Test 3 Flashcards
(166 cards)
1
Q
Cyclin complexes that operate through the animal cell cycle
A
Cdk1/CycB are not the only cyclin complex active throughout cell cycle
Cdk4,6/CycD complex mediates transition through restriction point
Cdk2/CycE complex mediates transition from G1 to S phase
2
Q
Name the steps to mitosis
A
Prophase
prometaphase
metaphase
anaphase
telophase
cytokinesis
3
Q
What is Immunofluorescence?
A
Use antibodies to visualize the presence of proteins and other molecules in cells and tissue, usually through a fluorescent molecule (immunofluorescence) or an enzymatic reporter bound to an antibody.
Can be used in conjunction with fluorescent stains like DAPI (stains DNA)
4
Q
What allows us to target multiple antigens in the same sample?
A
Double or multicolored labelling
5
Q
What triggers changes in cellular organelles and mitotic structures?
A
Mitotic kinases
6
Q
Describe how Mitotic kinases trigger changes in cellular organelles and mitotic structures
A
MPF/Cyclin B-Cdk1 activates other mitotic protein kinases like Aurora kinase
Aurora kinase then phosphorylates and activates Polo-like kinase
These kinases function in a positive feedback loop and play multiple roles during mitosis
7
Q
What organizes sister chromatids as they progressively condense through the cell cycle?
A
Condensin and cohesins
8
Q
Describe how Condensin and cohesins organize sister chromatids as they progressively condense through the cell cycle
A
Cohesin binds DNA during S phase, maintain linkages between sister chromatids during DNA replication
Condensins are activated and replace cohesins, cohesins only found near centromere
Condensins drive chromatin condensation by forming DNA loops
9
Q
What is one of the triggering events of prophase?
A
CDK1 phosphorylation of nuclear lamins
10
Q
Describe CDK1 phosphorylation of nuclear lamins
A
Phosphorylation of nuclear lamins by Cdk1/cyclin B dissociation of lamina polymers into free lamin dimers
Integral nuclear envelope proteins are absorbed into ER, which will be distributed to daughter cells
Golgi also breaks into small vesicles that are absorbed into ER or distributed to daughter cells
11
Q
Describe the mitotic spindle
A
The mitotic spindle is composed of microtubules. These microtubules attach to and reposition chromosomes during mitosis
12
Q
How is the movement of spindle poles mediated?
A
By molecular motors – kinesins (+) and dyneins (-)
13
Q
What are the different components of the mitotic spindle?
A
Interpolar microtubules
astral microtubules
kinectocore microtubules
14
Q
What is a kinectocore? What are some of its functions?
A
Dense protein complex associated with centromeres that serves as attachment site of kinetochore microtubules
The kinetochore also senses tension from attached microtubules and regulates progression through mitosis
15
Q
What do spindle microtubules do?
A
“search and capture” the kinetochore through an intrinsic property called dynamic instability
16
Q
What is the metaphase plate?
A
The region in the middle of the cell between both spindle poles where chromosomes align during metaphase
17
Q
Describe the spindle assembly checkpoint
A
APC/C activity is blocked by mitotic checkpoint complex (MCC), which is assembled through the activity of unattached kinetochores
Kinetochore attachment to spindle fibers inhibits MCC formation, leading to APC/C ubiquitinating both the MPF (specifically cyclin B) and securin, an inhibitory subunit of separase
Separase then degrades cohesin, separating sister chromatids leading to the initiation of anaphase
18
Q
What are the forces that separate chromosomes on the spindle? Describe them.
A
Astral microtubules pull spindles towards the plasma membrane
Interpolar microtubules slide past one another, pushing spindles apart
Kinesins associated with kinetochores depolymerize kinetochore microtubules, shortening them, leading to chromosomes being “pulled” towards spindles
19
Q
Describe Anaphase
A
Inactivation of Cdk1(through proteasomal breakdown of cyclin B) leads to chromosome decondensation and cytokinesis
20
Q
What does cytokinesis involve?
A
the formation of a contractile actin ring, the cytokinetic furrow
21
Q
Cytokinesis initiates following what?
A
the inactivation of Cdk1
22
Q
What is the formation of contractile ring mediated by?
