TI - Results Endogenous CK-B Translocates to Phagocytic Cups in Microglia and Macrophages . AB - 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fully synchronously throughout the culture , but at later time points , the concentrated staining dissipated ( unpublished data ) , indicating that CK-B associated only transiently with phagosome structures . Phalloidin staining demonstrated an almost complete overlap with CK-B encircling the zymosan particles in the phagosome ( Figure 1B and 1F ) . To assess whether CK-B is actually locally bound within the cup area , microglia were permeabilized with saponin before fixation to remove most of the unbound cytosolic protein . Strikingly , a fraction of endogenous CK-B remained associated with the actin-rich area ( Figure 1C and 1D ) . These data indicate that part of the CK-B molecules in the endogenous pool partition into sites of active F-actin remodeling . To further verify the general validity of this picture , we analyzed the behavior of endogenous or exogenously transfected CK-B in the murine macrophage cell line , RAW 264.7 . As anticipated , we also observed in this cell a uniform cytosolic distribution of endogenous CK-B and coaccumulation with F-actin at nascent phagosomes ( Figure 1G and 1H ) . To compensate for the rather weak endogenous CK-B staining in RAW 264.7 cells , we also produced pools of cells with a higher CK-B steady-state level by transduction with retroviral vectors to enhance immunofluorescent detection . Again , prominent accumulation of CK-B together with F-actin appeared in the phagosome ( Figure 1H and 1J ) . Notably , in RAW 264.7 cells with an overall high global CK-B level , we noticed CK-B accumulation at the distal tips of filopodia ( Figure 1K and 1L ) . This phenomenon has been observed for a number of other proteins involved in cytoskeletal rearrangement , dynamic adhesion and phagocytosis , including myosin-X , myosin-VII and vasodilator-stimulated PHOSphoprotein ( VASP ) [20 , 21 , 32] .