Evidence that the phosphatidylinositol cycle is linked to cell motility

Experimental

Cell Research

174 (1988) 1-15

SPECIAL ARTICLE Evidence

that the Phosphatidylinositol to Cell Motility I. LASSING

Department of Zoological

Cycle Is Linked

and U. LINDBERG’

Cell Biology, The Wenner-Gren Institute, University of Stockholm, S-11345 Stockholm, Sweden

Transmembrane signaling via specific ligandireceptor interactions induces the immediate polymerization of actin and formation of microlilament assemblies close to the plasma membrane. The profilin: actin complex appears to provide the actin for this filament formation. A clue to the nature of the regulatory mechanism involved was recently found in that phosphatidylinositol4,Sbisphosphate can bind to protilin, dissociate the profilactin complex, and thus liberate actin for polymerization. This suggests that the phosphatidylinositol (PI) cycle, which plays important roles in cellular regulation, also might control microfilament-based motility. We show here that neomycin, a drug which has a high affinity for phosphoinositides and in vivo interferes with the PI cycle, inhibits the polymerization of actin in platelets induced either by thrombin or by ADP. When ADP was used as agonist (but not in the case of thrombin) the induction of actin polymerization could also be blocked by the addition of aspirin. Introduction of Ca2+ mto platelets by the use of the ionophore Aznn or stimulation of protein kinase C (PkC) by the phorbol ester TPA did not induce actin polymerization; neither did the addition of a combination of these two agents. Retinoic acid which inhibits PkC was also without effect on thrombin-induced actin pOlymWiZation.

@ 1988 Academic

Press, Inc.

Certain transmembrane proteins appear to be directly linked to the submembraneous weave of microfilaments present in eukaryotic cells ([l], review) and there are many examples where external signals have immediate and profound effects on the organization and activity of these microfilaments. In several of these systems, direct biochemical analysis of the G/F-actin ratio of cell extracts has shown that in immediate conjunction to ligand/receptor interactions there is a rapid polymerization of actin [2-131. Changes in the gross morphology and ultrastructural organization of the cells caused by activation with specific agonists are also indicative of microtilament formation, and are often followed by a generalized stimulation of microfilament-based motility [14, 151. The specific ligand/receptor interactions involved here belong to the class which also induces an increased turnover in the phosphatidylinositol (PI) cycle and mobilization of Ca*+ (see Table 1) suggesting the possibility that there might be a link between this cycle and microfilament-based motility. This is supported by the finding that the microfilament precursor profilactin is specifically dissociated by phosphatidylinositol 4,5bisphosphate [16]. ’ To whom reprint requests should be addressed. 1

Copyright @ 1988 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827188 $03.00

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Platelets constitute an excellent model system for studying microfilament formation and functioning. Resting platelets have most of their actin (60-70%) in the unpolymerized precursor form, profilactin [17, 181. Stimulation of platelets with various agonists affecting the PI cycle induces a rapid polymerization of actin [2-4] and incorporation of formed filaments into supramolecular aggregates [4, 191. At the same time the profilactin level decreases and free profilin appears [19]. With thrombin, which is a potent platelet-activating agonist, the major part of the G+F-actin transition occurs within l&15 s after addition of the agonist. Receptor-mediated activation of the PI cycle appears to result in the immediate hydrolysis of PtdIns(4,5)PZ2 liberating InsP3 to the cytoplasm. The other product of the hydrolysis, diacylglycerol, remains in the lipid bilayer where it is phosphorylated to PhA which to a large extent is recycled to phosphatidylinositol (PtdIns) ([20], review). Neomycin binds to PtdIns, phosphatidylinositol 4-monophosphate (PtdIns(4)P), and PtdIns(4,5)P2 with increasing affinity in vitro [21], and in many cell systems it interferes with phosphatidylinositol metabolism [22-281. In hamster (NIL) fibroblasts it has been demonstrated that 0.7 mM neomycin blocks agonist-induced hydrolysis of PtdIns(4,5)P2 and causes an accumulation of this phospholipid. At 2 mil4 it appears also to inhibit the futile cycles connecting the phosphatidylinositols [29]. In the present work we have used neomycin and other drugs which interfere with the phosphatidylinositol cycle on platelets to see if there is a simultaneous interference with agonist-induced microfilament formation. The results obtained together with earlier findings that PtdIns(4,5)P2 specifically dissociates profilactin in vitro, and that PtdIns(4,5)P2-derived InsP3 or diacylglycerol analogs have no effect, suggest that it is the generation of PtdIns(4,5)P2, and not the second messengers produced from it, that controls actin polymerization. MATERIALS

