(8/30/10) - Nucleus and Cell Cycle

== 1. Describe the functions of the nucleus.

2. Localize the functions of the nucleus to structural domains of the nucleus

a. Storage of information (in DNA)

i. Organized as Chromatin in nucleoplasm (nucleolar organizing DNA is found in nucleolus). Some heterochromatin is attached to the nuclear lamina.

b. Replication of the genome (DNA replication)

i. Nucleoplasm, each of the 46 chromosomes (in humans) has its own unique domain

c. DNA repair

i. nucleoplasm

d. RNA synthesis (transcription)

i. nucleoplasm

e. RNA processing

i. Distinct area of nucleoplasm from where transcription occurs. Introns are excised before export to nucleus. Cajal bodies are sites of processing and assembly of small nuclear RNA (snRNA)

f. Ribosome assembly

i. Nucleolus is where large and small subunits of ribosome put together, plus where 45S rRNA precursor synthesized and processed.

g. Transport of tRNAs, mRNAs and ribosomal subunits to the cytoplasm

i. Through nuclear pores regulated by nucleoporins. All RNAs leave nucleus in form of ribonucleoprotein complexes.

h. Selective protein uptake from the cytosol.

i. Histones, ribosomal proteins, RNA and DNA polymerases and transcription factors are all synthesized in cytosol and imported to nucleus after translation.

3. Describe how proteins and RNAs move between the nucleus and the cytosol.

a. The proteins/RNAs that need to cross the nuclear envelope have Nuclear Localization Signals (NLS) and/or Nuclear Export Signals (NES) which are recognized by carrier proteins known as karyopherins (exportins [take molecules out of nucleus] and importins [bring molecules into the nucleus]). This process happens in conjunction with a small G-protein, RAN, which assists the transport process by forming a gradient across the nuclear envelope with RAN-GDP in the cytoplasm and RAN-GTP in the nucleoplasm. b.

4. List three major types of RNA synthesized in the nucleus. a. mRNA (messenger RNA, used to code for proteins), rRNA (ribosomal RNA, forms complex with proteins to comprise two ribosomal subunits), tRNA (transfer RNA, specific for one of twenty amino acids). One tRNA codes for only one amino acid, but one amino acid may have many tRNAs.

5. Describe why the amount of DNA in the cell changes during the cell cycle and at what point in the cell cycle this occurs. a. In order for cells to divide, the DNA in the cell must first be duplicated so that it can be divided amongst the daughter cells. This replication of DNA happens during the S phase of interphase. The cell retains this extra amount of DNA during the G2 phase. During mitosis the DNA contents are split amongst the daughter cells so that each cell remains diploid. The cells then maintain this amount of DNA during the G1 phase.

6. Describe the structural organization of the interphase nucleus to include the nucleolus and the nuclear envelope. a. Nucleolus is not membrane bound, rather it is a distinct domain containing RNA and DNA and is responsible for the production of ribosomal subunits. It is spherical in shape. The nuclear envelope is comprised of two membranes enclosing a cisternal space, which is continuous with the rough endoplasmic reticulum. The inner membrane of the nuclear envelope is supported by the fibrous lamina (which is comprised of lamins A, B, C). The nuclear envelope also contains nuclear pores which allow for the passage of molecules from the nucleus to the cytoplasm and back (as the envelope itself is largely impermeable).

7. Describe three changes in nuclear structure that can be associated with cancer or programmed cell death. a. Transformation of a normal cell into a neoplastic (cancer cell) can be associated with increased amounts and distributions of heterochromatin, changes in nuclear shape, and size/shape of nucleoli. b. Apoptosis is associated with greatly increased amounts of heterochromatin (pyknosis) and nuclear fragmentation (as can be caused by radiation).

8. Describe the composition of chromatin and how it is organized in the interphase cell. a. Chromatin is comprised of histones and DNA. The elementary unit of the chromatin is the nucleosome (10-11 nm in diameter) which is a length of DNA (~200 pairs of DNA) wrapped around an octomeric protein complex made up of 2 each of H2A, H2B, H3, and H4. Histone H1 is located between these nucleosomes to promote further packing of the nucleosomes into 30 nm chromatin filaments. Chromatin is in two states, euchromatin, which stains poorly and is active in transcription, and heterochromatin, which stains nicely and is inactive. Histone acetylation can control the transition between the two states and can regulate transcription.

