J. Cell Sci. 37, 169-180 (1979)Printed in Great Britain © Company of Biologists Limited 1979

MOTILITY OF THE LIMULUS BLOOD CELL

PETER B. ARMSTRONGDepartment of Zoology, University of California, Davis, CA 95616, U.S.A. andMarine Biological Laboratory, Woods Hole, MA 02543, U.S.A.

SUMMARY

The sole cell type (the amoebocyte) found in the coelomic fluid of the horseshoe crab,Limulns polyphevrus can be stimulated to become motile by extravasation or trauma. Motilitywas studied using time-lapse microcinematography and direct microscopic examination of cellsin tissue culture and in gill leaflets isolated from young animals. Phase-contrast and Nomarskidifferential-interference contrast optics were employed. Both in culture and in the gills, motilecells showed 2 interconvertible morphological types: the contracted cell, which was compactand rounded and had a relatively small area of contact with the substratum, and a flattened formwith a larger area of contact. In both morphological types, motility involved the protrusion ofhyaline pseudopods followed by flow of granular endoplasm forward in the pseudopod. Cellularmotility in vivo (in the gill leaflet) was morphologically identical to that displayed in tissueculture. In culture, motility was unaffected by the nature of the substratum: cells were indis-tinguishable on fluid (peraffin oil) or solid (glass) substrata or on hydrophobic (paraffin oil,siliconized glass) or hydrophilic (clean glass) surfaces. Cells migrated and spread on agarsurfaces. Cell motility was unaffected by high concentrations (100 /tg/ml) of the microrubule-depolymerizing agent colcemid and was abolished by cytochalasin B at 1 /tg/ml.

INTRODUCTION

The active, pseudopod-directed motility of animal tissue cells is important for anumber of processes including embryonic morphogenesis (Trinkaus, 1976), woundhealing (Radice, 1977), inflammation (Marchesi, 1970) and intercellular invasion(Armstrong, 1977a). Most of our knowledge of tissue cell locomotion has come fromstudies of cells in tissue culture migrating on glass or plastic surfaces (Abercrombie,1973; Goldman, Pollard & Rosenbaum, 1976). Indeed, most tissues and organismsare too large and opaque for direct microscopic observation of cell migration in situ.It is, thus, important in those situations where it is possible to observe cell locomotionin vivo to compare motility in vivo with that displayed in tissue culture. Previousstudies have suggested that although motility in vivo and in culture is probablybasically similar, the morphology and locomotion of vertebrate tissue cells can beprofoundly affected by the nature of the substratum in culture (Elsdale & Bard, 1972;Harris, 1973) and marked differences are apparent in comparisons of cells in cultureand the same cells in vivo (Bard & Hay, 1975; Bard, Hay & Meller, 1975). The presentreport presents the results of a study that compared the details of cellular motility ofthe motile blood cells of the horseshoe crab Limuluspolyphemus on a variety of culturesubstrata and in the organism.

Address reprint requests to: Peter B. Armstrong, Department of Zoology, University ofCalifornia, Davis, CA 95616, U.S.A.

170 P. B. Armstrong

MATERIALS AND METHODS

Glassware was rendered endotoxin-free by incubation at 180 °C for 4 h. The motility ofamoebocytes on glass was studied by time-lapse microcinematographic and real-time obser-vation of cells emigrating from aggregates of amoebocytes explanted into thin double-coverslipchambers as described previously (Armstrong, 19776). Limulus serum or sterile, endotoxin-free3 % NaCl (Travenol Laboratories, Deerfield, 111.) buffered at pH 7-8 with 2 mM NaHCO,served as the culture medium. The motile behaviour of amoebocytes was studied in vivo insingle gill leaflets dissected from small (25-40 mm width) individuals. Isolated gill leaflets wereplaced on microscope slides in MBL-formula artificial seawater and a coverslip was placed overthe preparation. The walls of the gill leaflets of young animals are thin and very transparentand allow use of oil-immersion objectives. The amoebocytes remained actively motile forseveral hours in the preparation described above if the coverslip was sealed to prevent evapora-tion. All observations were made at room temperature.

RESULTS

Motility in vitro

The blood of Limulus contains a single cell type, the granular amoebocyte (Fig. 1).Although not adhesive in the intact organism, the cells develop ability to aggregatewith each other to construct multicellular masses ('amoebocyte tissue') followingextravasation. When 1-2-mm fragments of amoebocyte tissue were explanted intothe double-coverslip chamber used in these studies, cells emigrated from the explantonto the glass (Fig. 2). The morphology of the cells during their initial migration onthe glass was as a compact, rounded cell with abundant cytoplasmic granules. Thesecells protruded hyaline, granule-free pseudopods which were thick, with blunt orpointed tips (cf. Figs. 5, 9). Forward movement of the trailing portion of the cell wasaccompanied by the flow of cytoplasmic granules into the pseudopod. The trailingportion of the cell appeared to be constricted, resembling the uropod of vertebratelymphocytes and granulocytes (Fig. 5). Moving cells showed thin processes ('retrac-tion fibres') that connected the uropod with residual sites of attachment to the sub-stratum or the other cells.

