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ADHESION-DEPENDENT F-ACTIN PATTERN INAMOEBA PROTEUSAS A COMMON FEATURE OF AMOEBAE AND THE METAZOAN MOTILE CELLS
Cell Biology International, 1997, Vol. 21, No. 9, 565–573
ADHESION-DEPENDENT F-ACTIN PATTERN IN AMOEBA PROTEUS AS A COMMON FEATURE OF AMOEBAE AND THE METAZOAN MOTILE CELLS LUCYNA GRE q BECKA*, PAWEŁ POMORSKI, ANDRZEJ GRE q BECKI and ANNA ŁOPATOWSKA Department of Cell Biology, Nencki Institute of Experimental Biology, ul. Pasteura 3, PL-02093 Warszawa, Poland Accepted 22 August 1997
Adhesion and movement of Amoeba proteus are both dependent on the appropriate arrangement of the F-actin cytoskeleton and on the presence of the cell nucleus. In this study the F-actin organization was examined by routine FITC-phalloidin staining and confocal laser microscopy in intact amoebae and in their nucleated and anucleated fragments, at different levels of cell adherence to the substratum. In the adhering and migrating intact cells and nucleated cell fragments dot-like aggregates of F-actin are scattered over the ventral side at sites close to the substratum. In the case of de-adhesion of nucleated specimens this pattern disappears and F-actin is accumulated in the cell centre and/or dispersed in the cytoplasm. The same actin distribution, without ventral dots, is found in the anucleated fragments which usually fail to attach to the substratum. Re-adhesion of anucleated fragments, induced by a modified substratum or spontaneous, is accompanied by restoration of actin dots at the lower cell side. It is concluded that: (1) adhering specimens of A. proteus display the same dot-like actin pattern on the ventral cell side, as many metazoan motile cells; (2) organization or disorganization of this pattern may occur independently of the presence of the cell nucleus, under the control of cell 1997 Academic Press Limited adhesion to the substratum. K: Amoeba proteus; F-actin organization; cell adhesion; cell nucleus
INTRODUCTION In most motile cells the cytoskeleton is composed of three classes of filamentous polymers, but in A. proteus only the F-actin microfilaments, accompanied by actin binding proteins, are basic components of the cortical network essential for cell movement (Christiani et al., 1986). The spatial arrangement of the F-actin cytoskeleton in moving A. proteus has been thoroughly examined by many authors and many methods (see reviews by Grain, 1986; Gre¸becka, 1988; Stockem and Kłopocka, 1988; Gre¸becki, 1994), but the structural relations between the position of cell-tosubstratum attachment sites and the organization of the cytoskeleton, such as were shown in metazoan cells (e.g. by Ono et al., 1993), and signaling molecules between adhesive and motor proteins (Yamada and Geiger, 1997), have not yet been *To whom correspondence should be addressed. 1065–6995/97/090565+09 $25.00/0/cb970181
detected in amoebae. Nevertheless, structural connections and signal transmission between the substrate-bound surface areas of A. proteus and the cytoskeleton should be expected, since F-actin is retracted during locomotion toward the attachment sites (Gre¸becki, 1984, 1985, 1994). A. proteus may contact with the substratum at any point, but it is usually attached at the middle– anterior body region by contact pseudopodia or adhesive knobs protruding from the ventral surface. These were either observed in side-view (Bell and Jeon, 1963; Gre¸becki, 1976) or in the reflection interference microscope (Haberey, 1971; Opas, 1978). In the smaller amoebae, Naegleria gruberi and Acanthamoeba castellanii, the sites of interaction with glass also prevail at the middle cell region and are also interpreted as contact points established by micropseudopodia produced by the ventral cell surface (Preston and King, 1978; King et al., 1979, 1982, 1983). Recently, similar protrusions at the same location were described under the 1997 Academic Press Limited
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name of eupodia in Dictyostelium amoebae by Fukui and Inoue´ (1997). Amoeba proteus, which is considerably larger, moves faster and contacts with the substratum at a greater separation distance, may form relatively fewer but large and extremely alternating attachments (cf. Opas, 1987; Harris, 1994). Instability and alternation of the attachment points is also a characteristic of normal and transformed motile metazoan cells. Such a highly motile free-living cell as A. proteus could then be compared in this respect with those metazoan cells, since it represents a basically similar type of locomotion. In the present study we have detected actin patches distributed along the ventral surface of substratum-attached A. proteus and we demonstrate the role of adhesion in the organization of this F-actin pattern. MATERIALS AND METHODS The cultures of Amoeba proteus strain C were grown in Pringsheim medium and fed twice a week on Tetrahymena pyriformis. Experiments were run at room temperature (202C), 2–3 days after feeding. Amoebae were dissected in nucleated and anucleated parts with a De Fonbrune micromanipulator (Beaudouin, Paris, France). The intact amoebae and their nucleated and anucleated fragments were either kept on untreated glass or placed on glass slides incubated in a 1 mg/ml water solution of poly--lysine or 1 mg/ml of concanavalin A (both from Sigma, St Louis, MO, U.S.A.) in 1 acetic acid, and rinsed thereafter with Pringsheim medium. Amoebae were left on these substrata without feeding for different periods of time, up to 1 week on the untreated glass, up to 24 hours on the polylysine-coated surface and up to 72 hours on slides covered with the concanavalin. Their morphology and behaviour were kept under a regular microscopic control. Samples were fixed at different moments, depending on the kind of the substratum, type of the examined material and the behaviour of cells. The cells, after fixation in 3.5% paraformaldehyde and 0.3% acrolein in phosphate-buffered Pringsheim medium, were stained for F-actin with 1% FITC-conjugated phalloidin (Sigma, St Louis, MO, U.S.A.). They were examined with a Diaphot TMD microscope (Nikon, Tokyo, Japan) equipped for a parallel Nomarski DIC and fluorescence observation with 10 NA 0.25 and 20 NA 0.4 LWD objectives. The images, collected by a SIT video camera were proceeded for contrast enhance-
