FINE STRUCTURE OF AN ORGANELLE ASSOCIATED WITH THE NUCLEUS AND CYTOPLASMIC MICROTUBULES IN THE CELLULAR SLIME MOULD POLYSPHONDYLIUM VIOLACEUM

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1 J. Cell Sci. 18, (1975) 315 Printed in Great Britain FINE STRUCTURE OF AN ORGANELLE ASSOCIATED WITH THE NUCLEUS AND CYTOPLASMIC MICROTUBULES IN THE CELLULAR SLIME MOULD POLYSPHONDYLIUM VIOLACEUM U.-P. ROOS Laboratoire de Microbiologie, Dipartement de Biologie Vigitale, Umversiti de Geneve, 1211 Geneve 4, Switzerland SUMMARY Polysphondylium violaceum was grown in association with Escherichia colt. Vegetative amoebae and pseudoplasmodia were fixed under different conditions and processed for electron microscopy. An electron-opaque body (nucleus-associated body, NAB) lies in the cytoplasm near the tapered end of interphase nuclei. The NAB consists of a disk-shaped, multilayered core, approximately 200 nm in diameter and 150 nm thick, embedded in a granular matrix from which electron-opaque nodules protrude. The nodules are termination points of microtubules radiating from the NAB into the cytoplasm or running along the nucleus. On the average there are 16 nodules per NAB. One or two microtubules terminate in each nodule. Spindle pole bodies, arising by duplication of the NAB at the beginning of mitosis, are unstructured foci for spindle microtubules in mitotic cells. It is suggested that cytoplasmic microtubules do not determine cell shape, but they probably cause the tapering deformation of the nucleus. They may, furthermore, represent a storage form of subunits for utilization during the formation of the mitotic spindle. The nodules of the NAB are potential nucleation sites of cytoplasmic microtubules during interphase. Spindle pole bodies presumably acquire a microtubule-organizing capability by integration of the decondensed nodules. INTRODUCTION In the life cycle of the cellular slime moulds (Acrasiomycetes; Raper, 1973) one distinguishes a vegetative phase of cell multiplication and a reproductive phase involving cell aggregation and fruiting body formation (Bonner, 1967). For many years investigators of differentiation and morphogenesis have used cellular slime moulds as model systems and, as a consequence, a wealth of biochemical data has accumulated (review: Bonner, 1971). Ultrastructural observations, though less numerous, have also dealt almost exclusively with problems of differentiation (e.g. Gregg & Badman, 1970; Hohl & Hamamoto, 1969). Little is therefore known about the ultrastructural cytology of amoebae. In a recent report (Roos, 1975) on the ultrastructure of mitosis in vegetative amoebae of Polysphondylium violaceum I briefly described an organelle associated with the interphase nucleus and cytoplasmic microtubules. I proposed the term 'nucleus-

