Mitosis in Amoebae of the cellular slime mold (Mycetozoan) Protostelium mycophaga

Europ. J. Protisto!' 31, 148-160 (1995) May 26,1995

European .Journal of

PROTISTOLOGY

Mitosis in Amoebae of the Cellular Slime Mold (Mycetozoan) Protostelium mycophaga Bruno Guhl ' and Urs-Peter Roos Institut fOr Pflanzenbiologie, Universitat ZOrich, ZOrich, Switzerland

SUMMARY We investigated mitosis in amoebae of Protostelium mycophaga by video microscopy of live cells, by indirect immunofluorescence with antibodies against tubulins, and by transmission electron microscopy of ultrathin sections. Amoebae in interphase usually contain two microtubule centers (MCs) on opposite sides of the nucleus, from which microtubules (MTs) radiate into the cytoplasm. During prophase these MTs shorten to form two asters between which the mitotic spindle develops during prometaphase. Concomitantly, the nucleolus fragments, the numerous small chromosomes orient amphitelically in the spindle and congress to the spindle equator, and the asters diminish further until metaphase. The spindle is open and acentric, but with complex spindle pole bodies. Each sister-chromatid is attached to a single MT by a tiny, layered kinetochore. During anaphase and telophase, asters develop anew and enlarge to become the elaborate MT cytoskeletons of the daughter cells. Anaphase lasted 2 min on average (s.d. = 0.6 min, n = 4), during which the chromosomes moved poleward with a mean velocity of 4.0 urn/min (s.d. = 0.8 urn/min, n = 5). The intermingling of kinetochore MTs and the numerous non-kinetochore MTs allows for a sliding interaction between them, but depolymerisationdriven chromosome movement is also possible. The spindle elongated at a mean rate of 5.9 urn/min (s.d. =2.2 um/min, n =5), and the mean elongation factor was 2.4 in live cells. In immunofluorescence preparations the longest spindles were 3.5 times longer than the average metaphase spindle. Spindle elongation thus requires the growth of interzonal MTs that assemble as several bundles from an ample pool of tubulin. At the end of telophase the nuclear envelope is reconstructed from membrane vesicles and flattened cisternae that appose to the masses of decondensed chromosomes and nucleolar material.

Introduction The structure of the mitotic spindle in lower eucaryotes is remarkably diverse compared to higher plants and animals [15,21,54], which raises questions about the universality of the mechanisms of genome segregation [25, 35] and about the origin and evolution of mitosis [15, 21]. To judge from early light microscopic studies [4] mitotic diversity is even greater, calling for detailed investigations with modern methods that may well reveal some unorthodox types of nuclear division. 1 Current address: University Hospital, Zurich, Department of Pathology, Division of Cellular and Molecular Pathology.

0932-4739-95-0031-0148$3.50-0

Our systematic approach to the study of mitosis in cellular slime molds [14, 38, 42-45] has been to explore not only the architecture of the mitotic spindle, but also its dynamics in these lower eucaryotes with both closer and distant phylogenetic relationships [5, 8, 32, 51]. Whereas mitosis in Dictyostelium discoideum and Polysphondylium violaceum, two closely related dictyostelid cellular slime molds [32, 36], is known in great detail [17, 28, 31, 38, 42, 43], the amoeboflagellate Planoprotostelium aurantium is the only protostelid cellular slime mold in which nuclear and cell division have been studied in some detail [50]. Scant and preliminary observations [15, 50] indicate that mitosis in Protostelium mycophaga [33] is similar, although some differences must obviously © 1995 by Gustav Fischer Verlag, Stuttgart

Mitosis in Protostelium mycophaga . 149

occur, as this species is obligatorily aflagellate [32, 51]. Our ultrastructural observations and the additional information we gained from immunofluorescence and an analysis of video recordings of mitosis in live amoebae show that nuclear division in this so-called primitive eucaryote is remarkably similar to mitosis in higher a.nimal cells. This conclusion raises interesting quesnons about the evolution of mitosis and mitotic mechanisms. Material and Methods

