0CH 10 Cell Cycle & Cell Division:
The Cell Cycle: The cell cycle is a series of sequential events that result in duplication and equal distribution of DNA and other sub-cellular components of the mother cell into two daughter cells (four in the case of meiosis). As stated in the third point of the cell theory by Matthias Schleiden and Theodor Schwann, 1838, ‘all cells come from pre-existing cells’. It is the key to sustainability of the cellular organisms- be it the increase in number of cells in unicellular and multicellular organisms (growth), increase in the number of progeny directly through cell division as asexual reproduction in unisexual organisms, gametogenesis (meiosis) during sexual reproduction, growth of the zygote directly into new offspring (fungi, algae, etc.) or growth of the zygote into miniature offspring through intermediate embryogenesis stage. Moreover, it also has the key roles in the maintenance processes like wound repair, regeneration, tissue repair and numerous others in higher animals and plants.
The cell cycle in eukaryotes consists of two phases- the Interphase in which the cell prepares itself for a division, and the Mitotic phase (in somatic cells) or Meiotic phase (in reproductive cells) during which the genetic material (DNA) is distributed into resultant daughter cells. The distinction among different stages of mitotic or meiotic phase depends on morphological changes associated with nucleus and chromosomes. However, cell cycle of prokaryotes that lack a nucleus is divided into B (or ‘I’), C (C= chromosome) and D (D= division) periods to eliminate the need of their nucleus- dependent eukaryotic counterparts. The B or I period is equivalent to eukaryotic G1 phase, extending from end of preceding cell division to beginning or initiation of DNA replication. The cell also enlarges in size and prepares itself for a division in this very period. The C period is equivalent to eukaryotic S phase marked by DNA replication and nucleoid duplication. The D period is equivalent to eukaryotic G2+ Mitotic phase characterized by segregation of duplicated nucleoids at opposite poles of the cell followed by cell division. Instead of differences in their chromosome and other cellular architectures, chromosome condensation & decondensation and DNA replication processes, both the prokaryotes and eukaryotes share fundamentally similar cell cycle. In all the three domains of life, cell division consists of sequential events of DNA replication, chromosome condensation, chromosome segregation, cytokinesis and chromosome decondensation.
Mitosis: The Cell Cycle of Somatic Eukaryotic Cell: The cell cycle in a eukaryotic somatic cell is divided into Interphase and Mitotic phase. Both the phases are sequentially coordinated and regulated by a vast number of proteins in numerous feedback loops. The cell monitors phase-specific processes to ensure that, at the end of the cycle, each resultant daughter cells inherit a complete set of genetic material in proper condition.
Interphase: The period between two subsequent M phases is called interphase. It extends from the end of previous cell division to immediately before initiation of nuclear membrane disintegration. Under the influence of various stimuli in its microenvironment, a newly formed daughter cell may prefer not to divide any further or to enter next cycle of cell division. A cell is said to be in G0 (G zero) phase or quiescent phase if it’s not preparing for next cell division after completion of previous one. Alternatively, a cell in G1 phase enters G0 phase after it decides not to undergo another cell cycle. A fully differentiated cell like neurons and cardiac muscle cells are permanently arrested in G0 phase (sometimes, said to be in ‘quiescent phase’) at their functional maturity. Mature hepatocytes and some other cells remain temporarily arrested in G0 phase. These cells have intrinsic capacity to initiate cell cycle in the urge of replacing wear off cells and tissue repair. The cells undergoing rapid cell division, for example, those during embryogenesis, bypass G1 and G2 phases. The cells in this phase carry out normal metabolic activities to sustain normal functioning of the tissues, organs and the organism itself.
G1 Phase: A rapidly dividing cell enters G1 phase after completion of a previous cell cycle. A cell arrested at G0 also enters G1 phase on getting proper stimuli. During this phase, the cell makes all necessary preparations to process and regulate all the sequential events of the cell cycle- from DNA replication to cytokinesis. The cell increases in size. It synthesizes a wide array of mRNAs, proteins (especially histones) and other biomolecules. Different groups of mRNAs formed during this phase may be translated differentially in later phases as required. The cell proceeds to next phase (S- phase) only on the availability of optimum conditions like temperature, nutrients, and other essential biomolecules.
