Role of gelsolin interaction with actin in regulation and creation of actin nuclei in chemotactic peptide activated polymorphonuclear neutrophils.

In vitro Ca++ activates gelsolin to sever F-actin and form a gelsolin-actin (GA) complex at the+end of F-actin that is not dissociated by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) but is separated by EGTA+PIP/PIP2. The gelsolin blocks the+end on the actin filament, but the-end of the filament can still initiate actin polymerization. In thrombin activated platelets, evidence suggests that severing of F-actin by gelsolin increases GA complex, creates one-end actin nucleus and one cryptic+end actin nucleus per cut, and then dissociates to yield free+ends to nucleate rapid actin assembly. We examined the role of F-actin severing in creation and regulation of nuclei and polymerization in polymorphonuclear neutrophils (PMNs). At 2-s intervals after formyl peptide (FMLP) activation of endotoxin free (ETF) PMNs, change in GA complex was correlated with change in+end actin nuclei,-end actin nuclei, and F-actin content. GA complex was quantitated by electrophoretograms of proteins absorbed by antigelsolin from cells lysed in 10 mM EGTA,+end actin nuclei as cytochalasin (CD) sensitive and-end actin nuclei as CD insensitive increases in G-pyrenyl actin polymerization rates induced by the same PMNs, and F-actin content by NBDphallacidin binding to fixed cells. Thirty three percent of gelsolin was in GA complex in basal ETF PMNs; from 2-6 s, GA complexes dissociate (low = 15% at 10 s) and sequentially+end nuclei and F-actin content and then-end nuclei increase to a maximum at 10 s. At > s GA complex increase toward basal and + end nuclei and F-actin content returned toward basal. These kinetic data show gelsolin regulates availability of + end nuclei and actin polymerization in FMLP. However, absence of an initial increase in GA complex or - end nucleating activity shows FMLP activation does not cause gelsolin to sever F- or to bind G-actin to create cryptic + end nuclei in PMNs; the results suggest the + nucleus formation is gelsolin independent.     

Identification and purification of calcium ion dependent modulators of actin polymerization from bovine thyroid

We describe the purification of Ca2+-dependent actin modulator proteins from bovine thyroid using DNase I affinity chromatography and diethylaminoethylcellulose chromatography. The 40K actin modulator has been purified to 98% homogeneity. It is a single polypeptide chain with a molecular weight of approximately 40 000 and an isoelectric point of 8.1. Its amino acid composition is different from previously described actin-associated proteins and thyroid actin. On the basis of the centrifugation assay and the DNase I inhibition assay, the actin complexed with the 40K protein is G-actin in its conformation rather than F-actin oligomers. Substoichiometric concentrations of the 40K protein rapidly inhibit actin polymerization in the presence of physiological concentrations of Ca2+ and Mg2+. An 80K actin modulator also has been purified to 98% homogeneity. It is a single polypeptide chain with a molecular weight of approximately 80 000 and an isoelectric point of 6.35-7.0. Its amino acid composition is different from those of villin, gelsolin, and leukocyte actin polymerization inhibitor. On the basis of the DNase inhibition assay and the centrifugation assay, the nonprecipitable actin associated with the 80K protein was F-actin in its conformation. The 80K protein acts very efficiently as a Ca2+-dependent nucleator for actin assembly and reduces its viscosity. In addition to the 40K and 80K actin modulators, 91K and 95K actin-associated proteins were partially purified. The 91K-95K fraction has similar activity to the 80K protein regarding precipitation of F-actin. The 125I-G-actin polyacrylamide gel overlay technique [Snabes, M. C., Boyd, A.E., & Bryan, J. (1981) J. Cell Biol. 90, 809-812] revealed that both the 91K and 95K proteins bind 125I-actin after sodium dodecyl sulfate (NaDodSO4) electrophoresis while the 80K and 40K proteins do not. Thyroid 91K protein comigrated with a human platelet 91K actin binding protein on NaDodSO4 gels and may be similar to macrophage gelsolin. The 95K protein may be similar to villin, the intestinal cytoskeletal protein.     

Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein

Exposure of cryptic actin filament fast growing ends (barbed ends) initiates actin polymerization in stimulated human and mouse platelets. Gelsolin amplifies platelet actin assembly by severing F-actin and increasing the number of barbed ends. Actin filaments in stimulated platelets from transgenic gelsolin-null mice elongate their actin without severing. F-actin barbed end capping activity persists in human platelet extracts, depleted of gelsolin, and the heterodimeric capping protein (CP) accounts for this residual activity. 35% of the approximately 5 microM CP is associated with the insoluble actin cytoskeleton of the resting platelet. Since resting platelets have an F- actin barbed end concentration of approximately 0.5 microM, sufficient CP is bound to cap these ends. CP is released from OG-permeabilized platelets by treatment with phosphatidylinositol 4,5-bisphosphate or through activation of the thrombin receptor. However, the fraction of CP bound to the actin cytoskeleton of thrombin-stimulated mouse and human platelets increases rapidly to approximately 60% within 30 s. In resting platelets from transgenic mice lacking gelsolin, which have 33% more F-actin than gelsolin-positive cells, there is a corresponding increase in the amount of CP associated with the resting cytoskeleton but no change with stimulation. These findings demonstrate an interaction between the two major F-actin barbed end capping proteins of the platelet: gelsolin-dependent severing produces barbed ends that are capped by CP. Phosphatidylinositol 4,5-bisphosphate release of gelsolin and CP from platelet cytoskeleton provides a mechanism for mediating barbed end exposure. After actin assembly, CP reassociates with the new actin cytoskeleton.     

Effect of muscle tropomyosin on the kinetics of polymerization of muscle actin

At saturating concentrations, tropomyosin inhibited the rate of spontaneous polymerization of ATP-actin and also inhibited by 40% the rates of association and dissociation of actin monomers to and from filaments. However, tropomyosin had no effect on the critical concentrations of ATP-actin or ADP-actin. The tropomyosin-troponin complex, with or without Ca2+, had a similar effect as tropomyosin alone on the rate of polymerization of ATP-actin. Although tropomyosin binds to F-actin and not to G-actin, the absence of an effect on the actin critical concentration is probably explicable in terms of the highly cooperative nature of the binding of tropomyosin to F-actin and its very low affinity for a single F-actin subunit relative to the affinity of one actin subunit for another in F-actin.     

Actin crystal dynamics: structural implications for F-actin nucleation, polymerization, and branching mediated by the anti-parallel dimer

Actin filament nucleation, polymerization, and branching are crucial steps in many forms of cell motility, cell shape, and intracellular organelle movements in a wide range of organisms. Previous biochemical data suggests that an anti-parallel actin dimer can incorporate itself into growing filamentous actin (F-actin) and has a role in branching. Furthermore, it is a widespread belief that nucleation is spawned from an actin trimer complex. Here we present the structures of actin dimers and trimers in two tetragonal crystal systems P4(3)2(1)2 and P4(3). Both crystal systems formed by an induced condensation transformation of a previously reported orthorhombic crystal system P2(1)2(1)2(1). Comparison between the three crystal systems demonstrates the dynamics and flexibility of actin-actin interactions. The dimer and trimer actin rearrangements observed between the three crystal systems may provide insight to in vivo actin-actin interactions that occur during the nucleation, polymerization, and branching of F-actin.     

Ca2+-calmodulin regulates fesselin-induced actin polymerization

Fesselin is a proline-rich actin-binding protein that was isolated from avian smooth muscle. Fesselin bundles actin and accelerates actin polymerization by facilitating nucleation. We now show that this polymerization of actin can be regulated by Ca(2+)-calmodulin. Fesselin was shown to bind to immobilized calmodulin in the presence of Ca(2+). The fesselin-calmodulin interaction was confirmed by a Ca(2+)-dependent increase in 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS) fluorescence upon addition of fesselin to MIANS-labeled wheat germ calmodulin. The affinity was estimated to be approximately 10(9) M(-1). The affinity of Ca(2+)-calmodulin to the fesselin F-actin complex was approximately 10(8) M(-1). Calmodulin binding to fesselin appeared to be functionally significant. In the presence of fesselin and calmodulin, the polymerization of actin was Ca(2+)-dependent. Ca(2+)-free calmodulin either had no effect or enhanced the ability of fesselin to accelerate actin polymerization. Ca(2+)-calmodulin not only reversed the stimulatory effect of fesselin but reduced the rate of polymerization below that observed in the absence of fesselin. While Ca(2+)-calmodulin had a large effect on the interaction of fesselin with G-actin, the effect on F-actin was small. Neither the binding of fesselin to F-actin nor the subsequent bundling of F-actin was greatly affected by Ca(2+)-calmodulin. Fesselin may function as an actin-polymerizing factor that is regulated by Ca(2+) levels.     

