Types of mitosis in protozoa. Microtubule-organizing center.
The mechanisms of mitosis in higher eukaryotic organisms are very well studied; however, regarding protozoa, there are still many questions in need of an answer. Because of the complexity with which it carries out this process, many forms of mitosis exist, such as open orthomitosis, semi-open orthomitosis, semi-open pleuromitosis, closed intranuclear pleuromitosis, closed intranuclear orthomitosis, and closed extranuclear pleuromitosis. The fascinating aspect about the mitosis of Entamoeba histolytica trophozoites is that it falls out of the context of this classification, but not entirely. The Entamoeba histolytica trophozoites first carry out karyokinesis and then cytokinesis. The mitosis of this parasite is comprised of the following phases: prophase, metaphase, early and late anaphase, early and late telophase, and karyokinesis. The difference lies in the mechanism by which it carries out the distribution of the genetic material because it forms three mitotic spindles: two radial spindles that practically surround every group of chromosomes and one that we call inter microtubule-organizing centers (IMTOCs). The latter transports each group of chromosomes at each of the nucleus poles. Based on these observations, we propose that Entamoeba histolytica trophozoites carry out a type of mitosis we have called modified intranuclear pleuromitosis open.
- Entamoeba histolytica
- mitotic spindle
Mitosis in the cells of living beings guarantees the cells’ multiplication during the processes of tissue replacement and repair. However, in some protozoa it carries out the purpose of maintaining the species, such as in the case of
In order to understand how mitosis occurs in
2. The cell cycle of higher eukaryotes
The cell cycle phases have been divided into interface and M phase (mitosis). During the interface, the cell performs functions of the tissue in which it differentiated into (phenotype) in order to stay alive (G1 phase), duplicate its genetic material (S phase), and prepare for mitosis (G2 phase). During the G1 phase, the cell maintains its biochemical integrity, expresses its phenotype, and synthesizes elements necessary for the duplication of genetic material. In the S phase, the cell carefully duplicates its genetic material so that each chromosome is doubled. During the G2 phase, the cell prepares for the M phase. Sometimes, some cells that are in the G1 phase enter a state of latency or rest, known as the G0 phase . The G1 phase covers the end of the M phase up until the beginning of the S phase. The S phase follows at the end of the G1 phase and ends at the beginning of the G2 phase. The G2 phase starts at the end of the S phase and finishes at the beginning of mitosis (Figure 2).
The duration of the interface is longer than the M phase, which lasts only 1 h. However, in the embryonic cells of higher eukaryotic organisms, the S phase is reduced; consequently these cells’ cycle is short . The cell cycle of embryonic cells explains the accelerated cell multiplication and the rapid growth of the embryo. The cell cycle of fruit fly embryos lasts only 8 h in contrast to the cell cycle of mammals which lasts 24 h .
The duration of the cell cycle varies but the process is similar in all cases. It involves the preparation of the cell in order to give rise to a new organism, as it occurs in unicellular organisms, or to form two identical cells during embryonic development or cell regeneration. Although the two newly formed cells of multicellular organisms are identical, during their cell cycle, each one can modify its phenotype to specialize in a specific function, as it occurs during the advanced stages of the embryonic development of higher eukaryotic organisms .
Before a cell initiates mitosis, it needs to duplicate its genetic material and prepare optimal cytoplasmic conditions that will allow it to form two identical cells. Mitosis of higher eukaryotic cells begins with prophase (P), during which the chromatin gradually condenses until the duplicated chromosomes are visible, each with its two sister chromatids joined by the centromere. The microtubules of the cytoskeleton are disassembled, and the formation of the mitotic spindle begins in between the centrosomes that move away from each other. In this phase, the nucleolus is disorganized and not visible during the entire mitosis. Prometaphase (PM) begins abruptly with the disorganization of the nuclear envelope that remains in the form of small vesicles around the mitotic spindle during mitosis. Several protein complexes called kinetochores mature and assemble in the centromere of each chromatid. The fibers of the mitotic spindle that are attached to these structures are called microtubules of the kinetochore; the fibers that do not bind to the kinetochore are known as polar microtubules, and the fibers that are outside the spindle are called astral microtubules. When changing to metaphase (M), the microtubules of the kinetochore align the condensed chromosomes into an equatorial plate. The other end of the kinetochore microtubules attaches to the centrosome of each pole opposite the spindle. Anaphase (A) begins exactly at the moment where the kinetochore pair separates, aided by the microtubules of the mitotic spindle, and is directed toward the opposite poles of the nucleus. The polarization of the chromosomes produces shortening of the kinetochore microtubules, whereas the polar microtubules become longer. During telophase (T) the daughter chromosomes reach the poles of the nucleus, and the kinetochore microtubules disappear. The polar microtubules have lengthened further, and the nuclear envelope begins to organize around the daughter chromosomes. In this phase the nucleolus reappears . During cytokinesis, the cytoplasm divides, and the cell membrane is strangulated in the middle portion of the cell by myosin rings causing the cell to separate, forming two daughter cells (Figure 3).