After a period of time in culture (hours or days), the contracted cells flattened onthe glass (Figs. 2, 3). The initial flattening of granular cells was reversible, but oncedegranulation occurred, the cells flattened irreversibly (Fig. 2). The reversibly flattenedgranular cells were still actively motile, though less so than the unflattened cells(Fig. 3). Once degranulation and irreversible flattening occurred, the cells showedvery low levels of motile activity of lamellar processes and translocated either veryslowly or were completely stationary.

Effects of drugs on motility

The motile amoebocyte was immobilized by cytochalasin B at 1 /tg/ml but wasinsensitive to colcemid at 1, 10 and 100/tg/ml. The potency of the colcemid prep-aration was validated by demonstrating that, consistent with the report of Zimmer-man & Zimmerman (1967), it inhibited the first mitotic division of fertilized Arbaciaeggs at 10 /tg/ml. In colcemid, both the speed of locomotion and the morphology andbehaviour of motile cells was normal (Fig. 4). Granule flow was unaffected.

Motility of the Limulus blood cell 171

Fig. 1. Limulus blood cells in a freshly excised gill leaflet. The oval fully granular cellsin the centre of the field are unactivated wheras some of the cells along the margins ofthe field have already undergone degranulation. One is protruding a pseudopod(arrow), x 1309.Fig. 2. Limulus amoebocytes in culture, showing the 3 morphological states shown invitro: the contracted cell (c), the flattened granular cell (g), and the flattened degranu-lated cell (d). The plane of focus is the glass-culture medium interface. The pseudopodsof the 2 contracted cells lie out of focus, above the substratum (arrows), x 867.

Substrate requirements for motility in vitro

The contracted amoebocyte was capable of migrating and flattening on a variety ofsubstrata that do not support locomotion or spreading of vertebrate tissue cells,including siliconized glass, agar (Figs. 5, 6), and paraffin oil (Fig. 7) with eitherLimulus plasma or 3 % NaCl as the culture medium. Rates of motility on paraffin oilwere similar to those of cells migrating on glass. Amoebocytes also migrated throughthe gel (Levin, 1967) that formed when the contents of the amoebocyte granules werereleased from the cells into an environment containing bacterial endotoxin. (In mostexperiments, the cultures were maintained free of endotoxin to prevent the formationof gel.)

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Fig. 3. Sequential views of the same field of a culture containing principally flattenedgranular amoebocytes at 5-minute intervals. Some of the more rapidly moving cells areidentified by number in successive pictures. The contracted cells (1,2) move morerapidly than the flattened cells (3, 4). x 517.

Motility of the Limulus blood cell 173

Motility of cells in vivo

The active motility of the blood cells could be observed in situ in isolated gillleaflets maintained on a microscope slide (Fig. 8). The blood cells in the isolated gillswere best observed with a Plan 100/1.25 objective and Nomarski differential inter-ference-contrast optics. Under these conditions, the details of motility could beobserved almost as well as with cells in culture. In the intact organism, nearly all ofthe amoebocytes are in the immotile, unactivated state. Amoebocytes were observedin the intact animal as they circulated in the capillaries of the abdominal spines usingtransmitted illumination. Such cells were oval, lacked pseudopods and were non-adhering. This was also true of the blood cells contained in the gill leaflets just afterisolation (cf. Figs. 1, 8) or in gills of animals that had been preserved by injecting theintact animal with 4% formaldehyde-seawater prior to dissection of the gill leafletsfor observation under the microscope. Apparently the trauma of dissection of the gillleaflet induced a variable fraction of the blood cells in the gills to transform into themotile state. Some of these cells apparently were stimulated to aggregate as well,since nodules of amoebocyte tissue were present in the isolated gills. The transformedamoebocytes in isolated gills displayed the full range of morphological states thatwere present in vitro (e.g. the compact, rounded state (Fig. 9), the flattened granularstate, and the flattened degranulated state (Fig. 10)). The only discernible differencesin morphology were those observed with the degranulated cells: although degranu-lated cells often were as flattened as were degranulated cells in vitro (cf. Fig. 10), somewere spindleshaped, being attached only at either end of the cell (Fig. 11).

In the isolated gills, the transformed granular amoebocytes migrated over the innersurfaces of the walls of the gill leaflets and over the vertical pillars (Fig. 8) thatprevent upper and lower gill surfaces from coming together. Morphologically, cellmotility was identical to that observed in vitro, being accomplished by the protrusionof hyaline pseudopods followed by the flow of granular cytoplasm into those pseudo-pods (Fig. 9). The amoebocytes migrating in the isolated gill leaflet could be viewednot only from above but also from the side when they moved over the pillars thatkeep the lumen of the gill leaflet open. Observation of cells from the side revealed thathyaline pseudopods often were protruded initially from the antero-dorsal surface intothe fluid-filled space of the gill and only later were lowered to contact the substratum(Fig. 12).