S Phase (DNA Synthesis Phase): S phase or DNA Synthesis phase is marked by initiation of DNA replication. Each ‘origin of replication’ (oriC) on a chromosome of the eukaryotic genome remains associated with ORC (origin recognition complex, a multisubunit protein complex) throughout the cell cycle. In early G1 phase, a pair of inactive MCM protein (minichromosome maintenance protein, has helicase activity) is loaded onto ORC by cdc6 and cdt1 proteins forming a pre-replicative complex (preRC). This mechanism of allowing oriC to replicate only once per cell cycle is also known as licensing. S phase-specific cyclin-dependent kinases (S CDK) phosphorylates specific proteins that, in turn, activate MCM helicase and load rest of the replication machinery to preRC. A complete replication machinery includes DNA polymerases, primase, topoisomerases, single strand binding proteins and all other components required for DNA replication. Early S phase also synthesizes histones. Through several coordinated mechanisms, like inhibition of cdt1 by geminin, the cell inhibits the formation of new preRC in this phase. By doing so the cell ensures that licensing of replication of the same oriC is not repeated until next G1 phase. Thus, DNA is replicated only once at each oriC of the whole genome during S phase. On completion of DNA replication, the cell has doubled its chromosomes number as well as DNA quantity.
After replication, each chromosome has a newly synthesized identical copy intertwined with it. A pair of an identical chromosome are called sister chromatids. Several rings of cohesin proteins (member of ATPase called Structural Maintenance of Chromosome, SMC, protein) along the length of sister chromatids keep them paired. Sister chromatid togetherness is essential for proper segregation later in the cell cycle. Centrosome (in animal cells) duplicates during this phase. Like chromosomes, centrosome duplication occurs only once per cell cycle and follows semi conservative mechanism. The duplicated centrosomes remain paired throughout this phase.
G2 Phase (Gap 2): The cell enters G2 phase after successful and correct duplication of the genome. In this phase, the cell synthesizes several proteins, like microtubules, required in M phase. The cell also grows in size. The paired centrosomes dissociate and move away from each other that would form two poles. Centrosome disjunction initiates microtubule nucleation.
M Phase (Mitosis Phase): Walter Fleming coined the term ‘mitosis’ (Originally ‘Mitosen’ meaning ‘thread -like’ appearance of chromatids during this period of cell division). M phase consists of two distinct events- karyokinesis and cytokinesis. Karyokinesis i.e. division of nucleus is commonly known as mitosis. Cytokinesis is the division of a cell. Since the number of chromosomes in daughter cells is same as that of their parents, it’s also called ‘equational division’ of a cell. Based on the morphological changes in the nuclear envelope and relative arrangement of chromosomes under a light microscope, mitosis is sub- categorized into prophase, prometaphase, metaphase, anaphase, and telophase.
Prophase: Prophase is marked by condensation of chromosomes in the nucleus. The chromosomes first become visible under the light microscope as long thread-like structures. Phosphorylation of H2A histones by Aurora B and mitotic CDK triggers condensin proteins (SMC family) binding to chromatin. Coupled with topoisomerase II activity, condensin protein forms positively supercoiled and highly condensed chromosome. Simultaneously with condensation, Polo kinase and Aurora B kinase phosphorylates cohesin protein and cause cohesin rings dissociate from sister chromatids along their length except at centromere. Protein phosphatase 2 (PP2) associated with centromere prevents cohesin phosphorylation and maintains sister chromatids attachment at this point. As a result, sister chromatids appear distinct rod-like structures attached at centromere under a light microscope. In the animal cell, radial nucleation of spindle aster occurs at the centrosome in the cytoplasm. In plant cells and many other eukaryotic cells that lack centrosome, the spindle fibers formation is ‘self-organized’ without the need of centrosome. Nucleolus, endoplasmic reticulum, and Golgi body disappear. The end of prophase is marked by the onset of nuclear envelope disintegration.
Prometaphase: Prometaphase is marked by rapid disintegration of the nuclear envelope. Once nuclear envelope degrades, spindle fibers bind to sister chromatids at the kinetochore and along their length. The kinetochore is a button- like protein complex assembled at the centromere. It facilitates spindle fibers attachment to chromosome and mediates segregation during cell division. The nucleosomes in centromeric chromatin have CENP-A (Centromeric protein- A is an H3 variant) instead of H3 histone. CENP-A is a hallmark of kinetochore because it triggers assembly of more than hundred proteins to form functional kinetochore during different phases of cell cycle. Kinetochore consists of an inner layer that persists through out the cell cycle and an outer layer laid over it during prometaphase. The plus end of spindle fibers binds to Ndc80 protein in outer kinetochore layer. The plus end undergoes rapid polymerization and depolymerization causing its length increase or decreases, the phenomenon is called ‘dynamic instability’. The transition of a microtubule from growth to shrinkage is termed catastrophe, and that from shrinkage to growth is termed rescue. A centromere interacts along the length of microtubule while kinetochore searches and bind to the plus end of an interacting microtubule.
