contractile filopodia and in vivo movement in the tunic of the

J. Cell Sci. 15, 513-535 (1974) 513

Printed in Great Britain

CONTRACTILE FILOPODIA AND IN VIVO CELL

MOVEMENT IN THE TUNIC OF THE

ASCIDIAN, BOTRYLLUS SCHLOSSERI

C. S. IZZARDDepartment of Biological Sciences, State University of New York,Albany, New York 12222, U.S.A., and Marine Biological Laboratory,Woods Hole, Massachusetts 02543, U.S.A.

SUMMARY

The in vivo movement of one class of cells in the tunic of the ascidian Botryllns schlosseri hasbeen analysed using differential interference optics and time-lapse cinematography. Long (upto 200/tm), thin (0-35-0-5 /tm diameter) filopodia radiate from the cell-body into the matrixof the tunic. Movement of the cell-body consists of a series of short, jerky displacements withfrequent changes in direction between successive displacements. The net displacement of thecell may be extremely small when the displacements are short and frequently change direction,or considerable when successive displacements show a persistence of direction (up to 114/tmin 60 min). Deformation of the elastic cuticle covering the tunic at points of attachment of thefilopodia has been used to record qualitatively changes in tension in the filopodia. Correlationof the changes in tension with changes in length of the filopodia and movement of the cell-bodyhave permitted the following conclusions. Active contractions of filopodia (i.e. increase intension during shortening) stretch and move the cell-body. These movements exert a force ontrailing or opposing filopodia. Relaxations of filopodia (i.e. decrease in tension during lengthen-ing) result in small movements of the cell-body due to the recoil of tension in the cell-body andopposing filopodia. The position of the cell-body in space at any one instant in time is thereforethe resultant of the forces developed in all the filopodia. Movement results from unilateralmodulation of the tension developed in the filopodia. Active contractions play a more signifi-cant role in movement than relaxations.

INTRODUCTION

The number of in vivo studies that provide an insight into the changes in cell shapeunderlying the movement of a cell through an intact organism is limited. In contrast,the motility of cells derived from multicellular organisms has been studied extensivelyunder in vitro conditions. The factor limiting in vivo studies has been the inherentopacity of the organism. The extant work centres upon a limited number of trans-parent tissues such as the tail fin of amphibian larvae (Clark, 1912; Clark & Clark,1920, 1925, 1930; Speidel, 1933, 1935), the sea-urchin larva (Gustafson & Wolpert,1961, 1967) and the embryo of Fundulus (Trinkaus & Lentz, 1967; Trinkaus, 1973).The present work analyses the motility of one class of cells in the tunic of the ascidianBotryllus schlosseri (Pallas). The ascidian tunic is an ideal tissue for optical studies ofcell motility in vivo because the tissue lies external to the body of the organism and ishighly transparent in many species.

Botryllus was selected for study because the colony shows a marked tendency to33 C E L 15

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spread as a thin sheet over the substrate. The newly metamorphosed larva providesthe most suitable preparation for high-resolution optical studies; during meta-morphosis, the vascular ampullae of the larva expand radially across the substratedrawing the tunic into thin web-like sheets between pairs of ampullae (Fig. 7, p. 531).Using these preparations, it was possible to obtain high-resolution, high-contrastimages of the tunic cells with differential interference optics. The success of thisoptical technique for in vivo studies results from the small contribution provided byout-of-focus objects to the contrast of the image. Thus it is possible to opticallysection relatively thick specimens (see Allen, David & Nomarski, 1969; Padawer,1968; Metuzals & Izzard, 1969).

Initial observations demonstrated that the cells in the tunic of Botryllus can bedivided into 2 classes on the basis of their motile behaviour and associated pseudo-podia. One class, here termed filopodial cells, is characterized by long filopodiaradiating from the cell-body. The other class of cells can be broadly described as'amoeboid'; they move by the eruption of relatively small hyaline pseudopodia andby cytoplasmic flow towards, or into, the bases of the pseudopodia. The present studyis concerned solely with the filopodial cells. It aims to describe briefly the cell morpho-logy, to characterize the extent and pattern of cell movement, but primarily to examinethe forces and changes in cell shape that produce the movement. The analysis hasdemonstrated that extended filopodia actively contract and that the position of thecell-body in space at any one instant in time is the resultant of tensions developed inthe radiating filopodia. A preliminary report of this work has been presented elsewhere(Izzard, 1971).

MATERIAL AND METHODS

Material

Sexually mature colonies of Botryllus schlosseri (Pallas) were collected from the dock in EelPond, Woods Hole, and placed in large finger bowls supplied with a slow flow of seawater.Coverslips (no. i-J-) were suspended vertically around the periphery of the dish. As larvae werereleased, they attached in significant numbers to the coverslips. Within 2 h the coverslips couldbe removed. Then they were supported at an angle, with the metamorphosing zooids facingdown, in 450 slots cut into f-in. (0954-cm) Tygon tubing. The supported coverslips were placedin the sea table until required. Arrangement of the zooids on the lower surface of the coverslipminimizes the accumulation of bacteria and detritus on the glass surface over which the tunicspreads, thereby maintaining the greatest optical clarity of the preparations. The specimens areready for use within 4 h (Fig. 7) but can be used for up to 7 days.

Microscope preparations

The coverslip bearing the young zooid was inverted over a large coverslip (no. i£) and sup-ported on spacers cut from no. 2 coverslips or thin slides. The 2 coverslips and spacers weresealed together with a 1:1:1 mixture of Vaseline, lanolin and paraffin wax leaving 2 narrowopenings, one at either end. Seawater was exchanged through these openings every 1-2 h.Such preparations remained healthy for at least i o h o n the microscope at a room temperatureof 21-22 °C.