Dual regulation of actin rearrangement through lysophosphatidic acid receptor in neuroblast cell lines: actin depolymerization by Ca(2+)-alpha-actinin and polymerization by rho

Lysophosphatidic acid (LPA) is a potent lipid mediator with actions on many cell types. Morphological changes involving actin polymerization are mediated by at least two cognate G protein-coupled receptors, LPA(1)/EDG-2 or LPA(2)/EDG-4. Herein, we show that LPA can also induce actin depolymerization preceding actin polymerization within single TR mouse immortalized neuroblasts. Actin depolymerization resulted in immediate loss of membrane ruffling, whereas actin polymerization resulted in process retraction. Each pathway was found to be independent: depolymerization mediated by intracellular calcium mobilization, and alpha-actinin activity and polymerization mediated by the activation of the small Rho GTPase. alpha-Actinin-mediated depolymerization seems to be involved in growth cone collapse of primary neurons, indicating a physiological significance of LPA-induced actin depolymerization. Further evidence for dual regulation of actin rearrangement was found by heterologous retroviral transduction of either lpa(1) or lpa(2) in B103 cells that neither express LPA receptors nor respond to LPA, to confer both forms of LPA-induced actin rearrangements. These results suggest that diverging intracellular signals from a single type of LPA receptor could regulate actin depolymerization, as well as polymerization, within a single cell. This dual actin rearrangement may play a novel, important role in regulation of the neuronal morphology and motility during brain development.     

Myosin 1E coordinates actin assembly and cargo trafficking during clathrin-mediated endocytosis

Myosin 1E (Myo1E) is recruited to sites of clathrin-mediated endocytosis coincident with a burst of actin assembly. The recruitment dynamics and lifetime of Myo1E are similar to those of tagged actin polymerization regulatory proteins. Like inhibition of actin assembly, depletion of Myo1E causes reduced transferrin endocytosis and a significant delay in transferrin trafficking to perinuclear compartments, demonstrating an integral role for Myo1E in these actin-mediated steps. Mistargeting of GFP-Myo1E or its src-homology 3 domain to mitochondria results in appearance of WIP, WIRE, N-WASP, and actin filaments at the mitochondria, providing evidence for Myo1E's role in actin assembly regulation. These results suggest for mammalian cells, similar to budding yeast, interdependence in the recruitment of type I myosins, WIP/WIRE, and N-WASP to endocytic sites for Arp2/3 complex activation to assemble F-actin as endocytic vesicles are being formed.     

Novel PKCα-mediated phosphorylation site(s) on cofilin and their potential role in terminating histamine release

Using specific inhibitors, kinase-negative mutants, and small interfering RNA against protein kinase Cα (PKCα) or PKCβI, we find that PKCβI positively regulates degranulation in rat basophilic leukemia-2H3 cells, whereas PKCα negatively regulates degranulation. Mass spectrometric and mutagenic analyses reveal that PKCα phosphorylates cofilin at Ser-23 and/or Ser-24 during degranulation. Overexpression of a nonphosphorylatable form (S23,24A), but not that of a mutant-mimicking phosphorylated form (S23,24E), increases degranulation. Furthermore, the S23,24A mutant binds to F-actin and retains its depolymerizing and/or cleavage activity; conversely, the S23,24E mutant is unable to sever actin filaments, resulting in F-actin polymerization. In addition, the S23,24E mutant preferentially binds to the 14-3-3ζ protein. Fluorescence-activated cell sorting analysis with fluorescein isothiocyanate-phalloidin and simultaneous observation of degranulation, PKC translocation, and actin polymerization reveals that during degranulation, actin polymerization is dependent on PKCα activity. These results indicate that a novel PKCα-mediated phosphorylation event regulates cofilin by inhibiting its ability to depolymerize F-actin and bind to 14-3-3ζ, thereby promoting F-actin polymerization, which is necessary for cessation of degranulation.     

