Journal of Cell Science 113, 3329-3340 (2000) 3329 Printed in Great Britain © The Company of Biologists Limited 2000 JCS0645

Actinin-4 is preferentially involved in circular ruffling and macropinocytosis in mouse macrophages: analysis by fluorescence ratio imaging

Nobukazu Araki1,*, Tanenori Hatae1, Tesshi Yamada2 and Setsuo Hirohashi2 1Department of Anatomy, Kagawa Medical University, Miki, Kagawa 761-0793, Japan 2Pathology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan *Author for correspondence (e-mail: [email protected])

Accepted 3 July 2000

SUMMARY

We have applied fluorescence ratio imaging to the analysis gradually dissociated with their maturation. Consistent of an -binding protein concentration relative to F- with ratio imaging data, macrophages scrape-loaded with actin in macrophages, in order to explore the role of a anti-actinin-4 showed a more reduced rate of novel α-actinin isoform, actinin-4, relative to that of macropinocytosis than those loaded with anti-actinin-1. the classical isoform, actinin-1. Conventional Altogether, these results indicate that actinin-4 and immunofluorescence images showed that both isoforms actinin-1 contribute differently to F-actin dynamics, that were enriched in F-actin-rich regions such as cell surface actinin-4 is more preferentially involved in early stages of ruffles. However, ratio images further demonstrated that macropinocytosis than actinin-1. A similar redistribution actinin-4 concentrations relative to F-actin were higher in of actinin-4 was also observed during phagocytosis, peripheral inward curved ruffles and dorsal circular suggesting that actinin-4 may play the same role in the two ruffles, presumed precursor forms of macropinosomes, mechanistically analogous types of endocytosis, i.e. than in straight linear ruffles, while actinin-1 macropinocytosis and phagocytosis. concentrations were uniform among the different types of ruffles. Macropinosome pulse-labeling and chase experiments indicated that actinin-4 was also closely Key words: α-Actinin, Actin, Macropinocytosis, Ruffling, associated with newly formed macropinosomes and Phagocytosis, Image analysis