Contractile filopodia and cell movement 515

Microscopy and filming

The work was performed with a Zeiss Photomicroscope II equipped with differential inter-ference optics, and 40/0-85 achromat oil and 100/1-25 planachromat oil objectives. Extinctionfactors for both objectives with these preparations were routinely 400. The light source waseither tungsten or mercury arc respectively for the lower or higher power objectives. A wide-band green interference filter and heat reflexion filter (Calflex) were used with both light sources.The following films were employed: 35 mm, Adox KB-14 processed in Diafine, or KodakSO-410 processed in HC-110; 16 mm cine -negative, Recordak 7460 and 7457 (Eastman KodakCo.), respectively, for the lower- and higher-power work, both processed in Diafine to enhancethe speed and retain fine-grain characteristics of the film. Cine films were taken with anArrifiex 16 S camera wall-mounted above a Zeiss panchratic projector lens and driven by aSage Arri Animation Motor. An auxiliary specimen-shielding shutter was used for framingrates below 60 frames per min. Cine films were taken at 3, 12, 20 and 30 frames per min withthe lower-power objective. Rates of 3 and 12 frames per min were most useful in analysing thepattern of cell movement. Framing rates of 60 and 120 frames per min were used with the higher-power objective, the faster framing rate proving to be most useful for relating filopodial activityto cell movement. The exposure time in all cine films was 025s.

Film analysis

The films were projected with a Photo-Optical Data Analyzer Model 224A (L-W Photo Inc.,Van Nuys, California) via a 450 mirror on to a tracing desk. Cell displacement was analysedfrom the lower magnification films projected at a final magnification of 2000 x . The 6-minanalysis interval (see Fig. 1, p. 517) was chosen to give maximum detail of changes in directionand yet permit recordable movement in slower cells. Filopodial activity was analysed from thehigher magnification films projected to give a final magnification of 2800 times. Cell outlineswere traced at 10- or 15-s intervals and the position of the cell recorded relative to arbitraryreference axes on the tracing screen. In Figs. 3-6 (pp. 521-4), selected tracings are reproducedat equal distances along a horizontal axis using the original reference axes. The broken lines,joining common points at successive time intervals, therefore depict the relative displacementsof the tip and base of the filopodium and of the cell-body. The original reference axes areincluded in each figure. Areas for filming were selected midway between well spaced ampullaewhere contractions of the ampullae and zooid would not produce displacements of the tunicas a whole. Films showing such movement were not analysed.

RESULTS

General characteristics of filopodial cells

The tunic of Botryllus is fairly uniformly populated with cells (Fig. 7). The cell-bodies are separated from one another by spaces ranging in width from one to severaltimes their own diameter. The uniformity of the distribution is illustrated moreclearly in Fig. 8 - a typical differential-interference optical section of the tunic. Thecells in Fig. 8 are of the filopodial type, with the exception of 2 amoeboid cells; thisfrequency of the 2 types is typical of the tunic as a whole.

The filopodia are thin, numerous and radiate in all directions from the cell-body.In the plane of one optical section, 15 or more filopodia can arise from one cell-body;therefore this figure represents only a small fraction of the total number of filopodiaper cell. The filopodia are either noticeably straight or follow a gently curving paththrough the tunic. The full length of a filopodium is rarely seen in the plane of a single

33-2

516 C.S.Izzard

optical section but, by continually refocusing, filopodia have been traced for 200 fimfrom base to tip. The filopodia range from 0-35 to 0-5 /tm in diameter. Individualfilopodia appear smooth in outline and are very uniform in diameter throughout theirlength (Figs. 8, 11). Small granularities occur infrequently along the filopodia (Fig. 9)and are difficult to detect by direct observation. However, these granularities becomemore apparent in time-lapse cine films as a result of their movement along the filo-podia. The filopodia seldom branch except close to their origin from the cell-body(Figs. 9, 10). In this region small webs of cytoplasm advance and retract betweenthe bases of branched or closely spaced filopodia.

The shape of the cell in optical section reflects the distribution and orientation inspace of the filopodia. In approximately isodiametric cells the filopodia are evenlydistributed and radiate uniformly in space, their broadening bases giving a scallopedoutline to the profile of the cell (Figs. 8, 9). However, in angulate or elongate cellsgroups of filipodia extend along a few radii from the tapering extensions of the cell-body (Figs. 10, 11).

Filopodial cells are further characterized by the presence of large vacuoles in thecytoplasm. The number of vacuoles ranges from a few to many occupying the bulkof the cytoplasm. In optical section, the vacuoles appear circular or, where closelypacked, polygonal in outline (Figs. 9-11). The vacuolar nature of these cytoplasmicinclusions can be established by reference to the contrast generated in the differentialinterference image. Contrast is directly related to the gradient of optical path differenceacross the object in the direction of shear in the microscope. Therefore gradients ofopposite sign appear as opposite contrast relationships and, by reference to a knownchange in optical path difference in the specimen, the contrast relationships can becalibrated (Allen et al. 1969). Within the thickness of an optical section, the opticalpath of the cytoplasm is greater than the surrounding tunic. Therefore the reversedcontrast of these inclusions relative to the whole cell demonstrates their vacuolarnature.

With the exception of a small centrally placed nucleus (Fig. 9), other cell organellesare noticeably few in number. In cells containing a large number of vacuoles, a smallcap of cytoplasm containing mitochondria can be detected adjacent to the nucleus inthe optical sections.

Characteristics of cell movement

The filopodial cells show little movement when observed directly with 40/0-85 oildifferential-interference equipment. However, when cell movement is speeded up80-320 times by time-lapse cinematography, the filopodial cells display a dramaticmotile activity. The cell bodies, strung between groups of filopodia, exhibit jerkymovements. In some cells the displacements are short with frequent changes in direc-tion, whereas in the same cells during different periods of time, or in other cells, thedisplacements are of greater length and persistence of direction. Graphic analysis ofthe time-lapse films clearly illustrates these features of cell movement. The tracks inFig. 1 represent the displacement of the centre of the cell-body over a period of 90 min.The intervals between successive points on the tracks represent 6 min in real time.

Contractile filopodia and cell movement

Fig. 1. Displacement of filopodial cells in a 16-mm cine frame over a 90-min period.The points represent the position of the centre of the cell-body at 6-min intervals.The outline of each cell-body in its initial position is indicated, and the initiatingpoint of each track is marked by the letter identifying the cell. The broken cell-outlines and tracks indicate that the cell, although visible, was just out of the planeof optical section. The shorter tracks arise from the cell moving out of the frame(e.g. cell p) or out of focus (e.g. cells g, li).