Control of actin turnover by a salmonella invasion protein

Salmonella force their way into nonphagocytic host intestinal cells to initiate infection. Uptake is triggered by delivery into the target cell of bacterial effector proteins that stimulate cytoskeletal rearrangements and membrane ruffling. The Salmonella invasion protein A (SipA) effector is an actin binding protein that enhances uptake efficiency by promoting actin polymerization. SipA-bound actin filaments (F-actin) are also resistant to artificial disassembly in vitro. Using biochemical assays of actin dynamics and actin-based motility models, we demonstrate that SipA directly arrests cellular mechanisms of actin turnover. SipA inhibits ADF/cofilin-directed depolymerization both by preventing binding of ADF and cofilin and by displacing them from F-actin. SipA also protects F-actin from gelsolin-directed severing and reanneals gelsolin-severed F-actin fragments. These data suggest that SipA focuses host cytoskeletal reorganization by locally inhibiting both ADF/cofilin- and gelsolin-directed actin disassembly, while simultaneously stimulating pathogen-induced actin polymerization.     

Actin depolymerization factor/cofilin activation regulates actin polymerization and tension development in canine tracheal smooth muscle

The contractile activation of airway smooth muscle tissues stimulates actin polymerization, and the inhibition of actin polymerization inhibits tension development. Actin-depolymerizing factor (ADF) and cofilin are members of a family of actin-binding proteins that mediate the severing of F-actin when activated by dephosphorylation at serine 3. The role of ADF/cofilin activation in the regulation of actin dynamics and tension development during the contractile activation of smooth muscle was evaluated in intact canine tracheal smooth muscle tissues. Two-dimensional gel electrophoresis revealed that ADF and cofilin exist in similar proportions in the muscle tissues, and that approximately 40% of the total ADF/cofilin in unstimulated tissues is phosphorylated. Phospho-ADF/cofilin decreased concurrently with tension development in response to stimulation with acetylcholine (ACh) or potassium depolarization indicating the activation of ADF/cofilin. Expression of an inactive phospho-cofilin mimetic (cofilin S3E) but not wild type cofilin in the smooth muscle tissues inhibited endogenous ADF/cofilin dephosphorylation and ACh-induced actin polymerization. Expression of cofilin S3E in the tissues depressed tension development in response to ACh, but it did not affect myosin light chain phosphorylation. The ACh-induced dephosphorylation of ADF/cofilin required the Ca2+-dependent activation of calcineurin (PP2B). The results indicate that the activation of ADF/cofilin is regulated by contractile stimulation in tracheal smooth muscle and that cofilin activation is required for actin polymerization and tension development in response to contractile stimulation.     

Inorganic phosphate regulates the binding of cofilin to actin filaments

Inorganic phosphate (Pi) and cofilin/actin depolymerizing factor proteins have opposite effects on actin filament structure and dynamics. Pi stabilizes the subdomain 2 in F-actin and decreases the critical concentration for actin polymerization. Conversely, cofilin enhances disorder in subdomain 2, increases the critical concentration, and accelerates actin treadmilling. Here, we report that Pi inhibits the rate, but not the extent of cofilin binding to actin filaments. This inhibition is also significant at physiological concentrations of Pi, and more pronounced at low pH. Cofilin prevents conformational changes in F-actin induced by Pi, even at high Pi concentrations, probably because allosteric changes in the nucleotide cleft decrease the affinity of Pi to F-actin. Cofilin induced allosteric changes in the nucleotide cleft of F-actin are also indicated by an increase in fluorescence emission and a decrease in the accessibility of etheno-ADP to collisional quenchers. These changes transform the nucleotide cleft of F-actin to G-actin-like. Pi regulation of cofilin binding and the cofilin regulation of Pi binding to F-actin can be important aspects of actin based cell motility.     

WAVE2 regulates high-affinity integrin binding by recruiting vinculin and talin to the immunological synapse

T-cell-receptor (TCR)-mediated integrin activation is required for T-cell-antigen-presenting cell conjugation and adhesion to extracellular matrix components. While it has been demonstrated that the actin cytoskeleton and its regulators play an essential role in this process, no mechanism has been established which directly links TCR-induced actin polymerization to the activation of integrins. Here, we demonstrate that TCR stimulation results in WAVE2-ARP2/3-dependent F-actin nucleation and the formation of a complex containing WAVE2, ARP2/3, vinculin, and talin. The verprolin-connecting-acidic (VCA) domain of WAVE2 mediates the formation of the ARP2/3-vinculin-talin signaling complex and talin recruitment to the immunological synapse (IS). Interestingly, although vinculin is not required for F-actin or integrin accumulation at the IS, it is required for the recruitment of talin. In addition, RNA interference of either WAVE2 or vinculin inhibits activation-dependent induction of high-affinity integrin binding to VCAM-1. Overall, these findings demonstrate a mechanism in which signals from the TCR produce WAVE2-ARP2/3-mediated de novo actin polymerization, leading to integrin clustering and high-affinity binding through the recruitment of vinculin and talin.     

