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Virtual Lab Manual 6 -?Mitosis:

Using a toxic compound from the yew tree in cancer therapy

Synopsis

How can a toxic compound be used in medicine? Paclitaxel, isolated from yew trees, can kill large animals like horses but is also used in cancer therapy. In this simulation, you will learn how cells divide and how they are affected by poisonous paclitaxel.

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How is DNA packaged?

Start by diving into a blood sample to find out how DNA is packaged in an immersive animation of the cell. Find the DNA inside the nucleus and then zoom in from the chromosomes all the way into individual nucleotides.

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Use microscopy to study mitosis

Understand the different stages of mitosis through interactive graphics and quiz questions. Then, prepare a sample of onion cells to observe the phases of mitosis and find out how each phase contributes to the successful duplication of the cell. By now, you will understand enough about mitosis to replicate the process in a computer model.

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Test the effects of paclitaxel

Finally, combine all your new skills and knowledge to test the effect of paclitaxel on cultured cells. Will the compound inhibit or accelerate cell division? How will it affect cancer cells or animals who eat the yew tree?

Learning Objectives

At the end of this simulation, you will be able to…

●?Understand and visualize basic concepts about eukaryotic cells such as main cellular components and DNA packaging by immersive animations

●?Understand the key characteristics of the cell cycle′s different stages: interphase (G1, S and G2) and mitosis

●?Use different microscopy techniques to observe the phases of the mitosis and describe their main characteristics:

●?Prophase

●?Metaphase

●?Anaphase

●?Telophase

●?Understand the cell cycle checkpoints and the molecules that control them (cyclins and cyclin-dependent kinases)

●?List the main differences between mitosis and meiosis

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Techniques in Lab

●?Sample preparation

●?Light microscopy

●?Fluorescence microscopy

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Theory

Eukaryotic Cells

The cell is the basic biological unit of all known living organisms (Figure below). All cells consist of a cytoplasm contained within a cell membrane, sometimes called the plasma membrane. Beyond this, however, they can differ significantly, with major differences between prokaryotic (bacteria and archaea) and eukaryotic (plants, animals, and fungi) cells, in their structure and in the organelles they contain. Even cells within an organism can differ greatly facilitating all the different physiological functions required for life.

●?Cell membrane:?The cell membrane surrounds the cytoplasm of the cell which serves to separate and protect the cell from its environment. The membrane is composed of a double layer of phospholipids making it very flexible. It is able to host various proteins and is semi or selectively permeable.

●?Cytoplasm:?The cytoplasm contains all of the material in the cell excluding the cell nucleus. Comprised of the cytosol, a gel-like substance which is enclosed by the cell membrane, and all the other organelles.

●?Nucleus:?The nucleus is one of the many organelles found within a cell. Cells typically contain one nucleus each, although certain specialized cells may contain many, for example muscle cells, with others, such as red blood cells, containing none. The nucleus contains most of a cell’s DNA molecules organized as multiple linear DNA molecules known as chromosomes. The nucleus is kept separate from the cytoplasm by the nuclear envelope, another double layer of phospholipids, although this membrane is punctuated by nuclear pores.

●?Mitochondria:?Mitochondria are the powerhouses of the mammalian cell generating a supply of adenosine triphosphate (ATP), for use as a source of chemical energy. The number of mitochondria present in a cell gives an idea as to how much energy it requires, for example red blood cells have none whereas cells in the liver can contain thousands.

●?Rough endoplasmic reticulum:?The rough endoplasmic reticulum is studded with protein-producing ribosomes and is the major source of protein translation in the cell.

●?Golgi apparatus:?The golgi apparatus, or golgi body is an organelle found in most cells and is a continuation of the endomembrane system and functions to package proteins for dispersal throughout the cell, or even to the outside of the cell via secretory vesicles.