INTRODUCTION 1996; Bretscher and Lynch, 1985; Fujiwara et al., 1978; Furukawa and Fechheimer, 1994; Geiger and Singer, 1979; Ruffling, macropinocytosis and phagocytosis are forms of cell Meigs and Wang, 1986). However, it is not known if different motility mediated by filamentous actin (F-actin) isoforms of α-actinin have different subcellular localizations. polymerization and its reorganization (Swanson et al., 1999; Actinin-2 and -3, encoded by ACTN2 and ACTN3 Swanson and Watts, 1995). Such motility requires a variety of respectively, are muscle α-actinin which cross-link F-actin actin-binding proteins to sever, bundle and/or cross-link F- together in the region of the Z-discs of striated muscle cells actin (Matsudaira, 1991; Otto, 1994; Schmidt and Hall, 1998; (Beggs et al., 1992). Stossel, 1993). α-Actinin is one actin-binding protein which Actinin-4 is a recently characterized isoform of nonmuscle can cross-link F-actin into F-actin bundles or networks and also α-actinin (Honda et al., 1998). Using the monoclonal antibody connect F-actin to the plasma membrane (Djinovic´-Carugo et (mAb) HCC-Lu-632, which specifically recognized human al., 1999; Matsudaira, 1991; Otto, 1994). So far, four isoforms actinin-4, it was shown that the localization of actinin-4 was of human α-actinin have been identified and characterized at different from that of actinin-1 in cancer cell lines. Unlike the gene level. Actinin-1, encoded by ACTN1, is a classical actinin-1, actinin-4 did not localize to focal adhesion plaques isoform of nonmuscle α-actinin which primarily localizes to or adherens junctions; its cytoplasmic localization was closely focal adhesion plaques. It binds F-actin bundles (stress fibers) associated with enhanced motility of cancer cells and might to the plasma membrane via other proteins such as , predict the metastatic potential of human cancer. Interestingly, and (Chen and Singer, 1982; Honda et al., this isoform was found to be translocated into the nucleus in 1998; Langanger et al., 1984; Lazaride and Burridge, 1975; several cancer cell lines upon inhibition of phosphoinositide 3- Meigs and Wang, 1986). It was also reported that nonmuscle kinase (PI3-kinase) by wortmannin (Honda et al., 1998). These α-actinin localized in various F-actin-based structures findings suggested that actinin-1 and actinin-4 might play including stress fibers, ruffling membranes, phagocytic cups, distinct roles in cellular motility and stability (adhesion) and cleavage furrows, receptor capping sites and contractile might be regulated by different cascades. vacuoles of Dictyostelium discoideum (Allen and Aderem, Expression of actinin-4 mRNA was detected in almost all 3330 N. Araki and others human tissues, but its precise intracellular localization in cells 4 to that of rhodamine phalloidin-labeled F-actin could indicate other than cancer cells has not yet been examined. It is of how much functional contribution actinin-4 made to F-actin- particular interest to determine actinin-4 localization in highly bundling, without the effect of F-actin content. We demonstrate motile cells such as macrophages, since actinin-4 seems to be here that actinin-4 preferentially participates in circular ruffling implicated in cell motility rather than . and has a role in macropinocytosis and phagocytosis in mouse Macrophages show active membrane ruffling and macrophages. macropinocytosis as well as phagocytosis. The ruffling can be divided into at least two types: cell edge ruffling and dorsal surface ruffling (Ridley, 1994; Swanson and Watts, 1995). MATERIALS AND METHODS Most ruffles are continuously initiated at the cell edge. The cell edge lamellipodial ruffling seems to be associated with cell Reagents and cells spreading, locomotion and chemotaxis. Some edge ruffles Monoclonal antibody (mAb) against actinin-4 (Clone HCC-Lu-632 undergo a smooth centipetal movement, known as retrograde mouse IgM) was produced as previously described (Honda et al., flow (Heath and Holifield, 1991; Mitchison and Cramer, 1996), 1998). Rhodamine phalloidin, Alexa 488-anti-rat IgG and fixable × 3 then shift to dorsal surface ruffles. The dorsal surface ruffling fluorescein-dextran Mr 10 10 (FDx10) were purchased from Molecular Probes, Inc. (Eugene, OR). Bovine serum albumin (BSA, is closely correlated with macropinocytic activity. In particular, α circular ruffles formed on the dorsal surface are known to be Fraction V), anti- -actinin (actinin-1) mAb (Clone BM-75.2 mouse IgM), Texas Red anti-mouse IgM, FITC-anti-mouse IgM, horseradish precursor forms of macropinosomes (Swanson and Watts, peroxidase (HRP)-anti-mouse IgM, Agarose anti-mouse IgM were 1995). Thus, each type of ruffling seems to be associated with from Sigma Chemical Co. (St Louis, MO). Rabbit anti-cathepsin D a different cell function and controlled by a different signaling serum was a gift from Dr Sadaki Yokota (Yamanashi Medical School, pathway (Allen et al., 1997; Cox et al., 1997; Hall, 1994; Japan). Anti-LAMP-1 (110 kDa lysosomal-associated membrane Ridley, 1994; Ridley et al., 1992), although both types of glycoprotein) mAb (Clone 1D4B) developed by Dr J. T. August was ruffles may be closely related to each other. Therefore, it would obtained from the Developmental Studies Hybridoma Bank be important to clarify any differences in the molecular maintained by the Department of Biological Sciences, University of composition and machinery among the morphological Iowa. Dulbecco’s-modified essential medium (DME) and fetal bovine phenotypes of ruffles. serum (FBS) were from Gibco BRL (Grand Island, NY). All other reagents were purchased from Wako Pure Chemical (Osaka, Japan), Fc-mediated phagocytosis, in which pseudopodial unless otherwise indicated. extensions of the cell surface cover opsonized particles and Bone marrow-derived macrophages were obtained from femurs of