A short sequence responsible for both phosphoinositide binding and actin binding activities of cofilin.

Cofilin is a widely distributed actin-modulating protein that has abilities to bind along the side of F-actin and to depolymerize F-actin. Both abilities of cofilin can be inhibited by phosphoinositides such as phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate (PIP2). We have previously shown that the synthetic dodecapeptide corresponding to Trp104-Met115 of cofilin is a potent inhibitor of actin polymerization (Yonezawa, N., Nishida, E., Iida, K., Kumagai, H., Yahara, I., and Sakai, H. (1991) J. Biol. Chem. 266, 10485-10489). In this study, we have found that the inhibitory effect of the synthetic dodecapeptide on actin polymerization is canceled specifically by phosphatidylinositol, phosphatidylinositol 4-monophosphate and PIP2. We further show that the dodecapeptide as well as cofilin binds to PIP2 molecules and inhibits PIP2 hydrolysis by phospholipase C. Thus, the actin-binding dodecapeptide sequence of cofilin may constitute a multifunctional domain in cofilin.     

F-actin-dependent translocation of the Rap1 GDP/GTP exchange factor RasGRP2

RasGRPs constitute a new group of diacylglycerol-dependent GDP/GTP exchange factors that activate Ras subfamily GTPases. Despite a common structure, Ras-GRPs diverge in their GTPase specificity, subcellular distribution, and downstream biological effects. The more divergent family member is RasGRP2, a Rap1-specific exchange factor with low affinity toward diacylglycerol. The regulation of RasGRP2 during signal transduction has remained elusive up to now. In this report, we show that the subcellular localization of Ras-GRP2 is highly dependent on actin dynamics. Thus, the induction of F-actin by cytoskeletal regulators such as Vav, Vav2, Dbl, and Rac1 leads to the shift of RasGRP2 from the cytosol to membrane ruffles and its co-localization with F-actin. Treatment of cells with cytoskeletal disrupting drugs abolishes this effect, leading to an abnormal localization of RasGRP2 in cytoplasmic clusters of actin. The use of Rac1 effector mutants indicates that the RasGRP2 translocation is linked exclusively to actin polymerization and is independent of other pathways such as p21-activated kinase JNK, or superoxide production. Biochemical experiments demonstrate that the translocation of RasGRP2 to membrane ruffles is mediated by the direct association of this protein with F-actin, a property contained within its 150 first amino acids. Finally, we show that the RasGRP2/F-actin interaction promotes the regionalized activation of Rap1 in juxtamembrane areas of the cell. These results reveal a novel function of the actin cytoskeleton in mediating the spatial activation of Ras subfamily GTPases through the selective recruitment of GDP/GTP exchange factors.     

A role for the actin cytoskeleton in the initiation and maintenance of store-mediated calcium entry in human platelets. Evidence for conformational coupling

The nature of the mechanism underlying store-mediated Ca(2+) entry has been investigated in human platelets through a combination of cytoskeletal modifications. Inhibition of actin polymerization by cytochalasin D or latrunculin A had a biphasic time-dependent effect on Ca(2+) entry, showing an initial potentiation followed by inhibition of Ca(2+) entry. Moreover, addition of these agents after induction of store-mediated Ca(2+) entry inhibited the Ca(2+) influx mechanism. Jasplakinolide, which reorganizes actin filaments into a tight cortical layer adjacent to the plasma membrane, prevented activation of store-mediated Ca(2+) entry but did not modify this process after its activation. In addition, jasplakinolide prevented cytochalasin D-induced inhibition of store-mediated Ca(2+) entry. Calyculin A, an inhibitor of protein serine/threonine phosphatases 1 and 2 which activates translocation of existing F-actin to the cell periphery without inducing actin polymerization, also prevented activation of store-mediated Ca(2+) entry. Finally, inhibition of vesicular transport with brefeldin A inhibited activation of store-mediated Ca(2+) entry but did not alter this mechanism once initiated. These data suggest that store-mediated Ca(2+) entry in platelets may be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, which shows close parallels to the events mediating secretion.     

