The anchoring of nuclei by cytoplasmic microtubules in Acetabularia

The giant unicellular alga Acetabularia enters the reproductive phase of growth when the cap (umbrella) has completed its expansion. The primary nucleus in the rhizoid then divides, generating several hundred secondary nuclei which migrate into the rays of the cap, where they assume fixed positions approximately equidistant from one another. Cyst formation follows, in which the cytoplasm of the rays cleaves into uniformly sized cysts, each containing a single secondary nucleus (Hämmerling, 1931; Schultze, 1939).

These events were studied both in living cells and in fixed material, and the effects of colchicine and vinblastine sulphate observed. This communication will be concerned with the distribution and function of the cytoplasmic microtubules which were found in the region of the secondary nuclei.

Cultures of Acetabularia mediterranea were maintained in a mixture of 2 parts Erdschreiber medium (Hämmerling, 1931) and one part of the artificial medium of Shephard (1971). Ade-quate growth could be obtained with either medium, but the mixture gave increased growth rates.

A variety of fixation conditions for light and electron microscopy were explored, and of these the following was found to give the best results: Fixation with 2·5% glutaraldehyde (Ladd Research Industries, Burlington, Vt., U.S.A.) 0·1 M sodium phosphate buffer pH 7·8 (prepared in half-strength seawater) for 0·5 h at room temperature then for 2·5 h at 2 °C. (The precipitate formed in this mixture was centrifuged out, leaving a solution of pH 7·4) Three washes of 1 h or more in cold 0·05 M phosphate buffer prepared in 0·75-strength seawater were followed by post-fixation in 1 % osmium tetroxide in the wash solution for 1 h in the cold, and a further 0·5-h wash. Dehydration through a cold ethanol series was followed by infiltration with Durcupan A.C.M. (Fluka A.G. Switzerland) or with the low-viscosity embedding medium of Spurr (Polysciences, Inc., Rydol, Penn., U.S.A.). Sections were cut at 2 μm for light microscopy and in some cases stained with 0·05 % toluidine blue. For electron microscopy, sections 3–4 μm in thickness were cut, mounted in the appropriate epoxy resin and examined with the phase-contrast microscope. Areas selected for thin sectioning were mounted on pre-cast resin blocks as described elsewhere (Woodcock & Bell, 1967) and cut with a Reichert OM-2 ultramicrotome. After staining with uranyl acetate and lead citrate, the sections were examined with a Philips 300 electron microscope.

Colchicine was obtained from Sigma Chemical Co. (St Louis, Mo., U.S.A.), and vinblastine sulphate was a gift from Eli Lilly Co. (Indianapolis, Ind., U.S.A.).

The morphological and cytological events leading up to and including cyst formation in Acetabularia have been described by Schultze (1939). A loss of green colour from the stem was the first indication of approaching encystment, and was concurrent with the division of the primary nucleus. One to 3 days after the paling of the stem, regularly spaced colourless areas (the ‘weisse Flecke’ of Schultze, 1939) appeared in the cytoplasm of the rays, each area marking the site of a secondary nucleus (Figs. 3, 4). Development continued with the enlargement of these areas, the contraction of the protoplast, and the almost complete cessation of cytoplasmic streaming. Encystment occurred 1–3 days after the first appearance of the colourless areas (Figs. 3, 4).

In section, each colourless area was seen to comprise a weakly basophilic nucleus surrounded by a layer of cytoplasm 2–3 μm in depth which was free from chloroplasts (Figs. 5–7). (It is this displacement of chloroplasts which makes the positions of the secondary nuclei visible as colourless areas in the green rays.) At the time the areas first appeared, the nuclei were spherical and about 5 μm in diameter, but as development continued they increased in size and assumed a distinct lenticular form. At cyst formation, each secondary nucleus was 10–15 μm in diameter and about 7μm in thickness. A number of strongly basophilic particles closely adpressed to the nuclei could be seen throughout their development (Figs. 5–7).

With the electron microscope, the organelle-free cytoplasm was seen to comprise a mass of smooth membranes containing many free ribosome clusters. The basophilic particles appeared as dense, granular material, spaced 0·1–0·5 μm from the nuclear membrane (Figs. 7–9). Many microtubule profiles were also seen in the cytoplasm (Figs. 8–10); some were associated with the nuclear membrane, some traversed the basophilic aggregates (Fig. 9), and some ran close to the cell membrane adjacent to the cell wall (Fig. 10). Sections perpendicular to the surface of the ray, and through the centre of the nucleus, encountered 100–300 microtubules, about 70% of which were cut perpendicular to their axes (Fig. 1), while, in similarly oriented sections through the edge of the nucleus, about 90% of the microtubules were cut parallel to their axes. Sections cut tangential to the ray surface also showed a preponderance of longitudinally cut microtubules (Fig. 2).

All the profiles of colourless areas examined were consistent with an arrangement of microtubules encircling the secondary nucleus in planes approximately tangential to the cell wall, with the cell membrane and nuclear membrane important attachment areas. Until serial sections are completed, it will not be possible to determine the length of individual microtubules, or to demonstrate continuity between the microtubules associated with the cell membrane, and those attached to the nuclear membrane.

In addition to those surrounding the nuclei, a second system of tubules was present in the ray cytoplasm. This consisted of loose bundles of 5–10 microtubules which radiated from the nuclei into the organelle-containing cytoplasm (Fig. 1). Some of these bundles were seen to extend 30 μm (one third of the distance between adjacent nuclei) into the cytoplasm. The number of radiating bundles per nucleus, and their total length, remain to be determined.

Both systems of microtubules, and the basophilic particles, disappeared within 1–3 days of cyst formation. Microtubules were not seen in immature caps (before the arrival of the secondary nuclei), nor have they been reported in the stems of Acetabularia.