The association of microtubule from one pole to one sister chromatid and those from opposite pole to the other chromatid of the pair is called amphoteric attachment. The spindle fibers from opposite poles pull their cognate sister chromatids towards their respective poles creating a ‘bi-polar tension’. Chromosomal passenger complex, CPC (Aurora B kinase complex with its regulatory proteins) associated with outer kinetochore, senses bipolar tension and simultaneously guides chromosome movement towards the central equatorial plate. All other attachments including merotelic (the same chromatid is attached to microtubules from both poles), monotelic (only one of the sister chromatids in the pair is attached to microtubule) and syntelic attachments (both sister chromatids are attached to microtubules from the same pole) are unstable and removed. Several motor proteins associated with kinetochore (ex.-) and arm of chromatids (ex.-) mediate chromosome movement during mitosis. Finally, all the sister chromatid pairs align at the equatorial plate at the end of prometaphase.
Metaphase: Once all the sister chromatid pairs align at the equatorial plate, the cell is said to be in metaphase. The hypothetical plane of alignment of all pairs of sister chromatids during metaphase is called metaphase plate. The metaphase microtubules may be categorized into astral, chromosomal and polar microtubules depending on interacting among themselves as well as with chromosomes and kinetochore. Astral microtubules radiate outside the spindle apparatus (the assembly of microtubules actively participating in chromosomal segregation). These microtubules help positioning the spindle apparatus in correct orientation. The microtubules extending from centrosome to a kinetochore is called chromosomal or kinetochore microtubules. These microtubules correctly position and carry the chromosomes for segregation. The number of kinetochore microtubules per sister chromatid varies from species to species, for example, it’s one in yeast and 20-30 in mammalian cells. The group of several microtubules bound to a kinetochore is also referred to as ‘spindle fibers’. The microtubules extending from one pole to beyond the metaphase plate towards the opposite pole without binding kinetochore are called polar or interpolar microtubules. Polar microtubules from opposite poles appear overlapping each other. These microtubules are crucial for mechanical integrity of the spindle apparatus.
The movements of chromosomes during and after alignment at metaphase plate is a result of simultaneously coordinated forces namely polar winds (polar ejection force), poleward flux (microtubule flux) and kinetochore- generated poleward force. Polar ejection force arises due to attachment of plus end of interpolar microtubules to chromosome arms through motor proteins (kinesin-4 and kinesin-10). Due to enhanced rate of polymerization at plus end, microtubules push the chromosome away from the spindle pole. This force helps in positioning the chromosomes towards the equatorial plate during prometaphase and metaphase. Poleward flux arises due to overall net depolymerisation of microtubules at minus end. Thus, this force ‘pulls’ the chromosome towards the spindle pole. Simultaneously with aiding chromosome alignment, it has key roles in segregation of sister chromatids during anaphase. Kinetochore- generated poleward force arises due to plus end depolymerisation of kinetochore associated microtubule. Some proteins in outer kinetochore layer cause this depolymerisation in ATP independent manner. The chromosome is displaced toward the spindle pole under the influence of this force. It also helps in chromosome alignment and segregation of sister chromatids during anaphase.
Colchicine, a natural secondary metabolite first extracted from plant Colchicum autumnal (common name- meadow saffron) acts at spindle poison. It inhibits microtubule polymerization as well as depolymerisation by binding to tubulin subunits. Thus colchicine treatment arrests the spindle apparatus as it is. At metaphase, the ‘frozen’ spindle apparatus along with maximally condensed chromosomes is useful in karyotyping and detecting several chromosomal disorders. Colchicine is also used to treat gout.
Anaphase: Beginning of anaphase is characterized by centromere splitting and separation of sister chromatids into two independent chromosomes. Securin protein keeps separase enzyme inactivated till metaphase. Once all sister chromatids align at the metaphase plate, anaphase promoting complex (APC)/C is phosphorylated and activated by Cdc20 (cell division control 20, protein). Separase enzyme, when free from its cognate inhibitor securin, degrades cohesin ring complexes (scc1 subunit) at centromere. As a result, both the sister chromatids separate from each other. The separated sister chromatids are now referred as chromosomes for not being attached to their ‘sisters’.
The chromosomes move towards their spindle pole as cognate microtubule depolymerizes both at its minus and plus end. The movement of chromosomes to spindle pole is called Anaphase A. Simultaneously to Anaphase A, the spindle poles also move further away from each other, the process being cal