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ment, noise reduction and background subtraction, using an Argus 10 real-time image processor (Hamamatsu, Hamamatsu City, Japan). The processed images were collected using a Pevon frame grabber and stored in PC 386 memory. A confocal laser scanning microscope, Molecular Dynamics CLSM Phoibos 1000 (Sarastro, Ypsilanti, U.S.A.), based on a Nikon Optiphot microscope, was used, with 20 and 40 objectives, for producing series of horizontal optical sections, three-dimensional reconstructions and the vertical cross-section reconstructions. Threedimensional reconstructions were based on 20 optical sections forming images composed of 512512 pixels each and were processed on a Silicon Graphics Personal Iris workstation. For cross-section reconstructions maximum intensity voxels were shown in every direction of view. Surface reconstructions were prepared by shading based on a surface normals algorithm, with surface recognition based on the minimum found in the image histogram between background and object maxima.
RESULTS The cells were incubated on the untreated glass (1–168 hours), on the glass covered with polylysine (1–24 hours) or with concanavalin (24–72 hours). The intact cells and the nucleated and anucleated cell fragments of A. proteus manifested a variable degree of adhesion depending on the kind of the substratum and the time of incubation. They were fixed and stained with FITC-phalloidin at three levels of cell–substratum adherence: (1) nonadhering specimens, floating in the medium before being collected for preparation; (2) moderately adhering specimens, formerly attached to the untreated or concanavalin-coated glass but detaching during the preparation; (3) firmly adhering specimens, remaining on the polylysine-coated surface after fixation and staining. Specimens incubated on the untreated glass Intact amoebae and nucleated fragments incubated on the untreated glass during 1–24 hours are moderately adhering and migrate well. The nucleated fragments differ from undamaged amoebae only by the cytoplasm volume. They start locomoting immediately after the operation and their F-actin spatial arrangement is generally the same as in the undamaged cells. In the intact, normally moving
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specimens the FITC-phalloidin staining reveals the peripheral actin layer (Fig. 1) and F-actin dots of different size dispersed at the ventral side of the cell, adjacent to the glass. These dots are localized with different density along the cell length (Fig. 2). Most often they are accumulated at the middle– anterior cell region (Fig. 3), and sometimes near the uroidal part of the amoeba. After a longer incubation on the untreated glass (without feeding) the locomotion rate decreases and finally, after one week, the intact amoebae as well as their nucleated fragments become motionless and non-adhering (Gre¸becka et al., 1997). In such specimens F-actin is accumulated at the cell centre, mainly around the vacuoles. The typical distinct submembraneous F-actin layer is replaced by F-actin dispersed in the cytoplasm. The cell shape after such a long incubation becomes heterotactic (Fig. 4), or rounded. The anucleated fragments are generally considered as non-adhering (Clark, 1942; Lorch, 1969; Gre¸becka et al., 1995, 1997). They detach from the substratum immediately after dissection and usually remain unattached, rounded and motionless, for several hours of stay on the untreated glass. In the non-adhering anucleated fragments the F-actin is accumulated in the central region (Fig. 5). On the second and following days some of them may produce pseudopodia and transiently readhere to the substrate. This partial regulation of shape and motility is accompanied by the reappearence of F-actin at the ventral surface (Fig. 11b). After 7 days of incubation the anucleated fragments again become unattached, motionless and round. Actin is dispersed in the cytoplasm, without aggregates at the ventral side (Fig. 6), similar to its distribution in the nonadhering nucleated specimens after the same period of time. Specimens incubated on the polylysine-coated glass The intact amoebae as well as their nucleated fragments, placed on the glass coated with polylysine move slower than on the untreated glass (cf. Kołodziejczyk et al., 1995; Gre¸becka et al., 1997). Already after 1 hour their rate of locomotion is considerably decreased and their flattened shape indicates an increase of the strength of adhesion. At this stage F-actin, besides a strong concentration in the posterior cell part and around the cell nucleus, presents a pattern of many dots more loosely dispersed along the middle and anterior regions of advancing pseudopodia (Fig. 7). After 24 hours of
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incubation on the polylysine substratum, the majority of cells become motionless and firmly adhering, not detaching during fixation. They are usually round, corrugated, and their peripheral hyaline layers are enlarged. In such specimens the F-actin accumulates in numerous small foci, which may be scattered over the whole ventral side and form a distinct ring along the adhering periphery of these fragments (Fig. 8). The anucleated fragments, which during the first hours after operation cannot attach to the untreated glass, adhere more strongly to the polylysine-coated surface. After 24 hours of incubation almost all of them are attached to the substratum, similar to the nucleated specimens. Their adhesion is, however, more moderated since they can move under the influence of external stimuli. In the anucleated fragments adhering to the polylysine-coated glass F-actin is dispersed in the form of small dots or larger areas and the peripheral actin layer may be reconstructed, as it is demonstrated in Fig. 9 and elsewhere (Gre¸becka et al., 1997). In general, the number and form of F-actin aggregates on the ventral part of anucleated fragments adhering to polylysine is comparable with the pattern seen in intact amoebae and their nucleated fragments under the same