IDENTIFICATION OF A MICROFILAMENT- ENRICHED, MOTILE … · 2005-08-21 · IDENTIFICATION OF A MICROFILAMENT-ENRICHED, MOTILE DOMAIN IN AMOEBOFLAGELLATES OF PHYSARUM POLYCEPHALUM KATHRYN

J. Cell Set. 54, 1-21 (1982)Printed in Great Britain © Company of Biologist! Limited 1982

IDENTIFICATION OF A MICROFILAMENT-

ENRICHED, MOTILE DOMAIN IN

AMOEBOFLAGELLATES OF

PHYSARUM POLYCEPHALUM

KATHRYN PAGHDepartment of Anatomy, Duke University Medical Center, Durluan, North Carolina27710, U.S.A. AND

MARK R. ADELMAN*Department of Anatomy, Uniformed Services University of the Health Sciences,Bethesda, 4301 Jones Bridge Road, Maryland 20014, U.S.A.

SUMMARY

A distinctive, transient cytoplasmic domain was identified in haploid cells of Physarumpolycephalum that are transforming from amoebae into flagellate swimming cells. As revealed bylight microscopy, this region, termed the ridge, is a flattened cytoplasmic extension thatdisplays a specific form of motility, characterized by undulations that propagate rapidly downits long axis. The ridge was further examined by scanning electron microscopy of whole cells,and by transmission electron microscopy of serial sections and negative stain preparations ofamoeboflagellate cytoskeletons. We identified three distinct components possibly related toridge motility: a short flagellum closely associated with the ridge surface, an array of micro-tubules that is part of a complex microtubular apparatus and a network of filaments that formsa well-defined laminar core within the ridge.

Labelling studies with heavy meromyosin subfragment 1 (S-i) indicated that a major portionof the filament network is actin. Peripheral microfilaments appeared to have a uniform orienta-tion in that arrowheads pointed towards the cell body. Other ordered filament arrangementswere apparent with a modified extraction protocol using phalloidin. Similar microfilamentorganization has been reported in motile regions of other systems, such as the lamellipodia oftissue culture cells. However, the ridge can be distinguished from such other motile regions inthat it forms independently of contact with a substratum and is only present in a specificregion of amoeboflagellates. Moreover, the ridge appears at a specific time during the course ofthe transformation.

The ridge constitutes a new system for studying the structural basis of cell motility. Thearrangements of actin- and microtubule-containing ridge components suggest several testablehypotheses concerning their involvement in cytoskeletal or motile functions.

INTRODUCTION

Motility is manifested in strikingly diverse forms in different organisms and atdifferent stages within the life history of a single cell. A number of studies indicatethat morphogenetic events involving changes in cell shape or the rearrangements ofcytoplasmic components can be generated by seemingly simple means, such as linearcontractile events (Schroeder, 1973; Bumside, 1976); polymerization of actin (Tilney,

• Author for correspondence.

2 K. Pagh and M. R. Adelman

Hatano, Ishikawa & Mooseker, 1973; Begg & Rebhun, 1979); or changes in distribu-tion of microtubules (Burnside, 1973; Warren, 1974). Thus far, well-developedmolecular explanations have been offered primarily for motility involving highlyordered, specialized structures such as the sarcomere of muscle (Huxley, 1957), themicrovilli of epithelial cells (Mooseker, 1976) and of oocytes (Begg & Rebhun, 1979),and the acrosomal process of certain sperm cells (Tilney, 1978; Tilney, Kiehart,Sardet & Tilney, 1978). However, many important forms of motility involve lesswell-ordered structures and do not appear to be due to single motile events nor toinvolve obvious molecular interconversions (Komnick, Stockem & Wolfarth-Botterman, 1973; Trinkhaus, 1976). A well-known example is the spreading andtranslocation of tissue culture cells on a substratum (Abercrombie, Heaysman &Pegrum, 1970). The cytoplasmic domains associated with these apparently morecomplicated movements (e.g. the lamellipodia of fibroblasts and leukocytes) consistprimarily of networks of cytoplasmic filaments (Small & Celis, 1978; Lazarides &Revel, 1979). It has been suggested that one or a few general mechanisms underliedifferent forms of cell motility (Huxley, 1973). A detailed understanding of the basisfor the observed differences will come in part from more extensive analyses of thediverse motile regions that are characteristic of a variety of eukaryotic cells.

The amoeboflagellate transformation of myxomycete slime moulds is well-suited toanalyses of cytoplasmic domains and structures involved in motility. A variety ofmotile behaviours are expressed as amoebae undergo a sequence of changes leading tothe development of flagellate swimming cells (Schuster, 1965; Jacobson, Johnke &Adelman, 1976). In Physarum polycephahim, the transformation can be made toproceed synchronously (Jacobson & Adelman, 1975), and both motility (Jacobson& Dove, 1975) and transformation-defective mutants (Mir, Del Castillo & Wright,1979; D. N. Jacobson, unpublished) have been obtained. Assembly of flagella and theelaboration of complex arrays of microtubules accompany changes in cell shape andthe onset of certain movements associated with the transformation (Schuster, 1965;Aldrich, 1968; Wright, Moisand & Mir, 1979; Wright, Mir & Moisand, 1980).