Organisms and Cultures J?r. E.W. Spiegel, Department of Botany and Microbiology, University of Arkansas, Fayetteville, kindly supplied initial cultures of P. mycophaga. We maintained stock cultures with th~ yeast Rhodotorula mucilaginosa (also supplied by Dr. Spiegel) as food organism in Petri dishes on an agar medium we modified from Bonner's [6] formula by reducing peptone and glucose to 1120 of the original concentration. After sporulation the agar cultures were stored at ca. 4°C. Fresh agar cultures were set up every 6-8 weeks by inoculation with a piec~ of agar of c~. 1 em- that contained numerous fruiting bodies, or by pounng 1-2 ml from a liquid culture into a nutrient Petri dish. To obtain high cell numbers, we grew amoebae in liquid medium [50]. The inoculum for 30 ml of medium in a 200ml Erlenmeyer flask was a densely grown piece of agar or 1-5 ml of a log-phase or stationary liquid culture. Cultures were shaken on a reciprocal shaker at 100 cycles/min at 18-20°C with artificial lighting during the day.

Immunofluorescence Amoebae from liquid cultures were harvested and largely separated from the food organisms by centrifugation in fresh medium. Four ml of an exponentially growing culture (1622 h) were centrifuged for 50 sec at 350 g, the cells were resuspended in fresh medium and centrifuged again for 5-10 sec at 650 g, a step that was repeated once. The amoebae were seeded onto Teflon~masked "Microprint" glass slides (Dynatech, Embrach, SWItzerland) in a moist Petri dish and left for 5-10 min at room temperature to settle and attach. .Amoebae were permeabilized, prefixed, and incubated w~th the monoclonal anti-tubulin antibody TATl [48) and "':Ith ~n FITS=-labelled secondary goat anti-mouse IgG (Nordie, Biogenzia Lemania, Lausanne, Switzerland) as described [14]. Cells ",:ere stained for DNA with DAPI [22] at 0.04 Ilg! ml TBS or with Hoechst 33258 [53] at O.4Ilg!ml in McIlvaine buffer, pH 7.4 and embedded in a 1 + 1 mixture of Gomori phosph~te ~uffer [10] and glycerol containing 1 mg!ml p-phenylenediamine [18]. Coverslips were placed on the slides sealed with nail polish, and the preparations were stored in the dark at -20°C, unless examined at once. The preparations were examined with a Zeiss Photomikroskop II equipped for epifluorescence and phase contrast (d. [14,40,41]). A 100x 1.3, N.A. Neofluar oil immersion objective was used throughout. Microphotographs were exposed on. Ilford XP-l 400 ASA film, which was developed commercially by the C-41 process.

Live Observations and Video Microscopy Amoebae were harvested as described (see the section Immunofluorescence), seeded onto coverslips in a moist Petri dish, and left for 5-10 min at room temperature to settle and attach. Observation chambers were prepared as described [41]. Preparations were examined in phase contrast (Neofluar 100x oil, 1.3 N.A. objective) on a Zeiss Photomikroskop III. T~ prevent heat damage and to increase contrast the light was filtered through a Calflex Bl/K2 heat absorption filter (Balzers, Liechtenstein) and a green broad-band interference filter (Zeiss, Switzerland). Images were captured with a Panasonic WV-1850/g video camera (Newvicon tube) and displayed on a Panasonic WV 5411 BIW monitor. Recordings were made on 114-inch U-matic tape with a Sony VO-5800 PS video recorder. Date, time and stop watch were provided by a Panasonic time-date-generator. Mitotic spindles were outlined against the cytoplasm as an area from which mitochondria, digestive vacuoles, and vesi~les were excluded. At the higher resolution of the running Image we could thus determine with confidence the position of the spindle poles as indicated in the photographs. To measure chromosome movement and spindle elongation during playback we switched the monitor to underscan and traced ~he contours of the nucleus, chromosomes and spindle poles m the freeze-frame mode on acetate sheets. The magnification factor was determined on recordings of a micrometer scale. For the diagrams depicting chromosome movement and spindle elongation half the distance between the chromosomes and .half the spind.le len~th were measured. Velocities during the interval of acnve spindle elongation (d. [42]) were computed from the regression lines.