2 316 U.-P. Roos associated body' or NAB for this organelle and presented evidence that it duplicates during prophase of mitosis, and that the 2 resulting units function as spindle pole bodies (SPBs, Aist & Williams, 1972) during the later stages of division. The present report is a more detailed account of the fine structure and possible function of the NAB in interphase cells of P. violaceum. MATERIALS AND METHODS A clone of Polysphondylium violaceum Brefeld was cultured in association with Esclierichia coli B. Vegetative amoebae were grown in. Sussman's (1961) liquid medium. Cultures were incubated for 24 or 48 h on a reciprocal shaker at 25 C in the dark. Three fixations were carried out. (i) Amoebae were harvested by low-speed centrifugation and resuspended in 3-3 % glutaraldehyde in 0-07 M SOrensen's (1912) phosphate buffer, ph 6-8. Fixation was for 1 h at room temperature. Amoebae were then rinsed in 2 changes of buffer and postfixed in buffered, 1 % osmium tetroxide for 1 h at room temperature. Following a buffer rinse the amoebae were embedded in 2 % agar, the pellets diced, rinsed in 3 changes of distilled water and prestained in 2 % aqueous uranyl acetate for 30 min at room temperature, (ii) Glutaraldehyde, buffered as above, was added to culture flasks to a final concentration of 3-2 %. Fixation was for 1-5 h at room temperature with intermittent agitation. Postfixation with osmium tetroxide and embedding in agar were done as described above. Prestaining in 2 % uranyl acetate was for 1 h at approximately 5 C. (iii) Amoebae were harvested by low-speed centrifugation, prefixed in 4 % p-formaldehyde in distilled water, ph 6-8, for 1 h at room temperature and rinsed in cold, distilled water, ph 68, for 1 h at room temperature and rinsed in cold, distilled water for 4 h (Ris, 1971). This was followed by a 30-min fixation at room temperature in 3 % glutaraldehyde in distilled water, ph 6-8, 2 changes of water and postfixation in 1 % osmium tetroxide in distilled water, ph 6-8, for 1 h at room temperature. Amoebae were embedded in agar and prestained overnight in cold 2 % uranyl acetate. Pseudoplasmodia were obtained by inoculating Bonner's (1967) solid medium in sterile Petri dishes with E. coli B and spores of P. violaceum. Cultures were incubated at C for 6 days in the dark. Petri dishes were flooded with 2 % glutaraldehyde in S6rensen's buffer, ph 6-8, the pseudoplasmodia transferred to vials and fixed for 30 min at room temperature. Following a rinse in 2 changes of buffer they were postfixed for 1 h in 2 % osmium tetroxide at room temperature. Prestaining in uranyl acetate was for 30 min at room temperature. Specimens were dehydrated in a graded series of cold ethanol and embedded in Spurr's (1969) medium, or transferred to propylene oxide and embedded in Epon. Sections in the silver range were cut with a diamond knife on a Reichert Om U-2 ultramicrotome and placed on single-hole grids coated with Formvar and carbon. Serial sections were transferred according to the technique of Galey&Nilsson(i966). The sections were stained with aqueous uranyl acetate (Watson, 1958) or hot, alcoholic uranyl acetate (Locke & Krishnan, 1971), followed by lead citrate (Reynolds, 1963), and examined in an AEI EM 6 B electron microscope at 60 kv, with objective apertures of 25 or 50 fim. The microscope was calibrated with a carbon replica grating. Three-dimensional reconstruction from serial sections was made from electron micrographs enlarged photographically to times. Features of interest were traced on acetate sheet overlays. These were affixed to 5-mm-thick Plexiglass plates representing 50-nm sections. The plates were stacked and aligned whereby microtubules and membranous elements served as reference points. In 2 cases the NAB alone was cut out of each Plexiglass plate and the elements were glued together, yielding a 3-dimensional model. OBSERVATIONS AND RESULTS Most amoebae are uninucleate. Interphase nuclei are typically broad and round at one end and tapered at the other (Fig. 2). The electron-opaque NAB always lies near the tapered end. In binucleate amoebae each nucleus is associated with one NAB. The shape of NABs as seen in section varies from approximately rectangular (Figs. 3, 4, 7)

3 Fine structure of Polysphondylium 317 to circular (Figs. 11, 12). A zone around the NAB is occupied by numerous small vesicles and membrane cisternae (Figs. 2, 3, 9-14). This feature is so characteristic in vegetative amoebae that the region of the NAB can be immediately located, even in sections in which it is not itself present. Cytoplasmic microtubules radiate from the NAB (Figs. 4, 7-14)- In many sections a few microtubules run parallel and very close to the nuclear envelope (Fig. 3). Table 1. Dimensions of the core of nucleus-associated bodies (NABs) (Pooled values from vegetative amoebae fixed according to any of the 3 schedules.) No. of NABs measured Mean and S.D. (nm) Circular profiles (cf. Figs. 9-14) diameter* ±25-0 Rectangular profiles (cf. Figs. 4, 15-20) width 14 I4S"S± I 3"S length ±34-7 Each value is the average of 2 measurements made at right angles. Note: If serial sections were available the greatest dimensions were used. At higher magnification one can distinguish a very electron-opaque core embedded in a granular matrix from which nodules protrude on all sides (Figs. 3, 7, 9-14). Depending on the angle of sectioning the core is approximately rectangular (Figs. 3, 7) or circular (Figs. 11, 12). The mean diameter of circular profiles is 200 nm, while the mean width and length of rectangular profiles are anc * 2I2-5 n" 1 ) respectively (Table 1). Microtubules terminate in the nodules, rather than the core (Figs. 9-14). Counts from serial sections of 9 NABs revealed an average of 16 nodules per NAB. One or two microtubules terminate in each nodule (Figs. 9-14, 23). Spindle pole bodies in mitotic cells are ring-shaped (cf. Roos, 1975). They are electron-opaque, but not structured like NABs (Figs. 5, 6). No core, matrix, or nodules are discernible. Astral and spindle microtubules, more numerous than the cytoplasmic microtubules in interphase cells, terminate overall in the SPBs, rather than at strictly localized points (Figs. 5, 6). In interphase cells fixed under conditions giving good overall preservation a faint pattern of bands is sometimes visible in rectangular, but not in circular, profiles of the core (cf. Figs. 7, 8, 10-13). A. much more distinct pattern of light and dark bands and lines is seen in cells fixed under suboptimal conditions (Fig. 4), particularly after prefixation with unbuffered />-formaldehyde (Figs ). The pattern is bilaterally symmetrical (Fig. 22). Two electron-opaque bands, approximately 9 nm wide, mark the lateral boundaries of the core. Less electron-opaque bands, approximately 25 nm wide, adjoin on the inside. Each of these bands is subdivided by an apparently broken, electron-opaque line. The middle part of the core consists of an electronopaque band approximately 62 nm wide, which is subdivided longitudinally by 2