AND

METHODS

Thrornbin, prostacyclin (PC&), 12-O-tetradecanoylphorbol-13-acetate (TPA), 4a-phorbol 12,13didecanoate (4a-PDD), and phorbol 12,13-dibutyrate (P(Bu),), neomycin sulfate, acetyl salicylic acid, retinoic acid, deoxyribonuclease I (DNase I), and phosphatidic acid were from Sigma. The DNase I was further purified according to Markey [30]. Adenosine diphosphate (ADP) (PL Biochemicals) was purified by ion exchange chromatography using a Mono Q column and FPLC system obtained from Pharmacia (Sweden). The column was eluted with a gradient of NaCl (O-O.4 M) in 5 mM potassium phosphate buffer, pH 7.3. Carrier-free [32P]orthophosphate was from NEN. Platelers. Fresh platelet-rich plasma from human volunteers was obtained from the Blood Centre, Sbdersjukhuset, Stockholm. The platelets were separated from plasma by chromatography at room temperature on Sepharose 2B (Pharmacia) in plastic columns. The buffer used contained 14.5 mM Tris-HCI, pH 7.4 (or 10 nu’t4 potassium phosphate, pH 7.4), 1 mM MgC12, 5.4 mM KCl, 0.126 M NaCl, 0.1 mM EGTA, 0.1% (w/v) glucose, 0.8% (w/v) human serum albumin (Kabi, Sweden), and 3 rig/ml of PGIz. The use of PG12 [31] in gel filtration [32] seems to be the most reliable method for preparing resting platelets as judged by both morphological criteria (a high proportion of fully discoid platelets) * EGTA, ethylene glycol bis@aminoethyl ether)-h’,N,N’,iV’-tetraacetic acid; ADP, adenosine PtdIns(4)P, phosphatidylinositol 4-monophosphate; diphosphate; PtdIns, phosphatidylinositol; PtdIns(4,5)P,, phosphatidylinositol 4,5-bisphosphate; Ins, inositol; InsP,, inositol monophosphate; InsPt, inositol bisphosphate; InsP,, inositol trisphosphate; TPA, 12-0-tetradecanoylphorbol-13-acetate; 4a-PDD, 4a-phorbol 12,13-didecanoate; Pi, phorbol 12,13-dibutyrate; PG12, prostacyclin; DNase I, deoxyribonucleate 5’-oligonucleotidohydrolase (EC 3.1.21.1).

