Passage:Centrosomes

Passage:Centrosomes

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Of the organelles found in the eukaryotic cell, the centrosome serves as a microtuble organizing center (MTOC) in animal cells. Originally discovered in 1988 by Theodor Boveri, it was thought to be a special organelle for cell division and it is believed to have evolved only in the metazoan lineage of eukaryotic cells. Recent research however has shown that the centrosome is not necessary for mitotic division.


Centrosomes are generally composed of two orthogonally arranged centrioles surrounded by an amorphous mass of pericentriolar material (PCM). The PCM contains proteins responsible for microtubule nucleation and anchoring including γ-tubulin, pericentrin and ninein. Each centriole generally comprises nine triplet microtubule blades in a pinwheel structure as well as centrin, cenexin and tektin.


Centrosomes are often associated with the nuclear membrane however during mitosis the centrosome nucleated microtubles interact with chromatin to build the mitotic spindle. The centrosome is duplicated only and exactly once per cell cycle during S phase. The replication however does not involve template reading or assembly, the mother centriole simply aids in the accumulation of materials required for the production of a daughter centriole.


Interestingly, centrosomes play an essential role in forming cilia and flagella however they are often not necessary for general development. Development of the fruit fly Drosophila is largely normal when centrioles are absent due to a mutation in a gene required for their duplication. The final knockout organism remains relatively morphologous to the wildtype but lacks functional sensory receptor cells. In the absence of a centrosome the microtubules of the spindle are focused by motors allowing the formation of a bipolar spindle and thus allowing for regular separation of chromatic material. Catastrophic cellular failures generally only occur when checkpoint defective cells are used for centrosome experiments.


Experiment 1

Cdk2, is essential to centrosome replication. When activated through phosphorylation, a conformational change exposes the kinase active site allowing it to in turn phosphorylate other cell cycle proteins, some of which are used in centrosome replication. Deletions of either the N-terminal or C-terminal coding region of cdk2 were performed in a paramecium cell line. The first produced paramecium deficient in centrosomes while the second produced paramecium unable to suppress centrosome replication and thus over produced centrosomes. Both strains were capable of surviving for prolonged periods of time, however the N-terminal deletion strain showed no signs of cilia and failed to avoid chemical gradients of toxic substances.

Experiment 2

In a second experiment, human respiratory epithelium cells were grown on a fibroblast cell layer providing a connective tissue support for the artificial epithelium. Initial experiments however failed until the cells were grown in conjunction with a small quantity of Goblet cells which are naturally found in the respiratory tract and secrete mucus. The plate was then treated with a centrosome inhibitor and a saline control. The cells were then incubated for six hours with gentle rotation and a continuous wash of fresh nutrient media.


1. Mitochondria and bacteria have all of the following features in common except?

Circular DNA
→ Mitochondria have genomes similar to bacteria but lack most of the genes.
Membrane bound and integral proteins
→ Both have proteins associated with their membranes
Replication through binary fission
→ Both mitochondia and bacteria exhibit similar division processes.
Mitochondria and most bacteria can survive in an oxygen environment or outside the cell.
→ Mitochondria cannot survive on their own, they have become completely dependent on their host.

2. The most likely observation to be made of the cells in experiment two when washed with the inhibitor is?

a dead colony
a loss in columnar structure
a loss in cilia
→ The passage states that development is often unaltered and that cilia are dependent n centrosomes
no change

3. The most likely reason for the requirement of Goblet cells in experiment two is?

the epithelial cells require a paracrine signal
→ paracrine signals are signals sent over short distances from one cell type to another.
the epithelial cells require a juxtacrine, or cell-cell contact, signal
→ The passage says that only a few Goblet cells are required thus it is unlikely all the epithelial cells can make contact to them.
the epithelial cells require an autocrine signal
→ An autocrine signal would imply that the epithelial cells are sending a signal to themselves.
the epithelial cells require an endocrine signal
→ An endocrine signal would imply cells distantly far away in the body are sending a signal, this is obviously not the case.

4. During mitosis, the step in which centrosomes pull apart the chromatids is called?

Prophase
Telephase
Metaphase
Anaphase

5. Cystic kidney disease is caused by a recessive centrosome mutation and affects 4% of the population. What is the frequency of the dominant allele in the population?

0.02
0.04
0.96
0.98
→ Firstly one must recognize that the disease is caused by having two copies of the recessive allele. Using the Hardy-Weinburg population genetics formula, q2 is the homozygous recessive frequency, and is given in the problem as 4%, thus q2=0.04. It follows then that, q, the frequency of the mutated allele, is 0.02. The frequency of the other allele then is simply p = 1-q, 1-0.02 = .98.

6. Assuming a healthy diploid spermatocyte with initially one centrosome undergoes meosis, how many centrioles will be present once the spermatids have been formed?

2
4
8
16
→ If we consider just centrosomes, then initially we have one, but in order to split into two cells a second centrosome must be formed. This then leads to one centrosome in each of the two haploid cells. Each one of these cells, must then repeat the process of making a second centrosome, and then dividing. Thus at the end, we have four cells (this is meosis) each with one centrosome. If we now consider what a centrosome is, we know from the passage that each centrosome consists of two centrioles, thus there are a total of eight centrioles in the four spermatids.

7. Which of the following in regards to the structure of cdk2 is most likely to be true?

The C-terminal must contain the kinase site
→ According to the passage, centrosomes were produced in the C-terminal deletion strain, and the centrosomes require cdk2 activity, thus this answer must be false
The C-terminal must contain the site of phosphorylation
→ While the C-terminal must contain a region that blocks and unblocks for activity, it does not necessarily have to be the site of phosphorylation, that could be on the site where the C-terminal region moves to.
The N-terminal must contain the kinase site
→ According to the passage, removing the N-terminal produces cells which lack centrosomes, thus it is most likely that this is because the kinase acticity is lost in these cells.
The N-terminal must contain a phosphate
→ This is meaningless, where the phosphorylation occurs is not specified and cannot be inferred.

8. Which statement best describes the function of the rough endoplasmic reticulum?

Specific transport and signaling systems
Synthesis and assembly of membrane and secreted proteins
Production of energy during oxidative phosphorylation
Processing of membrane and secreted proteins, including glycosylation
→ The rough ER get's its name from the numerous ribosomes attatched to it, which in turn function to produce proteins which will become embedded into the lipid layer and later transported to the various membranes of the cell.

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