In this paper, we provide evidence that a previously unrecognized, actin-enrichedcytoplasmic domain arises during the amoeboflagellate transformation. This domainappears to be associated with motility in P. polycephalum amoeboflagellates. Thedistinctive motile, morphological, spatial and temporal features of this domain make itparticularly suitable for further investigations of actin and microtubule functions inmovement. Portions of this work have appeared in abstract form (Pagh & Adelman,1979, 1980).

MATERIALS AND METHODS

Cell cultures and transformation conditions

P. polycephalum strain 911 (Jacobson, 1979) was used unless otherwise noted. Amoebae werecultured on2 %agar containing 01 M-sucroseando-oi M-phosphocitrate buffer (o-oi M-K,HPO4brought to pH 5 with o-oi M-citric acid). Mixtures of live and formalin-killed Escherichia coli Bwere added as a food source. The amoebal inoculum (approx. 10' cells/Petri dish) and bacterialconcentrations (approx. io10 E. coli/dish) were such that the stationary phase of growthwas reached after about 72 h at 26 °C (D. N. Jacobson, personal communication).

Motile domain in Physarum amoeboflagellates 3

Stationary-phase amoebae were induced to transform by suspension in buffer, usuallyco i M-phosphocitrate (pH 5 or 6) containing 0-04-0-1 M-sucrose. Occasionally other trans-formation solutions were used; these included deionized water, o-oi M-phosphocitrate (pH 7)and o-oi M-Tris (pH 7-5) with or without 0-04 M-sucrose. For most studies, amoebae wereharvested from agar plates and allowed to transform in suspension at room temperature(20-23 °C)- Synchronous transforming populations (Jacobson & Adelman, 197s) were used forultra8tructural studies of cytoskeletons. These were obtained as will be described in detailelsewhere (unpublished data). Briefly, flagellate swimming cells were collected from trans-forming cultures, concentrated by centrifugation and resuspended in buffer before replating onbacteria-free agar. Following reversion to amoeboid form but before encystment commenced,cells were induced to transform a second time as described above. For cytoskeletal studies,populations 16-22 min into the second transformation were used; 'early transforming popula-tions' refers to those at 10-15

Light microscopy and cinemicrography

Transforming cells were pipetted onto coverslips and observed using a Zeiss R.A. microscopeequipped with Nomarski optics. The behaviour of cells transforming at room temperature wasrecorded on Plus X Reversal film using a 40 x Nomarski objective and an Industrial Cameratime-lapse set-up (Baltimore Instruments, Baltimore, MD), operating at a rate of 1 frame/i -25 s.Measurements of the speed of ridge undulations were made using a Lafayette film analyser byprojecting the image onto tracing paper and measuring the distance travelled by one undulationfrom one frame to the next, usually over a total of 5 frames. The speed reported was based onanalysis of 5 cells; at least 2 separate undulatory waves were followed in each cell.

For photomicroscopy and quantitation of transformation kinetics cells transforming insuspension were fixed with 2 % glutaraldehyde (Electron Microscopy Sciences) in 0-04 M-8odium cacodylate buffer (pH 7). Photographs were taken on Kodak Pan X film using a ZeissUltraphot 2 with a 100 x Nomarski oil immersion objective.

Scanning electron microscopy

Suspended amoebae were allowed to settle onto coverslips and were fixed with 3 % glutar-aldehyde in 005 M-sodium cacodylate buffer (pH 7) containing 1 mM-CaClt. They were thenpost-fixed with 1 % OsO4 in the same buffer for 1 h at room temperature, dehydrated throughethanol, passed into Freon 113 and critical-point dried. After gold /palladium coating, speci-mens were examined in a JEOL JSM-T20 microscope.

Thin-section electron microscopy

Transforming cells were fixed for up to 2 h at room temperature with 2 % glutaraldehyde insodium cacodylate or phosphate buffer (0-075 M> pH 7), both containing 3 mM-MgCl,. Tannicacid (0-2 %) was either present in the initial fixative solution or added after 30 min. Cells werepost-fixed for 30 min at 4 °C with 0-1-0-4% OsO4 in either of the above buffers at pH 6;stained en bloc with 1 % uranyl acetate for 60 min at room temperature; dehydrated through agraded series of ethanol into propylene oxide and embedded in Epon/Araldite according toMollenhauer (1964). Because cells do not adhere to each other, they must be centrifuged ateach preparative step.

Serial sections were picked up on Formvar films and mounted on slot grids (Kubai, 1973),stained with uranyl acetate and lead citrate, and carbon-coated. Micrographs were taken onKodalith LR Estar base 70 mm roll film using a Siemens 101 microscope or on Kodak ElectronImage Plates using a Philips 300 microscope.