Electron Microscopy of Ultrathin Sections Amoebae attached to coverslips were fixed and embedded as described [14]. Selection of embedded cells by light microscopy and mount.ing for sectioning were carried out according to [41]. We sectioned one amoeba in prophase, three in prometaphase, six in metaphase, one in ana-telophase, and two in telophase. Serial ultrathin sections were cut with a diamond knife on a Reichert OmU-3 or Ultracut ultramicrotome picked up on slot grids coated with formvar and carbon' and stained with uranyl acetate and lead citrate. The sections were examined in a Hitachi HU-llE or H-7000 transmission electron microscope at 50, 60, or 75 kYo

Results

Formation and Breakdown of the Mitotic Spindle Most interphase amoebae have two microtubule centers (MCs) opposite each other and close to the nucleus. In prophase of mitosis (Fig. 1 a-c) the roughly circular contour of the nucleus was as clear as in interphase but the cytoplasmic complex of MTs (CMTC) was red~ced to dense asters consisting of short MTs, many of which coursed from the two MCs along the surface of the nucleus towards the future equatorial plane (Fig. 1 a, c, d, f). The condensed chromosomes were visible in the center of the nucleus prior to the breakdown of the nucl~olus, which occurred towards the end of prophase (FIg. 1 b, e, f). Prometaphase spindles were fusiform or ellipsoidal (Fig. 1 g). At this stage no clear

150 . B. Guhl and D.-P. Roos

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Mitosis in Protostelium mycophaga . 151

contour of the nucleus was visible in phase contrast (Fig. 1 i), an indication that the nuclear envelope had dissolved. Asters at the spindle poles were very small (Fig. 1 g). Chromosomes were grouped in the center of the spindle, but not precisely aligned in an equatorial plate (Fig. 1 h, i). Metaphase spindles had broader poles, without asters in most cells, and brighter strands were discernible between the spindle equator and the poles (Fig. 1 j). Chromosomes were assembled in a compact metaphase plate (Fig. 1 k, I). Among the many cells examined we found a few amoebae with tripolar metaphase spindles (Fig. 1 m-o). In these the three poles were in a single optical plane (Fig. 1 m) and the Y-shaped chromosome configuration included approximately equal angles between the three arms (Fig. 1 n, 0). No earlier or later stages of tripolar mitoses occurred in our preparations, nor did we observe any mitotic figures with more than three poles. In anaphase spindles the intensity of the MT-fluorescence varied considerably along the spindle axis. It was very bright between the spindle poles and the chromosomes, weaker in the interzone (Figs. 1 p, 2 a). In later stages of anaphase and in telophase the interzone fluorescence was even dimmer, except for the MT strands connecting the two half-spindles (Fig. 2 a, d). Whereas aster-less spindles of early anaphase resembled metaphase spindles except for their greater length (Fig. 1 p), asters were an increasingly dominant fea-

ture from late anaphase on (Fig. 2a,d,g,j). In amoebae in cytokinesis the astral MTs coursed through the entire cytoplasm of the future daughter cells, whereas few MT strands, some of which were curved, connected these (Fig. 2 g, j).

Spindle Dynamics in Live Cells In live amoebae the nucleus with its nucleolus, the mitotic spindle and the chromosomes were clearly visible (Fig. 3), but spindle pole organelles were rarely detectable. As a rule, the amoebae rounded up at the beginning of mitosis and ceased migration. Prophase nuclei lost their clear demarkation from the cytoplasm (Fig. 3a) and as the nucleolus disintegrated, the condensed chromosomes appeared (Fig. 3a, b). We recorded prometaphase in two amoebae: congression of chromosomes to the spindle equator was completed within just over two minutes. During anaphase the groups of daughter chromosomes migrated poleward as strictly ordered plaques with a constant velocity (Figs. 3 d-f, 4). During telophase the edges of these plaques were bent poleward (e.g., upper arrow in Fig. 3f). During cytokinesis the chromosomes decondensed as the nucleolus reappeared (Fig. 3 h, i). In three of the five cells analyzed quantitatively (Table 1), spindle length varied only slightly prior to the

Table 1. Parameters of chromosome movement and spindle elongation in P. mycophaga Spindle elongation Cell no.

Interval I (min)

1 2 3 4 5 x s.d."

