Protoplasma 120, 36~42 (1984)

O $MA �9 by Springer-Verlag 1984

The Behaviour of Kinetochore Microtubules During Meiosis in the Fungus Saprolegnia

KENJI TANAKA a n d I. BRENT HEATH*

Institute of Medical Mycology, Nagoya University School of Medicine, Nagoya, and Department of Biology, York University, Toronto

Received March 21, 1983 Accepted July 19, 1983

Summary

In Saprolegnia, kinetochore microtubules persist throughout the mitotic nuclear cycle but, whilst present at leptotene, they disappear coincidently with the formation of synaptonemal complexes at pachytene and reform at metaphase I. In some other fungi chromosomal segregation is random in meiosis and non-random in mitosis. The attachment of chromosomes to persistent kinetochore microtubules in mitosis, but not meiosis, in Saprolegnia provides a plausible explanation for such behaviour. At metaphase I each bivalent is connected to the spindle by 2 laterally paired kinetochore microtubules whereas at metaphase II (as in mitosis) each univalent bears only one kinetochore microtubule, thus showing that all kinetochores are fully active at all stages of meiosis.

Keywords: Kinetochores; Mitosis; Microtubules; Meiosis; Saprolegnia.

1. Introduction

In many respects mitosis, and other events of the nuclear cycle, in the fungi follow similar patterns to those found in other organisms. Because some fungi are highly amenable to genetic analysis and others, with diminutive spindles, are useful for ultrastructural analysis, these organisms are used extensively for investigations of diverse aspects of the cellular and molecular biology of the eukaryotic nuclear cycle.

* Correspondence and Reprints: Department of Biology, York University, 4700 Keele Street, Downsview, Ontario, Canada M3J 1P3.

However, recent data indicate that some aspects of some fungal nuclear cycles are distinctly different from the cycles of other organisms. For example, during mitosis in Saccharomyces (WILLIAMSON and FENNELL 1980) and Aspergillus (RoSENBERGER and KESSEL 1968)

�9 the chromosomes do not assort randomly; parental chromosomes segregate as an intact group, separate from the daughter group. In contrast, during meiosis both organisms exhibit normal random segregation which must follow from meiotic recombination. The basis for these contrasting behaviours is unknown. A second, possibly related, peculiarity of some fungal nuclear cycles is that at least some part of the mitotic spindle apparatus persists throughout the cycle (e.g., PETERSON and RIs 1976, HEATH 1980a, HEATH and RETHORET 1980, 1981, 1982). In Saprolegnia it is specifically the kinetochore microtubules which persist (HEATH 1980a, HEATH and RETHORET 1981). If persistent kinetochore microtubules and non-random mitotic segregation were related phenomenon, one would expect to see differences in the mitotic versus the meiotic nuclear cycles. The only data pertinent to this question are the observations of ZICKLER and OLSON (1975) who showed coexisting synaptonemal complexes and persisting spindles during meiotic prophase in Saccharomyces. At face value this would suggest that the persistent spindle is a common feature of both mitosis and meiosis and its persistence in itself is not the cause of the differential segregation pattern.

K. TANAKA and I. B. HEATH: The Behaviour of Kinetochore Microtubules During Meiosis in the Fungus Saprolegnia 37

However, it is the kinetochore microtubules that are the crucial ones in this context and the lack of clarity of yeast nuclear structure in general precludes unambiguous identification of kinetochore versus non- kinetochore microtubules in Saccharomyees. The problem is less acute in Saprolegnia where the identification is typically more clear. As part of our investigation into meiosis in Saprolegnia (TANAKA e t al. 1982) we have examined prophase to see if the kinetochore microtubules persist throughout synapsis. In addition we have observed variations in the number of kinetochore microtubules per chromosome at metaphase in mitosis, meiosis I and meiosis II. These variations may be significant in the context of kinetochore-microtubule interactions and kinetochore- microtubule number versus load and or velocity as discussed by MoENs (1979) and LIN et al. (1981).

2. Materials and Methods

The organism, cultural conditions and electron microscope preparative and analytical procedures are all identical to those described previously ((TANAKA et al. 1982).

3. Results

Pre-meiotic nuclei in antheridia show both the P1 (nucleus with one pair of centrioles) and P2/1 (nucleus with two pairs of centrioles and a single array of kinetochore microtubules) configurations as described by HEATH and RETI~ORET (1981), with approximately 42 kinetochore microtubules adjacent to the single (P1) or double (P2) pairs ofcentrioles (Figs. 1-3). In contrast to spindle formation during mitosis when centriole migration and spindle microtubule polymerization are coincident processes (HEATH 1980 a), during meiotic prophase the centriole pairs separate prior to spindle development. For example, we observed separations of 1.0 gm at leptotene and 3.1 and 4.1 gm by pachytene. During centriole migration the pre-prophase (Ga, S, and G2) array of 42 kinetochore microtubules separates into two groups of approximately 21 each and these groups begin to migrate with the centrioles. Thus, at leptotene when chromatin condensation is beginning and lateral elements are appearing, prior to any synaptonemal complex formation, we observed 17 and 21 kinetochore microtubules adjacent to the migrating centrioles (Figs. 4 and 5). However, by pachytene when synaptonemal complexes are present and variously completely formed, the kinetochore microtubules are

absent (Figs. 6 and 7). We have observed 14 pachytene nuclei and all lacked kinetochore microtubules, either associated with the centrioles or anywhere else in the nucleus.