In order to investigate the function of these transitory microtubules, caps at various stages of cyst formation were placed in colchicine or vinblastine sulphate solutions, both of which are known to bind to, and in some cases cause depolymerization of, microtubules (Robbins & Gonatas, 1964; Shelanski & Taylor, 1967; Borisy & Taylor, 1967a, b;Marantz, Ventilla & Shelanski, 1969). The effect of these drugs on caps in which colourless areas had been present for 12–24 h, before the start of cyst formation, is shown in Table 1. Administration of the drugs at an earlier stage in development had similar effects, but treatment during the actual cleavage of the cytoplasm was ineffective. A similar inhibition of cyst formation in Acetabularia by 10⁻³ M colchicine was reported by Werz (1969, 1970).

The loss of the colourless areas took place by the gradual encroachment of chloroplasts into the cytoplasm between the nuclei and the periphery of the ray. In 10⁻² M colchicine, this took place very rapidly, the effect being clearly noticeable after 5 min, and complete within 30–60 min (Figs. 11–14). Such migrations were, however, too restricted to be termed cytoplasmic streaming. After 1–2 h in 10⁻² M colchicine, the nuclei began to move from their fixed positions, and after 24 h most of them had congregated at the distal ends of the rays.

Microscopic examination of caps treated with colchicine showed that the microtubules were indeed absent. After 1 h in 10⁻² M colchicine, the nuclei were still close to the cell wall, but chloroplasts had approached to within 0·5 μm on all sides. After 24 h, the nuclei had moved away from the wall to the vacuolar side of the cytoplasm, and were surrounded by a layer of cytoplasm only 0·2–0·5 μm thick (Fig. 15). A few small basophilic particles remained, but these had rounded off, and did not show the close association with the nuclear membrane characteristic of untreated cells. Abnormal cyst formation produced by treatment with 2·5 × 10⁻³ M colchicine for 24 h is shown in Fig. 16.

Microtubules have been assigned many functions (Porter, 1966), including the establishment and maintenance of cell form (Borisy & Taylor, 1967a), the orientation of cellulose microfibrils (Newcomb & Bonnett, 1965; Pickett-Heaps, 1967), and the movement of chromosomes (Mclntosh, Heppler & Van Wie, 1969). In Acetabularia, the loss of the microtubules surrounding the secondary nuclei inhibits cyst formation, and is accompanied by 2 distinct cytological events. These are, the invasion of the colourless areas by plastids and mitochondria, and the migration of the secondary nuclei from their fixed positions.

The observation that cyst formation can in certain instances proceed in the absence of colourless areas, suggests that the latter are not of primary importance. If, for example, caps are cold-treated (2 °C) just prior to cyst formation, the colourless areas disappear. (Low temperatures are known to cause microtubule breakdown; see Tilney & Porter, 1967.) On warming to 25 °C, the colourless areas redevelop over several hours but, in some rays, cyst formation begins before their reappearance. Thus it seems likely that, of the 2 cytological events induced by colchicine, the migration of the nuclei from their fixed positions plays the more important role in the inhibition of cyst formation. On the basis of the evidence presented above, it is proposed that a major function of the microtubules which surround the secondary nuclei of Acetabularia, is to anchor them at fixed positions in the ray cytoplasm. This function is probably mediated by the association of microtubules both with the cell and nuclear membranes. Loss of these fixed positions would upset the regular inter-nuclear spacing, and either abnormal, irregularly sized cysts would be produced (Fig. 16), or cyst formation would be inhibited.

A similar anchoring function was proposed by Kiermayer (1968 a, b) for a group of microtubules adjacent to the post-telophase nucleus of Micrasterias denticulata. Here, also, colchicine caused the nuclei to wander, and cell organization was subsequently disrupted.

In Acetabularia, the evidence suggests that it is the secondary nuclei themselves which directly control the synthesis of microtubules. Before the arrival of the secondary nuclei, no microtubules were observed in the cap, whereas each secondary nucleus, while still in the rhizoid, already had a few short microtubules (and small basophilic particles) associated with it. Boloukhère (1970) has also observed cytoplasmic microtubules in the rhizoid at this stage.

In a study of the long-term effects of 10⁻³ M colchicine on Acetabularia, Werz (1969) showed that both cap and cyst formation were delayed or inhibited by the drug, but not stem elongation or cap enlargement. From these results, it was concluded that changes in the morphogenetic direction of the cell were blocked by colchicine, but that, once a morphogenetic pattern was established, colchicine sensitivity was lost. In the case of cyst formation, it is now possible to define the effect of colchicine with more precision: it causes the loss of the cytoplasmic microtubules which anchor the secondary nuclei in position.

The function of the second system of microtubules remains obscure. It is possible that the bundles of radiating tubules confer added positional stability to the nuclei, or they may have a role in the cleavage process. A third attractive possibility is that the interaction of microtubules from adjacent nuclei determines their equidistant spacing.

Further work is in progress to examine these possibilities and to study the composition and function of the basophilic particles, whose presence is correlated with that of the microtubules.

In A. mediterranea, microtubules have an important function in the organization of the multinucleate, coenocytic protoplasm of the cap. Perhaps a similar role for microtubules may be anticipated in other systems where a multinucleate protoplasm develops into an ordered, multicellular structure.

I am grateful to Marilyn J. Cozzens and Larry H. Clouse for assistance with the ultrastructural portion of this study and to Brian L. Jensen for help with the photographic reproductions. This investigation was supported by the United States Public Health Services, Grant Number GMO6637.

A preliminary account of this work was presented at the 10th Annual Meeting of the American Society for Cell Biology, 1970.