Cell extraction and negative staining

Samples (0-5-1-0 ml) of suspensions containing approximately 10' synchronized trans-forming cells in o-oi M-phosphocitrate buffer (pH 6-o), plus 0-04 M-sucrose were diluted 1 :1with a stock solution of 10 mM-PIPES, 50 mM-KCl, 50 mM-NaCl, 4 mM-MgCl, and 4 min-EGTA (pH 6-8), containing 0-5-1-0% Triton X100. Dimethylsulphoxide (10%) was included


in the extraction buffer when maximal preservation of microtubular organization was desired.Cells were extracted for 30-60 s at room temperature with gentle mixing.

In some cases, phalloidin (Sigma) was included in the extraction buffer at a final concentra-tion of 50-100 fig/ml of cell suspension (the stock phalloidin solution, 1 fig/ml in deionizedH|O, was stored at —20 CC). When it was desirable to extract for prolonged periods (up to5 min in phalloidin-containing buffer), the extraction was performed on ice and in the presenceof phenylmethylsulphonyl fluoride (Sigma), at a concentration of 0-5 mM (the stock solutionwas 50 mM in isopropyl alcohol).

The cell lysate was fixed by adding glutaraldehyde to give a final concentration of 2 % andafter 20 min, the suspension was centrifuged (approx. 300 g, for 10 min at 4 °C). The pellet wasrinsed twice with o-i M-ammonium acetate (pH 7). The concentrated suspension was appliedto carbon-coated grids and stained with a solution containing 2 % uranyl acetate and 0-2 /Jg/mlcytochrome c. Examination and photography were carried out with a Philips 300 microscopeoperating at 80 kV.

Subfragment S-i labelling

Rabbit muscle myosin was prepared essentially according to Holtzer & Lowey (1959); thetwice-precipitated protein was subjected to high-speed centrifugation in the presence ofMgATP. The myosin was stored in 50% glycerol at — 20 °C prior to being used for preparationof heavy-meromyosin subfragment-1 (S-i) by the chymotryptic digestion methods of Weeds &Pope (1977). For S-i labelling of cytoskeletons, equal volumes of cell lysate and S-i (1*7 mg/ml)were mixed and incubated for 3 min on ice. Partial rinsing prior to fixation was achieved by4-fold dilution with half-strength extraction buffer, minus detergent. As a control, MgATP wasadded to the rinse solution at a final concentration of o*i mM.

RESULTS

P. polycephalum amoebae transform into smoothly swimming flagellate cells within60 to 90 min following their suspension in buffer. Our observations of many aspectsof the transformation are in general agreement with descriptions previously reportedfor P. polycephalum (Wright et al. 1979) and for other slime moulds (reviewed byGray & Alexopoulos, 1968). Comparison of amoebae (Fig. 1) with a fully transformedcell (Fig. 2) serves to summarize the changes involved in the transformation: (1) a cellof highly variable shape, approximately 10 fim. in diameter, acquires a relativelystable, elongate shape, averaging 15-16 /im. in length; (2) cytoplasmic organelles areredistributed, e.g. the nucleus is relocated to a cone-shaped process at the anteriorregion of the cell; (3) flagella assemble, one anterior flagellum, (20 /tm long) and ashorter, posteriorly directed flagellum, which lies adjacent to the cell body (see Figs.5, 6). In addition, intracellular differentiation occurs as is discussed below.

Identification of a well-defined cytoplasmic domain in amoeboflagellates

A transforming culture includes cells having morphological and motile character-istics that span the spectrum between amoeboid and flagellate types. A subpopulationof these cells, which we refer to as amoeboflagellates, is distinguished by a cytoplasmicdifferentiation that is not found in amoebae and is only present as a remnant in fullytransformed cells (Fig. 2). This is a sheet-like cytoplasmic extension spanning thelength of the cell body (Figs. 3, 4). Amoeboflagellates often show distinct curvature oftheir long axes and whenever such curvature can be defined, the extension is invariablyfound on the convex side (Fig. 3). We have termed this region the ridge because of its

Motile domain in Physarum amoebofiagellates

Figs. 1-5. Nomarski light micrographs of P. polycephalum haploid cells fixed at differentstages in the amoeboflagellate transformation. Compare the 2 amoebae in Fig. 1 withthe fully transformed swimming cell in Fig. 2. Transforming amoebofiagellates (Figs.3-5) show the ridge (r) as a flattened process located along one side of the cell body.The ridge is absent from amoebae (Fig. 1) and only seen in remnant form in fullytransformed cells (arrow in Fig. 2). The asterisks in Figs. 3, 4 indicate 2 types ofsurface deformations characteristic of the ridge (see text). Alignment of the ridge withthe posterior flagellum (j>f) is evident when the ridge is viewed edge on in opticallysectioned cells (Fig. 5). n, nucleus; af, anterior flagellum. Bar, 5 fun.

appearance when viewed edge-on (Fig. 5) in the light microscope. However, the ridgeis not simply a small deformation of the cell surface. It constitutes a well-definedcytoplasmic domain that comprises up to approximately 25 % of the projection areaof cells imaged as in Figs. 3 and 4.