enclose them to form phagosomes, occurs by regulated actin C3H HeJ mice as previously described (Swanson, 1989). After 6 or polymerization and reorganization. We recently distinguished 7 days of culture, macrophages were harvested from dishes and plated two component activities of phagocytosis: pseudopod onto 12 mm circular coverslips in 24-well culture dishes, or 10 cm extension to form the phagocytic cup and a purse-string-like dishes, then incubated overnight in medium lacking M-CSF (DME- contraction that closes the phagosome, and also showed that 10F: DME with 10% heat-inactivated FBS). several different classes of are present in phagosomes Other cultures, a human vulval epidermoid cancer cell line A431, (Araki et al., 1996; Swanson et al., 1999). Accordingly, the a human monocytic leukemia cell line HL-60 and a human histiocytic characteristic membrane transformations such as extension, lymphoma cell line U937, were grown in 10-cm dishes in DME-10F. curvature and contraction must have distinct mechanisms Activation of macropinocytosis and endocytic labeling regulated by different kinds or isoforms of mechanochemical Thirty minutes before experiments, DME-10F was replaced with proteins, including myosins and actin-binding proteins. Ringer’s buffer (RB) consisting of 155 mM NaCl, 5 mM KCl, 1 mM α Therefore, we have focused on the possibility that each - MgCl2, 2 mM Na2HPO4, 10 mM glucose, 10 mM Hepes, pH 7.2 and actinin isoform may contribute differently to cellular functions 0.5 mg/ml BSA. including ruffling, macropinocytosis and phagocytosis. Macropinocytosis was stimulated by the addition of human Since most actin-binding proteins are concentrated in the F- recombinant M-CSF (2,000 unit/ml) to the macrophage culture (Araki actin-enriched regions, the amount of each is greatly affected et al., 1996; Racoosin and Swanson, 1992; Swanson, 1989). To label by the amount of F-actin. This fact is disadvantageous when macropinosomes, some macrophages were incubated in RB comparing the functional relationship between actin and actin- containing 1 mg/ml fixable FDx10 and M-CSF (2,000 unit/ml) for 2- 5 minutes at 37°C, followed by a brief rinse in PBS and fixation as binding proteins in different structures containing different described previously (Araki et al., 1996; Araki and Swanson, 1998). amounts of F-actin. In fact, even though conventional For FDx10 pulse/chase experiments, macrophages were pulse-labeled fluorescence microscopy could demonstrate the localization of with 1.0 mg/ml fixable FDx10 for 2 minutes as described above, then F-actin-associated proteins, it is hard to know the protein quickly rinsed in RB and chased in FDx10-free RB + M-CSF for 0- concentrations relative to F-actin. Moreover, conventional 10 minutes at 37°C. Some macrophage cultures were fed with 2 µm fluorescence microscopy and biochemical analyses may diameter latex (polystyrene) beads (Polyscience, Inc., Warrington, overlook the possibility that some actin-binding proteins are PA) and incubated for 5-30 minutes at 37°C to allow phagocytosis. associated with F-actin at a high rate in some regions where a Western blot and immunoprecipitation small amount of F-actin is present. Therefore, we have newly applied the ratio imaging technique to the analysis of the Cells were washed in cold PBS and scraped with a rubber policeman in lysis buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM EDTA, functional relationship between an actin-binding protein and F- 1% Triton X-100, 1 mg/ml NaN3, 0.5 mM Na3VO4, 1 mM PMSF, 1 actin, since ratio imaging can avoid the effects of other µg/ml leupeptin, 1 µg/ml pepstatin A) and homogenized on ice in a parameters such as cytoplasmic volume and F-actin amount Dounce homogenizer with a tight-fitting pestle. After 30 minutes (Araki and Hatae, 2000; Dunn and Maxfield, 1998). The ratio extraction on ice, the sample was centrifuged at 12,000 g for 15 minutes of the fluorescence intensity of FITC-immunolabeled actinin- and the supernatant was recovered. For western blot analysis, the cell Actinin-4/F-actin ratio in macrophages 3331 lysates (50 µg protein) were separated by 10% SDS-PAGE (Laemmli, Antibody scrape-loading and spectrofluorometric analysis 1970) and transferred to nitrocellulose membrane (Advantech Toyo, of macropinocytosis Japan). Actinin-4 and -1 proteins were revealed using mAb HCC-Lu- Macrophages were scrape-loaded with either an antibody against the 632 and mAb BM-75.2, respectively. After incubation with primary central rod of actinin-4 or -1, control mouse myeloma IgM, or PBS antibodies for 3 hours at room temperature, the blots were detected only, as previously described (McNeil et al., 1984; Swanson et al., using HRP-conjugated anti-mouse IgM and TMB substrate (Kirkegaard 1999). Briefly, macrophages cultured on a 35-mm dish were scrape- & Perry Laboratories, Inc., Gaithersburg, MD). loaded by first washing with PBS, then replacing it with 50 µl of IgM For immunoprecipitation, the macrophage cell lysate was incubated (0.5 mg/ml in PBS) and scraping the cells off the dish with a rubber with anti-actinin-1 (BM-75.2) mAb overnight at 4°C, then agarose- policeman. Cells were washed in PBS then replated on a 24-well dish conjugated anti-mouse IgM was added. After 3 hours incubation at at a cell density of 2.5×105 cells/well. After 60 minutes incubation in 4°C, the agarose was washed 4 times with Tris-buffered saline (TBS) DME-10F, macrophages were incubated with FDx10 (1.0 mg/ml in containing 1% Triton X-100 and 0.2% Tween-20, then lysed in DME-10F), a fluid-phase pinocytic probe, in the presence of M-CSF Laemmli buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 5% glycerol, (2,000 U). Although 2-5 minutes labeling of FDx10 was used to label 715 mM 2-mercaptoethanol, 0.0025% bromophenol blue). The only macropinosomes in other morphological experiments, we chose sample was separated by SDS-PAGE and analyzed by western blotting a 30 minute labeling time for spectrofluorometry, since the as described above. signal/noise ratio of FDx10 at 5 minutes was not high enough to assess the inhibitory effect. Then, macrophages were thoroughly washed and Immunofluorescence microscopy and ratio image analysis lysed in a lysis buffer. The amount of FDx10 in the cell lysate was Macrophages cultured on glass coverslips were usually fixed with 4% measured by a fluorescence spectrophotometer (Hitachi 650-40) as paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, 5% sucrose for previously described (Araki et al., 1996; Araki and Swanson, 1998). 