Cell cycle analysis

In 1838 Schleiden postulated that all plants are composed of cells1; then in the following year together with Theodor Schwann he formulated the cell theory of life. In 1855 Rudolf Virchow published his now-famous aphorism omnis cellula e cellula "every cell stems from another cell”2. These conclusions are thought to represent the creation of the cell theory of life. Schleiden stated that the first phase of the generation of a new cell is the formation of a crystallised nucleus. In which is a substance he called a “cytoblast”, which gets progressively larger to become a new cell. Following this observation Robert Remak (1815–1865), Rudolf Virchow (1821–1902) and Albert Kölliker (1817–1905) presented evidence to suggest that new cells are created as a result of “scission of pre-existing cells”3.

At the time of the cell theory the cell was thought to be composed of a wall or simple membrane, and fluid called protoplasm (now known as cytoplasm) and the nucleus. Within the nucleus a number of structures (ribbons, bands and threads) were seen to appear during cell division4. As these objects could be easily stained Walther Flemming called them chromatin. Flemming observed chromatins splitting of chromatins in salamander cells. He termed this process Mitosis in 18824.

Mitosis

Mitosis is the process by which a cell separates its chromosomes into two new cells. Following the splitting of the chromosomes the cell undergoes cytokinesis, splitting the cell organelles. Together the mitosis and cytokinesis form the mitotic (M) phase of the cell cycle. The M phase is only a small proportion of the cell cycle. The longest part of the cell cycle is interphase. Interphase is the process by which the cell prepares itself for the M phase of the cell cycle. There are three phases to interphase; G1 (first gap), S (synthesis) and G2 (second gap). During G1 the cell grows by creating proteins and organelles. During the S phase of interphase the chromosomes are synthesised duplicating the number of chromosomes. During G2 the cell continues to grow larger.

Mitosis is not just one single process, and is in fact separated into several phases that have been clearly defined.

Prophase

The first phase of Mitosis is Prophase. Before the M phase of the cell cycle the genetic material inside the nucleus are in a loosely bundled coil called chromatin. At the onset of prophase the chromatins condense to form structures called chromosomes. As the genetic material has been duplicated in S phase the chromosome has two sister chromatids bound by a centromere.

Outside of the nucleus is a very important structure; the centrosome. The centromere is made up of a pair of centrioles. The centrosome organises the microtubular network of a cell. Each cell gets one centrosome from its parent cell; this single structure is duplicated before mitosis begins. The centrosomes create microtubules from a substance called tubulin. The two centrosomes are then split apart and moved to opposite sides of the cell.

Prometaphase

At this stage the envelope surrounding the nucleus breaks down and microtubules begin to enter the nuclear space5. At each side of the centromere is ...

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Prometaphase

At this stage the envelope surrounding the nucleus breaks down and microtubules begin to enter the nuclear space5. At each side of the centromere is a structure called a kinetochore. This can be thought of a “ring” on to which a “hook”, (the microtubules) can attach too in an ATP driven process6.

When the microtubules attach to chromosomes, they attach to a specialized structure called the kinetochore is formed on the centromeres called microtubules. The microtubules enter the nuclear region. Centrosomes complete migration to opposite poles

Metaphase

Following the attachment of the chromosome to the kinetochore, the centrioles pull the chromosomes to the centre of the cell. The chromosomes line up at the equatorial plane or metaphase plate7.

Anaphase

When the chromosomes are lined up at the equatorial plane two events occur; the proteins binding the sister chromatids break down. The separated sister chromosomes are then pulled apart by the shortening of the microtubules. The centrioles and attached chromosomes are then pulled to opposite poles of the cell.

Telophase

This process of the cell cycle brings everything together. The sister chromatids join together to form single chromosomes. The chromosomes disappear and are no longer visible. The spindles break down and the cells begin to split.

Cytokinesis

The centre of the cell contracts pinching the cell into two new daughter cells.

Images used are from ref 8.