conditions. Serial optical sections through the anucleated fragments adhering to the polylysine-coated glass, generated by the confocal microscope (Fig. 9) and their three-dimensional reconstructions (Fig. 10), strongly suggest that F-actin aggregates revealed at the ventral side of amoebae and their fragments are formed at the sites of most close contact of the cell surface with the substratum. Specimens incubated on the concanavalin-coated glass The anucleated fragments of amoebae after 24 hours of incubation on the substratum coated with concanavalin A are spread flat on the surface. The adhering frontal edge is formed by a fan of many short truncated knobs, which produces a halfrosette shape of the whole fragments. The adhesion seems however to be weaker than on the polylysine, since these fragments are detached during fixation. Optical sections produced by the confocal laser microscopy demonstrate aggregates of F-actin close to the substratum, especially on the surface of knobs forming the fan-like frontal edge and beneath the central cell part (Fig. 11a, arrowheads). The control anucleated fragments, incubated 24 hours on the untreated glass, also show
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Cell Biology International, Vol. 21, No. 9, 1997
Cell Biology International, Vol. 21, No. 9, 1997
the presence of actin at the ventral surface, but have a different shape (Fig. 11b). After 72 hours the major part of anucleated fragments of amoebae cease to migrate and detach from the glass coated with concanavalin. In such cells prominent F-actin structures are revealed in the cell interior, but aggregations at the ventral surface are no longer observed (Fig. 11c). DISCUSSION Adhesion of metazoan cells to the substratum leads to concomitant rearrangement of the cytoskeleton (Willingham et al., 1977; Opas, 1987, 1994; Korohoda and Kajstura, 1988; Way and Weeds, 1990; Fukui et al., 1991). It may be associated with actin polymerization (Southwick et al., 1989; Wang et al., 1993; Cramer et al., 1994) and accumulation of actin filaments adjacent to the substratum Cramer and Mitchison, 1993). In polymorphonuclear leukocytes, many small foci of F-actin and F-actin-containing protrusions were found on the lower surface adjacent to the substratum, at the initial and advanced stages of attachment (Berlin and Oliver, 1978; Boyles and Bainton, 1979; Southwick et al., 1989; Yu¨ru¨ker and Niggli, 1992). Short protrusions contacting with the substratum and rich in filamentous actin were also demonstrated in avian osteoclasts (Marchisio et al., 1984). F-actin foci were also found in association with the substratum-attached membrane of macrophages adhering to glass, polystyrene and formvar-carbon and their transmembrane connec-
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tion with the substratum has been postulated (Trotter, 1981; Amato et al., 1983). It was later demonstrated by confocal laser microscopy that these F-actin aggregates in macrophages are in fact connected to the glass-adherent membrane proteins (Ono et al., 1991, 1993). It was suggested that F-actin accumulates owing to adhesive trapping of proteins diffusing in the plane of the membrane and connected to the cytoskeleton compounds (Gingell and Owens, 1992; Weber et al., 1995). More probably, however, clustering of adhesion receptors leads to a local concentration of signaling molecules and eventually results in the aggregation of cytoskeletal proteins (Yamada and Geiger, 1997). In general, the F-actin dots at the substratum attachment sites were described in cells of monocytic origin, and also in a variety of transformed cells by Carley et al. (1981, 1983), Carley and Webb (1983), and Carley (1985). According to Marchisio et al. (1987) this pattern of F-actin organization seems to be related to the high motility of these two cell categories. Among the free-living amoebae, the actin patches on the ventral side were as yet revealed in the cytoskeleton of substratum-attached Acanthamoeba, extracted by detergents and examined in fluorescence microscopy and high voltage EM (T. M. Preston, personal communication). In the eupodia which anchor Dictyostelium amoebae to the substratum a high concentration of F-actin, myosin-II and á-actinin were revealed by indirect immunofluorescence (Fukui and Inoue´, 1997). In our present study of A. proteus we found dot-like
Figs 1–8. Fig. 1. Peripheral F-actin layer (arrowheads) in an intact specimen of A. proteus on untreated glass. T-F, tail-front orientation; N, cell nucleus. Routine fluorescence microscopy after FITC-phalloidin staining. Focused at a half of cell thickness. Bar 25 ìm. Fig. 2. F-actin dots scattered at the ventral side of an undamaged complete cell of A. proteus moving on the untreated glass. FITC-phalloidin staining and routine FM. Focus at the substratum level. Bar 50 ìm. Fig. 3. Accumulation of F-actin dots at the middle–anterior region of the ventral side, which is the usual adhesion zone of the migrating amoebae. FITC-phalloidin staining and routine FM. Focus at the substratum level. Bar 25 ìm. Fig. 4. Aggregation of F-actin at the cell centre of undamaged amoebae, which have lost substratum adherence and motility, and assumed a heterotactic shape after 1 week of incubation on glass. CLSM. (a) Optical section at the substratum level. (b) Three-dimensional reconstruction of the same specimen. Bar 50 ìm. Fig. 5. Disorganized F-actin pattern in an anucleated fragment of the amoeba after 24 hours on untreated glass. Phalloidin staining and routine FM. Bar 50 ìm. Fig. 6. CLSM optical sections of an anucleated fragment after 168 hours on untreated glass. (a) Absence of F-actin at the substratum level. (b) Dispersed actin at the equatorial level of the fragment. (c) Absence of F-actin at the top level. Bar 50 ìm. Fig. 7. F-actin dots in a nucleated fragment strongly adhering to polylysine substratum and locomoting, after 1 hour of incubation. CLSM sections close to the substratum (a), at the nucleus level (b), and above it (c). Bar 50 ìm. Fig. 8. F-actin distribution in a strongly adhering non-locomoting nucleated fragment after 24 hours on the polylysine. CLSM sections, at the substratum level (a) and 18 ìm higher (b). Bar 50 ìm.