●?Lysosome and peroxisome:?The lysosome and peroxisome can be thought of as the recycling centers of the cell. Both rich in enzymes they are responsible for breaking down many kinds of biomolecules into their constituent parts for later reuse. Peroxisomes can be thought of as hazardous waste recycling centers as a major function is to reduce the damaging reactive oxygen species into harmless waste products.

Animal cells differ massively in size, appearance and function but some factors are conserved. All cells are comprised of a cytoplasm surrounded by a cell membrane. Most cells also contain a nucleus which contains a complete copy of an individual's DNA as well as other structures such as the energy-producing mitochondria and protein-producing rough endoplasmic reticulum.

Cell Division

The only way to make a new cell is to duplicate a cell that already exists. Three main functions of cell division are:

●?Reproduction

●?Growth and development

●?Tissue renewal

The fundamental function of the cell cycle?is to accurately copy the enormous length of DNA in the chromosome, and to segregate the copies precisely into two genetically identical daughter cells. In order to divide, a cell must first go through a series of events:

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signal.?The cell receives a signal for cell division related to the needs of the entire organism.

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Replication. The genetic material that makes up an organism is called the genome. Eukaryotic genomes consist of a number of DNA molecules that are enormous in length (almost 2 m in human cells). All of this DNA must be copied so that each daughter cell has a complete genome.

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Segregation.?The newly replicated chromosomes come in pairs that are called sister chromatids. A mechanism, mitosis, will segregate the sister chromatid into two new nuclei.

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Cytokinesis. The division of cytoplasm, cytokinesis, is preceded by the division of the genetic material in the nucleus, i.e. mitosis.

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Cell culture

Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. That is why cell culture is always an in vitro procedure. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been established.

????Primary culture

????Primary culture refers to the stage of the culture after the cells are isolated from the tissue and proliferated under the appropriate conditions until they occupy all the available substrate (i.e., reach confluence). At this stage, the cells have to be subcultured (i.e., passaged) and transferred to a new vessel with fresh growth medium to provide more room for continued growth.

????Cell line

????After the first subculture, the primary culture becomes known as a cell line or subclone. Cell lines derived from primary cultures have a limited life span (i.e., they are finite; see below), and as they are passaged, cells with the highest growth capacity predominate, resulting in a degree of genotypic and phenotypic uniformity in the population.

????Stem cell

????A stem cell is a single cell that can replicate itself or differentiate into other cell types. Stem cells are unspecialized, meaning they do not have any tissue-specific structures that allow them to perform specialized functions.

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Eukaryotic DNA Packaging

Eukaryotic DNA?is enormous in length, and therefore must be tightly packed in order to be manageable. DNA is wrapped around a protein called histone, forming bead-like units called nucleosomes. A number of nucleosomes form a string-like structure called chromatin. When cells are not dividing, the thin and long chromatin fiber is less densely packed. Prior to cell division, DNA replication takes place. After DNA replication, the chromatins are highly condensed into short and thick chromosomes.?Each duplicated chromosome consists of two sister chromatids, attached to each other by a protein complex called cohesins. The centromere?acts as the link between two sister chromatids. In the later stages of cell division, the sister chromatids will separate into two new nuclei. Remember, once the sister chromatids separate, they are no longer called sister chromatids, but become individual chromosomes.

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Every eukaryotic species exhibits a typical number of chromosomes. Human somatic cells?contain 2 sets of chromosomes, making 46 chromosomes in total. However, human reproductive cells, or gametes, contain only one set of 23 chromosomes.

Cell Cycle

The cell cycle is composed of the mitotic (M) phase?and interphase?(Figure below). The mitotic phase, which includes both mitosis?and cytokinesis, is the shortest part of the cell cycle. Most of the time, the cells are in interphase, which is composed of three different subphases: the G? phase?(first gap), the S phase?(synthesis), and the G? phase?(second gap).