3. The cell cycle in protozoa
For their survival, unicellular organisms also need to duplicate their DNA, divide, and, thus, give origin to a new organism . Like in the cell cycle of higher eukaryotic organisms, in the protozoa interphase (I) and mitosis (M) also occur. The phases (G1), (S), and (G2) are also present in interphase. The phases of mitosis are the same as those in higher eukaryotic cells . The duration of the M phase, as well as each of its phases, depends on the culture conditions. However, the beginning of the S phase and its duration and culmination are evidently regulated by the genetic material . For example, the cell cycle of the
The presence of cyclin-dependent protein kinases related to human cdc2 in some parasites such as
The cell cycle can be interrupted experimentally with drugs that inhibit the activity of proteasomes such as lactacystin which maintains the procyclic forms of
Mitosis is the main type of nuclear division of protozoa. The fundamental characteristic of mitosis is that the two copies of chromosomes or chromatids are equally distributed between the two daughter nuclei. Consequently, each daughter nucleus receives a complete series of chromosomes. The details of mitotic mechanisms vary widely, particularly in lower eukaryotes including protozoa. In protozoa the level of development of the spindle and centrioles (or structures that are functionally similar to them, such as the microtubule-organizing centers (MTOC)) and the behavior of the nucleolus during mitosis vary widely. In spite of the variants, the two chromatids of each replicated chromosome migrate toward the daughter nucleus while conserving the fundamental characteristic of mitosis .
The mitosis observed in protozoa has been classified according to the site in which the formation of the mitotic spindle occurs, the appearance of a complete spindle or formation of two half spindles, and the disintegration, or not, of the nuclear envelope. If the mitotic spindle forms inside the nucleus, it is an intranuclear mitosis, whereas if the spindle forms outside the nucleus, it is said to be an extranuclear mitosis. If a complete spindle is formed, it is an orthomitosis; on the other hand, if two half spindles are formed, it is called pleuromitosis . Finally, if the nuclear envelope remains intact, it is a closed mitosis; if it partially disintegrates, it is said to be a semi-open mitosis, but if it disintegrates completely, it is called a eumitosis, which is equivalent to the mitosis of the higher eukaryotic cells . The combination of these events, during the reproduction of the protozoa, allows the mitosis to be classified into six types: (1) open orthomitosis, (2) semi-open orthomitosis, (3) semi-open pleuromitosis, (4) intranuclear pleuromitosis, (5) intranuclear orthomitosis, and (6) extranuclear pleuromitosis  (Table 1).
|Types of mitosis||Spindle||Nuclear envelope||MTOC||Nucleolus||Chromatin|
|Open orthomitosis||Bipolar, axial, and symmetrical||Disorganized and not observable||Yes||Disorganized and not observable||Condensed|
|Semi-open orthomitosis||Bipolar and symmetric||Perforated at the core poles||Yes||¿?||Less condensed|
|Semi-open Pleuromitosis||Two identical hemi-spindles||Perforated near spindle formation||Yes||¿?||Less condensed|
|Closed intranuclear pleuromitosis||Two intranuclear symmetrical hemi-spindles||Whole||Yes||Yes||Less condensed|
|Closed intranuclear orthomitosis||Axial, bipolar, symmetrical, and intranuclear||Whole||Yes||¿?||Condensed|
|Closed extranuclear pleuromitosis||Two extranuclear mitotic hemi-spindles||Whole||Yes||Remains||Condensed|
The structures similar to the centrioles of kingdom Protista, currently known as MTOC, have received different names such as centrosphere, rhizoplast, spindle polar body, kinetosome, atractophores, or organelle associated with the nucleus . Even though they present great diversity in their arrangement, they adequately participate in the spatial organization and behavior of microtubules during the cell cycle. In
Apparently there is just one MTOC in this protozoan. However, the binding of recombinant anti-tubulin γ antibodies, from the amoeba, at the MTOC site, suggests that this structure is doubled and polarized, respectively, during anaphase and telophase .
The chromatin of the protozoa during interphase is in a decondensed and condensed form. In some protozoa, chromatin is dispersed, while in others it is condensed forming peripheral groups, reticular fibers, individual chromosomes, chromocenters, karyosome, or a dense mass that occupies the whole nucleus .
The level of compaction of chromatin mainly varies from one species to another. (i) Finely dispersed chromatin has been found in protozoa that have very large nuclei as seen in
Due to the fact that some protozoa have chromatin organized in a similar way to that of prokaryotes, it has led to classify this phylum in (a) mesocarion and (b) eukaryotes. Both types have a well-defined nuclear envelope, but the former possess chromatin arranged in fibers aggregated in a similar way to bacteria .
