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What makes up the cell membrane?
- Phospolipid bilayer
- Cholesterol (animals)
- Carbs (euk)
What kinds of proteins make up the cell membrane?
- Integral (Transmembrane)
What part of a protein is glycosylated?
The portion facing the extracellular matrix
What are the functions of the membrane?
- Selective barrier
- In eukaryotes, formation of organelles/compartments
- Localization of enzymatic reactions
- Cell-cell communication
- Transmission/reception of signals
- Receive stimuli
- Site of ECM attachment
Where does glycoslyation occur, and what is the destination?
- Cell membrane (i.e. not organelles)
What does flippase do?
It "evens out" enzymes since most are on cytosol side
What is a lipid raft?
A "microdomain" of the plasma membrane which aggregates proteins and phospholpids for transportation.
How does a lipid raft travel?
Do proteins for organelles get glycosylated? Why or why not?
- Glycosylation is for cell-cell recognition
What is an advantage of a compartment/organelle?
- Useful for specialized enzymatic reactions.
- Greater rate of collisions for reactions to occur.
What is the minimum length of a transmembrane protein?
What are some functions of transmembrane proteins on the cytoplasmic side?
- Intrinsic or associated enzymatic activity
- Provide cell with shape
What are functions of transmembrane proteins on the outside?
- Receptors for soluble ligands
- ECM attachment (e.g. integrins)
- Cell-cell attachment (e.g. cadherins)
How are gates/channels activated?
Hormones or action potentials
What are types of secondary structures for transmembrane proteins?
- Alpha helices
- Beta barrels
For alpha helices in a gate, where is the hydrophobic side?
Facing the outside surface of the gate
What transports water thru the membrane?
What is the function of membrane cholesterol?
Provides membrane fluidity
What types of molecular movement are there in the membrane?
- Rotation on axis
- Lateral (sideways)
- Flip-flopping (from one side to the other)
Describe flip-flopping movement in a membrane.
- Requires energy and is thus uncommon
- Requires the flippase enzyme
- Proteins are too large for this movement
What can cross a membrane by simple diffusion?
CO2 and O2
What happens if a cell membrane has no fluidity?
The cell dies
What are some ways to alter membrane fluidity?
- Change length of hydrocarbon tails
- Change saturation
- Change cholesterol content
- Change temperature
What does an increase in cholesterol do?
What does an increase in temp do?
What does an increase in saturation do?
What does an increase in hydrocarbon tail length do?
What is the most common phospholipid in cell membranes?
Why does the phospholipid bilayer form a sphere and not a plane?
It's more energetically favorable.
Where are membrane phosholipids synthesized?
How are phospholipds moved to the other side of a membrane?
What is a ligand?
A signal that is received by the cell usually resulting in a response.
What is a receptor?
A feature on the cell which receives a ligand
How does a receptor respond?
- Intrinsically as an enzyme.
- Associated with an enzyme.
- With the cytoskeleton directly.
What are some responses to a ligand being received?
- Gene expression
What are effector proteins?
They are proteins activated by intracellular signalling proteins
What are three types of effector proteins?
- Metabolic enzyme
- Gene regulatory protein
- Cytoskeletal protein (altered cell shape or movement)
What types of interactions are there between effectors and cells?
- Direct contact between transmembrane molecules of two neighboring cells
- Paracrine system (local)
- Synaptic (neurotransmitters)
- Cytonemes (thin cytoplasmic extensions releasing hormones)
- Endocrine (via bloodstream)
Which type of signalling requires the most signal molecules?
Which type of signalling is not soluble?
- Contact dependent
- Maybe Cytonemes?
What are three types of signalling responses?
- Quick; < 1 hour; protein de/activation; synaptic
- Slow; 18-24 hours; transcription/translation; endocrine
- Single cell or group in development; autocrine
What are four end-results of a cell in response to a signal?
- Grow + Divide
Describe how acetylcholine can cause two different reponses.
- Heart muscle - decreased rate and force of contraction
- Skeletal muscle - contraction
What are two types of signals wrt to water?
- Soluble - interact with transmembrane receptors
- Insoluble - interact with cytoplasmic receptors
Non-soluble signals affect ___.
