5.8 Mitotic nuclear division
Prior to the nuclear division cycle, interphase (vegetative or S-phase) fungal nuclei look more-or-less spherical and have diffuse chromatin in the light microscope. Nuclei that are migrating through the hypha are elongated and have a highly visible nucleolus. As the nucleus enters its division cycle nuclear volume decreases and chromatin condenses. The nucleoli remains evident in dividing fungal nuclei until late in the mitotic division (the stage called anaphase) and are then quickly restored and steadily enlarge until daughter nuclei are formed.
Fungal mitotic divisions are intranuclear: in this ‘closed mitosis’ the division spindle forms inside the nucleus. This is quite different from the ‘open mitosis’ seen in most animals and plants where the nuclear envelope disassembles and microtubules invade the nuclear space to form the division spindle. When the division spindle is formed within an intact nuclear membrane progress of the division is more difficult to see and study, but it does not appear to affect the biological consequences of the mitotic division.
The typical mitosis proceeds through a series of morphologically distinct stages: interphase, prophase, metaphase, anaphase, telophase, ending with cytokinesis, when the cell divides to produce two identical daughter cells. In animals and plants cytokinesis usually occurs in conjunction with mitosis. As we have mentioned, mitosis may occur independently of the branching and septation that are the equivalent of cytokinesis in filamentous fungi, but we will return to this topic later [CLICK HERE to see it now].
During the process of mitosis the newly-replicated paired chromatids condense and attach to fibres of the division spindle that then pull the sister chromatids to opposite ends of the spindle. The division spindle is an organelle in its own right. It consists of the two spindle poles, each containing a centrosome made up of two centrioles (in animals) or spindle pole body (SPB, in fungi) that manage the spindle fibres, which are microtubules, connecting the poles with specialised regions of the chromosomes called kinetochores, protein structures assembled on the centromeres of the chromatids (plants make do without a discrete organising centre, but all eukaryotes use γ-tubulin to anchor and initiate microtubule assembly).
All of the preparation for mitosis is done during interphase, the spindle apparatus is assembled during prophase. In metaphase the condensed chromosomes are aligned near the spindle equator (forming the metaphase plate), then the chromosomes are moved towards the poles as the spindle elongates in anaphase. Finally, the daughter nuclei are separated in some way; in animals and plants the cell divides near the spindle equator (= cytokinesis); this may also apply in fungi (fission yeast Schizosaccharomyces), or one nucleus may migrate into a bud (budding yeast, Saccharomyces), into a newly formed branch or conidium or other spore (many filamentous fungi), or into a ‘new’ volume of cytoplasm in the hypha which may, or may not, be separated off by septation (many filamentous fungi).
In their daughter cytoplasm the nuclei enter interphase; this whole series of events is called the cell cycle. There are networks of regulatory mechanisms, the crucial ones being known as checkpoints, which coordinate the timing and progress of events in mitosis. For example, the spindle checkpoint inhibits the progress of anaphase until all of the kinetochores are attached to spindle microtubules. We will describe the cell cycle later [CLICK HERE to see it now], but we first have to describe the basic machinery of mitosis.
Many features of nuclear and chromatid movement in mitosis are common to all eukaryotes and extensive study of mitosis in fungi, Aspergillus nidulans in particular, has contributed greatly to our understanding of eukaryote mitosis (Morris, 2000). Nuclei are very mobile, being pulled around by an attached organelle: in the fungi by the SPBs and in other eukaryotes by the centrosomes, and chromatids are pulled around by kinetochores assembled on their centromere regions. The centromere typically contains hundreds to thousands of kilobases of some tandemly repeated DNA sequences. Analysis of these repeats in organisms from insects, plants and fungi to mammals and other vertebrates reveals no obvious conservation of sequence, suggesting that a universal centromere sequence does not exist, or has at least has so far defied recognition.
Centromere characteristics depend less on the sequence and more on what is done to these regions as DNA molecules and proteins are assembled on them and the kinetochore is constructed. Kinetochores are assemblies of about 45 different proteins, including a histone H3 variant (called CENP-A or CenH3) which is specialised to helping kinetochore binding to centromeric DNA. Other kinetochore proteins attach to spindle microtubules, and there are motor proteins, including both dynein and dynactin (a regulator of dynein) generally, and three kinesins in S. cerevisiae, which generate forces that move chromosomes during mitosis. Other proteins monitor microtubule attachment and the tension between sister kinetochores and activate the spindle checkpoint to arrest the cell cycle when either of these is absent.