518 C.S.Izzard

The initiating point of each track is marked by the letter identifying the specific cell.The outline of the cell in its initial position is included in the figure so that comparisoncan be made between displacement and cell size (see legend for further explanations).Cells a and o showed virtually no net displacement over the 90-min analysis period;the last point on each track lies within the outline of the cell in its initial position.However, throughout the analysis period the cell-bodies underwent short displace-ments during each analysis interval but exhibited virtually no persistence in directionbetween consecutive analysis intervals. As a result, the cells appeared to oscillateabout a point in space. Other cells (e.g. cells c and n) underwent larger displacementsbut similarly showed very little net displacement in 90 min because again there waslittle persistence in direction between consecutive analysis intervals. Thus these cellsexhibited oscillations of larger amplitude about a point in space. Greater persistencein direction shown in the latter parts of the tracks for cells/, k and / resulted in signifi-cant net displacements in 90 min.

When analysis is continued at 6-min intervals for a period of 7-5 h real time thepattern of movement illustrated in Fig. 1 persists. However, the net linear displace-ment of most cells from their initial positions is shown to be extensive. Fig. 2 depictsthe continued movement of the cells in Fig. 1 over a period of 7-5 h. For the sake ofgraphical clarity this extended analysis has been summarized in Fig. 2 as linear dis-placements over a series of 90-min intervals. Thus the first 2 points for each of thecells a to q summarize the movement depicted in Fig. 1 as a single linear displacement.The second and third points in each track summarize the linear displacement over thesecond 90-min analysis period. In order to give a more complete impression of cellmovement over the 7-5 h period, the frame includes the tracks of cells entering thefield of view either from the sides or by vertical migration into the plane of opticalsection. These cells are identified by the letters r to w. (For further explanation seelegend.)

The extent of directed cell movement over 7"5-h analysis periods is highly variable(Fig. 2). Cell o, which showed little net displacement in the first 90-min period (Fig. 1),likewise showed movement but little persistence in direction throughout the followingfour 90-min periods. Cell a behaved similarly during the first 4 analysis periods, butshowed a greater persistence of direction between subsequent 6-min analysisintervals resulting in a net linear displacement of 38 /tm in the last 90-min period. Thegreater persistence in direction, shown by cells / , k and I in the first 90-min period(Fig. 1), continued throughout the subsequent periods that they remained in the fieldof view and resulted in large net linear displacements. Similarly cells t, v and w,entering the field later in the analysis, exhibited large linear displacements duringthe extended analysis periods (Fig. 2). These displacements again resulted frommarked persistence in the direction of movement between successive 6-min analysisintervals.

The following conclusions have been drawn from these analyses. First, the extentto which individual filopodial cells migrate through the tunic is highly variable.Secondly, all filopodial cells show a high degree of motile activity whether or not thisresults in significant movement through the tunic. Thirdly, directed movement


25 /im

Fig. 2. Displacement of cells in Fig. 1 over a 7'5-h period. The points represent theposition of the cell-body at 90-min intervals. The initiating point is marked by theidentifying letter. Thus for cells a to q the first 2 points summarize, as a single net lineardisplacement, the movement depicted in Fig. 1. The successive points summarize acontinued series of such analyses. Cells r to tv entered the cine frame subsequent tothe first 90-min period. In this figure, the broken lines indicate that the cell eitherentered or left the analysis field by vertical or horizontal migration during a 90-minanalysis period. Segments of the track, which include broken lines, therefore representless than a 90-min period.

520 C. S. Izzard

through the tunic results primarily from persistence in the direction of movement andnot from presence as opposed to absence of motile activity.

Since the extent of movement of the filopodial cells varies widely, estimates havebeen made only of the maximal linear displacement for selected time intervals. Theseestimates do not represent true maximal rates or instantaneous velocities because, asis clear from the above account of movement, the cell may have moved over a muchgreater distance during the time interval than is indicated by the linear displacement.It is important, therefore, that the values be recorded as linear distance per timeinterval and not converted to distance per unit time. The need to express the rates inthis manner is well illustrated by cell c (Fig. i) in which the linear displacement overthe whole 90-min analysis period is no greater than the linear displacement for mostof the 6-min analysis intervals. Maximal linear displacements recorded in this mannerwere as follows: 26 /tm in 6 min; 114/tm in 60 min; 103 /tm in 90 min; and 97 /tmin3h.

Cell movement as a function of changes in cell shape

The basic motile activity of the filopodial cells is a continual small-scale displace-ment of the cell-body in space. This activity results in large net displacements if thereis persistence in direction of the small-scale displacements. Therefore to account forcell movement it is pertinent to look for the changes in cell shape and the forces thatproduce the small-scale displacements. When speeded-up by time-lapse cinemato-graphy these displacements appear to result from an active pull exerted by the filopodia.This pull would require active development of tension in the filopodia. The positionof the cell-body in space at any one instant in time therefore would be the resultantof the tensions developed in all the filopodia. Any imbalance in the distribution oftension in the filopodia, resulting from a unilateral active contraction or relaxation,would cause the cell-body to move. The direction of movement would be towardsfilopodia developing tension 01 away from filopodia undergoing relaxation. Persistencein the direction of movement would result from a continued unilateral change intension.

The appearance of the cells in high-resolution micrographs indicates that the filo-podia are under tension and exert a force on the cell-body. For example, manyfilopodia are straight, appear taut, and at their bases merge into conical projections ofthe cell-body (Figs. 9-11). As described in a preceding section, the shape of the cell-body conforms to the distribution of filopodia; triangular or elongate cells bear groupsof straight filopodia at their apices or extremities (Figs. 10, 11) and the cell-bodyoften exhibits a smooth concave contour between filopodia suggesting a tension curve(Figs. 9-11). However, more direct evidence is required to demonstrate that tensionexists in the filopodia and that a unilateral, active, increase in tension, or a relaxationof tension, results in cell movement. This evidence is presented below and wasobtained by recording the movement of cells lying close to the surface of the tunic.

