Lectures 48-57 Flashcards
How do you tell apart the phases of mitosis?
In human somatic cells, the cell cycle has four phases. The S phase is when DNA synthesis occurs. The M phase is when cell division or mitosis occurs. Between these two are two additional “Gap” or “Growth” phases: G1 and G2. These cell cycle phases can be largely distinguished by DNA content. Cells in the G1 phase have a 2N DNA content whereas cells in the G2 phase (after DNA synthesis is complete) have a 4N DNA
What is prophase?
The centrosomes or microtubule organizing centers are duplicated during S phase. In prophase, the centrosomes move to the poles. In addition, the nuclear membrane breaks down and chromosomes condense. Each pair of sister chromatids are held together by the centromere.
What is prometaphase?
In prometaphase, the spindle fibers form. These consist of three types. Astral microtubules
What is metaphase?
In metaphase, there is alignment of chromosomes in the center of the mitotic spindle. Ty[pically, karyotyping is done at this stage
What is anaphase?
In anaphase, sister chromatids separate and move to opposite poles, as the cell elongates.
What is telaphase and cytokinesis?
In telophase, the nuclear membrane re—forms, the chromosomes decondense, and the spindle disappears. Cytokineses then occurs, driven by actin filaments, resulting in the separation of
What is meiosis?
Meiosis is the process of reductive cell division for germ cells. Germ cells have a 1N DNA content and are haploid, whereas somatic cells have a 2N DNA content
What happens in Meiosis I?
There is a pairing and separation of homologues. The resulting cell divisions result in daughter cells containing one maternal copy or one paternal
What happens in Meiosis II?
There is a pairing and separation of sister chromatids.
When does crossing over occur and what is it?
During Meiosis I, chromosomal crossing—over occurs in which segments of homologous chromosomes exchange genetic material. This is a process of recombination that results in limitless genetic diversity. This explains why meiosis does not occur by simple separation of homologues, but rather involves DNA synthesis plus two distinct cell divisions, meiosis I and meiosis II.
What is the synaptomeal complex?
The synaptonemal complex is highly ordered and consists of 2 lateral elements and a central element which facilitates recombination between the parental and maternal homologues.
What are chiasmata?
Chiasma represent remnants of the synaptonemeal complex that play an important functions. They physically hold homologues together until segregation occurs, much like centromeres do for sister chromatids.
What keeps X and Y chromosomes together?
There is a small region of homology between the X and Y chromosomes that allows their pairing. Crossing—over will only occur in this small region. The main function is to allow physically keeping the X and Y chromosomes together until segregation is ready to occur.
What is non-disjunction and when does it occur?
Non—disjunction happens when homologues fail to separate in Meiosis I. Resulting germ cells lead to abnormal embryos, most of which most die. Some survive resulting in developmental abnormalities such as Down’s syndrome, where there is an extra copy of chromosome 21. One set of homologues does not separate in meiosis I and then meiosis II occurs normally. all gametes have an abnormal # of chromosomes either one too many or one too little. Al;though nondis can occur in meiosis II as well, it is far less likely since this is a more tightly controlled process.
What do microfilaments and actin do during mitosis?
The centrosomes or microtubule organizing centers are replicated in prophase. In pro metaphase the spindle fibers form. There are 3 types. Astral microtubules position the mitotic spindle, kinectochore microtubules are attached to the chromosomes, and polar microtubules interdigitate with those emanating from the opposite poles. Cytokinesis is driven by actin filaments.
What are the two major ways by which genetic reassortment occurs during meiosis?
Independent assortment and crossing over.
How do you calculate the mitotic index and what is it?
The mitotic index is used to calculate the number of cells undergoing cell division, It is calculated as: Mitotic index=number of mitotic cells/total number of cells
How does the Ki-67 antigen work?
The Ki-67 antigen is expressed in cells undergoing active division. It can be detected using an antibody in immunohistochemistry. It is commonly used in fixed and embedded tissues samples to identify cells that are proliferating. Brown stain.