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aggregates of F-actin scattered on the ventral cell side (Figs 2, 3). They seem to be related to the discrete cell-to-substratum contact sites, since they accompany only the adhesion state. We neither found such F-actin spots in the anucleated fragments failing to adhere to the glass (Figs 5, 6), nor in nucleated specimens detached from the glass after incubation extended up to several days (Fig. 4). In such non-adhering specimens F-actin is mainly accumulated around vacuoles at the cell centre and/or dispersed in the cytoplasm. The increase of the cell–substratum attraction force by the polylysine seems to increase the number of F-actin foci at the lower side of nucleated fragments (Figs 7, 8), and it induces their formation in the anucleated ones (Gre¸becka et al., 1997). The position of F-actin aggregates in the cytoplasm examined in serial optical sections from the substratum plane to the top of the cells, vertical cross-sections and three-dimensional reconstructions produced by the confocal laser microscope (Figs 9, 10), confirmed their distribution at the ventral side of fragments as well as their contact with cell surface areas that touch with the substratum. It might be argued that adhesion promoted by the polylysine is excessively strong and nonspecific. As a matter of fact, Kołodziejczyk et al. (1995) attribute the polylysine effect on A. proteus, as in other cases, to electrostatic forces acting in the cell–substratum separation space. For that reason the experiments with the anucleated fragments were repeated with a milder adhesion-promoting factor acting through the known specific receptors. Concanavalin A has been chosen because (1) it moderately raises the adhesion of amoebae, Naegleria gruberi (Preston and O’Dell, 1980) and A. proteus (Kłopocka et al., 1996), and (2) the concanavalin receptors are abundant in the mucous layer covering the cell surface of A. proteus (Kukulies et al., 1984; Topf and Stockem, 1996).
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Our results demonstrated that anucleated fragments of amoebae attached to the concanavalincoated surface also accumulate F-actin at sites of the closest cell–substratum contact (Fig. 11a). The peculiar cell morphology and actin aggregation in patches, larger but fewer than dots on the untreated glass or polylysine substratum, may be related to the tendency of concanavalin to capping and inducing at least the first stages of pinocytosis in A. proteus (Prusch, 1981; Kukulies et al., 1984). After 72 hours the ventral actin aggregates disappear and actin concentrates in the cell interior (Fig. 11c). It is interesting to note in this respect that earlier enucleation experiments (Chatterjee, 1984) have suggested that the pool of concanavalin receptors on the surface of A. proteus declines dramatically after about 72 hours. Our present results demonstrate a close analogy of the adhesion-dependent actin organization in a free-living unicellular organism, Amoeba proteus, and in metazoan cells. We think, as Marchisio et al. (1987), that it should be interpreted as the adhesion pattern specifically related to the motility. Moreover, this pattern normally found in the intact locomoting amoebae, is also reconstructed by the anucleated fragments in the case of their readhesion to the substratum. It means that in the absence of the cell nucleus the adhesion may independently control the F-actin organization in the region adjacent to the substratum. Therefore, the role of the cell nucleus in the locomotion of amoebae would rather consist in the preservation of the F-actin network than in a direct regulation of its arrangement.
ACKNOWLEDGEMENTS This study was partially supported by grant 0453/ p2/93/04 from the Committee of Scientific Research (KBN).
Figs 9–11. Fig. 9. CLSM optical sections of an anucleated fragment incubated 24 hours on the polylysine. Note large actin-rich areas at the substratum level (0), distinct cortical actin layer at the higher level (12 ìm), and less regular actin aggregates in two upper sections (24 and 36 ìm). Bar 50 ìm. Fig. 10. Vertical cross-section (a) and three-dimensional reconstruction (b) of F-actin distribution in the same specimen as in Fig. 9. It demonstrates the contiguity of actin-rich structures at the ventral side of the specimen to the substratum. Fig. 11. CLSM optical sections of anucleated fragments incubated 24 hours on the concanavalin-coated glass (a) and untreated glass (b), both taken at the substratum level, and an equatorial section of a fragment incubated 72 hours on the concanavalin substratum (c). Arrowheads in a indicate F-actin patches clustered at the frontal adhesive knobs and under the middle body region. Bar 50 ìm.