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The goal of interphase is to grow by producing proteins and cytoplasmic organelle such as the endoplasmic reticulum. DNA is replicated during the S phase only. During the M phase, the replicated chromosomes are segregated into individual nuclei (mitosis), and the cell splits into two (cytokinesis).

Cell division occurs during the M phase whereby the replicated chromosomes from the S phase are split in half into individual nuclei, followed by cytokinesis where the cell itself splits in half forming two daughter cells.

A cell doesn’t continuously divide. Some cells, such as nerve cells and muscle cells, do not divide at all or they do it at a very low rate in mature humans. Cell division?rates and timing are crucial for development, growth and maintenance. Breakdown in cell cycle control plays a major role in cancer development.

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S phase

During the S phase, the chromosomes and chromatin protein that govern various aspects of chromosomes are duplicated accurately. Chromosome duplication is triggered by the activation of S-Cdk.

Cytokinesis

During cytokinesis, a contractile ring of actin and myosin divide the cytoplasm into two.

Mitosis

During mitosis, the sister chromatids are segregated into a pair of identical daughter nuclei (Figure below).

●?Prophase: During prophase, the sister chromatids are condensed. The centrosome outside of the nucleus will separate. The microtubule will form between the centrosomes (poles) to make the mitotic spindle.

●?Prometaphase: The nuclear envelope breaks down. The spindle can now attach to the chromosomes via the kinetochore.

●?Metaphase: The chromosomes are aligned at the equator. The kinetochore microtubule connects sister chromatids to opposite poles.

●?Anaphase: The sister chromatids separate to form two daughter chromosomes, and each is pulled slowly toward two opposite poles.

●?Telophase: The two sets of daughter chromosomes arrive at the poles and decondense. A new nuclear envelope begins to form around each set.

Mitosis results in two diploid cells. Diploid?refers to cells, nuclei, or organisms containing two sets of chromosomes (2n). Mitosis is one of two cell division?types. The other type of cell division is meiosis. Learn more about mitosis and meiosis comparison.

Mitosis consists of five key stages, with interphase being the resting or growth stage. Briefly, during prophase the sister chromatids condense and a centrosome forms outside of the nucleus beginning to form the mitotic spindle. During prometaphase the nuclear envelope breaks down and the chromosomes can connect to the mitotic spindle. During metaphase the chromosomes align along the center of the cell with sister chromatids connected to opposite poles of the spindle. These chromatids are then separated to form distinct chromosomes as each is pulled towards the opposite pole. Finally, during telophase the chromosomes decondense and a new nuclear envelope begins to form.

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Cell Cycle Checkpoint

The cell cycle?is controlled at three checkpoints (the G?, or restriction point, M, and the G? checkpoint) by both external and internal signals (Figure below). These signals report whether crucial cellular processes that should have occurred by that point have been completed correctly. If all is in order, the cell may proceed to the next cell cycle phase; if not, the cell will exit the cell cycle and enter the G0, or non-dividing, phase.

G? checkpoint.?This is the first checkpoint, also known as the restriction point. This checkpoint happens in the late G1 phase, when the cell begins to enter the cell cycle and chromosome duplication. Cells pass the G1 checkpoint when they are stimulated by appropriate external growth factors.

G? checkpoint. This checkpoint checks for damage to DNA after it has been replicated.

M checkpoint.?The metaphase (M) checkpoint checks that the mitotic spindles/ microtubules are properly attached to the kinetochore. If the cell passes this checkpoint, sister-chromatids will begin to separate, leading to the completion of mitosis and cytokinesis.

There are three checkpoints present in the cell cycle which control its progression. The first checkpoint G1, or the restriction checkpoint, occurs during normal cell growth and is passed when certain growth factors are present. The G2 checkpoint assesses for DNA damage following replication. Finally, the M checkpoint checks that the mitotic spindles and microtubules are properly attached to the kinetochore.