Chromosome velocity

Spindle length (11m) start

end

dF

elongation factor

velocity

(urn/min]

1.8 1.3 n.a.! 2.4 2.5

4.2 4.8 4.7 3.3 3.1

8.6 9.2 8.8 8.6 8.0

20.6 21.8 n.d. 18.4 21.4

12.0 12.6 n.d. 8.9 13.4

2.4 2.4 n.d. 2.1 2.7

5.6 7.3 8.9 3.4 4.2

2.0 0.6

4.0 0.8

8.6 0.4

20.6 1.5

11.7 2.0

2.4 0.2

5.9 2.2

(urn/min)

I interval of active spindle elongation as defined in Material and Methods length increase 3 velocities determined for part of anaphase only 4 standard deviation 2

.... Fig. 1. Prophase to early anaphase of mitosis in amoebae of P. mycophaga. Tubulin immunofluorescence (a, d, g, j, m, p), DNA fluorescence (b, e, h, k, n, q), and phase contrast (c, f, i, I, 0, r). - a-r-C, Early prophase. Rather short, straight MTs radiate from two nucleus-associated MCs, but q~ite a few curved MTs hug the nucleus (a). The nuclear contour is distinct as in interphase and the centrally located nucleolus IS opaque (c), but the condensed chromosomes clearly identify this stage as prophase (b). d-f. Late prophase. There are fewer astral MTs than in earlier stages and a spindle-shaped cage encloses the nucleus (d). The nu:cleol~s has ,disappeared a~d the chromosomes (e) a,re ~Iso, visible in the phase contrast image (f). - g-i. Prometaphase. The spindle IS lentil-shaped and ItS poles are marked by diminutive asters (g, arrowheads). The chromosomes are grouped in the center of the spindle, but not neatly aligned at the spindle equator (h, i).- i-I. Metaphase. The spindle is oval and there are no asters at its ~oles (j, arrowheads). The chromoso~es are ~~atly aligned at the spindle equator (k, I). - m-o. Tripolar metaphase. The three spindle poles ~arr~whe~ds) occupy median positions between the arms of the Y-shaped chromosome configuration.p-r. Early anaphase. ThISspindle IS also oval (p) and even broader at the poles (arrowheads) than the metaphase spindle of i-I. A narrow gap (arrow) separates the two sets of daughter chromosomes (q, r). Bar = 10 11 m.

152 . B. Guhl and D.-P. Roos

Mitosis in Protostelium mycophaga . 153

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Fig. 4. Representative diagram depict ing elongation of the half-spindles (.-.) and poleward movement of chromosomes (0-0) in cell no. 1 of Table 1. Time 0 is the beginning of anaphase. Arrows indicate the beginning and end of the phase of active spindle elongation and the regression lines for this interval are superimposed on the data points (for details see Material and Methods). Recording began in prometaph ase. The velocity of spindle elongation decreased , whereas chromosome velocity was constant during the interval of active spindle elongation.

beginning of anaphase (e.g., as in Fig. 4). The mean for this period was 9.3 11m (range 8.1 to 11.5 11m). In two amoebae the velocity of spindle elongation was constant during the period of active elongation, but in two other cells spindle elongation was biphasic, the velocity in the first part being greater than in the second part (Fig. 4). We therefore computed the velocity from the slope of the regression line of the first part. In cell no. 3 we could follow spindle elongation only for part of anaphase and it is from this interval that we computed the velocity. In all the five amoebae analysed, chromosome velocity was greater than the velocity of spindle elongation during the entire interval of active spindle elongation. This result corroborates the visual observation that the chromosomes moved poleward. They were in fact close to the spindle poles by the end of the active elongation. During telophase and cytokinesis the spindle elongated further, but only by a few 11m and at a much reduced rate. The completion of cytokinesis did not lag far behind the completion of active spindle elongation, as exemplified by one amoeba that terminated division 3.5 min after the end of anaphase.