A
Aurora and Polo-like kinases
23
Q
Describe cytokinesis in plant cells
A
During cytokinesis in plants, vesicles will fuse to form a cell plate, which grows to form plasma membrane between adjacent cells
Cell wall growth follows cell plate extension
24
Q
What is cancer the result of?
A
uncontrolled cell growth
25
What is cancer is characterized by?
loss of cell cycle regulation, usually resulting from mutations in cell cycle regulatory genes
26
What is a tumor?
Any abnormal proliferation of cells
27
What is the difference between a benign and malignant tumor?
Benign tumors do not spread throughout the body (metastasis) and are confined to a specific area. Their growth can still result in deleterious effects for the individual.
Malignant tumors are capable invading surrounding tissues and metastasizing to other locations in the body.
28
Why do tumor cells often have abnormal appearances?
as a result of the accumulation of mutations.
29
What are the three main groups of cancers and describe them.
Carcinoma - ~90% of human cancers, cancers of epithelial cells
Sarcoma – cancers of connective tissues (muscle, bone, cartilage, fibrous tissues)
Leukemia and lymphoma – cancers of blood-forming cells(leukemias) and immune cells (lymphomas)
30
What are the three steps to the development of cancer?
Tumor initiation, tumor progression, and clonal selection.
The transformation of a healthy cell into a tumor cell is usually a multi-step process resulting for a series of accumulated mutations
31
Describe tumor initiation
mutations cause abnormal proliferation of a single cell – forms a clonal population that ultimately gives rise to a tumor or neoplasm
32
Describe tumor progression
additional mutations accumulate in tumor population of cells – may confer an advantage to tumor cells, such as increased growth/proliferation
33
Describe clonal selection
cells with increased growth/proliferation in tumor population are selected for – their cell lineage becomes dominant cell type in tumor
34
True or false: Cancer cells often lack properties seen in healthy cells
True
35
What do healthy cells generally exhibit that leads to quiescence? Entering G0 of cell cycle
density-dependent inhibition and contact inhibition
36
What is a characteristic of tumor proliferation?
Tumor cells often exhibit proliferation that is not inhibited by contact with other cells or cell density, leading to abnormal cell growth and altered cell migration phenotypes
37
True or false:
Many tumor cells exhibit increased adhesion to the extracellular matrix, and some secrete proteases to actively degrade extracellular matrix proteins
False. Tumors exhibit decreased adhesion to the cellular matrix
38
What do cancer cells often produce and secrete, and what does this do?
Cancer cells often produce and secrete growth factors – which leads to autocrine growth stimulation
39
What is angiogenesis?
Angiogenesis is the process of new blood vessels forming from existing blood vessels, this can make the tumore even more ingrained in your body and these vessels provide nutrients and oxygen to the growing tumor – can also lead to metastasis
40
What can carcinogens do?
introduce mutations that cause cancer
41
Give examples of carcinogens
asbestos, radon, formaldehyde, UV light
42
Give two examples of tumor viruses
human papillomavirus (HPV) and Rous sarcoma virus (RSV)
43
What is an oncogene?
genes capable of inducing cancer
44
what do oncogenes derive from?
Oncogenes derive from mutations in proto-oncogenes – genes that drive cell proliferation
45
Give an example of a proto-onco gene
Viral raf oncogene has regulatory domain replaced by a partially deleted viral domain (Gag), leading to constitutive activity
46
How were oncogenes first discovered?
first identified through studies of tumor viruses
47
How did we get direct evidence for cellular oncogenes?
Through gene transfer.
DNA extracted from human cancer cells was introduced to healthy mouse cells in cell culture.
Mouse cells were transformed into tumor cells, indicating that DNA extracted contained mutated oncogenes
48
How do oncogenes develop?
Develop from proto-oncogenes through mutations.
49
Describe mutations in ras genes
Mutations in ras genes are involved in approximately 30% of all human malignancies, including about 50% of colon and 25% of lung carcinomas
Normal vs. oncogenic forms of ras may differ by as little as a single nucleotide (point mutation)
50
Describe altered expression of c-myc gene
Altered expression of the c-myc proto-oncogene can result from a translocation of c-myc from chromosome 8 to chromosome 14, leading to different regulatory elements controlling the expression of c-myc
51
True or false:
Many proto-oncogenes function in cell-signaling pathways
True
52
Give examples of proteins involved in growth factor/RTK/MAPK signal transduction pathways that can become oncogenes through mutation.