What are examples of non-soluble signals?
Vitamin D, estrogen, testosterone, cortisol, estradiol, retinoic acid, and thhroxine
Non-soluble signals have a ___ effect due to ___.
- their duration in the blood
Non-soluble signals enter the cytoplasm by ___.
Non-soluble receptor responses include ___.
- Early - first 30 min
- Depends on type of protein and timing of synthesis
Gene expression regulation falls into two general categories:
All nuclear receptors bind as either ___ or ___.
An inactive receptor protein is bound to ___ proteins.
Proteins in the primary response can ____.
Activate other proteins for a delayed/secondary response.
Describe responses to testosterone.
- Early - male characteristics in development
- Delayed - muscle growth
Describe responses to estrogen.
- Early - female characteristics
- Delayed - retention of bone mass
What are three classes of cell-surface receptors?
- Ion-channel-coupled (open channel)
- G-protein-coupled (G activates enzyme)
- Enzyme-coupled (intrinsic and associated enzymatic)
What are three types of "players" that affect the cell?
- First messengers - ligands such as hormones
- Intracellular signaling proteins
- Second messengers - not unique to one pathway
What are examples of second messengers?
cAMP, cGMP, 1,2-diacylglycerol (DAG), IP3, Ca+2
What second messengers require ATP?
What second messengers are derived from phospholipids
Name various proteins/molecules in signalling pathways/cascades.
Anchoring, amplifier, integrator, modular, relay, scaffold, transducer
Describe an anchoring protein.
Anchors proteins to a structure at a precise location where needed.
Describe amplifier proteins.
Greatly increases signal they receive.
Describe Integrator proteins.
Combine signals from two or more pathways before forwarding.
Describe modular proteins.
Modify the activity of signaling proteins to regulate signal strength.
Describe relay proteins.
Pass messages to the next signaling component in the pathway.
Describe scaffold proteins.
Bind to multiple signaling proteins together in a functional complex for quicker and more efficient interaction.
Describe transducer proteins.
Convert singal to a different form.
Describe the structure of a G Protein-linked receptor (or G protein-coupled).
- Seven transmembrane spanning domains.
- Large cytoplasmic region that associates/activates with trimeric G proteins
Does the GPCR have intrinsic enzymatic activity?
What are 4 classic downstream targets of G Proteins that regulate different effectors?
- Adenylyl cyclases
- Ion channels
- Protein kinases
What are the basic subunits of a G protein?
alpha, beta, gamma
What about G proteins might explain highly specific responses?
What part of the G protein binds with GDP?
The alpha subunit
When activated, the beta/gamma complex can ___.
move and activate other targets.
What else provides specific signaling specifity?
- Cell-specific receptors, G isoforms, and effectors
- Amount of receptors, G isoforms, and effectors
- Organization of signaling cascades
- Accessory proteins
How do accessory proteins regulate G protein action?
They regulate the strength, efficiency, and specificity of the transmitted signal.
What are some examples of accessory proteins?
- GAP-43 - promotes GDP dissociation
- AGS3 - stabilizes G-alpha-GDP interaction
- Tubulin - directly transfers GTP to G-alpha
What are three types of accessory proteins?
- Activators of G protein signaling (AGS)
- Regulator of G protein signaling (RGS)
- Inhibitors of GDP dissociation
Describe activators of G protein signaling.
Can activate G proteins without the use of a receptor
Describe regulators of G protein signaling.
- Accelerate the GTPase activity of specific G-alpha subunits.
- Quick inactivation - hydrolysis
What is the typical end of a pathway?
What are other roles associated with G proteins?
- Golgi stability (alternative binding partners)
- Cell polarity in the fruit fly and nematode
- Neurite outgrowth and path-finding
What do G protein-linked receptors activate?
- Adenylyl cyclase
- Phospholipase C-beta
What effect does the activation of adenylyl cyclase typically have?
- Increase of cyclic AMP concentration in the cytosol.
- This rise activates PKA.
- PKA enters the nucleus and phosphorylates CREB.
- CREB recruits CBP, and both stimulate gene transcription.