Active contraction and cell movement. In order to demonstrate that active contractionof the filopodia occurs and results in cell movement, it was necessary to establish thatthe following conditions apply: (1) filopodia must be attached at some point along


their length in order to transmit tension to the cell-body; (2) attached filopodia mustshorten as the cell-body moves; (3) shortening must result from an active contractionand not from a passive elastic recoil of tension imposed on the filopodia; and (4)shortening must occur along the length of the filopodium, and not just at its base asa result of the filopodium being absorbed into the cell-body.

Fig. 3. Active contraction of attached filopodium. A, the vertically oriented filopodiumwas attached at its tip to the cuticle. The portion of the cuticle that presented a sharpprofile in the optical section showed a slight indentation indicating tension in thefilopodium. B, the filopodium has shortened, producing increased indentation of thecuticle and slight stretching of the cell-body, c, the transformations in B have pro-gressed further and an increase in diameter of the filopodium is apparent, D, thecontinued shortening of the filopodium and indentation of the cuticle demonstratethe active nature of the contraction which has now displaced the cell-body as a whole.Interval between tracings 40 8.

Establishment of these conditions required preparations in which the full lengthof the filopodium could be observed. The filopodia of cells lying deep within the tunicwere unsuitable because they are long and seldom lie throughout their length withinthe plane of the optical section. In addition, they show no obvious points of attach-ment to structures in the tunic except for occasional attachments to other filopodia.However, cells lying close to the surface of the tunic provided filopodia that weresuitable for these studies. Short filopodia extend from these cells to the surface of thetunic and, if an area is selected where the surface of the tunic lies perpendicular to theplane of optical section, many of these filopodia lie in the plane of optical section(Figs. 9, 10).

New filopodia arise from the superficial cells, lengthen, and contact the undersurfaceof the cuticle that covers the tunic externally. The cuticle appears as a discretestructure 0-5 /im thick in differential interference micrographs (Figs. 9, 10, 12-14)and exhibits a dense fibrous structure and characteristic papillate surface contour inelectron micrographs (C. S. Izzard, unpublished results). Attachment of the tip of afilopodium to the cuticle is demonstrated by the progressive indentation of the cuticlewith time at the point of contact (Figs. 3, 12-14). The attached filopodia can beobserved to straighten and shorten (Fig. 13). Their decrease in length is accompaniedby movement of the cell-body towards the point of attachment (Figs. 12, 13), by aninitial stretching and subsequent movement of the cell-body (Fig. 3), or simply by

522 C. S. Izzard

stretching of the cell-body (Fig. 14). In each case, a net displacement of the cell-bodyoccurs.

The degree of indentation of the cuticle has been used as a qualitative measure oftension in an attached filopodium since the cuticle and underlying tunic are elasticwithin the range of operative forces. Their elastic property is demonstrated by therecoil of the cuticle to its original profile when contact between a filopodium and theindented cuticle breaks. During the shortening of the attached filopodium and move-ment of the cell-body the indentation of the cuticle increases slightly (Fig. 12) ormarkedly (Figs. 3, 13, 14). Hence tension in the filopodium increases during theshortening. This means that the filopodia are actively generating a force and thereforethat the shortening represents an active contraction. In the examples illustrated inFigs. 3 and 12-14, cell movement could not have resulted from elastic recoil of tensionalready present in the attached filopodia because such a recoil would be paralleled bya reduction of tension in the filopodia and hence regression of the indentation of thecuticle. In fact, when the indentation of the cuticle was observed to recoil, the filo-podia lengthened and the cell-body moved away from the point of attachment of thefilopodia (see Fig. 14 and below).

It has not been possible to demonstrate conclusively in every case examined thatshortening occurs primarily along the length of the filopodium. Structures that wouldprovide suitable reference marks along the filopodium are noticeably absent, and thesmall granularities present on some filopodia undergo centripetal movement irre-spective of whether or not the filopodium is shortening. However, by reference tosmall filopodial branches near the base of a filopodium, shortening can be demon-strated to occur along the length, rather than solely at the base, of the attachedfilopodium (Fig. 12). An increase in the diameter of the shortening filopodium wouldprovide further evidence for a contraction along the length of the filopodium. Whenthe events are speeded-up by time-lapse cinematography, a slight increase in diametercan be observed in contracting filopodia. However, unless the shortening is consider-able (up to 50 % of the original length) the increase in diameter is insufficient to bemeasured accurately or represented graphically. An example in which the increase indiameter was perfectly clear is illustrated in Fig. 3.

These events associated with movement of cells lying close to the surface of thetunic demonstrate that the filopodia form attachments and, through an active develop-ment of tension, contract to pull the cell-body through the tunic. The forward move-ment of the cell-body must transmit tension to and produce a commensurate changein length of and tension in the trailing filopodia. Indeed these filopodia frequentlyappear taut (Fig. 12). However, analysis of a large number of examples in which thecell-body moved away from the cuticle failed to show a clear-cut stretching of theseattached trailing filopodia, i.e. a parallel increase in length and tension. Nevertheless,in the example in Fig. 4 movement of the cell-body away from the cuticle was paral-leled by an increase in tension in the attached filopodium. Although the attachedfilopodium did not stretch, the increase in length of the cell-body and increase intension are interpreted to have resulted from active contraction of the leading filo-podia. The failure of the trailing filopodium to stretch in this example can be explained


in any of 3 ways: the filopodium was in a rigid condition; it was itself undergoingcontraction and therefore could transmit the increasing load imposed by the leadingfilopodia; or its short length did not permit a detectable level of strain (see Discussionfor the latter point). The example, however, does demonstrate that the contractileevents in one set of filopodia are reflected at least in the tension present in opposingfilopodia.

Fig. 4. Increase in tension in trailing filopodium. A, the cuticle was slightly indentedindicating tension in the attached filopodium. B, the cell-body has stretched and movedaway from the cuticle. The attached filopodium has not increased in length buttransmits a greater tension to the cuticle. Interval between tracings, 2 min.