9. List the four phases of the cell cycle. a. M phase ((mitosis) nuclear and (cytokinesis) cellular division), G1, S phase (DNA replication), G2,

10. Describe the critical events that occur in each phase of the cell cycle.

a. G1

i. Cell grows

ii. Commitment step à once here cell must complete cycle or undergo apoptosis b. S phase

i. DNA replicates

ii. Cell continues to grow c. G2

i. Cell grows d. M phase

i. Comprised of two subphases – mitosis and cytokinesis

ii. Mitosis comprised of prophase, metaphase, anaphase, and telophase à division of nucleus

iii. Cytokinesis is the division of the cytoplasm, during which reformation of the nuclear envelope occurs.

iv. Phosphorylation of the lamins targets the nuclear lamina for disassembly. Subsequent dephosphorylation triggers reassembly of the nuclear lamina and the nuclear envelope.

11. Describe the two phases of the cell cycle at which most cancer treatments are targeted. Explain how a therapy targeted at the S phase of the cell cycle must differ from a therapy targeted to the M phase of the cell cycle. Be able to list two drug targets for each of these two phases of the cell cycle. a. The two phases at which most cancer treatments are targeted are DNA synthesis (S phase) and mitosis (M phase). b. A therapy targeted at the S phase most likely inhibits the ability of the cancer cell to replicate its DNA by inhibiting one of the many proteins that regulate the process. A therapy targeted at the M phase likely intereferes with the microtubule dynamics and the proper segregation of chromosomes into daughter cells. c. Methotrexate (5-fluorouracil, a nucleoside analog) and Topoisomerase II inhibitors affect S phase. d. Vinblastine and Taxol interfere with microtubule dynamics.

12. List the major cell cycle check points and the function of each. a. Three types of checkpoints b. The checkpoints ensure that DNA repair has happened before S phase and all DNA has been accurately replicated before M phase. c. A spindle checkpoint ensures that all the chromosomes have been properly aligned on the metaphase plate before anaphase. d. DNA damage checkpoint ensures during both G phases that the DNA has been properly repaired before proceeding to the next phase e. DNA replication checkpoint ensures that all DNA has been replicated during the S phase before moving on.

13. Describe how the cell cycle checkpoints can be employed to fight cancer. a. If a cell cycle checkpoint is activated for extended periods of time, apoptosis is activated. Cancer therapies then might target ways to damage cancer cells’ DNA beyond repair or prevent proper alignment along the metaphase plate in such a way that cell cycle checkpoints are activated for extended periods of time, and apoptosis is activated.

14. Explain the mechanism by which radiation works as a cancer treatment. a. Radiation (and chemotherapy) work to damage DNA beyond repair such that cell cycle checkpoints are activated for extended periods of time, and apoptosis of cancer cells is signaled. However, these therapies are not specific, and many normal cells are also killed. But, as cancer cells have the faster replication rates, when compared to normal human cells, these cancer cells are more affected by the therapies.

15. Explain what happens when a cell cycle checkpoint is activated for an extended period of time and the cell is unable to correct the problem. a. When a cell cycle checkpoint is activated for an extended period of time, the cell is targeted for apoptosis. The signal for apoptosis can be internal or external, and in many cases is signaled for by the release of cytochrome c from the mitochondria. Apoptosis leads to the activation of proteolytic enzymes known as caspases.

16. Describe the manner in which the cell cycle is regulated. a. Cell cycle is largely regulated by CDK proteins. Cell cycle checkpoints ensure that the cell has completed everything it must do in one phase before moving on to the next. CDK-1 and cyclin B (M-cdk) regulate the transition into mitosis. CDK-2 and cyclin E regulate the transition into S phase.

17. Explain three ways in which the activity of a CDK (cyclin-dependent kinase) can be regulated. a. CDKs are phosphoproteins that are regulated by phosphorylation and by the binding of cyclins. b. Cyclins accumulate during the G2 phase of cell cycle and are degraded through proteolysis during anaphase. c. CDKs have both activating and inhibitory phosphorylations. d. In order to be active, CDK must be bound to cyclin B, have an activating phosphorylation, and lack an inhibitory phosphorylation.

18. Describe two methods by which cells can become multinucleated. a. Cells may become multinucleated by undergoing mitosis but not cytokinesis. b. Cells, such as skeletal muscle cells fuse together.

19. Describe some cell types in the body that can be multinucleated and why a cell might need more than one nucleus. a. Osteoclasts, skeletal muscle cell, and some hepatocytes (max two) have more than one nucleus. These cells might need more than one nucleus because they are so translationally active, and need to produce more viable mRNA to keep up with demand.