30 minutes at room temperature. For the combination of endocytic Macropinocytic activity was revealed as the amount of intracellularly labeling and immunofluorescence, 0.1% glutaraldehyde was added to accumulated FDx10 normalized by the total cell protein. the usual fixative, so that FDx10-labeling would be efficiently retained in macropinosomes. After fixation, cells were rinsed four times 5 Scanning EM observation minutes in PBS and two times 5 minutes in 0.25% NH4Cl in PBS, to Macrophages were cultivated on plastic sheets and stimulated with M- quench free-aldehyde groups. The fixed cells were treated with a CSF for 5 minutes before fixation. Cells were fixed with 2% blocking solution consisting of 1% normal goat serum and 0.5% BSA glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, containing 6% in permeabilizing buffer (0.25% Triton X-100 or 1% saponin in PBS). sucrose for 1 hour at room temperature. Fixed cells were rinsed, The first and second antibodies were diluted in the blocking solution postfixed with 1% osmium tetroxide and conventionally processed for and incubated at room temperature for 90 minutes and 60 minutes, scanning electron microscopy as previously described (Araki et al., respectively. As primary antibodies, we used mouse anti-actinin-4 1996). Specimens were observed by a Hitachi S-900 SEM. mAb HCC-Lu-632 at 1:20 dilution, mouse anti-actinin-1 mAb BM- 75.2 (1:100 dilution), rat anti-LAMP-1 mAb 1D4B (1:20 dilution) and rabbit anti-cathepsin D polyclonal antibody (1:500 dilution). As negative controls, normal mouse IgM or normal rabbit serum at the RESULTS same concentration was substituted for the specific antibody. As secondary antibodies we used FITC or Texas red-conjugated anti- Specificity of antibody mouse IgM, Alexa 488-conjugated anti-rat IgG and FITC-conjugated Specific reactivity of mAb HCC-Lu-632 with human actinin- anti-rabbit IgG (all 1:250 dilution). The indirect immunofluorescence 4 was previously determined using human epithelial cell lines protocol was previously described in detail (Araki and Swanson, including A431, GST-fusion proteins expressed in E. coli and 1998). To visualize F-actin, rhodamine-phalloidin (5 units/ml) was added to the secondary antibody dilution. We carefully determined the in vitro translation products of cDNA of actinin-4 and actinin- optimal dilution of antibodies and phalloidin by titration and their 1 (Honda et al., 1998). To confirm its reactivity with mouse saturation kinetics. The specimen coverslips were mounted on glass actinin-4, western blot and immunoprecipitation analyses were slides using Antifade (Molecular Probes, Inc., Eugene, OR) and performed using mouse macrophage lysates as well as human observed by a confocal laser microscope (Olympus GB200) or an cell lines. Western blot analysis revealed that mAb HCC-Lu- epifluorescence microscope (Nikon TE300). 632 reacted with a single protein of approx. 100 kDa in the For digital image analysis, 8 bit confocal images of both fluorescein whole cell lysate of mouse macrophages as well as human and rhodamine were obtained by simultaneous dual wavelength epithelial cell line A431, monocytic cell line HL-60 and excitation (488 nm and 568 nm, respectively). Laser intensity, aperture histiocytic cell line U937 (Fig. 1A). Although monoclonal size, gain and black level settings were carefully determined so that antibody against chick actinin-1 (mAb BM-75.2) also detected each signal was enough to be visualized, but any of the pixels in the interest area were not saturated. We confirmed that FITC and a single band at the same molecular mass (Fig. 1B), mAb BM- rhodamine images were exactly aligned in the same focal plane; FITC 75.2 immunoprecipitable products were not reacted with mAb and rhodamine signals were detected only in the 488 nm channel and HCC-Lu-632 (Fig. 1C). Normal mouse IgM, used as a control, the 568 nm channel, respectively; and there was little signal in the did not detect 100 kDa protein in any cell lysates nor mAb BM- other channel (Araki and Hatae, 2000). Also, essentially the same 75.2 immunoprecipitable products (data not shown). These results were obtained when FITC- and rhodamine-labeling were indicate that mAb HCC-Lu-632 cross-reacts with mouse replaced with Texas red- and NBD-labeling, respectively. These actinin-4, but not with mouse actinin-1. images saved as TIFF files were processed by MetaMorph Imaging System (Universal Imaging Co., West Chester, PA) to get the ratio Definition of circular ruffles and macropinosomes images of actinin-4/F-actin and sometimes the reversal (F- actin/actinin-4). The ratio images were shown as spectrum Scanning EM displayed the many ruffles that formed at the pseudocolor corresponding to the ratio value. Quantitative and peripheral edge and the dorsal surface of M-CSF-stimulated statistical analysis of ratio values was carried out at more than 150 macrophages. The morphology of the dorsal ruffles was varied different regions of ruffles in 30 cells using the line scan program of from straight to curved or circular (Fig. 2A). As previously MetaMorph software. shown by phase-contrast time-lapse video microscopy of living 3332 N. Araki and others