The ridge exhibits several characteristic forms of movement as will be presented indetail elsewhere (K. Pagh, unpublished data). Particularly striking are wave-like

K. Pagh and M. R. Adelman

Figs. 6, 7. Scanning electron micrographs of transforming amoeboflagellates. In allcells (Fig. 6), there is a close association of the ridge (r) and posterior flagellum (pf).In some cells (Fig. 7), the ridge is highly folded and exhibits fingerUke processes ofdifferent lengths along its edge. The spacing of these processes is often quite regularas shown in the inset, af, anterior flagellum. Bar, 1 fan; inset bar, 1 /(m.

8Motile domain in Physarum amoeboflagellates

Figs. 8, 9. Thin sections of amoeboflagellates showing the appearance of the ridge indifferent planes of section.

Fig. 8. This section is comparable to a section of a cell such as shown in Fig. 3 cutparallel to the plane of the page. The anterior of the cell is to the left. Anteriorportions of the ridge (r) contain ribosome-excluding material, pb, basal body ofposterior flagellum; ab, basal body of anterior flagellum; arrow, several microtubuleaof the microtubular apparatus. Bar, 1 fim.

Fig. 9. The ridge is sectioned roughly perpendicular to Fig. 8. The plane of sectionis indicated in the inset. The cell's anterior-posterior axis, determined by examiningserial sections, is from left to right. The ribosome-excluding material forms a con-tinuous laminar core of fairly uniform thickness. The lamina and cell surface areupfolded in one region (arrowhead). Bar, 1 fim.

K. Posh and M. R. Adelman

10A

pf


undulations that propagate at an approximate rate of 1 ftm/s, moving from anterior toposterior. On live cells 2 such undulatory waves, similar in appearance to the deforma-tions seen in fixed cells (Fig. 4), rnay be present simultaneously on the surface of theridge. In addition to undulations, the ridge often manifests propagating angulardeformations such as the one shown in Fig. 3. It should be pointed out that amoebo-flagellates show a variety of movements other than those mentioned here in connectionwith the ridge. Among these are propagating surface constrictions of the cell body,which are sometimes, but not always, coincident with the undulatory motions of theridge.

In scanning electron micrographs, the ridge either appears as a rather smooth,sheet-like process (Fig. 6) or it has a sinuous, folded appearance (Fig. 7); becausemany of the folds are perpendicular to the long axis of the ridge, we believe them tobe a manifestation of one type of undulation observed in living cells. At the free edgeof the ridge, finger-like protuberances of variable length often occur at rather regularintervals (Fig. 7 and inset). Such protuberances are absent from all other regions ofthe amoeboflagellate cell surface.

The observations described thus far show that the ridge is an external specializationeasily recognized by light microscopy and scanning electron microscopy of intactcells. Internally, the ridge also comprises a distinctive cytoplasmic region clearlyidentifiable using ultrastructural criteria. In thin sections, ribosome-excluding,filamentous material is concentrated within the ridge (Figs. 8, IOA, B). Serial sectionstaken in different planes (see insets to Figs. 9, 10) show that this material comprises afairly uniform lamina approximately 0-2 fim thick (Fig. 9) that serves as the core ofthe entire flattened extension and usually extends somewhat into the cell body. Whenthe thickness of the ridge exceeds that of the lamina, the latter underlies the plasmamembrane only at the outermost margin of the ridge. In sections more or less parallelto the long axis of the ridge, this lamina usually displays a wavy contour that occasion-ally is quite pronounced and may correspond with upfoldings of the cell surface(Fig. 9).

Fig\ 10. This figure shows a series of sections from a single amoeboflagellate cutperpendicular to the long axis at levels corresponding approximately to A-F in theinset, A and B include the most anterior portion of the ridge, which is recognizableby its filamentous core (Jc). The remaining sections include regions of the cellanterior to the ridge. The posterior flagellum (pf) is external to the cell along most of itslength but closely applied to the plasma membrane. Its terminus is shown in Awhere only an axonemal doublet remains. Anterior to the ridge, the flagellum isincorporated into the cell body; its basal body (pb) is shown in F. Microtubulararrays are numbered in accordance with Wright et al. (1979). MTA 5 (5) is acomplex of 4 microtubules that closely underlies the plasma membrane beneath theposterior flagellum. Its spatial relationship with the ridge is shown in A and B. MTA 5originates in association with the basal body of the posterior flagellum (F). MTA 3 (3)and 4 (4) are 2 other small arrays that begin anterior to F and are never seen in theridge proper. Portions of MTA 1 (1) encircling the nucleus are shown in c; portionsof MTA 2 (2) encircling the cone-shaped process of the cell are shown in F. Anotherelement of the microtubular apparatus in the anterior-most portion of the cellappears in cross-section (arrow in D); this structure has a striated appearancewhen sectioned longitudinally (K. Pagh, unpublished). Bar, 0-5 (im.