Hypothesis

As interphase involves the synthesis of the cells genetic material and cellular proteins this will be longest stage of the cell cycle. Therefore most cells observed will be in interphase and this will take the most proportion of the 24 hour cycle.

Null Hypothesis

There is no difference to the time spent in each stage of the cell cycle. Each phase will have an equal proportion of time allocated to it out of a 24 hour period.

Equipment

The equipment used was a described in the NEC handout

Method

The method was conducted as described in the NEC handout

Risk Assessment

Results

Cell cycle diagram

Angle of segment in pie chart θ = total number of cells at the stage .

Total number of cells in root tip squash

= θ x 360°

Figure 1 shows the percentage of cells in each stage of the cell cycle

The cell cycle diagram tells you how long a cell stays in each stage of the cell cycle. The diagram shows that the largest proportion of time is spent interphase as apposed to the M phase of the cell cycle.

Figure 2 shows the time a cell spends in each stage of the cell cycle of mitosis out of a 24 hour period

Figure 2 shows the amount of time spent in each stage of the cell cycle. From the results out of a 24 hour cell cycle 17 hours and 50 minutes is spent in interphase. 3 hours 16 minutes is spent in prophase. 37 minutes is spent in metaphase. 46 minutes is spent in anaphase and 1 hour 32 minutes in Telophase.

The results of the ANOVA test show a p value of <0.01. This means that we can reject the null hypothesis and accept the hypothesis will more than 99% confidence. Therefore there is a significance difference in the amount of time that a cell spends in each phase of the cell cycle.

Evaluation

The length of the cell cycle is dependent on the type of cell. A liver cell for example takes about 22 hours, where some cells of the nervous system will remain in interphase for most of their life. Most of the time is spent in interphase “usually, cells will take between 5 and 6 hours to complete S phase. G2 is shorter, lasting only 3 to 4 hours in most cells. In sum, then, interphase generally takes between 18 and 20 hours. Mitosis, during which the cell makes preparations for and completes cell division, only takes about 2 hours”9.

Although the data that I have collected here is relative in each stage of the cell cycle, the actual amount of time spent in each stage is inaccurate. This inaccuracy is also increased by the absence of cells being identified as being in cytokinesis.

The difference in the results could be due to inaccuracies in the experiment. The manner in which the slide produced kills the cells. This means that you are observing the cells at a static phase. Also this experiment was done in the middle of the day. No account has therefore been made for what happens at night or early morning. Therefore to ensure that the data is more accurate more experiments need to be done at different times of the day. As the cell cycle is a continuous process it is very difficult to actually identify what phase of the cell cycle a cell is in. For example when is a cell in metaphase or prophase? It is sometimes difficult to identify the end stage of each phase of the cycle.

Bibliography

Schleiden, M. J. Arch. Anat. Physiol. Wiss. Med. 13, 137– 176 (1838).
Schwann, T. Mikroskopische Untersuchungen über die Übereinstimmung in der Struktur und dem Wachstum der Tiere und Pflanzen (Sander'schen Buchhandlung,Berlin, 1839).
Mayr, E. The Growth of the Biological Thought (Belknap, Cambridge, MA, 1982).
Flemming, W. Zellsubstanz, Kern und Zelltheilung (FCW Vogel, Leipzig, 1882).
Chan G, Liu S, Yen T (2005). "Kinetochore structure and function". Trends Cell Biol 15 (11): 589–98.
Maiato H, DeLuca J, Salmon E, Earnshaw W (2004). "The dynamic kinetochore-microtubule interface". J Cell Sci 117 (Pt 23): 5461–77.
Winey M, Mamay C, O'Toole E, Mastronarde D, Giddings T, McDonald K, McIntosh J (1995). "Three-dimensional ultrastructural analysis of the Saccharomyces cerevisiae mitotic spindle". J Cell Biol 129 (6): 1601–15.
http://www.biology.arizona.edu/cell_bio/tutorials/cell_cycle/cells3.html
http://www.sparknotes.com/biology/cellreproduction/cellcycle/section2.rhtml