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and biochemical measurements (low levels of tilamentous actin, correlating with the resting state). Platelets prepared this way are also characterized by a low degree of radiolabeling in phosphatidic acid even after a l-h incubation with [32P]orthophosphate. Samples of platelet suspensions (10’ platelets/ml) were incubated on a rocking table at room temperature with different drugs at concentrations and for times as indicated. Short-time stimulations were performed by adding the agonist and immediately vortexing the samples for a few seconds. The incubations were terminated by the addition of either ice-cold lysis buffer or acidified chloroform: methanol mixture as described below. For radiolabeling of phospholipids platelet suspensions were incubated with carrier-free [“Plorthophosphate (10 @/ml) at room temperature for the times indicated. Lipid analysis. Lipids were extracted from platelet samples (0.5 ml) by the addition of 1 ml of methanol : HCl,,,, (10 : 1) and vortexing the mixture for 30 s. Then 1.5 ml of chloroform and 0.5 ml of H20 was added followed by vortexing. The extraction mixture was left on ice for 30 min and then centrifuged for 10 min at 1200g to separate the phases. One milliliter of the chloroform phase was removed and saved, and the remaining sample was reextracted with 1 ml of fresh chloroform. One milliliter from the last chloroform phase was removed and pooled with the previously saved extract. The collected organic phases were dried under nitrogen. The lipids were redissolved in chloroform and spotted on Silica Gel 60 HPTLC plates (Merck) that had been precoated with 1% potassium oxalate in methanol : water (2 : 3) and activated at + 120°C for 30 min [21]. Phosphatidic acid (PhA from Sigma) and PtdIns (4)P and PtdIns(4,5)P2 purified from bovine brain 1211were used as markers. The plates were developed with chloroform : acetone : methanol : acetic acid :H20 (40 : 15 : 13 : 12 : 8) and stained with iodine vapor and 3% copper acetate in 8% phosphoric acid for 10 min at +18o”C [21]. The plates were autoradiographed using Cronex 4 X-ray film and Cronex Xtra Lite intensifying screen (DuPont). The exposure time was 18 h. Spots containing labeled phospholipids were scraped off and transferred to scintillation vials, and 0.25 ml of HZ0 was added and then 10 ml of methanol : toluene (1 : 1) containing PPO/POPOP (4 and 0.05 g, respectively, per 1000 ml). The radioactivity was analyzed in an Intertechnique scintillation counter. Actin measuremenrs. Unpolymerized and total actin in platelet extracts was determined with the DNase I inhibition assay [33]. Platelets were lysed by mixing the platelet samples with 0.1 vol of 10% Triton X-100, 20 mM MgCl?, 20 mM EGTA, 2 mM ATP, 1 mM dithiotreitol (DTT), and 0.2 mM phenylmethylsulfonyl fluoride (PMSF), pH 7.5. The lysate was kept on ice. To determine the amount of unpolymerized actin lysates were centrifuged at 8000g for 15 min at 4°C and then the supernatants were analyzed for DNase I inhibition. Supematants from unstimulated platelets contain detectable amounts of tilamentous actin, whereas practically all the filaments are incorporated into sedimentable aggregates after stimulation [19]. For the determination of total actin 100 pl of the lysate was mixed with 100 ul of 1.5 M guanidine-HCl, 1 mM CaCb, 1 mM ATP, and 20 mM Tris-HCl, pH 7.5, and incubated at 0°C for 5 min. During this time tilamentous actin depolymerizes and total actin can be determined as unpolymerized actin as above. At least two measurements were made on each sample. Analysis of platelet lysates with time after lysis showed that in extracts of stimulated platelets (no neomycin) the pool of unpolymerized actin increased continuously from the time of lysis (from 33 to 49 % of total actin during the first 6 min) indicating that a considerable depolymerization occurred in the extract. In extracts from neomycin-treated-stimulated platelets, on the other hand, only a small increase in unpolymerized actin was observed suggesting that neomycin somehow inhibits the depolymerization that occurs in the extract. There was no depolymerization of the preexisting actin filaments in extracts of resting platelets either. Thus either preexisting filaments differ in some sense from newly formed filaments or stimulation of the platelets also activates a depolymerization mechanism expressed in the extracts. This stresses the importance of analyzing the actin pools in platelet extracts as rapidly as possible after the preparation of the lysates, as also pointed out previously [2].

RESULTS Effect of Neomycin

on the PI Cycle in Platelets

The diacylglycerol formed after receptor-mediated hydrolysis of PtdIns(4,5)P2 is to a large extent reutilized for the formation of PtdIns. The first step towards this end is the phosphorylation of the DG to PhA. In resting platelets the PhA pool contains only small amounts of radioactivity even after prolonged incubation

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Fig. 1. Inhibition of thrombin-induced formation of phosphatidic acid by neomycin. Platelets prelabeled for 90 min with [3ZP]orthophosphate were incubated in the absence and presence of increasing concentrations of neomycin for 30 min after which they were stimulated with thrombin (0.5 units ml-‘) for 40 s. Lipids were extracted and analyzed as described under Materials and Methods. In the control platelets there was a fivefold increase in radioactivity in PhA after stimulation with thrombin. The level of radioactivity reached in the presence of neomycin is plotted here as percentage of the level reached in the absence of the drug.

(90 min) of the platelets with [32P]orthophosphate, in contrast to the polyphosphoinositides which are heavily labeled. After stimulation with thrombin, however, PtdIns(4,5)P2 is hydrolyzed and there is a rapid increase in labeled PhA indicating that the PI cycle has been activated [34]. This has been used here to analyze for the effect of neomycin on thrombin-induced turnover in the PI cycle. Stimulation of platelets with 0.5 units/ml of thrombin for 40 s resulted in a fivefold increase in the level of radioactivity in PhA. As shown in Fig. 1, preincubation of the platelets with increasing concentrations of neomycin caused a progressive suppression of the labeling of PhA suggesting a corresponding inhibition of the hydrolysis of PtdIns(4,5)P2. At 0.7 and 2 mM neomycin the inhibition was 43 and 77%, respectively. Platelets which already showed signs of shape change and aggregation before the experiment invariably had reduced levels of unpolymerized actin and incorporated large amounts of 32P into PhA during the prelabeling period. Such platelets were not used for further experimentation. Prelabeled platelets were also analyzed for radioactivity in the polyphosphoinositide pools before and after stimulation with 0.5 unit/ml of thrombin in the presence and absence of 2 mM neomycin. The time course of the changes in PtdIns(4,5)P2 and PtdIns(4)P are shown in Figs. 2A and 2B, respectively. For experimental details see the legend to the figure. In the control (- neomycin) both polyphosphoinositide pools initially rapidly went through a small increase in radioactivity after which they decreased. In the neomycin-treated platelets on the other hand, the radioactivity in PtdIns(4)P did not change at all after stimulation, and in the case of PtdIns(4,5)P2 there was an initial increase after which the radioactivity stayed at the elevated level. These results support the conclusion