Kinetochore structure and function are not fully understood yet, but in essence the dynein motor uses energy from ATP to ‘crawl’ up the microtubule towards the originating SPB. This motor activity, along with polymerisation and depolymerisation of microtubules, is what provides the pulling force necessary to separate the chromosomes.
Knowledge of nuclear migration in S. cerevisiae derives from study of bud formation. The SPB is a microtubule organising centre embedded in the nuclear envelope; it produces astral microtubules emanating from the nucleus into the surrounding cytoplasm and spindle microtubules within the nucleus. Nuclear movements during mitosis mirror the growing and shrinking rate of astral microtubules, consistent with the idea that the microtubules are pulling the nuclei into position.
Nuclear migrations seen in yeast are very short range compared with the scale of nuclear migration in filamentous fungi. Nuclei migrate through the cytoplasm toward the advancing hyphal tip as the fungal colony grows; in Gelasinospora tetrasperma, a typical nuclear migration rate of 4 mm h-1 through a newly-formed heterokaryon compares with a typical hyphal extension rate of only 0.7 mm h-1 (and in both cases we do mean millimetres). From early observations it was clear that nuclei are pulled from a point on the nuclear periphery, where the SPB is located. Observations on living fungi show that the nuclei move apart after mitosis, then migrate in the same direction, but at different rates, towards the hyphal tip, evening out their distribution along the hypha. The motive force for the movement in mitosis is an interaction between the SPB and the microtubules. The pulling force that moves interphase nuclei through the hyphal cytoplasm is a continuation of this process and depends on cytoplasmic dynein motor activity.
The first nuclear migration mutants were a by-product of a mitotic mutant search in Aspergillus nidulans; they were named nud (for nuclear distribution). Similar mutations of Neurospora crassa were named ropy (ro) because the hyphae resemble intertwined strands of rope. Many of the nud and ro genes are now known to encode structural subunits of, or components essential for the consistency, localisation or activity of, cytoplasmic dynein or dynactin [CLICK HERE to see it now].
In the first stage of anaphase, chromatids move to the poles of the spindle, and in the second stage of anaphase the two poles of the division spindle move further apart. Mitotic anaphase movements in filamentous fungi are randomised in relation to the long axis of the hypha, so the mitotic division spindle does not have a preferred orientation (which it would be expected to show if septation were mechanically linked to division). The final stage of mitosis, telophase, follows one of three patterns:
- median constriction, separating the entire nucleoplasm into the two daughter nuclei;
- a double constriction which incorporates only a portion of the parental nucleoplasm into each daughter nucleus, the rest being discarded and degraded;
- formation of new daughter nuclear membranes, separate from the parental one, enclosing the chromosomes and a small portion of the nucleoplasm into the daughter nuclei while the bulk of the parental nucleoplasm and its membrane are discarded and degraded.
In multinucleate hyphae, mitotic divisions are not usually synchronised. The first three or four rounds of mitosis are synchronised in germinating spores of Aspergillus nidulans, but mitotic synchrony degenerates, perhaps because of increased difficulty of effective cytoplasmic signalling throughout the juvenile mycelium, though may still occur locally. In some fungal tissues, such as mushroom stems, nuclear division is rapid and there is some synchronisation although whether this is controlled or coincidental is not clear.
However, the higher fungi do seem to have a looser connection between cell differentiation and nuclear number and ploidy than is usual in plants and animals. In the cultivated mushroom, Agaricus bisporus, cells of vegetative mycelial hyphae have 6–20 nuclei and those in the mushroom fruit body have an average of six nuclei, though cells in the mushroom stem have up to 32. In Coprinopsis cinerea (appearing in most publications under the name Coprinus cinereus), the vegetative dikaryon is absolutely regular with strictly two nuclei per compartment. However, cells of the fruit body stem can become multinucleate through a series of consecutive conjugate divisions, ending up with up to 156 nuclei, a peculiarity seen in other agarics. Species of Armillaria are unusual in having diploid tissues in the fruit body, so ploidy level and nuclear number are both variable.
Updated January 18, 2017