K. Pagh and M. R. Adelman

Motile domain in Physarum amoebofiagellates 11

Localization of the ridge relative to flagella and cytoplasmic microtubular arrays

Several observations suggest that the ridge has a specific location relative to overallcell morphology. As has been pointed out, the ridgfe is aligned with the long axis of thecell and is positioned on the convex side of cells that are curved. A consistent spatialrelationship also exists between the posterior flagellum and the ridge (Figs. 5, 6):the flagellum emanates from a position slightly anterior to the ridge and is closelyassociated along its length with the anterior portion of the ridge surface, the longaxis of flagellum and ridge being roughly parallel.

The flagella of myxomycetes are known to be associated in an organized mannerwith a complex cytoplasmic microtubular apparatus. These microtubules are alignedwith the long axis of the cell, originating near the flagellar basal bodies and continuingposterior for some distance past the nucleus (Schuster, 1965; Aldrich, 1968; Fig. 8).In studies of isolated flagellar complexes, Wright et al. (1979) demonstrated thepresence of 5 distinct microtubular arrays and deduced their intracellular arrange-ment. According to these workers, 2 of the 5 arrays are concentric, one (MTA 1according to their terminology) encircling the nucleus, the other (MTA 2) underlyingthe plasma membrane of the anterior portion of the cell. In addition, 3 smaller arrays(MTA 3, 4 and 5) were recognized and a model regarding their location within intactcells was proposed.

Since Wright and co-workers were using extensively extracted cell preparations inwhich the ridge was not recognizable, we investigated the arrangement of the micro-tubular apparatus within intact cells and its orientation relative to the position of theridge. Based on examination of serial sections of 7 amoeboflagellates and of numerousrandom sections, we are in general agreement with the 3-dimensional model of themicrotubular apparatus proposed by Wright and co-workers (unpublished data). Ofspecific interest to this study is the precise spatial relationship of the ridge with thesemicrotubular arrays and with the posterior flagellum, as is illustrated in sections cutperpendicular to the long axis of an amoeboflagellate (Fig. 10 A, B ; see accompanyingfigure legend for details). One of the smaller arrays is the only collection of micro-tubules found within the ridge proper. The array consists of 4 microtubules (Fig. IOB)that closely parallel the posterior flagellum after originating in association with itsbasal body (Fig. IOC-F). Based on the origin and number of microtubules, this groupcorresponds to MTA 5 of Wright and co-workers.

Figs. 11, 12. Low magnification views of Triton-extracted amoeboflagellates. Cyto-skeletons contain the ridge (r), anterior flagellum (of), posterior flagellum (pf) andMTA 5 (5).

Fig. 11. The arrows designate the course of MTA 5 before it enters the dense cellbody. The inset shows the 4 microtubules of MTA 5 (indicated by bracket) beneaththe posterior flagellum. Bar, 1 fim; inset bar, 0-5 fim.

Fig. 12. The array (MTA 5) of 4 microtubules continues posteriorly within theridge of this cell. The termination point of 3 of the microtubules is indicated (arrow-head) and shown at higher magnification in the inset. Note the waviness of theremaining microtubule that continues posteriorly. Bar, 1 fim; inset bar, 0-2 /an.

if. K. Pagh and M. R. Adelman

Fig. 13. Higher magnification image of a cytoskeleton showing filaments at themargin of the ridge. Bar, 0-5 fim.Fig. 14. Filaments detected within the ridge close to the cell body. Bar, 0-5 /*m.

Ultrastructural analysis of the ridge in detergent-extracted cells

The motile behaviour of the ridge together with the filamentous nature of itslaminar core suggested to us that the ridge might be enriched in actin. To test thispossiblity, we prepared detergent-extracted cytoskeletons of transforming cells withthe aim of identifying its filamentous constituents. Some modification of conventionalprocedures for preparation of cytoskeletons was necessary: because amoeboflagellatesdo not adhere strongly to a substrate, extraction was done with cells in suspension;

Figs. 15-17. Negative stain preparations of cytoskeletons incubated with S-i.Fig. 15. This low magnification view shows the increased density of a ridge (r)

treated with S-i. Filaments are visible within the cell body (arrows), af, anteriorflagellum. Bar, 1 fim.

Fig. 16. Filaments that emanate from the margin of the ridge are decorated withS-i in the form of arrowheads which point towards the cell body. Bar, 0-5 fim.