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Fig. 2. Effect of neomycin on the radioactivity in the polyphosphoinositide pools in platelets after stimulation with thrombin. Platelets were prelabeled with [“Plorthophosphate (90 min), after which neomycin (2 m&f) was added and the incubation was continued for 55 min. The platelets were then stimulated with 0.5 units ml-’ of thrombin. Samples were taken at the times indicated and processed for lipid analysis as described under Materials and Methods. The incorporation of 32Pinto the pools did not reach equilibrium during the prelabeling period. Therefore samples from the unstimulated platelets (? neomycin) were taken before, during, and after the actual experiment and the radioactivity found in the polyphosphoinositides from stimulated samples was corrected for the continued background incorporation. This set of controls also showed that neomycin slowed the incorporation into the PtdIns(4)P and PtdIns(4,5)P2 pools during the prelabeling period by 35 and 55 %, respectively. In the figure the radioactivity in the samples is expressed as percentage of the radioactivity in the unstimulated control at the time of the actual experiment. Three separate stimulation were performed for each time point. (A and B) The changes in PtdIns(4,5)Pz and PtdIns(4)P, respectively. The filled and open symbols represent plus and minus neomycin, respectively.

drawn from the PhA analyses that neomycin inhibits the hydrolysis of PtdIns(4,5)P2.

(as in the hamster fibroblasts

[29])

The Effect of Neomycin on Thrombin-Znduced Actin Polymerization

The time course of actin polymerization in platelets stimulated with 0.5 unit/ml of thrombin in the absence and presence of neomycin is shown in Fig. 3 A. After a short lag phase there was a rapid decrease in the level of unpolymerized actin as measured with the DNase inhibition assay indicating filament formation. The major part of the change occurred during the first 15 s of stimulation and the final level of polymerization was reached in about 30 s. In platelets treated with 2 m.M neomycin the decrease in unpolymerized actin was much slower indicating that the drug strongly inhibited the thrombin-induced polymerization of actin. Comparison of the maximal rates of the decreases in the pools of unpolymerized actin in the two cases suggests an 85 % inhibition of polymerization at this concentration of neomycin. Figure 3 B shows the results of analyses of thrombin-induced polymerization of actin (0.2 unit/ml of thrombin for 30 s) in platelets pretreated with increasing concentrations of neomycin. In the absence of neomycin the level of unpolymerized actin dropped from 60 to 30% of total actin. Neomycin at 0.7 mM inhibited this decrease by 35-50% and at 2 mM the inhibition was 60-85%.

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Fig. 3. Effect of neomycin on thrombin-induced actin polymerization. (A) Platelets were incubated with neomycin for 30 min and then stimulated with thrombin (0.5 units ml-‘) for the times indicated. Platelet lysates were analyzed for unpolymerized and total actin with the DNase inhibition assay (see Materials and Methods). The level of unpolymerized actin in the extract is expressed as percentage of total actin. Filled and open symbols represent plus and minus neomycin, respectively. (B) Here platelets were treated with neomycin for 60 min and then stimulated with thrombin (0.2 units ml-‘) for 30 s at varying concentrations of neomycin. Platelet lysates were analyzed as above and the actin polymerization is expressed as percentage of the G+F-actin shift seen in the absence of neomycin. The results of experiments on two separate batches of platelets are shown.