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ADHESION-DEPENDENT F-ACTIN PATTERN IN AMOEBA PROTEUS AS A COMMON FEATURE OF AMOEBAE AND THE METAZOAN MOTILE CELLS LUCYNA GRE q BECKA*, PAWEŁ POMORSKI, ANDRZEJ GRE q BECKI and ANNA ŁOPATOWSKA Department of Cell Biology, Nencki Institute of Experimental Biology, ul. Pasteura 3, PL-02093 Warszawa, Poland Accepted 22 August 1997
Adhesion and movement of Amoeba proteus are both dependent on the appropriate arrangement of the F-actin cytoskeleton and on the presence of the cell nucleus. In this study the F-actin organization was examined by routine FITC-phalloidin staining and confocal laser microscopy in intact amoebae and in their nucleated and anucleated fragments, at different levels of cell adherence to the substratum. In the adhering and migrating intact cells and nucleated cell fragments dot-like aggregates of F-actin are scattered over the ventral side at sites close to the substratum. In the case of de-adhesion of nucleated specimens this pattern disappears and F-actin is accumulated in the cell centre and/or dispersed in the cytoplasm. The same actin distribution, without ventral dots, is found in the anucleated fragments which usually fail to attach to the substratum. Re-adhesion of anucleated fragments, induced by a modified substratum or spontaneous, is accompanied by restoration of actin dots at the lower cell side. It is concluded that: (1) adhering specimens of A. proteus display the same dot-like actin pattern on the ventral cell side, as many metazoan motile cells; (2) organization or disorganization of this pattern may occur independently of the presence of the cell nucleus, under the control of cell 1997 Academic Press Limited adhesion to the substratum. K: Amoeba proteus; F-actin organization; cell adhesion; cell nucleus
INTRODUCTION In most motile cells the cytoskeleton is composed of three classes of filamentous polymers, but in A. proteus only the F-actin microfilaments, accompanied by actin binding proteins, are basic components of the cortical network essential for cell movement (Christiani et al., 1986). The spatial arrangement of the F-actin cytoskeleton in moving A. proteus has been thoroughly examined by many authors and many methods (see reviews by Grain, 1986; Gre¸becka, 1988; Stockem and Kłopocka, 1988; Gre¸becki, 1994), but the structural relations between the position of cell-tosubstratum attachment sites and the organization of the cytoskeleton, such as were shown in metazoan cells (e.g. by Ono et al., 1993), and signaling molecules between adhesive and motor proteins (Yamada and Geiger, 1997), have not yet been *To whom correspondence should be addressed. 1065–6995/97/090565+09 $25.00/0/cb970181
detected in amoebae. Nevertheless, structural connections and signal transmission between the substrate-bound surface areas of A. proteus and the cytoskeleton should be expected, since F-actin is retracted during locomotion toward the attachment sites (Gre¸becki, 1984, 1985, 1994). A. proteus may contact with the substratum at any point, but it is usually attached at the middle– anterior body region by contact pseudopodia or adhesive knobs protruding from the ventral surface. These were either observed in side-view (Bell and Jeon, 1963; Gre¸becki, 1976) or in the reflection interference microscope (Haberey, 1971; Opas, 1978). In the smaller amoebae, Naegleria gruberi and Acanthamoeba castellanii, the sites of interaction with glass also prevail at the middle cell region and are also interpreted as contact points established by micropseudopodia produced by the ventral cell surface (Preston and King, 1978; King et al., 1979, 1982, 1983). Recently, similar protrusions at the same location were described under the 1997 Academic Press Limited
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name of eupodia in Dictyostelium amoebae by Fukui and Inoue´ (1997). Amoeba proteus, which is considerably larger, moves faster and contacts with the substratum at a greater separation distance, may form relatively fewer but large and extremely alternating attachments (cf. Opas, 1987; Harris, 1994). Instability and alternation of the attachment points is also a characteristic of normal and transformed motile metazoan cells. Such a highly motile free-living cell as A. proteus could then be compared in this respect with those metazoan cells, since it represents a basically similar type of locomotion. In the present study we have detected actin patches distributed along the ventral surface of substratum-attached A. proteus and we demonstrate the role of adhesion in the organization of this F-actin pattern. MATERIALS AND METHODS The cultures of Amoeba proteus strain C were grown in Pringsheim medium and fed twice a week on Tetrahymena pyriformis. Experiments were run at room temperature (202C), 2–3 days after feeding. Amoebae were dissected in nucleated and anucleated parts with a De Fonbrune micromanipulator (Beaudouin, Paris, France). The intact amoebae and their nucleated and anucleated fragments were either kept on untreated glass or placed on glass slides incubated in a 1 mg/ml water solution of poly--lysine or 1 mg/ml of concanavalin A (both from Sigma, St Louis, MO, U.S.A.) in 1 acetic acid, and rinsed thereafter with Pringsheim medium. Amoebae were left on these substrata without feeding for different periods of time, up to 1 week on the untreated glass, up to 24 hours on the polylysine-coated surface and up to 72 hours on slides covered with the concanavalin. Their morphology and behaviour were kept under a regular microscopic control. Samples were fixed at different moments, depending on the kind of the substratum, type of the examined material and the behaviour of cells. The cells, after fixation in 3.5% paraformaldehyde and 0.3% acrolein in phosphate-buffered Pringsheim medium, were stained for F-actin with 1% FITC-conjugated phalloidin (Sigma, St Louis, MO, U.S.A.). They were examined with a Diaphot TMD microscope (Nikon, Tokyo, Japan) equipped for a parallel Nomarski DIC and fluorescence observation with 10 NA 0.25 and 20 NA 0.4 LWD objectives. The images, collected by a SIT video camera were proceeded for contrast enhance-