Describe Flow cyt of DNA content.
The flow cytometer is a machine that can measure the amount of fluorescence per individual cell.3For measuring DNA content, cells are incubated with a fluorescent dye called propidium iodide. Propidium iodide intercalates into the genomic DNA. A single cell suspension is run through the flow cytometer as shown in the figure. A laser shines a light with a specific wavelength on each individual cell. A detector measures fluorescence at a distinct wavelength. The number of cells with each amount of fluorescence is then quantitated. G1 cells have 1X fluorescence (in some arbitrary units). G2 and M cells having twice as much DNA are at 2X fluorescence and S phase cells are in between, ranging from 1-2X fluorescence The data is displayed as a histogram (see an example below).
What are cyclin dependent kinases?
Cyclin-dependent kinases consist of a catalytic subunit (Cdk) and a regulatory subunit (Cyclin). Cyclin-dependent kinases, as the name suggests, require a regulatory subunit called a cyclin for activity. These kinases phosphorylate on either serine or threonine that immediately precede a proline residue.
Multiple distinct cyclin/cdk complexes exist in human cells. Name the 4 kinds and where they act.
1) cyclin D/cdk 4 and cyclin D/cdk 6 during G1.
2) Cyclin E/cdk 2 transition from G1 to S phase
3) Cyclin A/cdk 2 S phase
4) Cyclin A/cdk1 and cyclin B/cdk 1 transition from G2 to M phase.
What two things does the binding of cyclin to cdk do?
The binding of the cyclin to the Cdk does two things. First, it provides part of the substrate binding site. Hence the same Cdk (for example Cdk2) will have a distinct substrate specificity depending upon whether it is bound by one cyclin or another (in the case of Cdk2, this would be Cyclin E versus Cyclin A). Second, the cyclin induces a conformational change that allows the substrate to access the catalytic site. Note that Cyclin binding is necessary but not sufficient for Cdk activation.
What is needed in addition to cyclin binding to activate a cdk?
CAK or Cdk Activating Kinase phosphorylates the catalytic subunit of all cyclin-dependent kinases on a conserved threonine residue (threonine 160). In addition to Cyclin binding, this modification is required for full activation of the Cyclin-dependent kinases.
How does binding of cyclin and phosphorylation with CAK cause a conformational change in cdk?
Prior to Cyclin binding, a part of the Cdk called the T-loop sits in the substrate binding site and prevents substrate binding. The binding of the Cyclin causes the T-loop to shift, partially exposing the substrate binding site. Phosphorylation of the T-loop on threonine 160 by CAK fully moves the T-loop so that the substrate binding site is completely exposed.
What inhibits cdm activity?
Phosphorylation by Wee1 at T14 and T15 leaves negative residues immediately next to the ATP binding pocket making it impossible for the negatively charged ATP to bind there. This renders the Cdk inactive.
What reverses Wee1’s activity?
These phosphates (left by wee1) must be removed by a phosphatase called Cdc25 in order for the Cdk to be fully active. There are three Cdc25C phosphatases in human cells: Cdc25A, Cdc25B, and Cdc25C.
How does positive feedback work for Cdk’s?
Cyclin-dependent kinases (a lready active) phosphorylate Cdc25 phosphatases and make them more active to remove the inhibitory phosphates. The Cdks also phosphorylate Wee1 and prevent it from adding the inhibitory phosphates. Thus, as cyclin-dependent kinases become active, there is positive feedback that further enhances their activity.
What are the two classes of protein inhibitors of CDK’s?
The CIP family and the INK4 family.
What does the CIP family do?
The CIP family of inhibitors of Cyclin-dependent kinases consist of three proteins: p21, p27, and p57. The CIP family inhibits all Cyclin-dependent kinases. he CIP family binds to the Cyclin-Cdk complex and induces a conformational change that prevents substrate binding.
What does the INK family do?
The INK4 family is specific for Cdk4 and Cdk6. The INK4 family consists of p15, p16, p18, and p19. The INK4 family binds only to the Cdk subunit, preventing it from interacting with the Cyclin.
