , as seen between active ECFP-CK-B and mutant ECFP-CK-B (C283S) -expressing cells , correlated better with the fraction of cells participating in phagocytosis than with the number of fully ingested particles per cell . The observation of the apparent delayed internalization of COZ particles in the RAW-CK-B (C283S) line ( Figure 7E ) suggests that after early binding requirements have been fulfilled , CK-B also promotes transition to the next phases of phagocytosis . Therefore , we propose that the CK-B -mediated modulation of actin polymerization is particularly relevant during early CR3 -mediated phagocytosis , for example by increasing the number of successful probing attempts for particle binding . Our data also suggest that endogenous levels of resident CK-B molecules are largely sufficient to saturate these requirements for zymosan and COZ phagocytosis . CR3 is also known as CD11b18 or alphaMbeta2 integrin . Because actin polymerization is pivotal in early adhesion events mediated by integrins [39] , a picture emerges in which actin behavior determines the efficiency by which CR3s can bind their target . Actin behavior may be less central for IgG -mediated binding events . Our finding that low concentrations of the actin polymerization inhibitor , cytochalasin D , reduced binding of complement opsonized particles more than IgG-opsonized particles is consistent with such a model . In conclusion , we have demonstrated that CK-B enhances phagocytosis of zymosan and COZ , likely via a specific synergistic role in mechanistic events involved in actin polymerization behavior . Although our data indicate that the enzyme's metabolic role is dominant , we cannot completely rule out a structural role of CK-B in phagocytosis at this point . Taken together with the finding that CK-B (C283S) was able to inhibit phagocytosis without decreasing the total CK activity , our data suggest that CK-B acts to steer the delicate local balance in ATP/ADP ratio , during formation of the phagocytic cup , around the time that pseudopod-filopodium extensions or CR3 -mediated adhesions are formed . Because we do see CK-B in filopodia and phagocytic cups in our RAW cells ( Figures 1G-1L and 4 ) , but also observe CK-B accumulation in dynamic actin structures of other cell types during adhesion to substratum , spreading , and crawling , ( eg , in neurons and astrocytes ; unpublished data ) , this raises the exciting possibility that CK-B facilitates rapid cytoskeletal dynamics in a broad range of specialized events that occur during tissue development and disease , including dendritic spine generation in brain [65] , formation of immune-synapses , or protrusion dynamics for cancer cell invasion .