Fig. 17. A portion of the ridge from a cytoskeleton treated with S-i, then rinsed inbuffer containing MgATP. The dissociation of S-i from ridge components is indi-cated by the reduced overall density of the ridge (note that MTA 5 is visible) andthe absence of arrowhead decoration of the (fewer) filaments that are clearly detectablein such preparations. MTA 5 (5)- Bar, 0-5 fim.

Motile domain in Physarum amoeboflagellates

15


because cytoskeletons were found to be labile after Triton Xioo permeabilization,extraction times were kept short and subsequent manipulations were minimized.

Despite the liability of the cytoskeletons, spatial relationships between manystructures are preserved as in intact cells (Figs. 11, 12). The ridge is readily identifiablebecause of its morphology, location on one side of the cell and position with respect tothe posterior flagellum. In addition, MTA 5 is visible in the most anterior portion ofthe ridge (Figs. 11, 12 and insets). In most cytoskeletons, two or three of the fourmicrotubules of MTA 5 terminate within the anterior half of the ridge (Fig. 12 andinset); such termination of microtubules was confirmed in serial sections of intactcells (not shown). The remaining one or two microtubules continue further along thelength of the ridge, tracing paths that vary from cell to cell in their extent and positionwithin the ridge. One notable feature of this array is that it is often curved or wavy(Fig. 11) as are its individual microtubules (Fig. 12).

The predominant component of the extracted ridge is a web of randomly orientedfilaments, measuring approximately 6-5 nm in diameter within negatively stainedcytoskeletons extracted for longer periods of time (see below) or when imaged bypositive staining. These filaments are distinguishable most easily at the edge of theridge (Fig. 13). In less-dense areas of the ridge it is sometimes possible to discernregions of loose association of the filaments in small bundles (Fig. 14).

Incubation of cytoskeletons with subfragment-i (S-i) resulted in an increased densityof the entire ridge (Fig. 15, cf. with Fig. n ) . Where filaments were seen in loosearray, particularly at the edge of the ridge, S-i was bound in the form of arrowheads,thus identifying the filaments as F-actin (Fig. 16). In all cases where free filament endswere evident, the arrowheads pointed toward the cell body, indicating that peripheralfilaments have a similar polarity. Since visualization of labelled filaments within theridge was more difficult due to its high density, controls were particularly important.The cytoskeletons are so fragile that extensive washing to remove non-specificlabelling is not possible. Our most useful control therefore involved the rinsing ofS-i-labelled cytoskeletons by dilution in buffer containing MgATP. Following suchtreatment, fewer filaments could be detected but those present were no longerdecorated; in addition, the density of the entire ridge was clearly reduced to a densitycomparable to that of unlabelled cytoskeletons (Fig. 17). This indicates that allportions of the ridge contain actin. We cannot rule out the possibility that some of thisactin exists in non-filamentous form nor that non-actin filaments might also be presentwithin the ridge.

We tested whether our extraction buffer, designed to stabilize microfilaments,artefactually promoted their polymerization. Our tests involved: (1) addition of°'5% glutaraldehyde directly into the extraction buffer; and (2) extraction of cells atlow (less than 1 rriM) Mg concentrations for very brief times (i.e. 5 s) before fixation.In both cases, ridge microfilaments were detected (not shown), supporting theconclusion that they are present in vivo.

To determine whether filament arrangements within the ridge were adequatelymaintained during preparation of cytoskeletons, phalloidin, a stabilizing agent forF-actin (Dancker, Low, Hasselbach & Wieland, 1975), was added to the extraction

Motile domain in Physarum amoeboflagellates

18

Figs. 18-19. Amoeboflagellates extracted with detergent in the presence of 100 figphalloidin per ml.

Fig. 18. Margin of the ridge containing tufts of peripheral filaments spaced at fairlyregular intervals. Inset: filaments converge to form a small process. Bar, 0-2 fim;inset bar, 0-2 fim.

Fig. 19. Low magnification view of a ridge (r) present in early transformingpopulations. The ridge is not fully expanded and contains thin processes orientedperpendicular to its edge. One process (arrow) is shown at higher magnification in theinset. Bar, 2 fim; inset bar, 0-2 fim.

i6 K. Pagh and M. R. Adelman

0 10 20 30Time (min)

Fig. 20. Formation of the anterior flagellum (open symbols) and the ridge (closedsymbols) in cell populations transforming in suspension (triangles) and on a sub-stratum (circles). Samples were fixed at the times indicated and ioo randomly-selected cells from each group were scored using a ioo x oil-immersion Nomarskiobjective.

buffer. In the presence of phalloidin at 50 /^g/ml, the ridge and the overall shape of thecytoskeleton appeared to be more stable when exposed to longer extraction times(i.e. 5 min) in comparison with cytoskeletons treated identically (see Materials andMethods for details) but in the absence of phalloidin. At 100 fig phalloidin per ml, thedetailed filament organization at the edge of the ridge often appeared more ordered;tufts of convergent peripheral filaments, spaced at fairly regular intervals were ob-served (Fig. 18 and inset). Rather regular filament bundles oriented perpendicular toand extending beyond the edge of the ridge were apparent in phalloidin-treatedcytoskeletons using cells from 'early transforming populations' (Fig. 19 and inset) butnot when cells from later in the transformation were examined.