DISCUSSION

Several processes are involved in the locomotion of animal tissue cells, includingextension of the pseudopods (Abercrombie, Heaysman &Pegrum, 1970 a), retractionof the trailing portions of the cell (Trinkaus, Betchaku & Krulikowski, 1971), themaking and breaking of adhesions with the substratum (Abercrombie & Dunn, 1975),and a continual reorganization of both plasma membrane and the cytoskeleton(Abercrombie, Heaysman & Pegrum, 19706). During locomotion, the Limulusamoebocyte extended pseudopods as hyaline, granule-free processes that were later

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Motility of the Limuhis blood cell 175

invaded by granule-filled endoplasm. Endoplasmic flow was involved with forwardmovement of the trailing portions of the cell. The existence of extensive cytoplasmicflow distinguishes the motility of the amoebocyte from that of many of the vertebratetissue cells that have been extensively studied in tissue culture, in which cytoplasmicflow is an insignificant feature of locomotion (Abercrombie, 1961). In this regard, theLimulus amoebocyte resembles the vertebrate leukocyte (Armstrong & Lackie, 1975;Zigmond, 1978). The lack of sensitivity of locomotion of the amoebocyte to pharmaco-logical doses of antimicrotubule agents is shared by vertebrate leukocytes (Band-mann, Norberg & Rydgren, 1974; Edelson & Fudenberg, 1973; Ramsey & Harris,1973). Vertebrate fibroblasts lose the polarized distribution of lamellar pseudopodsupon exposure to anti-microtubule agents with a concomitant reduction in locomotionof the cell (Goldman, 1971; Vasiliev et al. 1970).

Motility of the Limulus amoebocyte was surprisingly unaffected by the nature ofthe substratum. The morphology, spreading and motility of vertebrate tissue cellsare markedly different on hydrophobic and hydrophilic surfaces (Harris, 1973;Ivanova & Margolis, 1973) and these cells are unable to spread and move on fluidsurfaces such as paraffin oil (Harris, 1973). In contrast, the Limulus amoebocyteappeared to be unaffected in morphology or motility by any of the surfaces used in thepresent study.

Systematic comparisons of the motility of tissue cells under various in vitro andin vivo circumstances are very few in number, due in large measure to the limitednumber of situations where cells can be observed in vivo. Certain embryonic tissues,including whole teleost (Armstrong, 1978; Trinkaus, 1973), and sea-urchin (Gustafson& Wolpert, 1967) embryos, the chick embryo cornea (Bard & Hay, 1975), and thefrog tadpole tailfin (Billings-Gagliardi, 1977; Clark, Clark & Rex, 1936; Radice, 1977)have proven thin and transparent enough for the study of cell motility in vivo. Inadult organisms, the test cells of the tunicate (Izzard, 1974) and the motile blood cellsin the wing veins of the cockroach (Arnold, 1961) have been subjected to study. Onlyin the study of Bard & Hay (1975) have detailed comparisons been made of cellbehaviour in culture and in vivo. They reported that the motility of corneal mesen-chyme cells differed in the 2 situations. Cells which showed broad leading lamellaewhen migrating on glass exhibited thin pseudopods in the cornea. Ruffling activitywas readily apparent in vitro but was absent in situ.

Fig. 4. Emigration in vitro of cells from an amoebocyte tissue fragment in the presenceof ioo/tg/ml colcemid. Both the numbers of cells emigrating, the distances ofmovement and the morphologies of individual cells were the same as control culture.X259-Fig. 5. Contracted cells migrating on agar. The cells are indistinguishable from cellsmigrating on clean glass, u, uropod. x 1309.Fig. 6. Flat granular cell on agar. x 1309.Fig. 7. Cells migrating on siliconized glass and paraffin oil. A large droplet of paraffinoil occupies the centre of the field. Cell morphology is identical on the 2 substrata.Cells migrated freely and without pause from glass to oil (arrows) and from oil to glass.X206.

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Motility of the Limulus blood cell 177

The present study compared the motility of the Limulus amoebocyte in vivo (in theisolated gill leaflet) with its performance on a glass surface in tissue culture. The rangeof cellular morphologies was the same in culture and in the gill leaflet. The obser-vations indicated further that at the morphological level, the motility of the compact,rounded amoebocyte was indistinguishable in the 2 situations. Thick hyaline pseudo-pods were protruded from the anterior-dorsal surface and subsequently were loweredto contact the substratum. After protrusion, the pseudopods were invaded by endo-plasm. Active cytoplasmic streaming accompanied motility in both situations.Observations of cells in the gill leaflet provided an opportunity to observe cells in sideview. It was observed that pseudopodial protrusion was often accomplished withoutcontact between pseudopod and substratum, with these pseudopods being loweredto bring them into contact with the substratum only after full extension had beenaccomplished. This observation is interesting because it can account for the abilityof cells to overlap each other even though contact paralysis of pseudopodial extensionapparently operates in these cells (Armstrong, 19776) and because it suggests that theforces that extend the pseudopod are not those of a passive spreading (haptotaxis)onto local areas of high-energy surface (Carter, 1967, 1970), since the pseudopod wasextended into the plasma-filled space and was not in contact with the substratum.

This study was supported by Cancer Research Fund of the University of California and NSFGrant No. PCM77-18950.

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