Thus there seems to be a correspondence between the interference with the turnover in the PI cycle caused by neomycin and its effect on actin polymerization supporting the idea that the two processes might be linked. The Effect of Neomycin and Aspirin on ADP-Induced Polymerization of Actin

Adenosine diphosphate is a considerably weaker agonist with platelets causing a conversion of unpolymerized to filamentous actin which is only about 30% of that seen with thrombin. In this case neomycin effectively inhibited the induction of actin polymerization even at 0.7 mM as shown in Fig. 4 indicating the involvement of the PI cycle in mediating the ADP signal causing actin polymerization. Adenosine diphosphate, however, seems to work through a somewhat different mechanism as compared with thrombin, because in this case also aspirin inhibited the formation of actin filaments. At 500 uM aspirin abolished the ADP effect on actin (Fig. 5). At this concentration the drug effectively inhibits the formation of prostaglandin H2 [35] which otherwise is metabolized to prostaglandin endoperoxides and thromboxane AZ. Thus induction of actin polymerization in this case appears to be mediated by the products of the cyclooxygenase pathway working either directly on the polymerization mechanism or via their effect on receptors coupled to the PI cycle [36, 371. The latter explanation seems more likely considering the ffect of neomycin on the ADP-induced actin polymerization.

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Fig. 4. Effect of neomycin on ADP-induced actin polymerization. Platelets were incubated with neomycin as in Fig. 38. Stimulation was performed with 10 pkf ADP for 5 min after which lysates were prepared and analyzed for actin pools. Open symbols, no ADP.

The Effect of TPA, Ionophore A23187,and Retinoic Acid on Actin Polymerization

The tumor-promoting phorbol ester TPA has been shown to affect the motile activity of a variety of cell types [3&41]. In BSC-I cells it causes outgrowth of membrane lamellae and microspikes and a dramatic reorganization of proteins involved in the microtilament system [41]. The major target for TPA in platelets as well as in other cells is protein kinase C ([42], review) whose activity normally seems to be regulated by the diacylglycerol produced in receptor-stimulated PtdIns(4,5)P2 hydrolysis. Since the reported effects of TPA on the motile activity

a 5 0 0r 50, ,

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(FM)

Fig. 5. Effect of aspirin on ADP- and thrombin-induced actin polymerization. Platelets were incubated with aspirin for 10 min at room temperature, and then stimulated with either ADP (10 Q4) (A) or thrombin (0.5 units ml-‘) (W) for 30 s. Lysates and actin measurements were made as described (Materials and Methods). Open circle indicates the level of unpolymerized actin in unstimulated platelets.

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Fig. 6. Effect of phorbol esters on actin pools in platelets. (A) Platelets incubated with phorbol esters (dissolved in dimethylsulfoxide) in the presence of 10 tu’kfCa*+ for 1 min and then lysed. The G/F-actin ratio determined as described under Materials and Methods. Symbols: unstimulated platelets (0); plus 0.5% dimethylsulfoxide (a)); plus thrombin (0.2 units ml-‘) for 30 s (0); with TPA (two different batches) (O), with P(Bu), (A); and with 4a PDD (V). (B) Platelets treated with 10 nM (0) or 100 n&f (A) TPA for 10 and 30 s at 10m6 M (-) or lo-’ M Ca2+ (- - -). Platelets were also stimulated with thrombin (0.2 units ml-‘) for 30 s before (0) and after treatment with 100 nM TPA (A).

of cells seemed to suggest that activation of PkC had something to do with the induction of actin polymerization it was important to test this drug on platelets. Treatment of resting platelets with different phorbol esters (tumor promoting as well as nontumor promoting) for 30 s, at concentrations of the phorbol esters varying from 10 nM to 10 l&f did not cause polymerization of actin (Fig. 6A). With TPA there was instead a small increase in the level of unpolymerized actin indicating a limited depolymerization of preexisting filaments. Figure 6B shows that there was no effect of the phorbol esters (10 and 100 nM) on the level of unpolymerized actin in the extracts at either 10 or 30 s of treatment, and that the agents did not suppress subsequent induction of actin polymerization with thrombin. Increasing the incubation period from 30 s up to 9 min did not change this result (Fig. 7), nor did the addition of Ca2’ to the medium. It should be noted that the same platelets used in this experiment responded well to stimulation with thrombin as shown by the decrease in the level of unpolymerized actin from 60 to about 40 % both before and after the TPA treatment. Addition of the ionophore A23187at concentrations up to 0.1 @4 in the presence or absence of 50 QZ Ca2+ did not cause polymerization of actin (Fig. 7). Instead there was a slight increase of the G/F-actin ratio in the platelet extracts. Combination of AZ3i8, (0.1 Q4) with TPA (10 nM) did not affect the G/F-actin ratio either. At higher concentrations of the ionophore (0.2 and 0.3 uM,), however, the proportion of unpolymerized actin decreased somewhat (5 and 7 %, respectively). Retinoic acid, which is thought to be an inhibitor of PkC [43, 441, did not affect the stimulation of actin polymerization caused by thrombin (Fig. 7). These results argue that neither PkC nor Ca2+ is involved in regulating actin