Table 1. Quantitative analysis of actinin-4/F-actin ratio in straight linear, curved and circular ruffles Actinin-4/F-actin Ruffle types (locations) ratio values ± s.d. Straight linear or lamellipod-like ruffles (peripheral) 0.32±0.22 (n=55) Curved ruffles (peripheral and dorsal) 0.89±0.72 (n=62) Circular ruffles (dorsal) 0.91±0.44 (n=35)

More than 150 different ruffle regions selected at random in fifty M-CSF- stimulated macrophages and classified into three ruffle types: (1) straight linear ruffles at cell periphery, (2) curved ruffles seen at both cell periphery and dorsal surface and (3) circular ruffles at dorsal surface. Ratio value of each ruffle region was calculated as dividing actinin-4 (green channel) value by F-actin (red channel) value. Ratio values represent the average mean ± standard deviation (s.d.) of ratio values in number (n) of ruffle regions. Similar results were obtained from another independent experiment.

essentially the same features of α-actinin localization. We described only M-CSF-stimulated macrophages here, since these cells displayed more ruffles and the features of α-actinin localization were more pronounced. Conventional confocal images of actinin-4 immunofluorescence and rhodamine-phalloidin-stained F-actin showed that actinin-4 was abundant in F-actin-rich cell cortical regions such as ruffles of the cell margin and the dorsal surface of macrophages (Fig. 3A-C). However, the color merged image and its line scan quantitative analysis indicated that the intensities of both fluorescences were not always correlative Fig. 1. Antibody specificity to actinin-1 and -4. Western blot analysis (Fig. 3C,C′). A confocal image of actinin-4 of anti-actinin-4 mAb HCC-Lu-632 (A) and anti-actinin-1 mAb BM- immunofluorescence shows the total amount of actinin-4 in 75.2 (B) in various human cell lines: U937 (lane 1), HL-60 (lane 2), cytoplasm optically sliced at 0.5 µm-thickness, but does not A321 (lane 3) and mouse macrophages (lane 4). (C) The cell lysate reveal the relative amounts of actinin-4 to F-actin. The relative of mouse macrophages was immunoprecipitated with mAb BM-75.2 and agarose-anti-mouse IgM. The immunoprecipitable product was amounts of actinin-4 to F-actin were determined by separated by SDS-PAGE and analyzed by western blotting. fluorescence ratio imaging analysis. The ratio image Approximately 100-kDa protein of actinin-1 was detected by mAb topographically revealed that the actinin-4/F-actin ratio was BM-75.2 but not HCC-Lu-632, suggesting that HCC-Lu-632 does especially high at circular ruffles (Fig. 3D). Conversely, the not cross-react with mouse actinin-1. Molecular masses (in kDa) are actinin-4/F-actin ratio was lower at the peripheral edge ruffles. shown on the left. This can be confirmed by the reversal ratio (F-actin/actinin-4) image in which peripheral edge lamellipod-like ruffles showed a high ratio value (Fig. 3E). In peripheral edge ruffles, some macrophages (Araki et al., 1996; Swanson and Watts, 1995), regions curving inward showed a higher actinin-4/F-actin ratio circular ruffles are most frequently formed by inward curving than did straighter regions (Fig. 4A,B). These ratio images of lateral edge ruffles; these then close on top to become phase- indicate that actinin-4 contributes to F-actin-bundling more in bright macropinosomes. Newly formed macropinosomes move circular or curved ruffles than in straight ruffles such as flat toward the perinuclear region while gradually shrinking in size. lamellipodia. Circular ruffles were defined by a phase-dark rim, which Some vacuolar structures, presumably macropinosomes, distinguished them from the phase-bright macropinosomes were also labeled with anti-actinin-4 to variable degrees; such (Fig. 2B). Two to five minutes pulse-labeling with the fluid- structures did not contain abundant F-actin (Fig. 4A-C). Ratio phase probe FDx10 labeled macropinosomes but not circular imaging showed that the actinin-4/F-actin ratio was ruffles (Fig. 2C). Rhodamine-phalloidin labeling showed that considerably higher in the vicinity of macropinosome-like circular ruffles were rich in F-actin, but FDx10-labeled structures, owing to the diminished F-actin levels (Fig. 4B,C). macropinosomes had very little associated F-actin (Fig. 2D). Other cytoplasmic regions were weakly positive for actinin-4. This indicates that F-actin dissociated from macropinosomes These findings were further confirmed by a statistical shortly after circular ruffles close into intracellular organelles. analysis of ratio values in different regions of 30 cells (Table In this paper, we defined circular ruffles as phase-dark-lined, 1). By a statistical t-test analysis, it was shown that ratio values FDx10-unlabelable and F-actin-rich profiles; we defined in curved ruffles and circular ruffles were significantly higher macropinosomes as phase-bright, FDx10-labelable and F- than that in straight ruffles (P<0.01). The difference in ratio actin-poor profiles. values between curved ruffles and circular ruffles was not significant (P>0.1). Immunolocalization of actinin-4 and its ratio image Immunofluorescence using mAb BM-75.2, which analysis for F-actin recognizes actinin-1, a classical isoform of α-actinin, indicated Control and M-CSF-stimulated macrophages showed that actinin-1 was in all F-actin-containing structures. These Actinin-4/F-actin ratio in macrophages 3333 included peripheral edge ruffles, dorsal surface ruffles and LAMP-1 and cathepsin D. Fluorescence microscopy and circular ruffles (Fig. 4D). Since the fluorescence intensities of quantitative analysis revealed that more than half of the F-actin and actinin-1 correlated (Fig. 4F), the ratio image was macropinosomes labeled for actinin-4 were negative for less contrasted (Fig. 4E). Thus, actinin-1 appeared to localize LAMP-1 and were generally located at the cell periphery. The more uniformly, relative to F-actin. majority of LAMP-1-positive macropinosomes located at the Confocal images of optical sections containing the basal perinuclear region were relatively small (< c.a. 2 µm) and not plasma membrane also showed different localizations of two associated with actinin-4 (Fig. 7B-G). However, α-actinin isoforms. Unlike fibroblasts, smooth muscle cells and macropinosomes showing both actinin-4 and LAMP-1 labeling epithelial cell cultures, macrophages do not have stress fibers, were also occasionally seen at the perinuclear region. Counting but instead focal adhesion plaque-like structures corresponding of 200 actinin-4 associated macropinosomes indicated that to cell-substratum contact sites (Fig. 5). It was reported that 34.9% of total actinin-4-associated macropinosomes was these F-actin-rich adhesion structures, termed or F- positive for LAMP-1. Curiously, macropinosomes having both actin dots, included several actin-binding proteins such as vinculin, fimbrin, talin and α- actinin, similar to focal adhesion plaques (Correia et al., 1999; Marchisio et al., 1987). Our confocal observation clearly revealed that actinin-4 did not localized in podosomes (Fig. 5A,B), while actinin-1 did (Fig. 5C,D). Association of actinin-4 with macropinosomes To confirm the association of actinin-4 with macropinosomes, macrophages were pulse- labeled with FDx10 for 5 minutes, fixed and immunolabeled for actinin-4 using Texas red- labeled secondary antibody. Most FDx10- labeled macropinosomes that were relatively large and located at cell periphery were associated with actinin-4, although perinuclear pinosomes were scarcely labeled for actinin-4 (Fig. 6A-C). Such actinin-4-negative pinosomes might be macropinosomes at a later stage. The quantitative data of FDx10 pulse- labeling and chase experiments supported this finding. As shown in the graph in Fig. 7A, more than 70% of 0-2 minutes old (2-minutes pulse/no chase) newly formed macropinosomes showed the actinin-4 association. The actinin-4 positive fraction decreased with increasing macropinosome age (chase time). Less than 20% of 10-12 minutes old (2-minutes pulse/10-minutes chase) macropinosomes were immunolabeled for actinin-4. No association of actinin-1 with any FDx10-labeled macropinosomes was observed by confocal microscopy (Fig. 6D-F). Racoosin and Swanson (1993) reported that early macropinosomes matured to late macropinosomes and finally merged with tubular lysosomes, all within approximately 15 minutes. During the sequential processes of Fig. 2. Morphological definition of circular ruffle and macropinosome in M-CSF- macropinosome maturation and fusion with stimulated macrophages. (A) Scanning EM of an M-CSF-stimulated macrophage tubular lysosomes, delivery of LAMP proteins showing active ruffling at both the peripheral edge and the dorsal surface. Circular to macropinosomes precedes delivery of ruffles are seen on the dorsal surface (arrows). Circular ruffles are most frequently cathepsins (Racoosin and Swanson, 1993). In formed by inward curving of peripheral edge ruffles (arrowhead). (B) Phase-contrast microscopy shows circular ruffles appear as phase-dark circles (a) and macropinosomes order to clarify the stages of macropinosome are phase-bright (b). Corresponding fluorescence image (C) of fluid-phase endocytic maturation at which actinin-4 associates, we labeling by 2 minutes pulse of 1 mg/ml FDx10 shows that macropinosomes (b) are further characterized actinin-4-associated labeled with FDx10, but circular ruffles (a) are not. Rhodamine-phalloidin image (D) macropinosomes by dual immunolabeling with revealed that circular ruffles (a) unlabeled with FDx10 are enriched with F-actin, but the endosomal/lysosomal markers such as FDx10-labeled macropinosomes (b) have scant F-actin. Bars, 10 µm. 3334 N. Araki and others

Fig. 3. Confocal microscopy and image analysis of actinin-4 and F-actin in M-CSF-stimulated macrophages. Conventional confocal images show that actinin-4 (A) and F-actin (B) localize similarly. However, different fluorescence intensities of actinin-4 (green) and F-actin (red) generate color variation from red to yellow or green in a merged image (C). (C′) Line scan analysis (at the position of hatched line in C) indicating fluorescence intensities of actinin-4 (green) and F-actin (red). At a circular ruffle both fluorescence intensities are high, but only red fluorescence is prominent at the cell periphery. The ratio images demonstrate that actinin-4/F-actin is higher at circular ruffles (arrows, D) and the reversal ratio (F-actin/actinin-4) is higher at peripheral ruffles (arrowheads, E) indicating that actinin-4 is especially less, relative to F-actin. Confocal plane was taken to contain an optical slice of the dorsal surface. n, nucleus. Bar, 10 µm.