What are Cdk’s regulated by?
Cyclin binding, various phosphorylations (CAK, Wee1, Cdc25), and two classes of inhibitors (CIP, Ink4)
What are the two E3 ubiquitin ligases that are important for cell cycle progression?
The SCF (SKP-Cullin-F box) protein complex, and the The Anaphase Promoting Complex (APC).
What does the SCF (SKP-Cullin-F box) protein complex do?
G1-S. Cyclins D and E. The SCF (SKP-Cullin-F box) protein complex serves as the ubiquitin ligases for Cyclins that are important in the G1 to S transition (Cyclin D, Cyclin E). The SKP ligase is always active and it is phosphorylation of the cyclin that causes it to be recognized by the SCF, leading to its degradation.
What are the domains of the SCF?
Catalytic subunit=rbx1 or ro50
Scaffold=cul1
adapter= skp1
Variable component; Fbox protein
What does the The Anaphase Promoting Complex (APC) do?
M Phase. Cyclin B and Separase. The Anaphase Promoting Complex (APC) is the ubiquitin ligase for the Cyclins that are important for mitotic progression, mainly Cyclin B*. In contrast to SKP, APC does not require Cyclin B to be phosphorylated. Unlike SKP, APC is not always active but becomes activated by the binding of a co-factor either Cdh1 or Cdc20.**
What are the subunits of APC?
catalytic=apc11
scaffold= apc2
adaptor= multiple units
variable component= Cdh1 or Cdc20** the binding of this activates APC.
Can the G2 and M phases of the cell cycle be distinguished using flow cytometry? If not, what techniques can be used to do so?
Using just a general DNA probe like propidium iodide you can not differentiate between G2 and M using flow cytometry as both have 4N. However, using a more nuanced probe we can separate out which cells are in which faze. The presence of phosphorylated histone 3 on ser10 is a reliable marker to identify cells specifically in M phase. Therefore you can use a bivariate cell cycle kit that probes with both an anti-H3-ser10 antibody and PI to parse out which cells are in soley M versus G2.
Entry into S phase is determined by what?
The phosphorylation of pRB.
How does the transition into S phase occur?
Cdk phosphorylation disrupts the interaction of pRb with E2F. Growth factor signaling upregulates cyclin D transcription. Cyclin D/cdk4/6 phosphorylation of pRb, release of E2F. E2F then activates low level of target expression (especially cyclin E). Cyclin E/cdk2 phosphorylation of pRb. Robust E2F activation of genes important for S phase.
What triggers DNA synthesis?
CDK phosphorylation.
How does cdk phosphorylation pre-start?
Formation of the pre-replication complex (pre-RC) occurs during G1 phase (Cdk 6, Cdt1, Orc1, Orc 2-6). Cdk phosphorylation of proteins involved in DNA replication include licensing factors which ensure initiation occurs only once each round, then factors are degraded. Cdk phosphorylation also causes origin firing.
What mediates the entry into mitosis?
Entry into mitosis (G2 to M transition) is mediated by Cyclin B/Cdk1 phosphorylation.
What does active mitotic cdk-cyclin stimulate?
Nuclear envelope breakdown, chromosome condensation, mitotic spindle formation, and target protein degradation.
What is the signaling cascade that begins mitosis?
Cyclin B levels are increased due to transcriptional regulation (loss of a repressor) Cdc25 is phosphorylated in response to the completion of DNA replication. This activates cyclinB/cdk1. Its phosphorylation of substrates results in mitotic entry. **Its targets include Lamins A and C resulting in nuclear membrane breakdown and condensin which leads to chromatin condensation.
What is needed for the transition from M to G1 or to exit mitosis?
This exiting of mitosis requires the activation of APC.
How is APC activated and what does it do?
APC becomes activated because it is associated with a co-factor, either Cdc20 or Cdh1. APC has two main substrates. Degradation of Separase by APC results in sister chromatid separation. Degradation of Cyclin B results in mitotic exit. Chromosomes decondense and the nuclear membrane re-forms.