General conditions associated with ridge formation

Many motile, actin-enriched cellular domains such as the lamellipodia of tissueculture cells are induced to form by contact with a substratum (Trinkhaus, 1976). Toexplore whether ridge formation in amoeboflagellates is similarly promoted by sub-strate contact, we harvested amoebae from a single culture dish and allowed half thecells to transform in suspension and the other half on coverslips. In three replicate


experiments, the formation of the anterior flagellum and the ridge were studied as afunction of time. Fig. 20 illustrates that the ridge appears in transforming populationsunder both sets of conditions and that the time course of appearance is similar. In fact,the mean ti0 values for substrate and suspension conditions showed no statisticallysignificant difference at the 95 % confidence level (<60 = time in min at which half of apopulation has a ridge).

The kinetic data also show that there is temporal specificity in the formation of theridge with respect to the appearance of the anterior flagellum. Based on t60 values forboth sets of conditions studied, the ridge consistently formed after the anteriorflagellum with an average time difference of 6-i ± 1-4 min.

Finally, it should be noted that the ridge is a constant feature of the transformationin P. polycephalum. It is present in other strains examined (e.g. LU688, 437, 211) andforms reproducibly under a variety of conditions that are suitable for the transforma-tion (e.g. varying pH and osmolarity as described in Materials and Methods).

DISCUSSION

We have shown that a structurally distinct, cytoplasmic domain is present inamoeboflagellates of P. polycephalum. This specialized domain, termed the ridge, isdistinguishable from the rest of the cell on the basis of morphological criteria such asits sheet-like appearance, detailed surface morphology and well-defined laminar core.Furthermore, it is located on a specific part of the cell such that two other structures,a short flagellum and one of five microtubular arrays comprising the flagellar apparatus,are consistently associated with its surface and interior, respectively. In the light ofthese striking and regular features, it is perhaps surprising that this structure has notbeen recognized in previous studies of P. polycephalum (reviewed by Gray &Alexopoulos, 1968; Wakasugi & Ohta, 1973; Wright et al. 1979). Several factors mayaccount for this. First, the ridge is best seen with Nomarski optics (rather than phase-contrast) and in fixed cells. Although we consistently detected the ridge while examin-ing living cells and when viewing cine-records, our recognition of its overall mor-phology did not become clear until we studied fixed cells by light microscopy andexamined such cells in the scanning electron microscope. Furthermore, it should benoted that the ridge is a transient structure, present for about 1 h during the trans-formation and is only detectable in remnant form in the fully transformed swimmersupon which other workers have focused most attention. While the appearance of theridge might vary depending on exact culture and transformation conditions used bydifferent laboratories, our studies of several strains and a variety of transformationbuffers indicate that ridge formation is not contingent upon a limited set of conditions.

Our analyses show that the ridge is enriched in microfilaments and suggest thatthese are arranged in the form of a network. The heavy meromyosin subfragment-i(S-i) labelling of the microfilaments in cytoskeletons demonstrates that the filamentscontain actin. Studies by Isenberg & Wohlfarth-Bottermann (1976) of the endoplasmof P. polycephalum plasmodia raise the possibility that microfilaments may be arte-factually polymerized during cell lysis by the extraction buffers. However, in our


studies microfilaments were still detected under extraction conditions (low Mgconcentrations, presence of glutaraldehyde) that are less favourable to promotingfilament polymerization. Also our S-i-labelling studies reveal a uniform polarity offilaments at the edge of the ridge: it seems unlikely that this result would stem frompolymerization of actin subsequent to disruption of the cell surface. Furthermore, theordered tufts of filaments seen in phalloidin-stabilized cytoskeletons correlate interms of their spacing with the fingerlike stubs present at the edge of the ridge inintact cells (compare Fig. n with Fig. 7 inset). That the actin microfilaments areactually present as a network, and not in a much more ordered array that we havefailed to preserve, seems unlikely since we were able to detect ordered filamentbundles within ridges of early transforming populations.

It seems reasonable to propose that, in vivo, these microfilaments are enrichedwithin the laminar core of the ridge. We are unable to demonstrate convincingly thinfilaments in this region by thin-sectioning methods, even using procedures (e.g.tannic acid, low concentrations of OsO4) shown by others to preserve F-actin duringpreparation for thin-sectioning (Begg, Rodewald & Rebhun, 1978; Maupin-Szamier &Pollard, 1978). This difficulty may be due, in part to the network organization of thesefilaments. The filamentous substructure of the core in thin sections resembles actin-enriched regions of other cell types (Abercrombie et al. 1970; Boyles & Bainton, 1979).