LAMP-1 and actinin-4 were considerably large (>3 µm the these monoclonal antibodies may perturb F-actin bundling in most frequent size) in spite of the perinuclear location (Fig. living cells. To address the functional contribution of α-actinin 7E-G). Only a small percentage (3.75% of total actinin-4- isoforms to macropinocytosis more directly, we compared the associated macropinosomes) was positive for cathepsin D. effect of scrape-loaded antibodies (IgM) against actinin-4 and Association of actinin-4 with cathepsin D-positive tubular -1. After scrape-loading with IgM (0.5 mg/ml), cells were lysosomes was hardly seen. Taken together, these observations incubated with 1.0 mg/ml FDx10 for 30 minutes to allow fluid- indicate that the dissociation of actinin-4 from late phase pinocytic uptake. Anti-actinin-4 IgM loading macropinosomes begins before macropinosomes gain LAMP- significantly reduced intracellular accumulation of FDx10 by 1 and is nearly complete before macropinosomes become fluid-phase pinocytosis for 30 minutes, compared with anti- cathepsin D-positive by fusing with tubular lysosomes. actinin-1 IgM or control IgM (Fig. 8). Higher concentrations (1.0 mg/ml) of anti-actinin-1 and control IgM also did not show Effect of scrape-loaded anti-α-actinin antibodies on significant inhibition on macropinocytosis (data not shown). macropinocytosis Our previous study showed that both anti-actinin-4 (HCC-Lu- Involvement of actinin-4 in phagocytosis 632) and anti-actinin-1 (BM-75.2) monoclonal antibodies Macropinocytosis and phagocytosis are mechanistically similar recognized the central rod domain of α-actinin. Since the (Araki et al., 1996; Swanson and Watts, 1995), although they central rod accounts for antiparallel dimerization of α-actinin differ in quality of internalized substances; fluid and particles, to form a rod-shaped molecule with an actin-binding domain respectively. In phagocytosis, pseudopod extension along a at either end (Djinovic´-Carugo et al., 1999; Matsudaira, 1991), particle surface results in phagocytic cup formation. The Actinin-4/F-actin ratio in macrophages 3335

Fig. 4. Comparison of the F-actin-associated distribution of actinin-4 (A-C) and actinin-1(D-F) by confocal microscopy and ratio imaging analysis in the peripheral and dorsal surface of M-CSF-stimulated macrophages. (A,B) Actinin-4 concentrations relative to the F-actin amount considerably varied by the shape of ruffles. In peripheral ruffles, inward curving regions (arrowheads) show higher actinin-4/F-actin ratio than straighter regions. The actinin-4/F-actin ratio is also high around macropinosome-like structure (arrows, A and B), where a small amount of F- actin is associated (Line scan, C). (D) A merged image showing homogeneous colocalization of actinin-1 (green) and F-actin (red). (E) The ratio image of actinin-1/F-actin is less contrasted, because actinin-1 (green) and F-actin (red) fluorescent intensities are considerably correlative as shown by line scan analysis (F). n, nucleus. Bar, 10 µm. phagocytic cup then closes into the phagosome. Finally, the Consistent with a previous report (Allen and Aderem, 1996), phagosome becomes a phagolysosome by fusing with lysosomes. actinin-1 was also enriched around phagocytic cups. However, We observed F-actin and actinin-4 redistributions in ratio imaging showed that actinin-1/F-actin was not so high as macrophages during phagocytosis of latex beads. Latex beads actinin-4/F-actin. Moreover, actinin-1 was hardly seen around were fed to macrophages to allow phagocytosis for 10-30 intracellular phagosomes (not shown). minutes. Since latex beads could be confirmed by the bright- field diffraction mode of confocal microscopy, phagocytic cups or phagosomes were distinguishable from circular ruffles or DISCUSSION macropinosomes. Also, phagocytic cups could be distinguished from phagosomes, since F-actin was enriched in It is well known that α-actinin cross-links F-actin to form F- phagocytic cups, whereas F-actin was scarcely seen around actin bundles (stress fibers) and mediates membrane-F-actin phagosomes and phagolysosomes. Immunofluorescence by interactions via other proteins such as talin, vinculin and anti-actinin-4 showed that actinin-4 localized in phagocytic integrin. Nonmuscle α-actinin, presumed to be actinin-1, is cups and phagosomes 10 minutes after latex beads feeding primarily localized in focal adhesion structures of cultured (Fig. 9A-C). Ratio imaging indicated that actinin-4/F-actin was fibroblasts, epithelial cells (Honda et al., 1998; Mangeat and higher at phagocytic cups and some phagosomes than Burridge, 1984; Meigs and Wang, 1986) and macrophages straighter ruffles (Fig. 9D). However, after 30 minutes, most (Marchisio et al., 1987). It was also reported that α-actinin latex bead-containing structures, presumed to be at a late stage participated in -mediated cell-cell adhesion via α- of phagosomes or phagolysosomes, were unlabeled for actinin- catenin at adherens junctions in epithelial cells (Knudsen et 4 (data not shown). al., 1995). These adhesion complexes stabilize the cell on 3336 N. Araki and others

Fig. 5. Confocal microscopy showing the different localizations of actinin-4 and actinin-1 in basal adherent surface of macrophages. Actinin-4 is not associated with podosomes where F-actin is predominantly concentrated in dots (arrowheads, A and B), while actinin-1 localizes in such F-actin-rich podosomes (arrowheads, C and D). Bars, 10 µm.

Fig. 6. Confocal images of M-CSF- stimulated macrophages incubated with FDx10 for 5 minutes, fixed and immunolabeled for actinin-4 (A-C) or actinin-1 (D-F) using a Texas red- conjugated secondary antibody. Some macropinosomes labeled with FDx10 were associated with actinin-4 (arrows, A-C), but others which were smaller and located near the nucleus were not. No association of actinin-1 with FDx10-labeled macropinosomes was observed (arrows, D-F). (A,D) Fluorescein images showing macropinosomes labeled with FDx10. (B,E) Corresponding Texas red images showing immunolocalizations of actinin-4 (B) and actinin-1 (E). (C,F) Merged images of fluorescein and Texas red. n, nucleus. Bars, 10 µm. adherent substrates or among epithelial tissues. Glück et al. some immunofluorescence studies, α-actinin was also (1993, 1994) reported that transfection with actinin-1 cDNA observed in dynamically moving structures including leading suppressed cell motility and tumorigenicity. These studies edges, phagocytic cups, phagosomes and contractile vacuoles indicate that α-actinin is a structural component, maintaining of Dictyostelium discoideum (Allen and Aderem, 1996; cell location, rather than contributing to force-generation. In Furukawa and Fechheimer, 1994). However, it is possible that Actinin-4/F-actin ratio in macrophages 3337