We have not observed stages in kinetochore microtubule reformation but have analysed 3 metaphase-anaphase I nuclei. The only one of these for which we have a complete series of sections (Figs. 8 and 9) was 4.0gin long and contained 42 kinetochore microtubules per half spindle. In all 3 nuclei the kinetochore microtubules occurred in side-by-side pairs in each half spindle (Figs. 8-11). The center to center distance between the microtubules at their kinetochores was approcimately 70 nm but ranged between 65 and 75 nm. They diverged very little as they approached the spindle poles (Fig. 8) and were still typically recognizable as distinct pairs most of the way to the pole (Fig. 11). The average kinetochore microtubule length in the completely sectioned spindle was 1.68 gm (range 0.81gin to 2.58gm, standard deviation 0.44gm), thus the average inter-kinetochore distance would be 0.6gm (spindle length = 3.96gm, less 2 x 1.68~tm = 0.06gin) which is consistent with our interpretation that this nucleus was in early anaphase and explains the observed lack of amphitelic pairing (end to end pairing, i.e., kinetochore microtubules from opposite poles facing each other). Our series of the other two nuclei were too short and oblique to show amphitelic pairing even if it were present. The kinetochore microtubules shorten to the spindle poles during anaphase and telophase.

We lack stages between telophase I and metaphase II but we have observed a single metaphase II spindle which was serially sectioned parallel to its long axis through approximately 60% of its diameter (Figs. 12- 14). This spindle was 1.88 gm long and contained 11 kinetochore microtubules per half spindle. In contrast to metaphase I, the kinetochores in each half spindle occurred only singly (Figs. 12-14) as in mitosis. However, each kinetochore microtubule formed an amphitelic pair with another microtubule in the opposite half spindle (Figs. 12-14). The mean inter- kinetochore distance was 0.22~tm (range 0.10gm to 0.28 gin, standard deviation 0.058 gin) and the average kinetochore microtubule length was 0.87gm (range 0.30 gm to 1.53 gin, standard deviation 0.25 gm). Post metaphase II behaviour of kinetochore microtubules was not observed in detail but by the characteristic cloverleaf stage of late meiosis (as described by HOWARD and MOORE 1970) they formed short clusters around each centriole.

38 K. TANAKA and I. B. HEATH: The Behaviour of Kinetochore Microtubules During Meiosis in the Fungus Saprolegnia

Figs. 1 and 2. Sections 2 and 6 from a series through a pre-meiotic antheridial nucleus at the P2/1 stage showing the two pairs of centrioles (c) and some of the 44 short kinetochore microtubules (arrows) which were present at this stage, x 57,000

Fig. 3. One of a series of sections through the centriole-associated region of an early leptotene nucleus. The centriole pairs (c) have begun to migrate apart, the nucleoplasm has assumed a "washed out" appearance characteristic of meiosis and numerous short kinetochore microtubules (arrows) are still present, x 52,500 Figs. 4 and 5. Sections 5 and 15 from a series through a leptotene nucleus on which two of the centriole pairs (e) had migrated 1.0 gm apart. Note the abundant short kinetochore microtubules (arrows) associated with each pair of centrioles, x 45,600

Figs. 6 and 7. Sections 5 and 13 from a series through a pachytene nucleus on which the centriole pairs (c) were separated by 4.0 ~tm. Note the absence of kinetochore microtubules, abundant cytoplasmic microtubules emanating from the centrioles (arrows) and the paired synaptonemal

complexes (sc). x 38,000

K. TANAKA and I. B. HEATH: The Behaviour of Kinetochore Microtubules During Meiosis in the Fungus Saprolegnia 39

Figs. 8 and 9. Sections 6 and 19 from a series through an early anaphase I meiotic spindle. Laterally paired kinetochore microtubules running parallel with one another to the spindle poles are shown by the arrows, x 45,500

Fig. 10. Laterally paired kinetochore microtubules (arrows) from a metaphase I meiotic spindle, x 38,000

Fig. 11. View of a third metaphase or early anaphase meiotic I spindle, again showing IateralIy paired kinetochore microtubules (arrows). x 38,700