Relaxation and cell movement. Filopodia attached to the cuticle have been used toexamine the extent to which relaxation plays a part in cell movement. Relaxation ofa filopodium would involve a decrease in the developed tension and hence a stretchingof the filopodium by the load imposed through the elasticity of the cuticle and thetension in the cell-body and opposing filopodia. Relaxations of this form occur: forexample, in Fig. 5 the attached filopodium was under tension (Fig. 5 A) and in thecourse of 15 s (Fig. 5B) the indentation of the cuticle regressed, the filopodiumlengthened and the cell-body moved away from the attachment point. However,relaxation of attached filopodia is paralleled more frequently by a recoil of the cell-body (Figs. 6, 14) than by a uniform movement of the cell-body (Fig. 5), most prob-ably because of the resistance to movement offered by the tunic. Nevertheless, in bothcases a net displacement of the cell-body occurs. During relaxation the increase inlength of the filopodium is accompanied by a decrease in diameter. The change indiameter is readily apparent in the time-lapse films, and in the examples in Figs. 5and 6 could be represented graphically.

Other aspects of cell movement. Using the degree of indentation of the cuticle, it hasbeen possible to monitor changes in the tension in attached filopodia and to determinethat contractions and relaxations of the filopodia result in cell movement. The tensiondeveloped in a filopodium can alternately increase and decrease (Fig. 14), and thecycle may repeat many times. However, individual filopodia are not permanent

524 C. S. Izzard

Fig. 5. Relaxation of attached filopodium. A, the indentation of the cuticle indicatesthat the filopodium was under tension, a, the fiiopodium has lengthened, lost tension,and decreased slightly in diameter. This relaxation has permitted the cell-body tomove away from the point of attachment. Interval between tracings, 15 s.

A B C 10 jim

Fig. 6. Relaxation of attached filopodium. A, the attached filopodium was under con-siderable tension as evidenced by the large indentation of the tunic and the drawn-outappearance of the cell-body, B, C, the filopodium lengthened and decreased slightlyin diameter. The cuticle recoiled demonstrating a reduction in tension. This relaxationresulted in release of tension in and recoil of the cell-body. Interval between tracings,IS s.


structures. New filopodia continually form at the surface of the cell-body and extendat an average rate of 16 fim per min. The base of an extending filopodium arisesabruptly from the contour of the cell-body (Fig. 10) and only after the filopodiumhas formed an attachment and developed tension does the base develop into a typicalconical projection of the cell-body. Filopodia retract at an average rate of 9 /im permin. Retraction does not appear to be directly analogous to the contractions describedabove because no increase in diameter occurs. Instead the filopodium appears to besteadily resorbed at its base into the cell-body. Nevertheless, the extension andretraction of filopodia appears to be one factor that is related to the degree of per-sistence of the direction of movement. A phase of prolonged unidirectional movementis often, but not exclusively, accompanied by the extension of many new filopodiain the direction of movement and the retraction of many of the trailing filopodia. Inthis respect there appears to be some integration of filopodial activity in differentregions of the cell.

Movement of cells deep in the tunic. For cells deep within the tunic it is not possibleto perform a definitive analysis of movement comparable to that presented above forthe superficial cells. However, the following observations are consistent with theinterpretation that active contractions of filopodia move these cell-bodies, that suchdisplacements exert a force on trailing filopodia, and that relaxations of fiJopodia alsoresult in cell movement.

Filopodia from one cell frequently cross groups of filopodia from adjacent cells(Figs. 8, 11). When a cell-body advances the filopodia crossing the leading filopodiaare frequently bent towards the moving cell-body either prior to or during its move-ment. This counter displacement of structures lying ahead of the moving cell isindicative of an active contraction in the leading filopodia. Similarly, filopodia crossingtrailing filopodia are frequently bent in the direction of cell movement. In this casebending occurs at or after, but not prior to, the onset of movement of the cell-bodyindicating that the contractile tension in the leading filopodia is transmitted to thetrailing filopodia and the tunic. The same bent filopodia, whether they intersectleading or trailing filopodia, often recoil to their original positions. These recoils areparalleled either by a rapid snapping-back or more rapid advance of the cell-bodyrespectively in the case of intersection with leading or trailing filopodia. Thus relaxa-tion as well as the development of tension plays a significant role in the movement ofcells deep in the tunic.

DISCUSSION

The in vivo observations reported in this study clearly demonstrate that active con-tractions of filopodia serve to move cells through the matrix of the tunic in Botryllus.Since the filopodia radiate in 3 dimensions from the cell-body, the position of thecell-body in space at any one instant in time will be the resultant of forces developedin all the filopodia. The observations demonstrate that active unilateral changes in thetension developed by the filopodia result in movement of the cell-body. These changestake the form of either contractions or relaxations.

526 C. S. Izzard

The mechanism of cell motility elucidated in this work provides a definitive exampleof a fundamental mechanism that is becoming progressively recognized for manytissue cells and some protozoans, namely the extension, attachment and contraction ofa pseudopodium (Gustafson & Wolpert, 1967; Wolpert & Gingell, 1968; Trinkaus,1973; Mast, 1931; Wohlman & Allen, 1968; Watters, 1968). However, among tissuecells there are exceedingly few in vivo examples in which an increase in tension in theattached pseudopodium has been demonstrated to precede, or occur concurrentlywith, pseudopodial shortening and cell movement. The primary and secondarymesenchyme cells of the sea urchin provide the most clear-cut example (Gustafson &Kinnander, 1956; Dan & Okazaki, 1956; Kinnander & Gustafson, i960; Gustafson &Wolpert, 1961). The filopodia of these cells are extraordinarily similar to those in thetunic of Botryllus. In both cases the filopodia are 0-5 /tm or less in diameter and extendat essentially comparable rates (10 fim per min in the sea urchin and 16 /tm per minin Botryllus). The evidence for an increase in tension of the contracting filopodia ofthe sea urchin is essentially the same as that provided here for the tunic cells, namelythe drawing-out of a cone-shaped deformation of the respective substrata at the pointof attachment of the filopodium.