.... Fig. 2. Late anaphase to cytokinesi s in amoebae of P. mycopbaga. Tubulin immunofluorescence (a, d, g, j), DNA fluorescence (b, c, h, k), and phase contrast (c, f, i, I). - a-e. Late anaphase. Poleward of the chromosomes the tubulin fluorescence is bright, but only a few strands of MTs link the two half-spindles across the interzone. Asters are quite modest (a, arrowheads) . The groups of daughter chromosomes have segregated as plaques (b, c). - d-f. Telophase. The two shaggy half-spindles with large asters are connected by several strands of interzonal MTs (d). - g-i. Early cytokinesis. Aster-MTs invade the entire cytoplasmic territory of the nascent daughter cells, but a few interzonal MT-strands remain (g). A minor MT-center off to one side of the spindle is connected to a strand of MTs from each half-spindle (g, arrow). Daughter nuclei with centrally located chromosomes are clearly delimited from the cytoplasm (i). - j-1. Late cytokinesis . The MT-array of each nascent daughter cell consists of a large aster that is brighter on the polar than on the interzonal face. A few thin strands link the two asters (j, arrows). Bar = 10 11m. Fig. 3. Mitosis in a live amoeba. The time indicated in min and sec in the lower right corner of each frame refers to the end of metaphase. Arrowheads mark the spindle poles, arrows mark chromosome groups. - a, b. Prometaphase. The initially lumped chromosomes (a) congress and line up at the spindle equator (b). - c. Late metaphase. - d, e. Anaphase. - f. Telophase. - g. Late telophase. - h. Cytokinesi s with visible nuclear contours - i. The end of cytokinesis. The round daughter nuclei (n) are distinct. Nucleol i are present, but not yet completely re-formed . Bar = 5 11m.

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Figs. 5 and 6. Ultrastructural aspects of mitosis in P. mycophaga. - Fig. 5 a. Overview of a prometaphase spindle. The electronopaque, smal1 chromosomes are loosely grouped in the middle of the spindle. Granular nucleolar material extends poleward of the chromosomes; it is streaked longitudinally by the MTs. A spindle pole body (SPB) marks the upper spindle pole (arrow). Bar = 111m.- b. Kinetochore region of sister-chromatids in a non -adjacent, serial section to (a). At each kinetochore a single MT ends in fuzz at a small distance from the chromatin (arrowheads). Bar =0.25 11m. - c. Half-spindle of the large metaphase cell shown in the inset. Numerous MTs converge in the spindle pole area (upper right). A kinetochore is visible at the arrowhead; its MT (arrow) crosses another MT of unknown origin and extends far towards the spindle pole area . Bar = 0.5 11m. Inset: DIC image of the amoeba embedded in resin. Bar =10 11m. - d. Pole region of the metaphase cell shown in the inset. A complex SPB (arrow) sits at the pole among large dictyosomes. A dome of membrane cisternae and other elements crowns the polar face of the nucleolar material (stars). Bar =0.5 11m. Inset: DIC image of the amoeba embedded in resin. Bar =10 11m. - e. Overview of a spindle in ana -telophase. The chromosomes and nucleolar material (ch) have coalesced to form two irregularly shaped masses. Ribosomes are largely excluded from the space between these masses and the spindle poles (stars). The arrow points to the complex SPB at the upper spindle pole. Note the large dictyosomes at the lower pole (arrowhead). Bar = 111m.

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154 . B. Guhl and D.-P. Roos

Mitosis in Protostelium mycophaga . 155

156 . B. Guhl and D.-P. Roos

Fig. 6. Serial, non-adjacent section of the upper pole area of the ana-telophase shown in Fig. 5 e. Membrane cisternae and vesicles (arrows) have congregated at the periphery of the mass of chromosomes and nucleolar material (ch). A kinetochore (arrowhead) lies in a deep recess of this mass. Bar =0.5 urn, - b. Overview of a daughter nucleus (n) and spindle pole region in a telophase cell. The nuclear envelope has re-formed and the chromosomes are decondensed. Several large dictyosomes are grouped near the SPB (arrow). Bar = 1 urn, Inset: phase contrast micrograph of the cell in resin; the lower daughter nucleus was in a different plane of focus. Bar = 10 urn.