Cyclin and Cyclin Dependent Kinase

The cell cycle?is mainly regulated by two types of proteins: cyclin-dependent kinase (CDK)?and cyclin. CDKs are protein kinases. Protein kinases are enzymes that activate or inactivate other proteins by phosphorylating them. In order to be active, a CDK needs to form a complex with the correct cyclin.

One type of cyclin-CDK complex, MPF (which stands for “mitotic promoting factor” or “maturation promoting factor”), allows the cell to proceed from the G? checkpoint?into mitosis. At the G? checkpoint, MPF will phosphorylate a variety of proteins, triggering mitosis.

Animal cells have at least four types of cyclins: G1-phase cyclin, G1/S-phase cyclin, S-phase cyclin, and M-phase cyclin. These types of cyclins will form CDK complexes in relation to their name. For example, S-phase cyclin will bind to its specific CDK and form S-CDK. The CDKs are expressed constantly throughout the entire cycle. By contrast, cyclin is being produced and destroyed at various rates in different cell cycle phases. The rise and fall of cyclin determines the fluctuation of the cyclin-CDK complex.

The fluctuating activities of the different cyclin-CDK complexes are of major importance in controlling all of the stages of the cell cycles.

Each cyclin-CDK complex phosphorylates a different set of proteins. S-CDK will catalyze phosphorylation of the protein that initiates DNA replication. M-CDK phosphorylates condensing proteins, which are essential for chromosome condensation, as well as lamin proteins, which are responsible for the nuclear membrane network. M-CDK also stimulates the assembly of the mitotic spindle by phosphorylating protein for the microtubule.

Beside the cyclin-CDK complex, other extracellular signals regulate cell size and cell number. They can be divided into three major classes:

●?Mitogens:?stimulate cell division by triggering G1/S-CDK.

●?Growth factor:?promotes cell growth by triggering protein synthesis and other macromolecules, and prevents their degradation.

●?Survival factor:?promotes cell survival by suppressing apoptosis (programmed cell death).

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Microscopy

Scientists use microscopes (micro- = “small”; -scope = “to look at”) to study structures that are too small to see with the naked eye, like most cells. A microscope is an instrument that magnifies an object.

There are several different microscopy techniques:

●?Light microscopy?is the most common method.

●?Fluorescent microscopy?can be used to study specific cell structures at high contrast.

●?Electron microscopy?can produce images at a very high resolution.

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Two types of cell division serving a different purpose: cell divisions where chromosomes/ DNA are exactly replicated in new cells (mitosis)?and reduction division where the number of chromosomes is halved in the new cells (meiosis). Reduction division is essential if genetic information from each parent is to be contributed to the offspring. In both mitosis and meiosis, the chromosomes or chromatids are pulled to opposite poles of the cell by the mitotic spindle comprised of microtubules before cytokinesis.

Mitosis vs meiosis -?Summary of Differences:

1.?Mitosis forms diploid cells that have the same number of chromosomes as the parent, whereas meiosis forms haploid cells with half the original number of chromosomes.

2.?Mitosis produces somatic cells (all cells except sex cells) while meiosis produces sex cells, ie. egg or sperm cells.

3.?Mitosis includes one round of cell division, while meiosis contains two rounds of cell division.

4.?Stages of mitosis include interphase, prophase, metaphase, anaphase and telophase. Stages of meiosis include interphase, prophase I, metaphase I, anaphase I, telophase I, prophase II, metaphase II, anaphase II, and telophase II.

5.?In mitosis, homologs do not pair up, whereas in meiosis homologs do pair up.

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Paclitaxel

Paclitaxel was isolated from the bark of pacific yew (Taxus brevifolia). The precursor of the drug paclitaxel can be synthesized easily from the extract of the leaves of the European yew (Taxus baccata). Most parts of the tree are toxic, except the bright red aril surrounding the seed. Paclitaxel is now used to treat a number of types of cancer. Paclitaxel's mechanism of action involves interference with the normal breakdown of microtubules during cell division.

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