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ment, noise reduction and background subtraction, using an Argus 10 real-time image processor (Hamamatsu, Hamamatsu City, Japan). The processed images were collected using a Pevon frame grabber and stored in PC 386 memory. A confocal laser scanning microscope, Molecular Dynamics CLSM Phoibos 1000 (Sarastro, Ypsilanti, U.S.A.), based on a Nikon Optiphot microscope, was used, with 20 and 40 objectives, for producing series of horizontal optical sections, three-dimensional reconstructions and the vertical cross-section reconstructions. Threedimensional reconstructions were based on 20 optical sections forming images composed of 512512 pixels each and were processed on a Silicon Graphics Personal Iris workstation. For cross-section reconstructions maximum intensity voxels were shown in every direction of view. Surface reconstructions were prepared by shading based on a surface normals algorithm, with surface recognition based on the minimum found in the image histogram between background and object maxima.
RESULTS The cells were incubated on the untreated glass (1–168 hours), on the glass covered with polylysine (1–24 hours) or with concanavalin (24–72 hours). The intact cells and the nucleated and anucleated cell fragments of A. proteus manifested a variable degree of adhesion depending on the kind of the substratum and the time of incubation. They were fixed and stained with FITC-phalloidin at three levels of cell–substratum adherence: (1) nonadhering specimens, floating in the medium before being collected for preparation; (2) moderately adhering specimens, formerly attached to the untreated or concanavalin-coated glass but detaching during the preparation; (3) firmly adhering specimens, remaining on the polylysine-coated surface after fixation and staining. Specimens incubated on the untreated glass Intact amoebae and nucleated fragments incubated on the untreated glass during 1–24 hours are moderately adhering and migrate well. The nucleated fragments differ from undamaged amoebae only by the cytoplasm volume. They start locomoting immediately after the operation and their F-actin spatial arrangement is generally the same as in the undamaged cells. In the intact, normally moving
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specimens the FITC-phalloidin staining reveals the peripheral actin layer (Fig. 1) and F-actin dots of different size dispersed at the ventral side of the cell, adjacent to the glass. These dots are localized with different density along the cell length (Fig. 2). Most often they are accumulated at the middle– anterior cell region (Fig. 3), and sometimes near the uroidal part of the amoeba. After a longer incubation on the untreated glass (without feeding) the locomotion rate decreases and finally, after one week, the intact amoebae as well as their nucleated fragments become motionless and non-adhering (Gre¸becka et al., 1997). In such specimens F-actin is accumulated at the cell centre, mainly around the vacuoles. The typical distinct submembraneous F-actin layer is replaced by F-actin dispersed in the cytoplasm. The cell shape after such a long incubation becomes heterotactic (Fig. 4), or rounded. The anucleated fragments are generally considered as non-adhering (Clark, 1942; Lorch, 1969; Gre¸becka et al., 1995, 1997). They detach from the substratum immediately after dissection and usually remain unattached, rounded and motionless, for several hours of stay on the untreated glass. In the non-adhering anucleated fragments the F-actin is accumulated in the central region (Fig. 5). On the second and following days some of them may produce pseudopodia and transiently readhere to the substrate. This partial regulation of shape and motility is accompanied by the reappearence of F-actin at the ventral surface (Fig. 11b). After 7 days of incubation the anucleated fragments again become unattached, motionless and round. Actin is dispersed in the cytoplasm, without aggregates at the ventral side (Fig. 6), similar to its distribution in the nonadhering nucleated specimens after the same period of time. Specimens incubated on the polylysine-coated glass The intact amoebae as well as their nucleated fragments, placed on the glass coated with polylysine move slower than on the untreated glass (cf. Kołodziejczyk et al., 1995; Gre¸becka et al., 1997). Already after 1 hour their rate of locomotion is considerably decreased and their flattened shape indicates an increase of the strength of adhesion. At this stage F-actin, besides a strong concentration in the posterior cell part and around the cell nucleus, presents a pattern of many dots more loosely dispersed along the middle and anterior regions of advancing pseudopodia (Fig. 7). After 24 hours of
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incubation on the polylysine substratum, the majority of cells become motionless and firmly adhering, not detaching during fixation. They are usually round, corrugated, and their peripheral hyaline layers are enlarged. In such specimens the F-actin accumulates in numerous small foci, which may be scattered over the whole ventral side and form a distinct ring along the adhering periphery of these fragments (Fig. 8). The anucleated fragments, which during the first hours after operation cannot attach to the untreated glass, adhere more strongly to the polylysine-coated surface. After 24 hours of incubation almost all of them are attached to the substratum, similar to the nucleated specimens. Their adhesion is, however, more moderated since they can move under the influence of external stimuli. In the anucleated fragments adhering to the polylysine-coated glass F-actin is dispersed in the form of small dots or larger areas and the peripheral actin layer may be reconstructed, as it is demonstrated in Fig. 9 and elsewhere (Gre¸becka et al., 1997). In general, the number and form of F-actin aggregates on the ventral part of anucleated fragments adhering to polylysine is comparable with the pattern seen in intact amoebae and their nucleated fragments under