Ultrastructure The salient features of prometaphase and metaphase were the dissolved nuclear envelope, the dispersed nucleolus, polar "caps" consisting of membrane cisternae and vesicles, and spindle pole bodies (SPBs) with a complex, very electron-opaque core and two or more large dictyosomes close by (Fig. Sa, c, d). The body of the spindle made up of these components, although not enclosed by membranes, was thus clearly delimited from the surrounding cytoplasm (Fig. Sa). The small chromosomes (we estimate that there are approximately 40 in an amoeba of normal size) were embedded in the granular mass of nucleolar material that extended quite far towards the spindle poles (Fig. Sa, c, d). Longitudinally, this mass was broken up by "channels" within which the spindle MTs lay. Chromosomes were tethered to spindle MTs by the tiny kinetochores of sister-chromatids, each of which was clearly layered and bore a single MT (Fig. S b, c). Most chromosomes were located at the

spindle periphery and because of the considerable curvature of their kMTs thus imposed we could not follow these all the way to the poles, but that they extended quite far into the spindle pole area was illustrated by a few favorable sections (Fig. 5 c). In ana-telophase two masses composed of the chromosomes, nucleolar material, and polar caps were separated by a large interzone in which few MTs could be seen. Poleward, however, MTs were numerous, some of them clearly connected to kinetochores in deep recesses in the chromatin-nucleolus mass. At the periphery of this mass, apposed flattened vesicles and double membrane cisternae reflected re-formation of the nuclear envelope (Fig. 6 a). This process was completed by telophase, when the chromosomes had decondensed and filled the daughter nuclei, together with still dispersed nucleolar material (Fig. 6 b). The membrane caps were largely dispersed and few MTs occurred between the polar face of the daughter nuclei and the SPBs with associated large dictyosomes (Fig. 6 b).

Mitosis in Protostelium mycophaga . 157 Discussion

Spindle Formation, Structure, and Breakdown Most mitotic spindles of lower eucaryotes are closed or fenestrated and the few known open spindles are either centric or without pole organelles [15]. The mitotic spindle of P. mycophaga is open and acentric, but astral. This is unusual, for acentric spindles are commonly anastral, as exemplified by most spindles of higher plants (e.g. [49]). Furthermore, astral spindles with focused poles are usually centric, as are those of animals [24]. Thus, the spindle of P. mycophaga is unique in that it unites features of centric and acentric spindles. The complex SPBs [13] obviously play the role of MT-focussing organizing centers that centrosomes fulfill in centric spindles. The combination of data we obtained with the different techniques allows us to reconstruct mitotic events. When amoebae of P. mycophaga prepare for mitosis the CMTC is reduced from the cell periphery towards the MTOCs, presumably by disassembly of MTs at their free plus-ends [47]. We have obtained no evidence supporting a mechanism of MT separation from MTOCs and subsequent disassembly at their destabilized minus-ends, as claimed by Kitanishi-Yumura and Fukui [19] for Dictyostelium. In P. mycophaga the shortening of the cytoplasmic MTs during prophase produces two asters that lie opposite each other near the nucleus . We have described and discussed in detail the cycle of the MTOCs [13] and like to point out only that spindle MTOCs are structurally identical to interphase MTOCs and that duplication occurs during mitosis. P. mycophaga therefore counts among the minority of organisms that do not replicate their MTOCs shortly before spindle formation begins (see [15]). The most frequent interphase configuration of two MTOCs on opposite sides of the nucleus [13] obviates the need for duplication and migration just prior to or during prophase. Consequently, only a single mature SPB is located at each spindle pole, which probably prevents the formation of multipolar spindles (see [13] for a more detailed discussion). As spindle formation progresses in late prophase and prometaphase the asters diminish, obviously by further shortening of their component MTs. Concomitantly, the chromosomes condense, orient amphitelically, and congress at the spindle equator to form a distinct metaphase plate. The facile visibility of the chromosomes in the light microscope is a feature that distinguishes P. mycophaga (and also P. aurantium [50]) from D. discoideum and P. violaceum in which congression also occurs, as inferred from ultrastructural and immunofluorescence observations [31, 42-45] . The difference obviously arises from the behavior of the nucleolus: in the latter two species it disperses completely, fills the entire mitotic nucleus, and has a sufficiently similar refraction as the chromosomes to permit their visualization only with special video enhancement