Growth factors
Growth factor receptors (RTKs)
Ras
Raf
Intracellular kinases (MEK, ERK)
Transcription factors (Elk-1, AP-1)
53
Describe how the Wnt pathway involved proto-onco genes.
In the absence of Wnt, Beta-catenin is phosphorylated by destruction complex, targeting it for ubiquitination and degradation (E3 ubiquitin ligase pathway)
Wnt signaling through Frizzled regulates development and cell proliferation through regulation of gene expression
Mutations that prevent Beta-catenin degradation can convert it to an oncogene and lead to increased expression of target genes, including c-myc and Cyclin D1
54
True or false:
proto-oncogenes cannot regulate apoptosis
False
55
Describe how proto-onco genes can regulate apoptosis
Growth factor signaling through RTKs inhibits proapoptotic regulatory proteins like Bad and Bim
Akt also inhibits the proapoptotic FOXO transcription factors
Mutations in PI3K, Akt, or Bcl-2 can cause the cell to fail to undergo apoptosis in response to appropriate signals
56
How do tumor suppressor genes like Rb function to inhibit cell proliferation?
Rb is a repressor of E2F transcription factors which regulate expression of cell-cycle and S phase specific genes
Phosphorylation of Rb by Cdk4,6/Cyclin D complex leads to its dissociation, activating E2F and transcription of target genes
Mutations in Rb can lead to overexpression of cell cycle genes, driving tumor initiation
57
What is Rb
Retinoblastoma protein – first tumor suppressor gene identified
58
How were tumor suppressor genes hypothesized to exist?
Following cell fusion experiments that fused tumor cells and healthy cells
The fused cell was usually nontumorigenic, suggesting that genes present in the healthy cell could suppress tumor development and proliferation
59
How does Human papillomavirus (HPV) inhibit Rb and drive tumor development?
The viral HPV protein E7 binds to and inhibits Rb in HPV infected cells and can drive tumor development through post-translational inactivation of Rb, rather than mutational inactivation
60
What are the numbers on HPVs cancer rates?
HPV causes ~3% of all cancers in women and 2% of all cancers in men, including the majority of cervical cancer and oropharyngeal cancer
61
What is p53?
p53 is another key tumor suppressor gene activated in response to DNA damage.
p53 was the second tumor suppressor gene to be identified
62
What does p53 do?
p53 is phosphorylated and activated in response to DNA damage – induces expression of p21 (cell cycle arrest at G1-->S transition and/or
Bcl-2 family members PUMA and Noxa (apoptosis) depending on level of p53 induction.
63
How is p53 degradation mediated?
mediated by the E3 ubiquitin ligase MDM2, which can function as an oncogene if overexpressed
64
True or false:
Estimated that 65% of all cancers have mutations in p53 gene
False.
50% of cancers have mutations in p53 gene.
65
How does PTEN regulate cell proliferation?
through dephosphorylation of PIP3
66
What do mutations in PTEN lead to?
increased PIP3 levels and increased Akt signaling, inhibiting apoptosis
67
Describe how PTEN regulates cell proliferation through dephosphorylation of PIP3
PI 3-Kinase – often activated following growth factor binding to RTK; can also be activated by GPCR signaling
Phosphorylates PIP2 to PIP3 which acts as second messenger activating mTOR/Akt – regulate cell growth and cell survival (apoptosis)
Dephosphorylation of PIP3 to PIP2 is regulated by PTEN, a lipid phosphatase that acts as a tumor suppressor
Mutations in PTEN can lead to increased PIP3 levels and increased Akt signaling, inhibiting apoptosis
68
Can Tumor suppressors and oncogenes can function in the same pathway?
Yes
69
What do stability genes do?
maintain integrity of the genome
70
What are two examples of stability genes?
BRCA1 and BRCA2
71
How do BRCA1 and BRCA2 maintain genome stability?
BRCA1 and BRCA2 act downstream of ATM/Chk2 activation and repair double-stranded breaks in DNA through homologous recombination. Mutations in BRCA1 and BRCA2 are associated with increased rates of mutation.
72
The viral Human Papillomavirus (HPV) protein E7 can bind to and inhibit the tumor suppressor gene ____________ to drive tumor development
Rb
73
What does the progression of tumor development result from?
usually results from multiple mutations accumulating in oncogenes and tumor suppressor genes
74
Are some genes more commonly mutated than others to cause cancer? roughly how many genes are commonly mutated to cause cancer?