How does the Cyclin A/cdk2 complex get activated and what does it do?
It is activated by E2F release from rRb. It has two functions. It binds to the polymerase complex to initiate origin firing. it also binds to Orc1, cdk6 and cdt1 to block reinitiation.
What is the concept of a checkpoint control?
an ordered series of events dependent on completion of a previous step. Loss of checkpoint control leads to uncontrolled proliferation (cancer).
What is the restriction point?
The point at which a cell becomes committed to DNA replication. It reaches the point do to extracellular signaling with growth factors but beyond this point they no longer need it.
What mediates the restriction point?
Phophorylation of pRb in response to growth factors.
How is the restriction point altered in cancer?
Often the problem with cancer is their entry into the cell cycle continuously with no appropriate stimulus. the checkpoint is lost and cells proliferate in an unregulated way.
What happens in the G1 checkpoint?
DNA damage causes up-regulation of the tumor suppressor p53. A kinase cascade results in phosphorylation of both p53 and its negative regulator Mdm2. Mdm2 is a
ubiquitin ligase for p53. Phosphorylation disrupts their binding and p53 levels rise. p53 is a transcription factor. One of its targets is the gene encoding p21, a cyclin-dependent kinase inhibitor. p21 binds to Cdk complexes and prevents phosphorylation of pRb, leading to arrest prior to S phase. p53 can also cause apoptosis.
What does Mdm2 do and what happens when it is phosphorylated?
Mdm2 is a negative regulator (ubiquitin ligase) of the p53 tumor suppressor. When it is phosphorylated in response to DNA damage, this disrupts its ability to bind which causes p53 levels to rise.
What is a target of p53 activated in the G1 checkpoint?
p21, a cyclin-dependent kinase inhibitor. p21 binds to Cdk complexes and prevents phosphorylation of pRb, leading to arrest prior to S phase. p53 can also cause apoptosis.
What happens in the G2 checkpoint?
For the G2 checkpoint, DNA damage triggers a kinase cascade that results in inhibitory phosphorylation of Cdc25. Thus, there is no activation of Cyclin B/Cdk1. There is also a role for p53 as the cyclin-dependent kinase inhibitor p21 can also inhibit Cyclin B/Cdk1.
How does unreplicated DNA prevent entry into M phase?
Cdc25 needs to be phosphorylated to be active. This phosphorylation does not occur until DNA synthesis is complete and a full 4N DNA content is achieved.
What does the spindle checkpoint do?
Arrests cells in mitosis until all chromosomes are properly aligned in metaphase. The checkpoint is triggered because of a disruption of the spindle and a loss of attachment of microtubules to the kinetochore. This causes a delay in activation of APC.
What are benign cells?
Benign cells are generally considered ones that are undergoing uncontrolled proliferation but this does not result in any manifest human disease. In contrast, malignant cells have unlimited cell renewal and result in what is generally considered to be cancer.
What are primary cells?
Cells with a finite lifespan that are directly removed from the organism.
What are immortalized cells?
cells which have an unlimited life span
What are transformed cells?
immortalized cells that have gained certain traits like anchorage independency or failure to stop growing in response to other cell contact.
What are tumorigenic cells?
Transformed cells that have the additional property of being able to form tumors in the animal.
How is the morphology of cancer cells different?
The morphology of cancer cells is spindle-shaped, often appearing refractile in the plight microscope. This is thought to be due to cytoskeletal changes including actin depolymerization.
How does cancer cell metabolism differ from normal cells?
Cancer cell metabolism is altered such that they rely on glycolysis for their ATP production whereas normal cells use the TCA cycle for this purpose. The enhanced glycolytic rate of tumor cells is not necessarily due to being in a low oxygen environment, but rather is a property intrinsic to the cancer cells themselves. This is referred to as the Warburg effect.
What is the Warburg effect?
The intrinsic property of cancer cells using glycolosis for ATP production, or having an enhanced glycolytic rate.
