Structural Biochemistry/Mitosis

Overview
Mitosis is the process by which two identical daughter cells are produced after nuclear divison and cytokinesis. It is the process by which cells replicate and occurs during the normal cell cycle, and should not be confused with meiosis. The following outlines the stages of mitosis.

Interphase

Interphase is technically not part of mitosis, but it is important because it is discussed as part of the cell cycle and precedes the process of mitosis. In interphase, the cell is in preparation for nuclear division. It is also the beginning stage, where DNA is synthised and proteins are made. C The cell undergoes the G1, S, and G2 phases of the cell cycle during interphase. G1 is recognized as the stage before synthesis of DNA. S phase is when DNA is synthesized, and the G2 phase is the stage immediately preceding the first stage of mitosis (prophase) during which the cell synthesizes proteins and grows.

Prophase

Prophase is the next step in the cell cycle and during prophase, the chromatin in the nucleus becomes compact and the nucleolus breaks down. The centrioles, or microtubule organizing centers for plant cells, move to opposite ends of the cell. Fibers extend from the centrioles to create the mitotic spindle.

Prometaphase

During this stage, the nuclear membrane degrades and proteins attach to the centromeres creating a new complex called kinetochores. The microtubules attach at this complex and the chromosome start moving toward the middle of the cell.

Metaphase

During metaphase, the chromosomes are aligned at the middle of the cell with the help of spindle fibers. This line known as the metaphase plate is a method of organization to ensure proper division of the cell and that each new daughter cell receives exactly one copy of each chromosome.

Anaphase

At this stage, the paired chromosomes are split by separation at the kinetochores to opposite sides of the cell. The chromosomes are now called sister chromatids.

Telophase

The chromatids are fully separated as they are at opposite ends of the cell. New membranes form around the perimeter of the daughter nuclei and the chromosomes are dispersed. The spindle fibers start to degrade and prepare for cytokinesis.

Cytokinesis

Cytokinesis is the process by which the cell partitions to make two complete, and identical daughter cells. The process is slightly different for animal and plant cells. In animal cells, cytokinesis is completed through the help of actin that pinches the cell into two equivalent cells. However, in plant cells a cell plate is formed in the middle of the cell to result in two equivalent daughter cells. This extra step is due to the rigid cell wall that is a characteristic of plant cells.

Cell Cycle Checkpoints
Although components that actually partake in cell division is very important, there are other components involved in regulating and signaling mitosis that are just as crucially important and are often overlooked.

 G1 Checkpoint 

Before a cell enters the S phase, it must pass the G1 checkpoint. This checkpoint decides whether the cell should undergo cell division, also known as mitosis, or enter into a rest phase and delay division. Whether the cell goes into the resting stage or cell division depends on its type. For example, liver cells only pass the G1 checkpoint to undergo cell division twice a year. If the cell stops at the G1 checkpoint, it will go into the G0 stage which is known as the resting stage. The G1 checkpoint in eukaryotes is controlled by the CDK inhibitor p16, or CKI p16. The purpose of this protein is to inhibit CDK4/6 which then prevents its interactions with cyclin D1. Inhibited interactions between CDK4/6 and cyclin D1 will prevent a cell from entering the cell cycle. When growth is induced, the cell proceeds from G0 to G1 to S phase due to the increased expression of cyclin D1 which then competitively binds to CDK4/6. When CDK 4/6-Cyclin D forms, this complex phosphorylates retinoblastoma, denoted Rb. Rb allows transcription factor E2F to express cyclin E, and the cyclin E interacts with CDK2 to allows cells to transition from the G1 phase to S phase. 6

 G2 Checkpoint 

Once the cell has passed the first checkpoint, G1, it must pass the G2 checkpoint before actually going into mitosis. G2 checkpoint occurs at the end of the G2 phase, prior to the M phase or the mitotic phase. This checkpoint is primarily to make sure that the cell is ready for mitosis. At this checkpoint, the cell is checked to see if it has a number of factors that determines if it is ready to undergo cell division. The CDKs present at the G2 checkpoint are initiated by the phosphorylation of the CDK via MPF, which is called the “Mitosis Promoting Factor”. At the G2 checkpoint, an activating phosphatase called CDC25 removes phosphates from MPF so that the MPF may promote mitosis. However, DNA tends to be damaged prior to mitosis. Therefore, the cell cycle is put in arrest by inactivating CDC25. It is important to inactivate the CDC25 so as to prevent the transmission of damaged DNA to daughter cells. 5