P53 and PI3K are more commonly mutated than others.
~150 genes are commonly mutated and contribute to tumor development
75
What does the accumulation of mutations lead to?
A progressive loss of cell cycle regulation and uncontrolled proliferation
76
What is associated with a better cancer outcome?
Early detection!
Screening can increase likelihood of identifying cancer early.
77
What can genetic testing do for cancer detection?
Genetic testing can identify individuals with mutations in cancer-causing genes (ex. BRCA1/2) who are at higher risk.
78
True or false:
Many drugs exist that target specific oncogenes
True
79
Give two examples of cancer immunotherapies
Checkpoint inhibitors: PD-1 – programmed cell death protein 1, inhibits T-cell activation when bound to PD-L1 or PD-L2.
Immune checkpoint inhibitors block this interaction.
CAR-T: chimeric antigen receptor T-cell
80
What are the three types of filaments in animal cells?
1. Actin
2. Microtubules
3. Intermediate filaments
81
What is the rough diamter of an actin filament?
7nm
82
True or false:
Actin is not highly conserved
False:
Actin is highly conserved – yeast actin is 90% identical to human actin at AA level
83
Describe how actin can form a polar filament.
Actin is a polar molecule that can oligomerize into polar filaments.
G actin – globular or monomeric actin
Assemble into dimers and trimers through head to tail interactions
Trimer represents a “seed”, nucleates actin filament
F actin – filamentous actin – helical structure
Actin has innate ATPase activity that regulates assembly and disassembly
84
Describe how actin’s ATPase ability regulates the assembly and disassembly of filaments
ADP-actin is less tightly bound and dissociates from the minus end
Actin bound to ATP (ATP-actin) rapidly associates with growing plus ends
ATP is hydrolyzed to ADP after ATP-actin is incorporated into filament
85
What is "treadmilling"?
ATP-actin added to plus end while ADP-actin dissociates from minus end
86
What are the accessory proteins that facilitate ATPase cycle and nucleation of actin?
Actin is able to form filaments innately, but filaments are regulated in the cell by accessory proteins
Formin – nucleates new actin filaments (formation of trimer)
Profilin – swaps ADP for ATP on actin monomers, exchange factor
87
What does the Arp2/3 complex do?
Arp2/3 – Actin related protein 2/3 – nucleates new actin filaments that branch off of existing filaments – creates branched actin structures
88
Where does the Arp2/3 complex bind?
Binds near intersection of ADP-actin and ATP-actin near plus end of filament
89
T/F:
A number of unique actin structures exist in cells
True
90
What do sheets of branched actin create?
lamellipodia
91
Describe how a series of proteins prevent assembly, disassembly, and motor access of actin
Plus end capping protein: Prevent further polymerization/extension of filament
Minus end capping protein: Prevent further depolymerization of filament
Tropomyosin: Stabilize actin filament and block access of molecular motors to actin filament
92
What is cofilin?
Cofilin is a filament severing enzyme which can increase polymerization and depolymerization.
severs actin filaments, creating new plus and minus ends that can grow/depolymerize
93
What do actin filaments assemble into?
Depending on the type of associated cross-linking protein, actin filaments assemble into:
parallel bundles
mesh-like networks
94
What is the actin cortex?
A meshwork of actin that is cross linked and associated with the plasma membrane
95
What is spectrin?
actin binding protein that also binds to phospholipids in plasma membrane – made of two polypeptide chains with actin binding domains (ABD) at each end
Provides structural rigidity to cell/maintain cellular morphology
96
What is Ankryn?
binds to both spectrin and transmembrane protein band 3 – links spectrin-actin network to plasma membrane
97
What are two examples of bundled actin arrays?
Filopodia and stress fibers
98
How is the extracellular environment linked with the actin cytoskeleton (stress fibers)?
Stress fibers – tropomyosin stabilized actin filaments crosslinked to alpha actinin, help to anchor cell to substrate/ECM
Stress fibers attach to integrins through the binding of talin and vinculin at sites called focal adhesions
This linkage provides adhesion to the ECM helps create traction force
99
T/F:
Focal adhesion remodel during cell migration
True
100
What is actin polymerization controlled by? Describe the process.