4 U.-P. Roos Fig. i. Diagrammatic interpretation of the NAB. The front side is cut to reveal the core. The matrix is stippled. Several nodules are drawn with their microtubules. Other microtubules project from nodules on the hidden face of the NAB. dark lines into 3 bands of approximately equal width. In a complete series of sections the bands and lines are fuzzy in the first and last sections (Figs. 15, 20) and most distinct in the middle sections (Figs. 17, 18). DISCUSSION Fig. 1 illustrates the most probable interpretation of the shape of the NAB, based on 3 series of tracings mounted as shown in Fig. 23 and 2 cut-out models as shown in Figs. 24 and 25. The multilayered core has the shape of a flat or, more rarely, concave or convex disk. The following arguments support this interpretation. Rectangular profiles represent transverse sections and circular profiles represent planar sections of the core. The difference between the length of rectangular profiles and the diameter of circular profiles is not significant (Table 1). There is also good agreement between the width of rectangular profiles (Table 1) and the thickness of the core estimated from serial planar sections (Figs. 9-14), if one assumes an average section thickness of nm. Finally, the cut-out models of the core have the shape of an oval disk (Figs. 24, 25). The deviation from the ideal circular disk may in this case be attributed to a slight overestimation of section thickness, which can only be approximately determined. For example, if one assumes an average section thickness of 40 nm instead of 50 nm the resulting model is more nearly a circular disk. The matrix in which the core of the NAB is embedded is lacking under certain

5 Fine structure of Polysphondylium 319 conditions of fixation (Fig. 4). Generally, however, it is arranged around the core in such a way that the NAB resembles a flattened sphere (Fig. 1). Microtubule proteins, the tubulins, have been isolated from a variety of cells and tissues (see review by Olmsted & Borisy, 1973). Under appropriate conditions microtubules can be assembled in vitro (e.g. Borisi & Olmsted, 1972; Dentler, Granett, Witman & Rosenbaum, 1974; Snell, Dentler, Haimo, Binder & Rosenbaum, 1974). The sine qua non for polymerization of microtubules in vitro from subunits seems to be the presence of nucleation centres (Borisy & Olmsted, 1972; Snell et al. 1974). In vivo experiments have also demonstrated the importance of nucleation centres for the regeneration of microtubules during recovery of cells following treatment with agents that depolymerize microtubules. Examples are the centriolar satellites of ectodermal cells of the sea urchin Arbacia punctulata (Tilney & Goddard, 1970), the centroplast of the protozoan Raphidiophrys (Tilney, 1971), and the rhizoplast and kineto-beak of the alga Ochromonas (Brown & Bouck, 1973). The common characteristic of such centres is that they appear as electron-opaque, structured or amorphous organelles or organelle appendages in thin sections. Morphologically similar structures have been identified in thin sections of a great number of cells and organisms (see review by Pickett-Heaps, 1969). The nodules of the NAB of P. violaceum resemble nucleation centres in that they are electron-opaque termination sites of microtubules. As a working hypothesis I have assumed that they play a role in the assembly of microtubules, and experiments designed to test this hypothesis are now under way. The role of cytoplasmic microtubules in P. violaceum is not obvious. They do not determine cell shape, for they occur almost exclusively near the NAB and the nucleus. They rarely project to the cell surface and they do not extend into the delicate pseudopodia. The cone-like array of cytoplasmic microtubules emanating from the NAB is probably responsible for the tapering of the interphase nucleus, but the significance of this is not clear. During mitosis, on the other hand, microtubules have a vital function, for they are an essential component of the spindle apparatus (Roos, 1975). It is possible that the cytoplasmic microtubules of interphase cells represent a storage form of microtubule subunits to be used for spindle formation during mitosis. The reason for the lack of nodules in the SPBs may be that they are integrated in a decondensed state, so that each SPB as a whole becomes a nucleation centre. Organization and accommodation of the numerous spindle microtubules may indeed depend on such an expansion and dispersion of the capability for nucleation. I gratefully acknowledge Dr H. Ris' helpful advice concerning ^-formaldehyde fixation and the excellent technical assistance of Miss Sara Mastelli. I am grateful to Prof. G. Turian for making available the facilities of his laboratory and to Dr R. Peck for critically reading the manuscript.