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Fig. 7. Effect of TPA, AZjt8,, and retinoic acid polymerization in platelets. Platelets in the presence of 50 pM Ca” were treated as described below, lysed, and analyzed for actin pools. Samples: I, resting platelets; 2, thrombin, 0.2 units ml-‘, 30 s; 3, TPA, 10 ~44, 30 s; 4, TPA, IO PM, 9 min; 5, TPA, 10 pM, 9 min, followed by thrombin (0.2 units ml-‘); 6, A.n,u. 0.1 @f, 30 s; 7, Ax,~,, 0.2 PM, 60 s; 8, Azx18,,0.3 pJ4, 90 s; 9, TPA, 10 @I, 30 s followed by A zj18,,0.1 @4, 30 s; 10, retinoic acid, 0.1 mM, 30 s followed by thrombin, 0.5 units ml-‘, 30 s.

polymerization. Consequently one of the major targets phosphorylated by PkC after platelet stimulation, the 40,000-D protein [45], should not be involved either. DISCUSSION In the advancing edge of motile cells membrane lamellae and microspikes form, translocate, and disappear in a cyclic fashion [46, 471. This dynamic activity appears to depend on a continuous reorganization of microfilament assemblies in close association with the inner face of the plasma membrane. The basic steps in this cyclic process seem to be (1) polymerization of actin from an unpolymerized precursor, profilactin, to filaments, (2) crosslinking of the filaments to form the supramolecular assemblies seen in the cell surface, (3) translocation of the microfilament assemblies (most likely a myosin-dependent process), and finally (4) depolymerization of the filaments to reform profilactin 148-501. The information given in Table 1 serves to illustrate the correlation that exists between activation of this motility cycle [2-131 and receptor-mediated activation of the PI cycle 15l-641. According to the current view the transmembrane signaling involved here causes an increased turnover in the PI cycle by activating a phosphodiesterase which hydrolyzes PtdIns(4,5)P2 to produce the two second messengers InsP3 and diacylglycerol. Inositol trisphosphate is thought to release Ca2+ ions from intracellular storage pools, and diacylglycerols apparently remains in the lipid bilayer where it activates protein kinase C ([20, 651, reviews). As a consequence of the receptor-mediated cell stimulation the intracellular concentration of free Ca2+ increases from about lo-’ to I-5x lop6 M [66, 671, and it would not be far-fetched

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TABLE

1

Systems where there is evidence for receptor-mediated stimulation of actin polymerization. The same ligandlreceptor interactions also cause increased turnover in the PI cycle as described in the references given

System Platelets

Ligand

Pancreatic /l-cells Lymphocytes

Thrombin ADP Glucose Con Ab

Neutrophils

FMLP’ ;;Gd

Sea urchin eggs Glial cells Swiss 3T3 cells

Fertilizatiorr’ PDGFg

Aa3, cells

EGFh

Immediate physiological change Disks+Spiky spheres Secretion Membrane folds Microspikes Membrane folds Microspikes Phagocytosis Microvillar fur Membrane folds Microspikes Macropinocytosis Membrane folds Microspikes

Actin polymerization DI” [2-4]

PI cycle activation 51, 521

DI [91

[34, [541 [551 [561

DI NDBphalloidin’ [lo-l31

157-601

DI [51

[611

DI [31

DI [61

LM, EM [15] LM [14]

[62, 631 [641

’ Polymerization assessed using the DNase I inhibition assay. b Lectin concanavalin A. ’ Chemotactic peptide f Met-Leu-Phe. d Immunoglobulin complex. e Polymerization assessed also using NDB-phalloidin. ’ Fertilization reaction elicited by adding either sperm concentrate or alkaline seawater. g Platelet-derived growth factor. f Epidermal growth factor.

to assume that actin polymerization is somehow regulated by Ca’+. However, as shown here direct introduction of Ca2+ by the use of the ionophore.AZ3ts7 to raise the Ca2+ concentrations to the levels reached with natural agonists [533 does not result in rapid polymerization of actin. Our experiences from analyses of actin pools in cell extracts also argue against Ca2+ as an effector inducing actin polymerization. Filments present in platelet extracts undergo rapid depolymeriG+F-actin zation at micromolar concentrations of Ca’+, and receptor-mediated transformations can be detected only if the extracts are made in buffers where the Ca*’ concentration is lowered to submicromolar levels by the addition of EGTA