Rho Family GTPase activity.
Rho activates Arp2/3 and formin to initiate new filaments as well as new branched filaments
Profilin promotes the exchange of ATP for ADP to extend filaments
Cofilin cleaves existing filaments to generate new plus ends
These growing actin filaments push (physical force) against the plasma membrane at the leading edge to drive cell motility
101
Describe how Cell-cell adhesions also link the actin cytoskeleton to adherens junctions
Adherens junctions – cell-cell junctions that link to actin filaments on the cytoplasmic face - mediates cell-cell adhesion
Form a continuous belt-like structure around certain cells known as an adhesion belt
Contact is mediated through transmembrane cadherin proteins that extend into the extracellular space and bind intracellular catenins
102
What are microvilli?
Microvilli – “small hairs” – small fingerlike cellular protrusions
Abundant in epithelial cells, greatly increase surface area
103
How are microvilli supported?
Microvilli are supported by parallel actin fibers linked together by villin and fimbrin
Microvilli are anchored to the actin cortex at a region called the terminal web
104
Give three cell surface projections that all rely on the remodeling of actin filaments
1. Pseudopodia – moderate width cellular extensions involved in phagocytosis and motility
2. Lamellipodia – broad, sheetlike extensions at the leading edge
3. Filipodia – thin projections that grow out of lamellipodia
105
Describe the composition of a muscle fiber
Muscle fibers are long, multinucleate cells that fuse together during development
Composed of myofibrils – bundles of actin and myosin filaments organized in chains of contractile units known as sarcomeres
106
How does muscle contraction occur?
Through highly organized and coordinated motor movement along actin.
Actin fibers sliding past myosin filaments, contracting the sarcomere, forms the basis of the sliding filament model
107
What type of myosin is present in muscle cells?
Myosin II. Globular head region binds to actin and hydrolyzes ATP
108
Describe how myosin arrays on a single muscle fiber
Multiple actin-myosin contact points along the muscle fiber prevent actin filaments from sliding back as myosin head groups dissociate/reassociate with different sites along the actin filament
109
Describe the contraction cycle of muscles.
ATP binding to myosin head group dissociates myosin from actin
ATP hydrolysis induces a conformational change in myosin, moving head group into “cocked” position (myosin still bound to ADP and Pi)
Myosin head group binds to new position on actin filament. Pi is released, initiating “power stroke”
“Power stroke” occurs and ADP is released. Myosin head group adopts original conformation, sliding filaments past one another
110
Describe how calcium triggers a change in tropomyosin and exposure of myosin binding sites
Muscle contraction is tied to calcium release from the sarcoplasmic reticulum – structure similar to smooth ER in skeletal muscle cells that stores of calcium Ca2+ ions
Nerve impulses signaling through the neuromuscular junction (NMJ) induce the release of calcium Ca2+ ions from the sarcoplasmic reticulum
Myosin binding sites on actin filaments are blocked by troponin/tropomyosin when calcium concentration is low
Calcium binding to troponin complex shifts troponin-tropomyosin complex, exposing myosin binding sites
111
True or false:
Non muscle cells organize their actin and myosin filaments into sarcomeres.
False. Only muscles have sarcomeres.
112
Describe why cytokinesis requires coordinated myosin contractility
Contractile ring composed of actin and myosin filaments
113
Describe how non-muscle myosin is activated by calcium/calmodulin
Ca2+ binding to calmodulin induces a conformational change and allows calmodulin to bind to myosin light-chain regulatory kinase (MLCK)
MLCK/calmodulin complex then phosphorylates regulatory light chain of myosin II, activating the protein
114
Describe other types of myosin paired to cargo or other intracellular components
Myosin I lacks a long tail and does not form dimers, instead the tail interacts with vesicles and other structures in the cell, which can be transported along actin filaments
Myosin V plays an important role transporting cargo towards the plus end of actin filaments, particularly in neurons
115
What do cell cycle checkpoints do?
Detect (and repair) DNA damage.
DNA damage checkpoints prevent cells with incomplete/damaged genomes from replicating – prevents abnormal cell function and tumor development
116
What are two primary mechanisms for DNA repair?