4. Discussion

D u r i n g the mi to t ic nuclear cycle o f Saprolegnia, the

k ine tochore mic ro tubu les persis t t h r o u g h o u t the cycle,

segregate into two equal g roups dur ing S phase and

increase in n u m b e r to fo rm amphi te l ic pa i rs dur ing

ear ly me taphase (HEATH and RETHORET 1981). The fact

tha t Saccharomyces shows somewha t s imilar behav iour

(P~TERSON and RIs 1976) and has n o n - r a n d o m mi to t ic

c h r o m o s o m e segregat ion (WILLIAMSON and FENNEI.L

1980) suggests tha t Saprolegnia m a y also show similar

segregat ion. Whi ls t we have so far fai led to ob ta in

direct evidence for, or against , this hypothesis , our

40 K. TANAKA and I. B. HEATH: The Behaviour of Kinetochore Microtubules During Meiosis in the Fungus Saprolegnia

Figs. 12-14. Three serial sections from a metaphase II meiotic spindle showing a single centriole (c) at one pole and kinetochore microtubules (arrows) which form amphitelic pairs but do not occur in lateral pairs. This partially sectioned spindle contained 10 and l l kinetochore

microtubules in the respective half spindles, x 57,000

K. TANAKA and I. B. HEATH: The Behaviour of Kinetochore Microtubules During Meiosis in the Fungus Saprolegnia 41

observations that the kinetochore microtubules behave differently (i.e., are lost) during meiotic prophase when the chromosomes may also behave differently (i.e., segregate randomly) provide a possible structural basis for the phenomenon. Such a correlation should obviously be sought in Saccharornyces where the data for chromosome behaviour are clearer. Irrespective of this phenomenon, the current observations also help clarify the mitotic behaviour of the chromosomes. In previous work on mitosis it was impossible to determine if the chromatin remained attached to the kinetochore microtubules throughout the mitotic cycle (HEATH 1980a, HEATH and RETHORET 1981). During meiotic prophase the chromosomes condense around the synaptonemal complexes (HEATH 1980 b, TANAKA et al. 1982) which do not retain any affinity for the centriole region of the nucleus (TANAKA et al. 1982).

The dissociation of the chromosomes from the kinetochore microtubules and the concomitant loss of the microtubules supports the argument that retention of the microtubules during the mitotic cycle indicates retention of a chromosome-kinetochore attachment during that cycle when they persist. It is possible that the specific loss of the kinetochore microtubules during pachytene is a fixation artifact. We believe this is unlikely because a) cytoplasmic microtubules are well preserved at this stage (Figs. 6 and 7) and b) a similar absence is detectable in pictures of the comparable stage in previous work on Saprolegnia meiosis (HOWARD and MOORE 1970, BEAKES and GAY 1977).

Data on kinetochore activity and the relative number of kinetochore microtubules at mitosis, meiosis I and meiosis II are rare but contradictory. For example MOENS (1979) reported that whilst all four kinetochores of Locusta tetrads were attached to kinetochore microtubules at metaphase I, the total number of microtubules running to each pole was not much more than that found on each kinetochore at mitosis. In contrast, LIN et al. (1981) found very close to double the number per kinetochore bundle at meiosis I relative to meiosis II. With the small number of kinetochore microtubules per chromatid in Saprolegnia, we find a very clear and simple situation wherein all kinetochores are attached to a single microtubule at late metaphase in both mitosis and meiosis. Thus we have one per chromatid at mitotis, two per half tetrad at meiosis I and 1 per half dyad at meiosis II. This shows that all of the kinetochores are active in nucleating or trapping kinetochore microtubules at all divisions. However, as argued by both MOENS (1979) and LIN et al. (1981), it would be unwise to conclude that the correlation of two

kinetochore microtubules per half tetrad and one per half dyad or chromatid indicated a load to number of microtubules correlation because there is more than a threefold variation in chromosome size (TANAKA et al. 1982) yet all have only one or two kinetochore microtubules at the relevant divisions. In contrast to this ambivalence with respect to spindle architecture and force production mechanisms, it is clear that the maintenance of parallel kinetochore microtubules all the way to the spindle poles during anaphase I is a strong argument against the zipper hypothesis (BAJER 1973) for chromosome to pole movement. It is interesting to note that whilst the chromatin at meiosis II appears to be more condensed than at mitosis (compare Figs. 12-14 with Fig. 6 of HEATH 1980 a), the inter-kinetochore distance of the paired kinetochores is very similar with means of 0.17 gm (range 0.09 gm to 0.28 gin) and 0.22gin (range 0.10gin to 0.28 gin) for mitosis (HEATH 1980a) and meiosis (present data) respectively. This suggests that inter-kinetochore distance is not regulated by degree of chromatin condensation.

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42 K. TANAKA and I. B. HEATH: The Behaviour of Kinetochore Microtubules During Meiosis in the Fungus Saprolegnia

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