6 320 JJ.-P. Roos REFERENCES AIST, J. R. & WILLIAMS, P. H. (1972). Ultrastructure and time course of mitosis in the fungus Fusarium oxysporum. J. Cell Biol. 55, BONNER, J. T. (1967). The Cellular Slime Molds (2nd ed.), 205 pp. Princeton, New Jersey: Princeton University Press. BONNER, J. T. (1971). Aggregation and differentiation in the cellular slime molds. A. Rev. Microbiol. 25, BORISY, G. G. & OLMSTED, J. B. (1972). Nucleated assembly of microtubules in porcine brain extracts. Science, N.Y. 177, BROWN, D. L. & BOUCK, G. B. (1973). Microtubule biogenesis and cell shape in Ochromonas. II. The role of nucleating sites in shape development. J. Cell Biol. 56, DENTLER, W. L., GRANETT, S., WITMAN, G. B. & ROSENBAUM, J. L. (1974). Directionality of brain microtubule assembly in vitro. Proc. natn. Acad. Sci. U.S.A. 71, GALEY, F. R. & NILSSON, S. E. G. (1966). A new method for transferring sections from the liquid surface of the trough through staining solutions to the supporting film of a grid. y. Ultrastruct. Res. 14, GREGG, J. H. & BADMAN, W. S. (1970). Morphogenesis and ultrastructure in Dictyostelitrm. Devi Biol. 22, HOHL, H. R. & HAMAMOTO, S. T. (1969). Ultrastructure of spore differentiation in Dictyostelitrm: the prespore vacuole. y. Ultrastruct. Res. 26, LOCKE, M. & KRISHNAN, N. (1971). Hot alcoholic phosphotungstic acid and uranyl acetate as routine stains for thick and thin sections, y. Cell Biol. 50, OLMSTED, J. B. & BORISY, G. G. (1973). Microtubules. A. Rev. Biochem. 42, PICKETT-HEAPS, J. D. (1969). The evolution of the mitotic apparatus: an attempt at comparative ultrastructural cytology in dividing plant cells. Cytobios 1, RAPER, K. B. (1973). Acrasiomycetes. In The Fungi, vol. 4B (ed. G. C. Ainsworth, F. K. Sparrow & A. S. Sussman), pp New York and London: Academic Press. REYNOLDS, E. S. (1963). The use of lead citrate at high ph as an electron-opaque stain in electron microscopy, y. Cell Biol. 17, RlS, H. (1971). A method for in situ demonstration of chromosomal nucleohistone fibers. nth A. Meeting Am. Soc. Cell Biol. (New Orleans), Sci. Demonstration. Roos, U.-P. (1975). Mitosis in the cellular slime mold PolysphondyHum violaceum. y. Cell Biol. 64, SNELL, W. J., DENTLER, W. L., HAIMO, L. T., BINDER, L. I. & ROSENBAUM, J. L. (1974). Assembly of chick brain tubulin on to isolated basal bodies of Chlamydomonas reinhardi. Science, N. Y. 185, SORENSEN, S. P. L. (1912). Uber die Messung und Bedeutung der Wasserstoffionenkonzentration bei biologischen Prozessen. Ergebn. Physiol. 12, SPURR, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy. y. Ultrastruct. Res. 26, SUSSMAN, M. (1961). Cultivation and serial transfer of the slime mould, Dictyostelium discoideum, in liquid nutrient medium, y. gen. Microbiol. 25, TILNEY, L. G. (1971). How microtubule patterns are generated. The relative importance of nucleation and bridging of microtubules in the formation of the axoneme of Raphidiophrys. y. Cell Biol. si, TILNEY, L. G. & GODDARD, J. (1970). Nucleating sites for the assembly of cytoplasmic microtubules in the ectodermal cells of blastulae of Arbacia punctulata. y. Cell Biol. 46, WATSON, M. L. (1958). Staining of tissue sections for electron microscopy with heavy metals. y. biophys. biochem. Cytol. 4, {Received 16 December 1974)