1. direct reversal of the chemical reaction responsible for DNA damage
2. removal of the damaged bases followed by their replacement with newly synthesized DNA
117
What are ATR and ATM?
kinases that recognize single strand (ATR) or double strand (ATM) breaks in DNA. ATR and ATM activate Chk1 or Chk2, kinases that phosphorylate and inhibit Cdc25 phosphatases
118
What does Cdc25 do?
removes inhibitory phosphorylations from Cdk1 and Cdk2, activating these kinases
119
What stabilizes p53 and prevents its degradation?
phophorylation
120
The p53 pathway is a major mediator of responses to stress and damage to the genome. Why.
P53 builds up and prevents the cell from progressing through a checkpoint. This is a method of cell cycle arrest.
Arrest gives the cell a chance to repair the double stranded break. If the cell cannot fix the break, higher levels of P53 will be generated and this typically leads to apoptosis.
A double stranded break leads to loss of a lot of genetic info.
121
Give three examples of DNA damage
Deamination- loss of amine group. Becomes uracil. This messes up complementary base pairing. Leads to distortion in DNA through improper base pairing.
UV light and radiation. Energy from this light can cause adjacent thymine resides to crosslink and interfere with base pairing. Can cause issues with transcription.
Carcinogens cause cancer. Causes distortion in structure
122
Describe how thymidine dimers can be reversed by photo reactivation
Energy from visible light used to reverse UV-induced dimerization of adjacent thymine residues
This repair mechanism is not present in mammals but is found in some other animals, as well as yeast and bacteria
123
Describe base excision repair of cytosine deamination
Base excision repair is the removal of a single damaged base from a DNA strand
DNA contains U caused by a deamination of C.
Uracil removed by DNA glycosylase, sugar-phosphate backbone remains intact, but creates an AP site.
The gap is then rapired via DNA pol and ligation.
124
Describe nucleotide excision of thymidine dimers
Damaged bases/bulky group additions induce distortion in DNA helical structure
Helicase separates DNA around lesion
Nucleotides surrounding lesion cleaved and removed by nucleases
Gap in molecule repaired by DNA polymerase and ligase
125
Describe correcting mismatched bases during DNA replication
Methylation distinguishes parent strand from daughter strand, MutS binds to mismatched base followed by MutL
MutH cleaves unmethylated DNA at GATC sequence
MutS and MutL, along with helicase and exonuclease, cleave and remove DNA around mismatch
126
Describe how DNA replication can continue through DNA damage lesions using special DNA polymerases
Polymerase stalls at DNA damage due to a thymidine dimer.
Specialized DNA polymerases are highly error prone and lack proofreading activity, but are recruited to synthesize across the lesion.
Normal replication then resumes, and the lesion i slater repaired via excision repair.
127
Describe repair by non-homologous end joining (NHEJ)
These double stranded breaks can be repaired by simply rejoining the ends of the broken molecule, but this frequently leads to the loss of bases around the site of damage.
Small insertions and deletions (indels) are common with NHEJ
128
Describe repair via homologous Recombination
Both strands of DNA at a double-strand break are digested by nucleases in the 5′ to 3′ direction. RecA binds to the overhanging 3′ ends, which then invade the other parental molecule by homologous base pairing.
The gaps are then filled by repair synthesis and sealed by ligation, yielding a crossed strand intermediate.
Cleavage and ligation of the crossed strands then yields recombinant molecules.
Undamaged homologous chromosome used as template to repair damaged chromosome
129
What is apoptosis?
A form of “programmed cell death” that prevents molecules in the cell from being released onto neighboring cells
In contrast to necrosis – accidental death of cell resulting from acute injury
130
What are apoptotic cells marked by?
exterior phosphatidylserine
131
Describe how phosphatidylserine functions during apoptosis
Phosphatidylserine is found in the inner plasma membrane in normal, healthy cells
Apoptotic cells “flip” phosphatidylserine to the outer membrane, where it is recognized by phagocytic cells
132
What are caspases?
promiscuous proteases that mediate apoptosis.
They cleave over 100 different cellular proteins.
133
What is the general pathway of apoptosis regulated by?