What is produced from the hydrolysis of PIP2?
- inositol 1,4,5-trisphosphate (IP3) - releases Ca2+ from the ER
- diacylglycerol (DAG) - helps to activate PKC
Describe how GPCRs increase cytosolic Ca2+ and activate PKC.
- PLC-beta is activated by G protein (via alpha, beta/gamma, or both).
- Two messenger molecules, P3 and DAG, produced from hydrolysis of PIP2.
- IP3 releases Ca2+ from ER
- Ca2+ and DAG activate PKC
What other purpose does the release of Ca2+ serve?
Prevents polyspermy by creating fertilization envelope
In the notch pathway, ___ and Jagged are ___.
Notch is composed of ___.
- NECD - Notch extracellular domain
- transmembrane domain
- NID - Notch intracellular domain
___ cleaves the NECD from the ___.
What is ADAM?
A desintegrin and metalloprotease
The NICD makes its way to the ___ and generally results in ___.
- transcription, cell division, differentiation
Hh are ___, a ___.
- family of secreted proteins
Hh generally functions in ___.
In adult cells Hh can lead to ___.
Hh homologues in vertebrates include ___.
sonic, desert, Indian Hh
What is the receptor for Hh?
What are the receptors for Hh in mammals?
Besides Patch and Hip, what else is needed for Hh pathway activation?
The ultimate target of Hh in the fruitfly is ___, a ___.
- cubitus interruptus (Ci)
- transcription factor
What vertebrate homologue most closely resembles Drosophila Hh?
How do Shh, Dhh, and Ihh differ?
Typically by potency - Shh>Ihh>Dhh
What role in develoment does Hh play?
cellular proliferation, growth, and axon path finding
What are examples of human developmental disorders from Hh?
- Greig cephalopolysyndactyly syndrome
- Pallister-Hall Syndrome
- Gorlin's syndrome
What are some cancers triggered by malfunctioning Hh?
- Basal cell carcinoma
- Small cell lung cncer
- Pancreatic cancer
What are some components of the Hh pathway?
- Patch(Ptc) - membrane receptor
- Smoothened (Smo) - intermembrane protein
- Intracellular Hh Signaling complex (HSC)
- Membrane receptor which activates Smo when bound to Hh.
- After binding, Ptc levels decrease as a result of endocytosis
In vertebrates, Ptc does not have ___, so it needs ___.
Intermembrane protein that when activated relays signals to HSC
In vertebrates, Smo is always ___.
at the cell membrane
- Coastal 2 (Co2) - kinesin-related protein
- Fused (Fu) - Ser/Threo kinase
- Supressor of fused (Su/Fu)
- Cubitus Interruptus (Ci)
What are three Ci homologs as activators in mammals?
Gli1, Gli2, Gli3
Since Gli is acts only as an activator, it does not get ___.
What happens to Ci when there is no Hh?
HSC truncates Ci which becomes a repressor
What happens when Hh binds to Ptc?
Production of Ci which becomes an activator
In the Drosophila wing imaginal disc, Ci is truncated where?
In all but cell fate 1 nearest the posterior
In the Drosophila wing imaginal disc, Hh concentration results in activation where?
In all but cell fate 5 (nearest the Anterior) which results in repressor.
In the Drosophila wing imaginal disc, how does Hh concentration affect expression?
[Hh] is proportional to activation
What is involved in Hh processing?
- Binding of cholesterol to C end
- Addition of palmitate to amino terminus
Describe the binding of Hh to cholesterol.
- Critical for target cell intake
- Critical for signal transduction after Hh binds to Ptc
- If binding is inhibited, Hh doesn't work
In the absence of Hh, Ptc ___.
blocks the phosphorylation and stability of Smo.
What type of receptor does Smoothened have?
G protein coupled receptor
When there is no Ptc, ___.
Smo is found in endosomal vesicles
Upon Hh binding to Ptc, ___.
Smo is released and localizes to the cell membrane
Smo multimers may be required for ___.
high level signaling
With Hh present, Ci__ is processed to ___.
- Weak activator - Ci^act
- Strong activator - Ci*
If no Hh is present, Ci__ is formed.