7 Fine structure of Polysphondylium Figs For legends see p. 322.

8 322 U.-P. Roos Figs Vegetative amoebae of P. violaceum. Fig. 2. An interphase nucleus (n) with its associated electron-opaque body (nab). Note the zone of vesicles surrounding the NAB. fv, food vacuole; no, nucleolus. x Fig. 3. The NAB of Fig. 2 at higher magnification. Microtubules radiate from the NAB into the cytoplasm or run along the nuclear envelope (single arrows). Four electron-opaque nodules, the termination sites of microtubules, can be distinguished (double arrows), n, nucleus, x Fig. 4. NAB of another interphase cell. A bilaterally symmetrical pattern of longitudinal, dark and light bands is recognizable, x Figs, s, 6. Sections no. 5 and 6 of a complete series of 8 sections through one spindle pole body (SPB) of a nucleus (n) in telophase of mitosis. Astral and spindle (arrow) microtubules originate from the unstructured, electron-opaque SPB. x Figs. 2, 3, s, 6, fixation (ii); Fig. 4, fixation (i) (see Materials and methods). Figs. 7, 8. Pseudoplasmodium in the pre-culmination stage. In both figures a banding pattern similar to that of Fig. 4 is recognizable, although it is less distinct. Fig. 7. NAB from a prespore cell. Note the 2 nodules (arrows), x Fig. 8. NAB from an immature stalk cell, x Figs Complete series of sections through the NAB of a vegetative amoeba in interphase (fixation (ii)). The NAB consists of a very electron-opaque, roughly circular core embedded in a granular matrix from which nodules protrude (Figs ). Similar nodules appear in the sections just above and below the core (Figs, g, 14). The nodules are termination points of microtubules (arrows), x

9 Fine structure of Polysphondylium 323

10 324 U.-P. Roos Figs Complete series of sections through the NAB of a vegetative amoeba in interphase (fixation (iii)). The NAB is occasionally slightly curved, as seen here. The core exhibits a delicate banding pattern. Microtubules are not preserved and the nodules (arrows) are larger than in the previous series (cf. Figs. 9-14). n, nucleus, x Figs. 21, 22. The NAB of Fig. 18 and an interpretative drawing. The banding pattern of the core is bilaterally symmetrical. The very electron-opaque middle part, approximately 62 nm wide, is subdivided by 2 parallel lines into 3 bands of approximately equal width. Less electron-opaque bands, each approximately 25 nm wide and subdivided by a dark line, lie on either side of the middle part. Electron-opaque bands approximately 9 nm wide mark the lateral boundaries of the core. Fig. 21 is x

11 Fine structure of Polysphondylium. 25 nm 9 nm

12 326 U.-P. Roos Fig. 23. Reconstruction, by means of acetate-sheet tracings and Plexiglass spacer plates, of the NAB shown in Figs Matrix and nodules are stippled. Microtubules (double lines) terminate in nodules. Circles and irregularly shaped curvilinear elements are profiles of vesicles used for alignment. Figs. 24, 25. Two views of a cut-out model of the core from the NAB shown in Figs The shape approximates to a disk. One unit on scale of Fig. 24 represents 100 nm. 25