the same conditions. Serial optical sections through the anucleated fragments adhering to the polylysine-coated glass, generated by the confocal microscope (Fig. 9) and their three-dimensional reconstructions (Fig. 10), strongly suggest that F-actin aggregates revealed at the ventral side of amoebae and their fragments are formed at the sites of most close contact of the cell surface with the substratum. Specimens incubated on the concanavalin-coated glass The anucleated fragments of amoebae after 24 hours of incubation on the substratum coated with concanavalin A are spread flat on the surface. The adhering frontal edge is formed by a fan of many short truncated knobs, which produces a halfrosette shape of the whole fragments. The adhesion seems however to be weaker than on the polylysine, since these fragments are detached during fixation. Optical sections produced by the confocal laser microscopy demonstrate aggregates of F-actin close to the substratum, especially on the surface of knobs forming the fan-like frontal edge and beneath the central cell part (Fig. 11a, arrowheads). The control anucleated fragments, incubated 24 hours on the untreated glass, also show
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the presence of actin at the ventral surface, but have a different shape (Fig. 11b). After 72 hours the major part of anucleated fragments of amoebae cease to migrate and detach from the glass coated with concanavalin. In such cells prominent F-actin structures are revealed in the cell interior, but aggregations at the ventral surface are no longer observed (Fig. 11c). DISCUSSION Adhesion of metazoan cells to the substratum leads to concomitant rearrangement of the cytoskeleton (Willingham et al., 1977; Opas, 1987, 1994; Korohoda and Kajstura, 1988; Way and Weeds, 1990; Fukui et al., 1991). It may be associated with actin polymerization (Southwick et al., 1989; Wang et al., 1993; Cramer et al., 1994) and accumulation of actin filaments adjacent to the substratum Cramer and Mitchison, 1993). In polymorphonuclear leukocytes, many small foci of F-actin and F-actin-containing protrusions were found on the lower surface adjacent to the substratum, at the initial and advanced stages of attachment (Berlin and Oliver, 1978; Boyles and Bainton, 1979; Southwick et al., 1989; Yu¨ru¨ker and Niggli, 1992). Short protrusions contacting with the substratum and rich in filamentous actin were also demonstrated in avian osteoclasts (Marchisio et al., 1984). F-actin foci were also found in association with the substratum-attached membrane of macrophages adhering to glass, polystyrene and formvar-carbon and their transmembrane connec-
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tion with the substratum has been postulated (Trotter, 1981; Amato et al., 1983). It was later demonstrated by confocal laser microscopy that these F-actin aggregates in macrophages are in fact connected to the glass-adherent membrane proteins (Ono et al., 1991, 1993). It was suggested that F-actin accumulates owing to adhesive trapping of proteins diffusing in the plane of the membrane and connected to the cytoskeleton compounds (Gingell and Owens, 1992; Weber et al., 1995). More probably, however, clustering of adhesion receptors leads to a local concentration of signaling molecules and eventually results in the aggregation of cytoskeletal proteins (Yamada and Geiger, 1997). In general, the F-actin dots at the substratum attachment sites were described in cells of monocytic origin, and also in a variety of transformed cells by Carley et al. (1981, 1983), Carley and Webb (1983), and Carley (1985). According to Marchisio et al. (1987) this pattern of F-actin organization seems to be related to the high motility of these two cell categories. Among the free-living amoebae, the actin patches on the ventral side were as yet revealed in the cytoskeleton of substratum-attached Acanthamoeba, extracted by detergents and examined in fluorescence microscopy and high voltage EM (T. M. Preston, personal communication). In the eupodia which anchor Dictyostelium amoebae to the substratum a high concentration of F-actin, myosin-II and á-actinin were revealed by indirect immunofluorescence (Fukui and Inoue´, 1997). In our present study of A. proteus we found dot-like
Figs 1–8. Fig. 1. Peripheral F-actin layer (arrowheads) in an intact specimen of A. proteus on untreated glass. T-F, tail-front orientation; N, cell nucleus. Routine fluorescence microscopy after FITC-phalloidin staining. Focused at a half of cell thickness. Bar 25 ìm. Fig. 2. F-actin dots scattered at the ventral side of an undamaged complete cell of A. proteus moving on the untreated glass. FITC-phalloidin staining and routine FM. Focus at the substratum level. Bar 50 ìm. Fig. 3. Accumulation of F-actin dots at the middle–anterior region of the ventral side, which is the usual adhesion zone of the migrating amoebae. FITC-phalloidin staining and routine FM. Focus at the substratum level. Bar 25 ìm. Fig. 4. Aggregation of F-actin at the cell centre of undamaged amoebae, which have lost substratum adherence and motility, and assumed a heterotactic shape after 1 week of incubation on glass. CLSM. (a) Optical section at the substratum level. (b) Three-dimensional reconstruction of the same specimen. Bar 50 ìm. Fig. 5. Disorganized F-actin pattern in an anucleated fragment of the amoeba after 24 hours on untreated glass. Phalloidin staining and routine FM. Bar 50 ìm. Fig. 6. CLSM optical sections of an anucleated fragment after 168 hours on untreated glass. (a) Absence of F-actin at the substratum level. (b) Dispersed actin at the equatorial level of the fragment. (c) Absence of F-actin at the top level. Bar 50 ìm. Fig. 7. F-actin dots in a nucleated fragment strongly adhering to polylysine substratum and locomoting, after 1 hour of incubation. CLSM sections close to the substratum (a), at the nucleus level (b), and above it (c). Bar 50 ìm. Fig. 8. F-actin distribution in a strongly adhering non-locomoting nucleated fragment after 24 hours on the polylysine. CLSM sections, at the substratum level (a) and 18 ìm higher (b). Bar 50 ìm.