How is Ci phosphorylated?
- Glycogen synthase Kinase 3-beta
- Casein Kinase 1-gamma
Upon phosphorylation, Ci is ___ by ___, a ___.
- Slimb (supernumerary limbs
- proteosome for cleavage
Describe Coastal 2 (Cos2)
- Possibly a microtubule-motor
- Interacts with Smo
- Responsible for moving Smo
How do Cos2 and Smo interact?
- Cos2 binds Fu to Smo
- Their interaction is critical for hh pathway signalling
Where does Cos2 move Smo?
- cell membrane upon Hh pathway activation.
- Intracellular vessicles in the absence of Hh ligand
Describe Fused (Fu)
- Has kinase activity, i.e. might phosphorylate Cos2 and Su(Fu) upon Hh pathway activation.
- Binds to Cos2 and Su(Fu) via carboxy terminus domain
Describe Suppressor of Fused
- May be antagonistic to Fu
- No a.a. homology to known proteins
- Binds Fu and Ci, but not Cos2
What evidence is there that Su(Fu) and Fu might be antagonistic?
Su(Fu)- and Fu- flies yield a wt phenotype
What are possible functions of Su(Fu)?
- May inhibit Ci activation
- Nuclear translocation of Ci
- Transcriptional regulation in vertebrates
Describe Hh pathway at no/low [Hh].
- Ptc on cell membrane repressing/sequestering Smo at vesicle with HSC-A inactive.
- HSC-R on MT picking up vesicle leading to truncated Ci^75 by Su(Fu).
- Ci^75 to nucleus as repressor
Describe Hh pathway at medium [Hh].
- Hh binds Ptc at cell membrane permitting Smo/HSC-A to go to cell membrane
- HSC-R not at MT
- HSC-A allows Ci^act into Nucleus for low activation with possible low amount of Ci^75.
Describe Hh pathway at high [Hh].
- Hh sequesters Ptc to vesicle permitting Smo Multimer/HSC-A to go to cell membrane
- Fu phosphorylates Cos2/SuFu
- SuFu leaves HSC
- Cos2 drives Ci to Smo
- HSC-R not present/inactivated.
- HSC-A allows Ci* to be processed untruncated into Nucleus by SuFu for high activation
Research shows that Hh signalling malfuction is responsible for:
- Formation of tumors
- Survival of tumors
In mammals, absence of Hh leads to ___.
- Gli forms MT-attached complex with Fu and SuFu
- Gli remains in cytoplasm
In mammals, if Hh is present, ___.
- Hh binds to Ptc
- Smo is activated (no longer supressed)
- Processing of Gli is activated
- Gli is translocated to nucleus
In mammals, the negative feedback regulators in the Hh pathway are ___.
Ptch, Hip, Gli
What genes are for cell proliferation?
- Cyclins D1 and D2 (mammalian) -> mitosis
What are three target proteins?
- Cyclin B - Mitotic P Factor (MPF) - nuclear translocation
- P21 inhibition - tumor supression
- PDGF pathway activation (MAPK) - cell division
What are two types of genetic problems w.r.t. cancer?
- LOF for tumor supression - Ptc and SuFu
- Overespression of oncogenes - Shh and Smo
LOF of SuFu leads to ___.
LOF of Ptc1 leads to ___.
cell nevus carcinoma
Heterozygous Ptc mice ___.
Blocking Smo blocks ___.
binding of Hh
What should be inhibited for cancer treatment?
What are inhibitors of Smo?
- Cyclopamine - binds to Smo, but difficult to synthesize and toxic
- KAAD - modified cyclopamine - less toxic
How do you inhibit Gli?
- Forskolin - PKA activator - used in different pathways as well
- RNAi - Not feasible for treatment
In connective tissue, the main stress-bearing component is the ___.
In epithelial tissue, the ___ form the main stress-bearing component.
cytoskeletons of the cells themselves (linked by anchoring junctions)
What is the purpose of the ECM?
- Provides scaffolding and support for tissues and cells.
- Signal transduction.
What makes up the ECM?
proteoglycans, collagen, laminim, fibronectin, and vitronectin.