Fig. 7. Characterization of actinin-4-associated macropinosomes. (A) Dissociation of actinin-4 from macropinosomes with increasing age. M-CSF stimulated macrophages were pulse-labeled with fixable FDx10 (1.0 mg/ml) for 2 minutes and chased as described above. Then, cells were fixed, immunostained with anti- actinin-4 and observed by phase-contrast and fluorescence microscopy. More than fifty FDx10 labeled, phase-bright macropinosomes were examined and the actinin-4-positive fraction of FDx10-labeled macropinosomes was scored for each chase time. The average of three time-course experiments±s.d. is displayed in the graph. (B-G) Dual immunofluorescence of actinin-4 (B,E) and LAMP-1 (C,F) and corresponding phase-contrast images (D,G). Actinin-4- associated circular ruffles (a) and large macropinosomes (b) located cell periphery are negative for LAMP-1. Most LAMP-1-positive macropinosomes (c) are not associated with actinin-4, although actinin-4 occasionally remained to be associated with LAMP-1-positive large compartments (d) in perinuclear regions. Bar, 10 µm. the antibodies against α-actinin that showed multiple localizations recognized multiple isoforms of the protein with 1200 the same molecular mass. In our observation on macrophages, actinin-1 was observed in all F-actin-containing structures, 1000 including dorsal and peripheral edge ruffles, phagocytic cups and basal podosomes. However, the ratio images indicated 800 that actinin-1 was distributed homogeneously in such structures, relative to F-actin. On the contrary, actinin-4 was 600 shown to be most concentrated in special regions of ruffles such as curved and circular ruffles on the dorsal surface of macrophages. Consistent with the previous findings in cancer protein FDx ng/mg 400 cells (Honda et al., 1998), actinin-4 was not concentrated in focal adhesion structures, called podosomes in macrophages, 200 as was actinin-1. Judging from the localization of actinin-4, it is unlikely that 0 actinin-4 contributes to stabilization of the cell on the adherent PBS IgM ACTN1 ACTN4 substrate. Conversely, actinin-4 preferentially localizes to Fig. 8. The effect of different scrape-loading treatments on the actively moving structures such as dorsal ruffles. This is macropinocytic activity in macrophages. Macrophages on 35 mm- consistent with the proposal that actinin-4 predicts the dishes were scrape-loaded with either a monoclonal antibody metastatic potential of cancer, since active membrane ruffling (IgM=0.5 mg/ml) against the central rod of actinin-4 or actinin-1, of cancer cells is considered to be a parameter of tumor cell control mouse myeloma IgM or PBS only. The macrophages were invasion and metastasis (Jiang, 1995). In an epithelial cancer incubated with FDx10 (1.0 mg/ml) for 30 minutes to allow cell line, actinin-4 was concentrated at the leading edge of a macropinocytosis. Accumulated FDx10 in cells was measured by a motile epithelial sheet during wound healing (Honda et al., spectrofluorometer as described in Materials and Methods. By the t- 1998). However, our ratio imaging demonstrated that actinin- test, scrape-loading actinin-4 antibody significantly reduced FDx10 accumulation taken up by macropinocytosis, compared with scrape- 4 was not concentrated in peripheral edge ruffles of loading actinin-1 antibody or control (P<0.05). Actinin-1 antibody macrophages. This discrepancy may be due to other or control IgM-loaded macrophages do not differ from those of PBS differences between cancer cells and phagocytic cells. In many only-loaded cells (P>0.1). cell types, including cancer cells, ruffling is observed in response to certain extracellular factors and leading edge lamellipod-like ruffles are required for directed . lamellipodial extension during cell migration and the In macrophages showing amoeboid patterns of movement, peripheral edge ruffling constitutively shown in macrophages however, edge ruffling is constitutively observable even when may be functionally different. Though peripheral edge ruffles they are not migrating. Moreover, PI3-kinase inhibitors of macrophages have a low actinin-4/F-actin ratio on the suppress growth factor-induced ruffling in some cell types average, some portions have a high actinin-4/F-actin ratio. This (Kotani et al., 1994; Wennstrom et al., 1994) but not in may imply that peripheral edge ruffling of macrophages macrophages (Araki et al., 1996). Thus, the leading edge consists of more than a single phenotype for different cell 3338 N. Araki and others