Trinkaus (1973) has described several different modes of movement for the deepcells of the gastrulating Fundulus embryo. One distinct mode involves the extensionof either long lobopodia or flattened cell protrusions and the subsequent shorteningof these structures as the rounded cell-body advances. The tip or small side branchesof the extension remain stationary during the shortening thus providing evidence forattachment at these points. Trinkaus carefully describes the phenomenon as a shorten-ing rather than a contraction of the extensions and, in reviewing the major in vivoexamples of similar movement, justifiably makes the point that one cannot determinefrom existing information whether the shortening results from an active contractionor elastic recoil from a stretched state. The examples to which he refers include themovement of tracheoles by the inferred contraction of filopodia of epidermal cells inRhodnius (Wigglesworth, 1959), the migration of mesenchyme cells from the primitivestreak of the chick by leading filopodia (Trelstad, Hay & Revel, 1967) (both examplesbased on fixed material), the extension of regenerating axons in the larval amphibiantail fin (Speidel, 1933, 1935), movement of retinal pigment cells during sorting-out inmixed aggregates (Trinkaus & Lentz, 1964), and the movement of dissociated spongecells undergoing reaggregation (Sindelar & Burnett, 1966).

In addition to these examples, attention should also be directed towards in vivostudies of cell movement in the tail fin of larval amphibia. Clark (1912), and Clark &Clark (1925) recorded slow but distinct movement of fibrocytes in the growing tail finof various amphibians. The cells are stellate in shape with broad-based processesbranching and tapering into extremely fine processes. They are embedded in an extra-cellular matrix and therefore are somewhat analogous to the filopodial cells inBotryllus. Based on recordings made every few hours, Clark (1912) concluded thatmovement was brought about by the extension on one side and withdrawal on theother side of cell processes with a shift of the main cell mass from retreating intoadvancing processes. He further concluded that the changes accompanying movement


could not result from purely mechanical pulling or pushing. Clark & Clark (1925)confirmed this conclusion. However, Speidel (1935) from time-lapse studies reportedthat the constant movement of the fibrocyte processes caused discernible movement ofintercellular fluids and recorded one instance in which the repeated shortening of aprocess caused a nerve fibre to bend back and forth several times a minute. Botheffects strongly suggest an active contraction of the fibrocyte processes. Recent time-lapse studies on the tail fin of Ambystoma tigrinum and Rana pipiens (C. S. Izzard,in preparation) demonstrate active contractions of the fibrocyte processes. Thesecontractions produce short, jerky movements of the main cell-body that are directlycomparable to the movements exhibited by cells a, c and o in Fig. 1 of this paper. Inthis context, the response of the fibrocytes to injury produced by the injection ofcroton oil into the tail fin is significant (Clark & Clark, 1920). Surrounding fibrocytesmigrate rapidly into the wound with their processes leading, suggesting that a direc-tional extension and contraction of the processes may be largely responsible for therapid movement. Although definite evidence is lacking for active contraction versuselastic recoil in many of these in vivo examples, the following discussion of the role ofrelaxation in the motility of the filopodial cells of Botryllus will serve to point out theminimal potential role of elastic recoils in these examples.

Relaxations were detected as a decrease in the tension present in attached filopodia(Figs. 5, 6, 14). The relaxation was paralleled by a lengthening of the filopodium anda concomitant shortening or more rarely movement of the whole cell-body. Theshortening or movement of the cell-body can be interpreted respectively as elasticrecoils resulting from tension present in the cell-body or opposing filopodia. The netmovement of the cell-body in these cases is very small. The small movements are tobe expected from the following considerations. Tension decreases during an elasticrecoil and shortening will cease when the declining tension equals the forces resistingshortening, namely the resistance of the matrix of the tunic to the movement of thecell-body and the residual tension in the relaxed opposing filopodia. In contrast, itwas noted that the increased tension associated with active contraction is maintainedor even further increased as the cell is stretched or totally displaced (Figs. 3, 12, 13).The maintenance of tension by the shortening filopodium can only result from acontinued active process, not from an elastic recoil. It is not surprising then, as wasfound, that displacements of the cell-body over distances equal to two or more timesits diameter were associated with active contractions of leading filopodia. In thiscontext, active contractions contribute more extensively to the net displacements ofthe filopodial cells than do relaxations coupled with elastic recoils. The same wouldbe expected for other similar examples of cell motility.

The failure to observe clear-cut examples of elastic recoil in attached filopodiarequires consideration since these recoils are implied in the preceding discussion. Inthe classical consideration of the relationship between stress and strain in a structure,strain is expressed independent of the actual length of the structure. However, theactual observed change in length, whether an increase or decrease, will be relateddirectly to the length of the structure. In these studies it was necessary to selectrelatively short filopodia in order to include their point of attachment and the cell-

528 C. S. Izzard

body in the field of view. It may be that the actual shortenings occurring in thesefilopodia were too small to detect. In the same context, the failure to observe a clear-cut example of stretching (Fig. 4) may be related to the shortness of the filopodiasuitable for study.

The filopodia of the tunic cells exhibit the basic properties of muscle, namely theability to develop tension and through shortening to perform work. The slow rate ofdevelopment of tension (Figs. 3, 14) renders them more similar to slow striatedmuscle fibres and smooth muscle fibres. However, a further comparison of the filo-podia with these muscle fibres on the basis of their respective mechanical propertieswould require experimental control of the active state of the filopodia and quantifica-tion of the forces developed (see Axelsson (1970) for discussion of mechanical proper-ties of smooth muscle). The basis for a more feasible line of comparison would be theultrastructural and chemical basis of the development of tension. The ultrastructurewill be reported in a subsequent paper.

Studies of cell motility in the tunic of other ascidians are limited in number. How-ever, since cells with filopodia appear to constitute a basic cell-type in the tunic ofmany ascidians, the mode of cell motility described for Botrylltis is probably commonto many ascidians. For example, Saint-Hilaire (1931) describes, in 18 of the 32 speciesthat he studied, stellate- or spindle-shaped cells bearing thin filopodia. Similarly,Brien (1930) and Peres (1948) in their accounts of the regeneration of the tunic inClaveKna describe the major cell-type as stellate, vacuolate, and possessing long thinfilopodia. Filopodia also have been reported to extend from vanadocytes in the tunicof Ascidia pygmaea (Kalk, 1963) and Phallusia mammillata (Endean, 1961). Thepotential role of these filopodia in motility was not recognized.