Our evidence that the ridge is a transient structure enriched in a network ofmicrofilaments underscores its similarity to motile regions of other cell types (e.g.platelets, Nachmias, 1980; leukocytes, Boyles & Bainton, 1979); fibroblasts, Lazarides& Revel, 1979). In particular, the ridge resembles the lamellipodia of fibroblastseven with respect to such fine details as the presence of fingerlike stubs at its edge anda similarly oriented, uniform polarity of microfilaments (Small & Celis, 1978;Lazarides & Revel, 1979). One major difference between these two structures mustbe noted; lamellipodial extension is induced by substrate contact (Abercrombie et al.1970), while our studies provide clear evidence that ridge formation in amoebo-flagellates is independent of this condition. If substrate adhesion proves to be anintegral part of the mechanisms for lamellar expansion, as has been suggested by some(Huxley, 1973; Small & Celis, 1978; Allen, 1980), the mechanisms underlying ridgeassembly must proceed by different means. Another difference between fibroblastlamellipodia and the amoeboflagellate ridge relates to their respective motility. Thelamellipodium undergoes ruffling in a direction parallel to its advancing front(reviewed by Trinkhaus, 1976). We have reported that, in living cells, the ridge showsdistinct undulatory movements in a direction parallel to its long axis.

The specific localization of actin, an array of microtubules and a flagellum relativeto the ridge raise the possibility that one or more of these structures plays a directmotile role in ridge movement. For example, MTA 5 could function as a smallaxostyle. Although cross-bridges were never observed by us or by other investigators(Wright et al. 1979), the stability of the array under the harsh extraction conditionsused in the latter investigation (Wright et al. 1979) may reflect intertubule linkage.The bends we see within MTA 5 are reminiscent of those in axostyles (Langford &Inoue, 1979). The significance of these bends is presently unclear, particularly


because individual microtubules also appear curved in our preparations of cyto-skeletons. Moreover, undulations occur in posterior portions of the ridge where atmost only two microtubules of MTA 5 have been detected in cytoskeletons or inserially sectioned intact cells (e.g. see Fig. 9). The role of these microtubules inmovement would be clarified by directly mapping the distribution of MTA 5 relativeto ridge undulations that have been preserved in critical-point dried cytoskeletons.

Both the extensiveness and waviness of the laminar core (Fig. 9) are features con-sistent with either a motile or a passive cytoskeletal role of actin in ridge movement.To distinguish between the alternatives, it will be important to determine if myosinis present within the ridge and to examine structural rearrangements of the ridgecytoskeleton that might be produced by MgATP-containing buffers.

With respect to the posterior flagellum, it is of interest that in cells possessing anundulating membrane (e.g. sperm, Swan, Linck, Ito & Fawcett, 1980; certainprotozoa, Holwill, 1966), the association of a flagellum with this motile structure iswell-documented. Further, Swan et al. (1980) have shown that undulatory movementscorrelate with flagellar activity. Any hypothesis attributing a similar role to theposterior flagellum of amoeboflagellates must take account of the fact that, whereasin these other organisms the flagellum interacts with a major portion of the undulatingmembrane either by adhering externally or by internalization, in P. polycephalum theposterior flagellum occupies only a small portion of the ridge's surface (see Fig. 6),and thus can be expected to have little effect on ridge motility. In this context, itshould also be noted that observations of living cells have led us as well as others(Aldrich, 1968) to question whether the posterior flagellum is actively motile. Theeffect of flagellar activity on ridge movement may be clarified through investigationsof another amoeboflagellate, Echinostelium minutum, which contains a ridge-likestructure (our unpublished observations) but which lacks a posteriorly directedflagellum (Haskins, 1978).

Several features of this newly recognized structure make it a valuable system forinvestigations of the structural basis of cell motility. The ridge forms reproducibly insynchronous transforming populations. Ridge motility is characterized by distincttypes of surface deformations that propagate unidirectionally. Microtubule- andactin-containing components are associated with the ridge in a regular manner. Theextensiveness of the laminar core of microfilaments suggests that actin plays animportant motile or cytoskeletal role in this system.

We wish to thank Drs J. D. Robertson, M. K. Reedy, R. B. Nicklas and S. Miller foraccess to their laboratory facilities. We are grateful to Drs D. N. Jacobson and D. Kubaifor many valuable discussions, technical advice and suggestions regarding the preparation ofthis manuscript. We also thank Drs S. Counce and A. Magid for their careful reading of themanuscript.

This work was supported by grant nos. 2-RO1-GM-2O141 and T32-GM-07184 from theNational Institutes of Health and by NSF grant 43510.


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IDENTIFICATION OF A MICROFILAMENT- ENRICHED, MOTILE … · 2005-08-21 · IDENTIFICATION OF A MICROFILAMENT-ENRICHED, MOTILE DOMAIN IN AMOEBOFLAGELLATES OF PHYSARUM POLYCEPHALUM KATHRYN