Fig. 9. Confocal microscopy showing immunolocalization of actinin-4 (A and green in C) and F-actin distribution (B and red in C) in a macrophage during phagocytosis of latex beads. Latex beads were fed to macrophages to allow phagocytosis for 10 minutes. Latex beads were confirmed by bright-field diffraction mode (not shown). All phagocytic organelles containing latex beads are indicated by three different- colored arrows. (D) The actinin-4/F-actin ratio is high near phagocytic cups (white arrows) and some presumed nascent phagosomes (yellow arrows), but low at perinuclear phagosomes or phagolysosomes (red arrows) and straight linear ruffles. n, nucleus. Bar, 10 µm. functions and some such as inward curving ruffles, are transit macropinosomes. However, our quantitative analysis by line forms from peripheral ruffles to dorsal circular ruffles. scan on a macropinosome (Fig. 4C) indicated that F-actin was Our study indicated that the actinin-4/F-actin ratio was high apparently associated with the macropinosome, although the around newly formed macropinosomes. Macropinosomes are amount of F-actin was much less than in circular ruffles. This formed from circular ruffles by closure of the ruffle’s tip. As finding is not surprising because F-actin facilitates endosome shown in Fig. 2, newly formed macropinosomes immediately trafficking and/or endosome/lysosome fusion (Durrbach et al., lost most of the associated F-actin, while circular ruffles were 1996). From our ratio image analysis, it is conceivable that enriched with F-actin. Nevertheless, actinin-4 retained a small actinin-4 predominantly cross-links F-actin and forms an amount of F-actin around macropinosomes for a short while. actinin-4-F-actin network surrounding macropinosomes. A This suggests that actinin-4 preferentially contributed to actin- previous report revealed that a cortical α-actinin-F-actin bundling around early macropinosomes. Newly formed network prevented deformation of cell shape against osmotic macropinosomes with variable sizes (often as large as 2-5 µm stress (Rivero et al., 1996). A rheological study with a torsion in diameter) move toward the perinuclear region, shrink in size pendulum showed that the viscoelastic properties of F-actin and ultimately merge with tubular lysosomes. During this gels cross-linked by α-actinin differed from gels cross-linked process, macropinosomes acquire lysosomal membrane by the 120 kDa gelation factors: α-actinin-F-actin networks glycoproteins such as LAMP-1 (<5 minutes) and then several responded to deformation with a strongly damped oscillation, minutes later the cation-independent mannose-6-receptor and whereas the gelation factor-F-actin networks reacted in a more markers of lysosomal content such as cathepsins derived from elastic, weakly damped way (Janssen et al., 1996). Moreover, tubular lysosomes (Racoosin and Swanson, 1993). Our FDx10 a mutation study using Dictyostelium indicated that lack of α- pulse/chase experiment showed that actinin-4 gradually actinin caused a significant reduction of the viscoelastic dissociated from macropinosomes. Comparing the time course response after deformation at high frequency (Eichinger et al., of actinin-4 dissociation in Fig. 7A with previous published 1996). These results suggest that α-actinin is responsible for data (Racoosin and Swanson, 1993), it appears that actinin-4 organizing the actin against fast and strong dissociation occurs during acquisition of LAMP proteins. impacts. Consistent with these properties of α-actinin-F-actin However, dissociation of actinin-4 from macropinosomes gels, it is considered that the actinin-4-F-actin network seemed to be unnecessary for LAMP-1 delivery to surrounding macropinosomes may provide a constructive brace macropinosomes, because some actinin-4 positive to maintain a large vacuolar structure in the cell. Larger macropinosomes (<35%) showed LAMP-1 in their vacuoles may require more structural resistance to the membranes. It is noteworthy that macropinosomes having both cytoplasmic pressure (viscosity) during centripetal movement. actinin-4 and LAMP-1 are generally larger than Although we cannot rule out the possibility that the actinin- macropinosomes having only LAMP-1. This implies that the 4–F-actin interaction may be directly involved in the actinin-4 association with the macropinosomal membrane may contraction (shrinkage) force of macropinosomes and/or the be dependent on not only the degree of macropinosome intracellular transport of macropinosomes, there has been no maturation but also the size of macropinosomes. report indicating that α-actinin is directly involved in such What is the role of actinin-4 around macropinosomes? It has force generating machinery. been known that F-actin immediately dissociates from the In the process of phagocytosis, actinin-4 was enriched in plasma membrane when circular ruffles close into phagocytic cups and some phagosomes but not in late Actinin-4/F-actin ratio in macrophages 3339 phagosomes or phagolysosomes. The relationship between macropinocytosis. A recent study using F-actin cross-linking actinin-4 and F-actin in phagocytic cups and phagosomes proteins mutant strains of Dictyostelium showed double seems to be comparable to that in circular ruffles and mutants lacking α-actinin and gelation factor ABP120 or 34 macropinosomes, respectively. Since the processes of kDa actin-bundling protein exhibited a reduced rate of fluid macropinocytosis and phagocytosis are mechanistically similar phase endocytosis, while single mutants lacking α-actinin did (Araki et al., 1996; Swanson et al., 1999; Swanson and Watts, not, suggesting that α-actinin function may be guaranteed by 1995), it seems likely that actinin-4 plays the same role in both more than a single molecule (Rivero et al., 1999). There is macropinocytosis and phagocytosis. Taken together, the likely some redundancy of function among these actin cross- localization of actinin-4 in macrophages correlated with linking proteins. Further studies using dominant negative actively moving structures involved in endocytosis but not in mutants or gene knockout of individual isoforms of actin- migratory movements, whereas actinin-4 in cancer cells is binding proteins and signal transduction-related molecules associated with cell migration. would be required for a full understanding their interplay and Actinin-4 shows a high degree of similarity to actinin-1 physiological roles in cell functions. (80% nucleotide and 86.7% amino acid similarity. The amino To our knowledge, this is a first report that reveals a acid sequences indicate several domains conserved among α- difference in molecular composition between dorsal circular or actinin family members, including actin-binding domains, curved ruffles and edge straight ruffles. The present study can pleckstrin-homology (PH) domains, central rod domains and provide insight into the distinct roles of α-actinin isoforms in two EF-hand calcium regulation domains. This indicates that specific cellular functions and a clue to resolving how ruffling both actinin-1 and actinin-4 actin-binding activities could be in special forms occurs. Studies of signaling mechanisms of commonly regulated by Ca2+ and phosphoinositides (Djinovic´- actinin-4 recruitment to specialized F-actin-containing Carugo et al., 1999; Honda et al., 1998; Matsudaira, 1991). structures such as circular ruffles are now in progress using Molecular properties such as molecular flexibility, the ability macrophages and other cell types. to bind other molecules and F-actin cross-linking angles may affect the cell structural configurations generated by F-actin gel The authors thank Dr Joel A. Swanson, University of Michigan network (Djinovic´-Carugo et al., 1999). However, such Medical School for critical reading of this manuscripts and helpful properties have not been obtained for actinin-1 or -4. advice, Drs M. Tokuda and K. Kawakami for help with Different localizations of α-actinin isoforms suggest that immunoprecipitation and cell culture, Dr S. Yokota for a gift of anti- their recruitment to specialized regions could be regulated by cathepsin D antibody and Drs M. Hamasaki, T. Ishida and T. different signals. Recruitment of actinin-4 to actively moving Toyosima for helpful discussion. Secretarial and technical services provided from Ms H. Yamamoto and Mr K. Yokoi are greatly structures should be more highly regulated by rapid signal appreciated. This study was supported by a Grant-in-Aid for Scientific transduction than actinin-1 which is present homogeneously, Research from Japan Society for the Promotion of Science and by a or constitutively at stable structures such as basal adhesion grant from the Ichiro Kanehara Foundation (to N.A.). podosomes. Honda et al. 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