[11, 41]. In Acytostelium leptosomum, another dietyostelid cellular slime mold, however, the chromosomes are also easily visible [14]. The small kinetochores, on which a single MT is inserted, resemble those of Ceratiomyxella tahitiensis [52] and P. aurantium [50], but also those of D. discoideum [31], P. violaceum [38], Physarum [46], and a number of fungi (reviewed in [15]). The MTOCs of P. mycophaga do not disappear during metaphase, as Spiegel [50] stated, but they function as SPBs throughout division (d. [13]). P. mycophaga is assumed to have evolved from a mastigote ancestor [51] by loss of flagella and centrioles. A search for common elements in the pole MTOCs of the centric spindle of P. aurantium and the acentric one of P. mycophaga could partly retrace this transition, but this would require a detailed reinvestigation of the spindle poles of P. aurantium. At metaphase the spindle of P. mycophaga actually consists of two half-spindles apparently joined at the equator, but numerous nkMTs must extend from one pole into the opposite half-spindle to hold the structure together. Such an organization has been verified in all spindles of similar appearance that have been analyzed quantitatively (review and references: [39]; see also [28]). In anaphase and telophase spindles this architectural aspect is more clearly evident: whereas kMTs are a minority, the more numerous nkMTs form two populations: ones that terminate in the interzone just behind the chromosomes and presumably contribute to spindle elongation only in the first stage of anaphase B, and others that form strands or bundles across the interzone and may drive spindle elongation further (see below). There is thus no central spindle in P. mycophaga as in the intranuclear mitosis of dictyostelid cellular slime molds [31, 38, 44]. The observation of interzonal MTs in ultrathin sections is difficult because of the large volume of the spindles, but our immunofluorescence results agree with Spiegel's description of scattered bundles of a few MTs [50]. These bundles disappear in the final stages of division, rather than being packed more tightly and gathered in a Flemming body as in animal cells [24]. Quite remarkable is the reappearance and growth of the asters during anaphase and telophase. The tubulin required for this massive increase in polymer quite obviously surpasses the amount freed by shortening of kMTs and the disassembly of some of the nkMTs that most likely occurs concomitantly (compare the situation in D. discoideum: [28]). As synthesis of new tubulin during mitosis is improbable [12] the conclusion is inevitable that amoebae of P. mycophaga maintain a tight control over the utilization of a large pool of tubulin. The distribution of nucleolar material among the chromosomes and poleward of the metaphase plate is similar to the situation described by Mignot and Brugerolle [29] for the heliozoan Dimorpha mutans. In P. mycophaga the nucleolar material is transported poleward in front of the chromosomes during ana-

158 . B. Guhl and D.-P. Roos

phase and telophase and it is subsequently included in the re-forming daughter nuclei. This passive transport mechanism may ensure that each daughter nucleus receives a stock of preribosomes and other necessary components for the rapid reconstitution of the nucleolus (d. [15]). Reconstruction of the nuclear envelope follows the classical animal pattern [24]: it begins with the apposition of membrane vesicles and cisternae, probably derived from the poleward vesicle caps, on the two masses of congregated nucleolar material and partially decondensed chromosomes. This process progresses from the MT-free periphery of the nascent daughter nuclei towards their center, where kMTs and nkMTs persist well into telophase. The location of kinetochores in deep recesses of the masses of nucleolar material and chromosomes indicates that forces other than those acting on the kinetochores are involved in segregation during late anaphase and telophase and justifies our conservative definition of active spindle elongation (see below).

dles. Aist and collaborators [2,3] (and references therein) concluded from observations and experiments on hyphal nuclei of Nectria haematococca (Fusarium solani) and PtK 2 cells, that asters pull on the spindle poles during anaphase B, exerting a major force for spindle elongation. Of course, such a mechanism requires that astral MTs be anchored in scaffold elements capable of exerting the necessary counterforce. Whereas one can envision such an anchoring for instance in the actin meshwork of a slime mold amoeba it is doubtful whether this would be sufficiently rigid, given the elastic deformability of these cells. Furthermore, the asters in P. mycophaga enlarge mostly after much of anaphase B has occurred. A pushing mechanism driving the spindle poles apart by the sliding interaction between interdigitating interzonal nkMTs [7] is certainly a serious alternative for anaphase B in P. mycophaga, but its verification must await testing by physical or chemical experimental manipulations.