 Mitotic Checkpoint of Spindle Assembly (SAC) 

An important process that regulates the cell cycle is called the spindle assembly checkpoint, also known as SAC. SAC is a cell cycle checkpoint that cells encounter before going into anaphase. Checkpoints are important to the cell cycle because they control the rate and extent to which a certain phase occurs. SAC specifically helps chromosomal stability and prevents cases of aneuploidy. 4

Protein Kinase involved in SAC: BUB1 and BUBR1

BUB1 (budding uninhibited by benzimidazole-1) and BUBR1 (budding uninhibited by benzimidazole-related 1) are proteins that play a key role in the establishment of central mitotic checkpoint. BUB1 and BUBR1 consist of three main regions. One of these regions is the C-terminus. This region is a catalytic serine/threonine protein domain. The other region is the N-terminal region which is conserved in BUBR1 and BUB1, as well as their homologs. The N-terminal region contains the kinetochore localization domain. The last region is a non-conserved region where BUB3 is known to bind. 3 Both BUB1 and BUBR1 are directed to the kinetochores via blinkin. Blinkin is a multiprotein macromolecular complex that works as a connecting dock between the kinetochores and BUB1/BUBR1. 7

BUB1 Functions

Although similar in structure, BUB1 and BUBR1 are paralogs because they have different functions and roles in the mitotic checkpoint. The role of BUB1 is primarily: 1) to establish and/or maintain the efficient bipolar attachment of spindle microtubules to the kinetochore of chromosomes and 2) for chromosome congression.

The primary role of the SAC is to ensure that chromosomes are being passed on to the next generation in a reliable manner by serving as the central surveillance mechanism. The SAC halts metaphase to anaphase transition as long as the kinetochore lacks bipolar attachment to the microtubule. Because of this, a highly sensitive signaling pathway is extremely crucial. BUB1 comes into play by acting as the master regulator of forming and signaling SAC. There are several other proteins (such as MAD1, MAD2, MAD3/BUBR1, BUB3, MPsp1) that are also a part of the checkpoint, but many of these proteins are known to interact with BUB1.

Because BUB1 is a multi-domain protein kinases, it has several domains that may function independently of one another. Aside from all the other functions, one of BUB1’s functions is to transport phosphate from ATP to different molecules. Thus, once SAC is activated, BUB1 phosphorylates APC/C’s coactivator called Cdc20. This causes a decrease in activity of APC/C, which is responsible for the metaphase to anaphase transition. APC/C acts in turn by priming BUB1 for degradation so that it can exit mitosis. The N-terminus is extremely vital for an efficient SAC because studies involving a mutation or a deletion of an exon that code for the N-terminus have led to chromosome segregation errors as well as chromosome instability and meager spindle checkpoint responses. The deletion of BUB1 in yeast cells increased the rate of chromosome missegregation, which verifies the role of BUB1 in SAC. 3

BUBR1 Function

BUBR1 has a vastly different role. Its roles are associated with fixing incorrectly attached or unattached kinetochores. BUBR1 also comes into play for chromosome alignment. BUBR1 helps stabilize the attachment of microtubules to the kinetochore on a chromosome so that segregation may occur efficiently. 3

Connections to Cancer

BUB1 and BUBR1 are important components in helping regulate cell division and the overall cell cycle. Any damages to BUB1 or BUBR1 would lead to a disturbed mitotic checkpoint. A disturbed mitotic checkpoint has been linked to many forms of cancer. The reason for this is because a mutation in the spindle checkpoint leads to cases of aneuploidy and chromosomal instability. Specifically, a reduced gene expression of the BUB1 gene or a mutation of the gene has been proven to correlate with the formation of tumors in the colon, breast, gastric, esophageal, and melanoma. Animal experiments have also pointed to the possibility of BUB1’s involvement in tumor formation. For example, mice with low BUB1 expression have shown an increase for tumor susceptibility. 3

References

1. http://www.biology.arizona.edu/cell_bio/tutorials/cell_cycle/cells3.html

2. http://www.cellsalive.com/mitosis.htm

3. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3061984/

4. http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=bub1

5. http://www.ncbi.nlm.nih.gov/pubmed/10856933

6. http://www.cellsignal.com/reference/pathway/Cell_Cycle_G1S.html

7. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3267040/

External Links For animations: http://www.khanacademy.org/science/biology/cell-division/v/phases-of-mitosis?playlist=Biology