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aggregates of F-actin scattered on the ventral cell side (Figs 2, 3). They seem to be related to the discrete cell-to-substratum contact sites, since they accompany only the adhesion state. We neither found such F-actin spots in the anucleated fragments failing to adhere to the glass (Figs 5, 6), nor in nucleated specimens detached from the glass after incubation extended up to several days (Fig. 4). In such non-adhering specimens F-actin is mainly accumulated around vacuoles at the cell centre and/or dispersed in the cytoplasm. The increase of the cell–substratum attraction force by the polylysine seems to increase the number of F-actin foci at the lower side of nucleated fragments (Figs 7, 8), and it induces their formation in the anucleated ones (Gre¸becka et al., 1997). The position of F-actin aggregates in the cytoplasm examined in serial optical sections from the substratum plane to the top of the cells, vertical cross-sections and three-dimensional reconstructions produced by the confocal laser microscope (Figs 9, 10), confirmed their distribution at the ventral side of fragments as well as their contact with cell surface areas that touch with the substratum. It might be argued that adhesion promoted by the polylysine is excessively strong and nonspecific. As a matter of fact, Kołodziejczyk et al. (1995) attribute the polylysine effect on A. proteus, as in other cases, to electrostatic forces acting in the cell–substratum separation space. For that reason the experiments with the anucleated fragments were repeated with a milder adhesion-promoting factor acting through the known specific receptors. Concanavalin A has been chosen because (1) it moderately raises the adhesion of amoebae, Naegleria gruberi (Preston and O’Dell, 1980) and A. proteus (Kłopocka et al., 1996), and (2) the concanavalin receptors are abundant in the mucous layer covering the cell surface of A. proteus (Kukulies et al., 1984; Topf and Stockem, 1996).
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Our results demonstrated that anucleated fragments of amoebae attached to the concanavalincoated surface also accumulate F-actin at sites of the closest cell–substratum contact (Fig. 11a). The peculiar cell morphology and actin aggregation in patches, larger but fewer than dots on the untreated glass or polylysine substratum, may be related to the tendency of concanavalin to capping and inducing at least the first stages of pinocytosis in A. proteus (Prusch, 1981; Kukulies et al., 1984). After 72 hours the ventral actin aggregates disappear and actin concentrates in the cell interior (Fig. 11c). It is interesting to note in this respect that earlier enucleation experiments (Chatterjee, 1984) have suggested that the pool of concanavalin receptors on the surface of A. proteus declines dramatically after about 72 hours. Our present results demonstrate a close analogy of the adhesion-dependent actin organization in a free-living unicellular organism, Amoeba proteus, and in metazoan cells. We think, as Marchisio et al. (1987), that it should be interpreted as the adhesion pattern specifically related to the motility. Moreover, this pattern normally found in the intact locomoting amoebae, is also reconstructed by the anucleated fragments in the case of their readhesion to the substratum. It means that in the absence of the cell nucleus the adhesion may independently control the F-actin organization in the region adjacent to the substratum. Therefore, the role of the cell nucleus in the locomotion of amoebae would rather consist in the preservation of the F-actin network than in a direct regulation of its arrangement.
ACKNOWLEDGEMENTS This study was partially supported by grant 0453/ p2/93/04 from the Committee of Scientific Research (KBN).
Figs 9–11. Fig. 9. CLSM optical sections of an anucleated fragment incubated 24 hours on the polylysine. Note large actin-rich areas at the substratum level (0), distinct cortical actin layer at the higher level (12 ìm), and less regular actin aggregates in two upper sections (24 and 36 ìm). Bar 50 ìm. Fig. 10. Vertical cross-section (a) and three-dimensional reconstruction (b) of F-actin distribution in the same specimen as in Fig. 9. It demonstrates the contiguity of actin-rich structures at the ventral side of the specimen to the substratum. Fig. 11. CLSM optical sections of anucleated fragments incubated 24 hours on the concanavalin-coated glass (a) and untreated glass (b), both taken at the substratum level, and an equatorial section of a fragment incubated 72 hours on the concanavalin substratum (c). Arrowheads in a indicate F-actin patches clustered at the frontal adhesive knobs and under the middle body region. Bar 50 ìm.
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