A quantitative study of the role of F-actin in producing neutrophil shape

Neutrophils change shape from round to polar and sequentially polymerize/depolymerize actin following chemotactic peptide activation in suspension. To study the relationship between changes in F-actin content and shape we altered the kinetics/extent of actin polymerization and depolymerization with tBOC peptide, cytochalasin D (CD), and low-dose FMLP, and determined the effect of these alterations on the temporal sequence of changes in neutrophil shape. F-actin was measured by FACS analysis of NBDphallacidin-stained cells and expressed as relative fluorescent intensity (RFI) compared to control (RFI = 1.00). Shape was determined by scanning electron microscopy. FMLP causes serial polymerization/depolymerization of actin (RFI = 1.00 +/- 0.04, 1.60 +/- 0.21, 1.10 +/- 0.18, and 1.05 +/- 0.14) associated with four distinct shapes (round-smooth, round-ruffled, blebbed, and polar) noted at 0, 30, 90, 300 sec respectively. Since blebbed and polar shapes appear concurrent with depolymerization and following polymerization, we determined whether depolymerization is required for polarization of cells. The kinetics of depolymerization were: (1) accelerated by tBOC addition at 45 sec, and (2) slowed by high concentrations of FMLP (greater than 10-7M) (300 sec RFI = 1.46). Neither change altered the time course of shape change. To determine whether duration of actin polymerization defines shape, polymerization was halted by addition of tBOC at 5, 10, 20, 30 sec after FMLP to block actin polymerization and shape was monitored at 300 sec. TBOC added 5-20 sec after FMLP limited neutrophil shape change to the blebbed form, while tBOC addition 30 sec following FMLP resulted in a polar shape at 300 sec. To determine whether the extent of actin polymerization affects the shape change sequence, polymerization was limited by (1) inhibition of polymerization with CD, (2) exposure of cells to low concentrations of FMLP (less than 10-9 M), and (3) interruption of polymerization with tBOC. Actin polymerization to RFI less than 1.35-fold basal results in blebbed shape; polymerization greater than 1.35-fold basal yields polar shape. The data show: (1) the human neutrophil demonstrates intermediate shapes when activated by chemotactic peptide, (2) depolymerization of F-actin does not determine shape, and (3) blebbed shape appears when actin polymerizes for greater than 5 sec; polar shape with polymerization greater than or equal to 30 sec to RFI greater than 1.35-fold basal. The data suggest actin polymerization is required for, and extent of polymerization determines, the shape of human neutrophils.     

Activation of protein kinase C in rat basophilic leukemia cells stimulates increased production of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate: correlation with actin polymerization.

Cross-linking of the immunoglobulin E receptor on rat basophilic leukemia (RBL)1 cells by multivalent antigen activates phosphatidylinositol (PI) kinase and phosphatidylinositol 4-phosphate (PIP) kinase leading to the increased production of PIP and phosphatidylinositol 4,5-bisphosphate (PIP2). Activators of protein kinase C (PKC), such as phorbol myristate acetate (PMA) and the synthetic diacylglycerol, 1,2-dioctanoyl-sn-glycerol (diC8), were found to have the same effect even though PMA and diC8 do not cause the activation of phospholipase C. Although the kinetics are different depending on the stimulant, activation of PKC using multivalent antigen, PMA or diC8 also causes the polymerization of actin and an increase in the F-actin content of the cells. In all cases, a good correlation was observed between F-actin levels, activation of PI and PIP kinases, and the increased production of PIP and PIP2. However, in the case of antigen, there is no correlation between actin polymerization and the total amount of PIP and PIP2. Staurosporine, an inhibitor of protein kinases, blocks the F-actin response and the increased synthesis of PIP and PIP2 with similar dose dependencies. Furthermore, depletion of PKC activity through long-term exposure to PMA, inhibited both the F-actin response and the increased synthesis of PIP and PIP2 induced by either DNP-BSA or diC8. These results suggest that activation of PKC precedes the activation of PI and PIP kinases and that under certain circumstances activation of the kinases and the increased synthesis of PIP and PIP2 may be involved in the polymerization of actin in RBL cells, possibly through the interaction of the polyphosphoinositides with actin-binding proteins such as gelsolin and profilin.     

Creatine Kinase–Mediated ATP Supply Fuels Actin-Based Events in Phagocytosis

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 β-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-γR–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.