LL 681. Furthermore studies of the properties of profilactin have shown that Ca2+ stabilizes rather than destabilizes the complex [69], and gelsolin which nucleates actin polymerization from G-actin in the presence of Ca2+ [70, 711 under similar conditions caused an apparent stabilization of profilactin [72]. Neither protilin nor actin seems to be phosphorylated during activation of platelets suggesting that there is no involvement of Ca’+-activated kinases (F. Markey, unpublished observation). That Ca2+ ions may not play a role in inducing actin polymerization

Phosphatidylinositol

/PIP,

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@ Polymerization

@ Crosslinking 0 @

TgnslOcatiOn

@ Depolymerization

Fig. 8. The hypothetical model linking the microfilament-based motility cycle to the phosphatidylinositol cycle. Circled numbers denote the four major steps in the cell motility cycle. PI, PtdIns; PIP, PtdIns(4)P; PIP*, PtdIns(4,5)P,; DG, diacylglycerol; PhA, phosphatidic acid; CDP-DG, cytidine diphosphate diacylglycerol; IP, InsP; IP2, InsP; IP3, InsP3. Futile cycles are thought to connect the inositides. The arrow indicates where the agonist is thought to act on a transmembrane receptor linked to the PtdIns(4,5)P2-phosphodiesterase. This point is discussed in the text. The possibility seems to exist that the initial event is a stimulation of the formation of PtdIns(4J)P instead of degradation. In this scheme the phosphodiesterase is envisioned to recognize the PIP,:profilin complex.

was also suggested recently from studies on rabbit neutrophils stimulated with the chemotactic peptide fMet-Leu-Phe [73]. The observations described above together with the fact that TPA was unable to stimulate actin polymerization in resting platelets argue against the products of agonist-induced hydrolysis of PtdIns(4,5)P2 being involved in the initiation of actin filament formation. This argument is strengthened by the fact that the DG analogs diolein and O-acetyldiacylglycerol did not have any effect on protilactin in vitro [16, 741. The effect of neomycin on the turnover in the PI cycle and on actin polymerization described here, together with the observations that profilactin is specifically dissociated by PtdIns(4,5)P2 in vitro [16, 741, rather suggest the possibility that generation of PtdIns(4,5)P2 in the inner leaflet of the plasma membrane recruits profilactin which then dissociates providing actin for filament formation. In the hypothetical model presented in Fig. 8 the ligand/receptor interaction controls microfilament-based motility by influencing the turnover in the PI cycle. As long as the PI cycle is switched on to produce PtdIns(4,5)P2 actin polymerizes forming microtilaments. After dissociation of the profilactin complex by PtdIns(4,5)P2 in vitro, profilin remains tightly bound to the lipid [ 161. Profilin from both calf spleen and human platelets bind tightly to PtdIns(4,5)P2 [74]. Whether

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there is a membrane-bound (in addition to a free) form of protilin in the cell, as indicated by these observations, has not yet been investigated. Since diolein and synthetic diacylglycerol do not bind profilin, agonist-induced hydrolysis of PtdIns(4,5)Pz might release profilin from the membrane making it available for later depolymerization of filaments. It is implied in the model that the phosphodiesterase hydrolyzing PtdIns(45)Pz might recognize a prolilin : PtdIns(4,5)P2 complex rather than the free phospholipid. If polymerization of actin occurs in response to the appearance of PtdIns(4,5)P2 in the membrane one would expect this phospholipid to be formed in amounts at least equimolar to the amounts of actin incorporated into filaments after activation. Estimates of these numbers can be made from the data available in the literature. The concentration of actin in platelets is about 1 mA4 [33], i.e., about 500 pmol of actin per IO9 platelets assuming a platelet volume of 5 fl [76]. Of this 60% is present in the unpolymerized form and of this about 50 % is engaged in filament formation during the initial 10-15 s of stimulation with thrombin, i.e., 150 pmol of unpolymerized actin per lo9 platelets are transformed into filaments. The amount of PtdIns(4,5)P2 can also be estimated from data in the literature. Determinations of the mass of InsP3 present at various times after stimulation showed that after 15 s of stimulation, IO9 platelets contained about 135 pmol of InsP3 [53]. However, in estimating the amount of InsP3 formed during this time period, one should also take into account the conversion of InsP3 to its degradation products InsP* and InsP [20]. In fact the increase in the InsPz pool seems to be three to five times that in the InsP3 pool [73, 521, suggesting that the agonistinduced hydrolysis of PtdIns(4,5)P2 might generate at least 400 pmol of InsP, during the first 15 s of stimulation, and possibly as much as 700 pmol. The drop in the PtdIns(4,5)P2 level that occurred during the same time period indicated that 170 pmol of PtdIns(4,5)P2 were hydrolyzed [53] provided that no net production of this phospholipid occurred after stimulation. Because much more InsP3 appeared to be produced than was accounted for by the decrease in the PtdIns(4,5)P, pool it seems that a considerable amount of PtdIns(4,5)P2 was produced and hydrolyzed during the first 15 s of stimulation. The estimates of the amount of InsP3 formed (400-700 pmol/lO’ platelets) suggest that at least 230 (400 minus 170) pmol of PtdIns(4,5)P2 are formed during the initial phase of stimulation and possibly as much as 530. This is important since it shows that formation of PtdIns(4,5)Pz very well might serve as a signal for actin polymerization. The initial expansion of the PtdIns(4,5)P2 pool seen here (Fig. 2) suggests that PtdIns(4,5)P2 actually might be synthesized at an increased rate right after stimulation with thrombin. Since the cortical weave of actin filaments is highly ordered (filaments running in parallel over long distances [48, 49]), and the filaments in the advancing edges of motile cells all seem to have their barbed ends at the front [78, 481, actin filament formation must be a highly regulated process. It is therefore reasonable to assume that there are specific mechanisms which locate the recruitment of actin for filament formation to the plasma membrane, and membrane-associated proteins which serve to nucleate actin polymerization or which hold already