Phylogenetic and Evolutionary Considerations Spindle Dynamics and Mechanisms Mitosis in P. mycophaga is orthodox [37], because anaphase A and anaphase B both occur. Duration of anaphase, chromosome velocity, and extent and rate of spindle elongation in P. mycophaga are in accord with the general observation that mitosis in lower eucaryotes with small spindles is short and velocities are high [15] (see also [1] and [23] for more recent examples and references). A favorite current model for anaphase A is depolymerization-driven kinetochore movement, whereby MT disassembly at the kinetochore is rate-limiting for the velocity [30]. Koshland et al. [20] showed that analogous movements in a model system in vitro can reach velocities of 10 urn/min, depending on the tubulin concentration, and Coue et al. [9] observed, with a different system in vitro, chromosome velocities up to 26 urn/min. Chromosome velocity in P. mycophaga, although at 4 urn/min as much as 10 times faster than in higher plants and animals [15], is compatible with these data and the above model for anaphase A, but the close proximity between kMTs and the numerous nkMTs also conforms to a sliding filament model [26, 27]. The velocity of spindle elongation in P. mycophaga is also high compared to that in higher eucaryotes, but it corresponds to the mean that Heath [15] computed for several lower eucaryotes. The spindle elongation factor of 2.4 that we measured from the video recordings is probably too low because we were careful to exclude passive spindle elongation. By comparison, the longest spindles in immunofluorescence preparations were approximately 3.5 times the average length of metaphase spindles. This bears consequences for anaphase B, because a mere sliding apart, by whatever mechanism, of the two half-spindles could at the most produce a twofold elongation. Some nkMTs must therefore elongate and we conclude that these form the interzonal bun-

Spiegel [50] noted, based on preliminary ultrastructural observations, that mitosis in P. mycophaga is identical to that in the closely related P. aurantium (see also [15]), except that the latter, being an amoeboflagellate, has a centric spindle. However, to set mitotic similarities and dissimilarities between the two species on firmer ground will require tubulin immunofluorescence data and observations on chromosome and spindle dynamics in live cells of P. aurantium. Our results confirm that gross spindle structure is similar in the two species, but in contrast to P. aurantium [50] the nucleolus of P. mycophaga does not disperse, its fragments remaining associated with the spindle and chromosomes. Among the mycetozoans open spindles are known only from the protostelids and myxamoebae of Physarum. Mitosis in the dictyostelid cellular slime molds is acentric, essentially closed, and composed of few kMTs and a central spindle that becomes a shaft during anaphase and telophase [17,28,31,38,42-45]. In these organisms, too, the MTs undergo cyclical changes as in P. mycophaga: the interphase complex of MTs breaks down before prophase and it is reconstructed as the asters enlarge during anaphase and telophase. Anaphase A and anaphase B both exist. Based on spindle fine structure the dictyostelid and protostelid mycetozoans thus do not seem to be closely related (d. [16, 51]), but the dynamics of the MT cytoskeleton and the mitotic spindle are similar in the two groups. Which mitotic features are primitive and which ones are more highly evolved is conjectural (e.g., [15, 16,21, 34,37]). The similarity between mitosis in P. mycophaga and that in metazoans may exemplify the high degree of diversification that mitosis achieved early during evolution, but alternatively it may also signify that this organism, which has been considered to belong to the more primitive mycetozoans [32] (see also [51]), is actually more highly evolved.

Mitosis in Protostelium mycophaga . 159 Acknowledgements We thank Dr. Fred Spiegel for supplying starter cultures and Mrs. Vreni Jenni for competent help with sectioning and electron microscopy. This investigation was supported in part by the Swiss National Science Foundation, by the Geigy-jubilaums-Stiftung, and by the jubilaumsspende fur die Universitat Zurich. We are also grateful to Prof. H. R. Hohl for continued support and infrastructures.

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Key words: Mitosis - Cellular slime mold - Protostelium - Video microscopy - Immunofluorescence - Ultrastructure Urs-Peter Roos, Institut fur Pflanzenbiologie, Universitat Zurich, Zollikerstr. 107, CH-8008 Zurich, Switzerland