Bcl-2 family member proteins regulate apoptosis through proapoptotic and antiapoptotic activity
134
Describe the activation of caspases by Mitochondrial cytochrome C
Cytochrome c is usually found between the inner and outer membranes in mitochondria
Bax and Bak create pores in mitochondrial outer membrane, allowing cytochrome c to be released into cytosol, where they interact with Apaf-1 and Caspase-9 to form the apoptosome
Caspase 9 in apoptosome then cleaves and activates procaspase-3, leading to caspase-3 activation and apoptosis
Caspase 9 is a key initiator caspase in mammalian cells
135
Describe P53 induced apoptosis following DNA damage
DNA damage leads to Chk2 activation and increased levels of p53
p53 induces transcription of pro-apoptotic target genes, whose expression leads to apoptosis
Level of p53 induction determine whether cell cycle arrest/repair or apoptosis will occur
136
Describe how PI-3K /Akt suppress apoptosis and supports cell survival
Growth factor signaling through RTKs inhibits proapoptotic regulatory proteins like Bad and Bim
Akt also inhibits the proapoptotic FOXO transcription factors
137
What are extracellular signaling pathways for apoptosis
TNF – Tumor necrosis factor
TNF induces TNF receptor trimerization and Caspase-8 recruitment and activation
Caspase-8 then cleaves and activates other caspases, while also cleaving and activating the proapoptotic regulatory protein Bid
Targets infected or cancer cells for apoptosis
138
What oncogenes lie within the PI-3K/Akt pathway to block apoptosis
Proto-oncogenes found in the PI3K/Akt pathway include
Growth factors
Growth factor receptors
PI3K
Akt
Bcl-2
139
What do early embryonic cell cycles don’t do?
incorporate G1 or G2 phases
140
Describe variation during animal development
- Early embryonic cell cycles don’t incorporate G1 or G2 phases
- Rapid alternation of DNA synthesis and mitosis
o It never grows, stays the same size as the oocyte
- Surplus of cytosolic content
141
What does Flow cytometry/ flow assisted cell sorter (FACS) do?
used to determine where cells are in the cell cycle based on DNA content
142
Describe how Flow cytometry/ flow assisted cell sorter (FACS) works
- Charge added to deflect cells into different test tubes based on fluorescent tag
- ID cells based on characteristics stains/antibodies to see presence or absence
o S has the most variable amount of DNA because cells split between 2n and 4n amount of DNA when replicating
o More cells in G1 because it’s a bigger phase than G2
143
What does passing the START regulatory point mean?
You committed to entering S phase and mitosis
o First identified in yeast cells
144
Why would the cell cycle arrest during the START regulatory point?
o Low nutrient availability, mating signals, cells not being the minimum size
145
If conditions are unfavorable for replication, what occurs?
cells exit cycle and enter G0 (quiescence)
146
What is quiescence?
state of exiting cell cycle, however, Cells can reenter cell cycle in response to signals (growth factors)
147
What is the restriction point?
like START regulatory point, but present in mammalian cells (START point in yeast cells)
- Regulated by external growth factors in animals
- START controlled by internal signals (cell size and nutrients)
- Passing restriction point commits cell to S phase and cell cycle
- Growth factors act on RTKS to activate MAPK and other growth pathways
148
Why are checkpoints present in replication?
ensure genome fidelity before proceeding through cell cycle. Ensures damaged DNA is not replicated and passed on daughter cells
149
What happens when you fail to pass a checkpoint?
cycle arrest and apoptosis
150
How is cell cycle regulated?
cyclins and CDKs
151
Describe Experiment 1 to discover CDKs: maturation promoting factor (MPF) first discovered during oocyte meiosis
-Progesterone treatment causes oocyte to progress from G2 to M phase
-Take cytoplasm from cell and inject in recipient oocyte in G2
o Cell progressed to M phase
o Shows there was some
cytoplasmic factor in donor
cell that drove recipient cell to
M phase
a. Discovery of maturation
promoting factor
152
Describe Experiment 2 to discover CDKs: temperature sensitive mutants discovered that stopped cell cycle progression at START/restriction point
- At high temperatures, cells arrested at START and stalled at G1
o Mutant phenotypes induced at high temperatures
Results from protein folding incorrectly at nonpermissive temperatures
153
Describe Experiment 3 to discover CDKs: discovery of proteins during sea urchin development that underwent cyclical production and degradation
Cyclins build up in cell through interphase and degraded during mitosis
o Cyclins drive mitosis, but
drops when mitosis happens
-Some cyclins are proto-oncogenes
o Genes that drive cell
growth/mitosis that can also
promote tumor development
when mutated
154
What is MPF?