Previous accounts, which provide significant details of the motility of tunic cells,deal primarily with amoeboid cells. For example, presumptive tunic cells move bythe eruption of blunt, hyaline lobopodia during their migration across the epidermisfrom the blood sinuses at metamorphosis (Seeliger, 1893; Cloney & Grimm, 1970)and during the regeneration of removed tunic (Brien, 1930; Peres, 1948). Similarly,the tunic cells to which Saint-Hilaire (1931) ascribes motile properties are phagocytesand various types of amoebocytes both exhibiting a range of pseudopodia distinct fromfilopodia. His lack of specific reference to the motility of the stellate filopodial cells isnoticeable. In Botryllus this omission may well be due to the marked difference in therates of movement of filopodial and amoeboid cells (the latter show net linear dis-placements of 46 /im in 6 min as compared with a maximal value of 26 /6m in 6 minfor the filopodial cells). It is worth noting that after migrating through the epidermisby lobopodial activity, the tunic cells in Clavelina put out fine filopodia and assumea stellate shape (Brien, 1930). Changes in the type of pseudopodium exhibited by thetunic cells were looked for, but not found, in this work. In this respect, the filopodialand amoeboid cells of Botryllus are distinct cell-types once within the tunic.

Clearly further studies on the in vivo motility of cells in the tunic of ascidians aremerited on the basis of the range of modes of motility, the optical suitability of thetissue, and the similarity of the tunic to more inaccessible, internal connective tissues.

This work was supported by USPHS grant GM 18853.


REFERENCES

ALLEN, R. D., DAVID, G. B. & NOMARSKI, G. (1969). The Zeiss-Nomarski differential inter-ference equipment for transmitted-light microscopy. Z. wiss. Mikrosk. 69, 193-221.

AXELSSON, J. (1970). Mechanical properties of smooth muscle, and the relationship betweenmechanical and electrical activity. In Smooth Muscle (ed. E. Biilbring, A. F. Brading,A. W. Jones & T. TOMITA), pp. 289—315. London: Arnold.

BRIEN, P. (1930). Contribution a l'dtude de la regeneration naturelle et experimental chez lesClavelinidae. Amth Soc. r. zool. Belg. 61, 19-112.

CLARK, E. R. (1912). Further observations on living growing lymphatics: their relation to themesenchyme cells. Am. J. Anat. 13, 351-379.

CLARK, E. R. & CLARK, E. L. (1920). Reactions of cells in the tail of amphibian larvae to in-jected croton oil (aseptic inflammation). Am. J. Anat. 27, 221-254.

CLARK, E. R. & CLARK, E. L. (1925). The development of adventitial (Rouget) cells on theblood capillaries of amphibian larvae. Am. J. Anat. 35, 239—264.

CLARK, E. R. & CLARK, E. L. (1930). Observations on the macrophages of living amphibianlarvae. Am. J. Anat. 46, 91-147.

CLONEY, R. A. & GRIMM, L. (1970). Transcellular emigration of blood cells during ascidianmetamorphosis. Z. Zellforsch. mikrosk. Anat. 107, 157-173.

DAN, K. & OKAZAKI, K. (1956). Cyto-embryological studies of sea urchins. III . Role of thesecondary mesenchyme cells in the formation of the primitive gut in sea urchin larvae. Biol.Bull. mar. biol. Lab., Woods Hole n o , 29-42.

ENDEAN, R. (1961). The test of the ascidian, Phallusia mammillata. Q. Jl microsc. Set. 102,107-117.

GUSTAFSON, T. & KINNANDER, H. (1956). Microaquaria for time-lapse cinematographic studiesof morphogenesis in swimming larvae and observations on sea urchin gastrulation. ExplCell Res. 11, 36-51.

GUSTAFSON, T. & WOLPERT, L. (1961). Studies on the cellular basis of morphogenesis in thesea urchin embryo. Expl Cell Res. 24, 64-79.

GOSTAFSON, T. & WOLPERT, L. (1967). Cellular movement and contact in sea urchin morpho-genesis. Biol. Rev. 42, 442-498.

IZZARD, C. S. (1971). Cell movement by filopod contraction in the tunic of Botryllus schlosseri.Am. Soc. Cell Biol. Abstracts p. 137.

KALK, M. (1963). Intracellular sites of activity in the histogenesis of tunicate vanadocytes.Q.Jl microsc. Sci. 104, 483-493.

KINNANDER, H. & GUSTAFSON, T. (i960). Further studies on the cellular basis of gastrulationin the sea urchin larva. Expl Cell Res. 19, 278-290.

MAST, S. O. (1931). Movement and response in Difflugia with special reference to the natureof the cytoplasmic contraction. Biol. Bull. mar. biol. Lab., Woods Hole 61, 223-241.

METUZALS, J. & IZZARD, C. S. (1969). Spatial patterns of thread-like elements in the axoplasmof the giant nerve fiber of the squid (Loligo pealii L.) as disclosed by differential interferencemicroscopy and electron microscopy. J. Cell Biol. 43, 456-479.

PADAWER, J. (1968). The Nomarski interference-contrast microscope. An experimental basisfor image interpretation. Jl R. microsc. Soc. 88, 305-349.

PERES, J. M. (1948). Recherches sur la genese et la re'ge'ne'ration de la tunique chez Clavelinalepadiformis Miiller. Arclis Anat. microsc. Morph. exp. 37, 230-260.

SAINT-HILAIRE, K. (193 I ) . Morphogenetische Untersuchungen des Ascidienmantels. Zool.Jahrb. (Anat.) 54, 435-608.

SEELICER, O. (1893). Einige Beobachtungen iiber die Bildung des ausseren Mantels der Tuni-caten. Z. wiss. Zool. 56, 488-505.

SINDELAR, W. F. & BURNETT, A. L. (1967). A time-lapse photographic analysis of sponge cellreaggregation. J. gen. Physiol. 50, 1089-1090.