Phosphatidylinositol

cycle and cell motility

13

existing actin filament (or oligomers) in such a way that actin monomers can be added onto them. Candidates for such functions do exist but it is still impossible to envision how they fit into the system. It is of special interest in this context, however, that the binding of the spectrin : actin : band 4.1 complex to glycophorin in red blood cells is strengthened by the presence of PtdIns(4,5)P2 in the membrane [79]. The spectrin: actin: band 4.1 complex in vitro causes the efficient nucleation of actin polymerization from profilactin in a Mg’+-containing milieu [80] indicating that the actin oligomer present in the complex provides free barbed ends onto which actin monomers can be added. Neither Ca2+ nor diacylglycerol seems to induce actin polymerization, but may play major roles in the later steps of the cell motility cycle (i.e., crosslinking, translocation, and depolymerization). a-Actinin, possibly involved in crosslinking of actin filaments and/or binding of filaments to the plasma membrane, interacts specifically with diolein in the presence of palmitic acid in vitro [81], and immunoprecipitates of platelet a-actinin contain diacylglycerol and palmitic acid [82]. Vinculin, present at increased concentrations where microtilaments attach to the plasma membrane [l], appears to be phosphorylated at serine and threonine residues by the diacylglycerol-stimulated protein kinase C [83]. Furthermore there is evidence that vinculin interacts specifically with phospholipid vesicles containing phosphatidylinositol [84], an interaction which seems to change the structure of vinculin. Since vinculin and a-actinin are related to the organization of actin filament assemblies close to the plasma membrane [l], this suggests that the second step of the cell motility cycle (Fig. 8) may also be controlled by components of the PI cycle. Evidence from studies on platelets suggests that later in the activation process there is a massive Ca2+ -dependent production of diacylglycerol directly from phosphatidylinositol [53, 85, 861. This indicates a mechanism by which the activities of a-actinin and vinculin may be indirectly regulated by Ca2’. Myosin, which most likely is involved in translocation of microfilament assemblies, appears to be regulated by Ca2+-dependent phosphorylation [87, 881, and the activities of profilin [55] and gelsolin [56, 571 in vitro with respect to actin filaments suggest that also depolymerization of filaments may be regulated by Ca2+. We are most grateful to Anna-Lena Kullgren for expert technical assistance. This work was supported by grants from the Swedish Cancer Society and from the Swedish Natural Science Research Council. Note added in proof. In neutrophils the effect of the chemotactic peptide, F-MetLeuPhe, on the PIcycle is transduced by a G-protein whose activity is inhibited by pertussis toxin through ADPribosylation (Yassin et al. (1985) J. Ce// Biol. 101, 182). In addition it has recently been found that pertussis toxin in this system inhibits the agonist-induced actin polymerization (Bengtsson et al. (1986) Eur. J. Cell Biol. 42, 338). This also tits well with the idea that PtdIns(4,5)P, formation and actin polymerization precede and lead up to the hydrolysis of PtdIns(4,5)P,.

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Received May 8, 1987

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