Maturation promoting factor. A complex of Cyclin B and Cdk1
155
Describe the Life cycle of Cdk1/Cyclin B: G2 to M transition
1. Cdk1 inactive when not bound to cyclins
2. Cyclin B synthesis upregulated in G2 to create MPF
a. More cyclin B = more complex
forms
3. Cdk1 phosphorylated at Thr161 (activating), Tyr15 (inhibiting), and Thr14 (inhibiting)
a. Adding phosphate activates all
three, but complex not active
because Tyr15 and Thr14
inhibitory effects active
4. Thr4 and Tyr15 dephosphorylated
a. Removing phosphate = inactive
Thr14 and Tyr15 = no
inhibition = activated complex
b. Another checkpoint
5. End of mitosis with Cyclin B polyubiquitinated by anaphase promoting complex/cyclosome APC/C
a. Cyclin B degraded to terminate
MPF activity
156
What are CDK inhibitors?
CDK inhibitors can function as tumor suppressors
o Inhibit cell proliferation and cell
growth
o Inhibitors mutated/
nonfunctional in cancer cells
157
How does cell signaling feeds into cell cycle
- Cyclin D rapidly degraded in G1 through APC/C polyubiquitination
o No growth factors present,
cyclin D levels fall
o Cells don’t progress through
G1 to S phase
1.Growth factors feed into Ras/Rak/MEK/ERK pathway to start cell cycle
2. Synthesis of D-type cyclins
3. Synthesis of Cyclin D is higher than degradation
158
Describe Moving from G1 through S phase
- P27 bind to inhibit Cdk2/Cyclin E complex activity
o Increased cyclin E synthesis
o P27 decrease through
reductions in transcription
and translation
-Cdk2/Cyclin E not bound and phosphorylate P27
o Phosphorylation = p27
polyubiquitinated and
degraded
- Activated Cdk2/Cyclin E complex block APC/C activity to prevent cyclin degraded
o Blocking APC/C activity
increase cyclin D too, increasing
cyclin D to increase cyclin E
transcription?
159
Describe how Cdk2/cyclin E is important for DNA replication
-MCM helicase binds to origins of replications/origin recognition complex (ORC) to form prereplication complex during G1
-Cdk2/Cyclin E and DDK phosphorylate MCM and proteins that associate with MCM to initiate DNA replication
160
What does RNA Primase do?
Makes the short RNA primers for both Okazaki fragments and leading strands.
- RNA primer binds, and DNA polymerase binds to 3’ OH RNA primer
- Primer removed by RNAase H and 5’ to 3’ exonuclease
o Gap in sugar-phosphate
backbone joined by DNA
ligase
-Both strands with continuous and discontinuous replication
161
What does PCNA do?
helps DNA polymerase associate with DNA during replication
162
Describe how PCNA helps DNA polymerase associate with DNA during replication
- protein RFC loads PCNA onto the
DNA
o RFC released to load PCNA
-PCNA loads DNA polymerase onto template at primer-template junction
-Ring structure of PCNA holds DNA polymerase in place as it moves down template synthesizing new strand
163
What do Single stranded binding proteins do?
stabilize exposed ssDNA
- Single stranded DNA binding proteins bind to unwound strands of DNA
o Stabilize molecule and protect
from degradation
o Prevent formation of
secondary structures
164
Certain DNA polymerases have proofreading properties to correct base mismatches. Describe this.
- Polymerase excises mismatches nucleotide via 3’-5’ exonuclease
- Synthesis proceeds with incorporation of correct base
165
Telomerase replicates the ends of chromosomes. Describe this
- DNA polymerases can’t replicate the 5’ ends of linear DNA molecules (telomeres)
- Telomerase maintains ends of chromosomes through reverse transcriptase activity
o Brings its own RNA template,
which is complementary to
telomere sequences
a. Uses template to
extend 3’ of one DNA
strand
- Primase and DNA polymerase then extend the complementary strand.
- Telomerase active in germ and stem cells, but some somatic cells lack telomerase activity
o Somatic cells telomeres
shorten progressively with
each round of cell division
a. Cell aging shorten ends,
and eventually shorten to
important DNA
b. Stop replicating after about
50 replications
166
True or false:
Replication origins spaced throughout genome
If only a single origin site existed in a mammalian genome, DNA replication would take 3 weeks