SPEIDEL, C. C. (1933). Studies of living nerves. II. Activities of amoeboid growth cones, sheathcells, and myelin segments, as revealed by prolonged observation of individual nerve fibresin frog tadpoles. Am. J. Anat. 52, 1-79.

SPEIDEL, C. C. (1935). Studies of living nerves. III . Phenomena of nerve irritation and recovery,degeneration and repair. J. comp. Neurol. 61, 1-80.

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TRELSTAD, R. L., HAY, E. D. & REVEL, J. P. (1967). Cell contact during early morphogenesisin the chick embryo. Devi Biol. 16, 78-106.

TRINKAUS, J. P. (1973). Surface activity and locomotion of Fundulus deep cells during blastulaand gastrula stages. Devi Biol. 30, 68-103.

TRINKAUS, J. P. & LENTZ, J. P. (1964). Direct observation of type-specific segregation in mixedcell aggregates. Devi Biol. 9, 115-136.

TRINKAUS, J. P. & LENTZ, T. L. (1967). Surface specializations of Fundulus cells and theirrelation to cell movements during gastrulation. J. Cell Biol. 32, 139—153.

WAITERS, C. (1968). Studies on the motility of the Heliozoa. I. The locomotion of Actino-sphaerium eichhorni and Actinophrys sp. J. Cell Sci. 3, 231-244.

WIGGLESWORTH, V. B. (1959). The role of the epidermal cells in the 'migration' of tracheolesin Rliodnius prolixus (Hemiptera). J. exp. Biol. 36, 632-640.

WOHLMAN, A. & ALLEN, R. D. (1968). Structural organization associated with pseudopodextension and contraction during cell locomotion in Difflugia. J. Cell Sci. 3, 105-114.

WOLPEHT, L. & GINGELL, D. (1968). Cell surface membrane and amoeboid movement. InAspects of Cellular Motility (ed. P. Miller), Symp. Soc. exp. Biol. 22, pp. 169—198. Cambridge:Cambridge University Press.

{Received 15 January 1974)

Fig. 7. Newly metamorphosed larva typical of those used in the study. Eight, radiallyoriented, vascular ampullae (a) are spreading across the coverslip from the relativelyopaque central zooid (z). The tunic (i), which encloses both zooid and ampullae, isdrawn out into thin web-like sheets between the ampullae. The dark bodies within thetunic are the cell-bodies of the tunic cells. Bright field 6'3/o-i6 planachromat.In Figs. 8-14 the direction of shear in the microscope is indicated by a large arrow inone corner.

Fig. 8. Optical section of tunic showing filopodial cells if) and 2 amoeboid cells (a)lying in the matrix of the tunic (t). The filopodia (small arrows) vary greatly in lengthbut not in diameter. They extend through the matrix of the tunic to interweave withthose from adjacent cells. The amoeboid cells (a) lack prominent filopodia, but developsmall conical or irregular hyaline pseudopodia. Differential interference optics40/085 achromat.

Fig. 9. Filopodial cell lying close to surface of tunic. The cuticle (c) forms a discretestructure at the surface of the tunic. Two of the filopodia branch near their bases(small arrows). Small granularities are present on the vertical branched fuopodium. Thecell-body is packed with polygonal vacuoles (v) and contains a small central nucleus (n).The refractile granules in the tunic are unidentified. Differential interference optics100/125 planachromat.


3-1-2

532 C. S. Izzard

Fig. io. Filopodial cell showing close correlation between the shape of the cell-bodyand distribution of straight, taut filopodia. The cell is approximately triangular inoutline. From one corner a pair of taut filopodia extend to and are attached to thecuticle (c). From the apex a single filopodium branches (small arrow) and extendsinto the tunic. Two straight filopodia run from the third corner. The filopodiummarked (/) was extending when the photograph was taken. Note that it arises abruptlyat its base from the contour of the cell-body in contrast to the other taut filopodia thatarise from small conical projections of the cell-body. The cytoplasm is densely filledwith polygonal vacuoles (v). Differential interference optics 100/1-25 planachromat.

Fig. 11. Group of filopodial cells lying deep within the tunic. Although attachmentsof the filopodia are not apparent, the shape of the cell-bodies again clearly reflects thedistribution of filopodia. The filopodia of adjacent cells interweave without anyindication of mutual adhesion. Note the smooth outline and uniformity in diameter ofthe filopodia. Vacuoles (v) are present in the cells. Differential interference optics100/125 planachromat.

534 C. S. Izzard

Figs. 12,13, 14. Three sets of timed 35-mm photographs illustrating the relationshipbetween cell movement and changes in length and tension of attached filopodia. Thenumbers state seconds elapsed since the first photograph in each set. Within the planeof optical section, filopodia extend from the cell-bodies to the cuticle. The latter runstransversely or obliquely across the top of each set. Differential interference optics100/1-25 planachromat.

Fig. 12. Zero seconds: the cuticle is slightly indented at its point of contact withthe extended filopodium. 30 and 60 seconds: the indentation of the cuticle increasesslightly as the cell-body moves towards the point of attachment of the shorteningfilopodium. Reference to the small projection on the filopodium (arrow) demonstratesthat shortening occurs along the lengtli and not solely at the base of the filopodium. Thedirecton of movement of the cell-body is not parallel to the long axis of the in-focusfilopodium but appears to follow the resultant that would be expected from the arrayof in-focus and slightly out-of-focus leading filopodia. The trailing filopodia appeartaut.

Fig. 13. Zero seconds: a group of 3 filopodia extend to an indentation of the cuticle.The indentation is primarily associated with the right and centre filopodia. The leftfilopodium is distinctly flexed. 30 and 60 seconds: the cell body has moved towards thecuticle, the indentation increased and all filopodia have shortened. The left filopodiumhas become noticeably straight and a pronounced indentation of the cuticle has formedat its tip.

Fig. 14. Zero seconds: a pair of straight filopodia extend to an indentation of thecuticle. 100 seconds: the cell body has elongated as the filopodia shortened and theindentation increased. 140 seconds: the cell-body has recoiled as the filopodia lengthenedand the indentation of the cuticle regressed. This cycle was repeated several times bythe same filopodia.

contractile filopodia and in vivo movement in the tunic of the

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