"filler" substance. Traps water. Binds cations.
Most abundant ECM component. Connective tissue.
Forms network of weblike structures that resist tensile forces.
Glycoproteins. Maintains cell shape.
Glycoprotein. Promotes cell adhesion and spreading.
What are two principles of tissue formation?
- Cells must be attached to each other.
- Cells must be attached to a scaffold.
Why must cells be attached to each other?
Protein-protein interaction between cells.
Why are cells attached to a scaffold?
- Cells secrete proteins and carbs which make up the ECM.
- Intermembrane proteins connect the ECM with the cell's cytoskeleton.
What types of proteins are used in cell adhesion?
Cadherins, selectins, integrins, Ig family
Tissue specific; dimerize; Ca+2 dependent; regulate cell shape and migration.
Cancer cells also change ___ expression.
Cadherins affect cell shape and migration via ___.
- Cell adhesion molecules that bind to sugars.
- Type of lectin.
In wound-clotting, what is selectin-dependent?
Weak adhesion and rolling.
In wound-clotting, what is integrin-dependent?
- strong adhesion and emigration
- Lets white blood cells exit capillary
What are three kinds of cell junctions?
Describe adhesive junctions.
- Desmosomes and adherens.
- Hold cells together in fixed positions w/in tissues.
- Ca+2 dependent.
What are two types of adhesive junctions?
Desmosomes and adherens (both Ca+2 dependent)
Describe the structure of desmosomes.
- Keratin intermediate filaments connected to plaque.
- Plaque composed of anchor proteins.
- Transmembrane cadherin adhesion proteins attached to plaque.
What makes up the transmembrane cadherin adhesion proteins?
desmoglein and desmocollin.
What proteins make up the plaque?
What is the purpose of the intermediate filaments attached to the plaque?
structural support (not movement)
Describe tight junctions.
- Seal space between cells.
- Prevent flow of molecules and ions thru EC space.
- Important for organs that store liquids.
What proteins make up tight junctions?
claudin and occluding
Describe gap junctions.
- Most common type of junction between animal cells.
- Form open channels between cells allowing ions and small molecules to pass.
- Useful for cell-cell communication.
- Open at low Ca+2 and low pH
What binds to gap junction to open the channel?
calmodulin (also binds to calcium)
What kind of molecule can pass thru a gap junction?
small (e.g. cAMP)
How is the ECM formed?
Secreted by the cells
- Used in cell-cell adhesion.
- Serve as attachment to ECM.
- Bind to specific ECM proteins.
Describe how collagen fibers are formed.
- Procollagen triple-helix formed in ER/Golgi complex.
- Single procollagen molecule out via secretory vessicle.
- Cleavage of propeptides.
- Thousands of collagen molecules form fibril in ECM.
- Aggregation of fibrils form collagen fiber.
What is the purpose of proteoglycans?
- Trap water and provide elasticity (e.g. skin).
- "Filler" substance.
- Hold ECM in place.
Describe structure of proteoglycans.
- 95% carbs by weight.
- glycosaminoglycan (GAG) is main component.
- Single polypeptide with hundreds of GAGs.
What holds the ECM in place?
Linkages of proteoglycans to cell membranes.
What are three types of interactions for proteoglycans?
- Binding to ECM.
- Integrins binding to proeins in ECM.
What are two adhesive glycoproteins?
fibronectin and laminin
What's the main purpose of Fibronectin?
Provides/maintains cell shape.
Describe fibronectin structure.
- Two large polypeptides (not identical) linked by disulfide bridges.
- Some domains bind to ECM.
- Other domains bind to membrane receptors.
How is fibronectin specificity determined?
By the a.a.'s flanking the RGD motif
What are integrins?
- Receptors that mediate attachment between cells and ECM/other cells.
- Critical for growth, hemostasis, and host defense.
- Interact with cytoskelton.
Describe integrin structure.
- Heterodimeric with alpha and beta subunits.
- Variable subunits - mammals have 18 alpha, 8 beta
- e.g. melanoma: alpha-v, beta-3
Describe the integrin receptor.
- Binds to soluble and attached ligands.
- Binding changes conformation of the dimer.