(a) Mesocarion protozoa include organisms of the
The number of chromosomes of protozoa has been determined by applying electrophoresis in a pulse field gradient (PFG) and obtaining karyotypes through cellular explosion. By means of the PFG, the DNA size of the chromosome in two species of fish microsporidia has been established. The molecular karyotype of
In general, the chromatin of the flagellates is compacted, although there are variations of condensation sometimes forming dense rods that touch the nuclear envelope . In functional terms, decondensed chromatin is considered transcriptionally active . Its arrangement is fibrillar with the formation of aggregates of granular appearance as it occurs in organisms of the
In the ciliated protozoa, the genetic material is stored in the macro- and micronucleus. The DNA contained in the macronucleus is transcriptionally active unlike the content in the micronucleus which is inactive . Both nuclei contain DNA and RNA . The chromatin of the macronucleus of the ciliated protozoa is commonly organized into numerous discrete masses called “small bodies”  although it has also been observed in the form of spongy masses , reticular formations, long chains of chromatin thin plexuses , discretely elongated bodies , compact spheres, spherical masses, and clear halo granules . The macronucleus of these protozoa contains a structure made visible by light microscope called the replication band, which is a specific site of DNA replication that migrates from the macronucleus and advances along the edge of this band generating the rearrangement of the chromatin in two zones: the proximal and the distal. The proximal reorganizes the chromatin for the synthesis of DNA, and the distal carries it out . Apparently in these organisms, chromosomal fragmentation and the elimination of internal sequences as a route of DNA processing occur .
4. Cell cycle of the trophozoites of
One of the main problems in studying the cell cycle and the mitosis of the trophozoites of
4.1 Mitosis in the trophozoites of
Studies on the organization of the nucleic acids of live trophozoites
Unlike the mitosis of the higher eukaryotic cells, the nuclear envelope of the
The permanence of the nuclear envelope of
During prophase, the oval nucleus shows 5 to 6 chromosomes and 16 to 18 small chromosomes. The chromosomes are observed to be arranged around the microtubule-organizing center (Figures 4b, 5c and d) in the center of the nucleus, and the RNA is located around the periphery in a ring shape .
In metaphase, the round nucleus increases in size, and the chromosomes move further away from the center of the nucleus. The side view of the duplicated chromosomes shows two parallel rows of round bodies. The RNA ring breaks and forms two to three oval portions located in opposite poles (Figure 4c).
Early and late anaphase is characterized by the separation of the chromosomes into two apparent equal parts, each with six chromosomes. In this phase, the nucleus is observed to be elongated with an unchanged RNA (Figure 4d and e).
In early and late telophase, the nucleus size is 21 μm. Each group of six chromosomes, with the respective small chromosomes, is located in the opposite poles of the nucleus arranged in a ring shape, while the RNA forms small condensations evenly distributed between the two daughter nuclei (Figure 4f,g, and h).
As mentioned at the beginning of this chapter, there are variations in the types and in the location of the mitotic spindles in different protozoa. By transmission electron microscopy  and immunofluorescence studies [39, 2], the mitotic spindle of
|Mitotic apparatus||Human cells ||E. histolytica trophozoites|
|Types||Bipolar, axial, and|
|Two radial and one inter MTOCs|
|Two radial are intranuclear,|
The inter MTOCs is intranuclear
and cytoplasmic 
Bipolar spindles 
|Origin of spindle|
|α and β||Cytoplasmic||Intranuclear and Cytoplasmic|
|Nuclear envelope||Disorganized during|
|Remains during mitosis|
mitosis and not observable
|No nucleolar structure has been|
|RNA||Disorganized and not|
|It remains condensed |
|Chromatin||Condensed in the metaphasic chromosomes||It remains condensed throughout mitosis|
|DNA||Condensed and Decondensed||It remain condensed|
|44 autosomes||24–32 |
|2 sex chromosomes||6 |
The MTOC of the protozoa is equivalent to the centrosome of the higher eukaryotic organisms (Table 2).
During prophase, the formation of many radially arranged microtubule fibers is observed in the MTOC located in the center of the nucleus  (Figures 6b and c, 7b and c). In metaphase, the MTOC is duplicated, and radial microtubule fibers directed toward the nuclear envelope (radial spindles) emerge from each one. MTOC fibers also arise from transverse microtubules that go from one MTOC to the other (spindle inter MTOC). It is possible to appreciate in the nuclei of the
After karyokinesis, cytokinesis begins; the trophozoite with two nuclei begins to divide by narrowing the cytoplasm in its middle part . This process is relatively slow and occurs gradually through stretching with intervals of rest. The cytoplasm of the middle part of the trophozoite thins until it forms a very thin filament which then breaks and results in two trophozoites of
The identification of β-tubulin bundles that surround the DNA nuclei in telophase suggests a mechanism of entrapment similar to a “hand-closure movement” that allows, along with the inter-MTOC spindle, the distribution of genetic material between the two newly formed nuclei.
The mitosis of