- Binding is Mn+2 dependent.
- Clustering occurs with other integrin receptors upon ligand binding.
What are the two types of integrin activation?
- Outside-in - info from outside to cell
- Inside-out - info from cell to outside
Integrins can bind to the ___, with a ___ of integrins.
same target, cluster
What is anoikis?
Cells cease to be bound to ECM
What are the typical results of integrin signalling?
Cell death, cell migration, cell shape change, cell division
What does integrin clustering do in normal cells?
Affects cell migration and differentiation.
What does integrin clustering do in cancer cells?
- Angiogenesis and metastasis
- Focal adhestion tyrosine kinase -> cell survival
- MAP Kinase -> differentiation, cell growth, apoptosis
What are the 4 stages of the cell cycle?
G1, S, G2, M
At what stage do fully differentiated cells stay arrested?
Where are the checkpoints?
G1/S and G2/M and in the middle of Mitosis
Cells increase in size in the ___ phase.
DNA replication occurs during the ___ phase.
After DNA is synthesized, growth occurs during the ___ phase.
Cell division occurs during the ___ phase.
What is needed to pass a checkpoint?
What are things that might prevent change to S phase?
- Unhealthy cell - e.g. starving.
- Heavily damaged DNA
What might happen if DNA is heavily damaged?
What else is needed to move to the M phase?
- Replicated DNA
- No more than 2 copies of chromosomes
What else is needed to pass from meta to ana?
Chromosomes must be attached to spindles
What are some methods for studying the cell cycle.
- Temp senstive mutants.
- Biochemical experiments with frogs.
- In vitro studies with tissue culture.
- BrdU pulse labeling.
- Flow cytometry.
Describe BrdU pulse labeling.
- BrdU is a thymidine analog.
- Anti-BrdU Abs are used to stain chromosomal DNA
- Provides estimate of duration of each phase
When is BrdU effective?
S and G2 (when chromsomes are duplicated)
How do you know cells are in M phase?
- Look at copies of chromsomes if they're labelled.
- Look for mitotic spindles.
A flow cytometer shows that most cells are in the ___ phase.
What regulates the cell cycle?
- Available space.
- Organ size.
- Proteins (e.g. cyclins)
What are some variations on the cell cycle?
- M -> M
- S -> M
- G1 -> S -> G2 (some plants and insects)
Experiments with fusion between S & G1 and M and G1 indicated what?
Something in S and M activate G1 (G1 is pushed into either of those phases)
Where does the MPF appear to be?
- In the cytoplasm.
- Found by removing cytoplasm from M cell and placing in G1 cell.
___ experiments with frog embryos show that MPF activity is ___.
What does MPF cause?
- Nuclear envelope breakdown.
- Chromosome condensation.
- Spindle fiber formation.
MPF activity is ___ during mitosis and disappears ___.
- at the end of mitosis
Activity of MPF is correlated with the presence of ___.
a protein called cyclin
MPF is composed of ___.
- p34 - CdK - phosphorylates
- p45 - cyclin - activates CdK
Describe the three classes of cyclin.
- G1/S - progression thru start.
- S - stimulate DNA replication.
- M - part of the MPF.
When are cyclins removed?
At the beginning of mitosis.
What cell cycle component is highly conserved?
CdKs, but not CKIs (inhibitors)
How is a CKI removed?
How are the inhibitory phosphates removed?
How is cyclin degraded?
Proteolysis by APC/C and cdc20 and ubiquitin
What regulates cyclin/CdK activity?
Wee1 inactivates CdK by ___.
Adding two inhibitory phosphates.
Cdc25 reactivates CdK by ___.
Removing an inhibitory phosphates.
CdK also has ___ which is added in ___ and removed in ___ and is required for activity of CdK.
- an activating phosphate
___, not ___, activates CdK.
- Cyclin concentration
Figure 17-21 - overview of cell-cycle control system.
Favorable extracellular environment ___.
DNA damage ___.
inhibits G1/S-CdK, S-CdK, M-CdK
Unrepicated DNA ___.
Chromosome unattached to spindle ___.
- Activates S-Phase.
- inhibits DNA re-replication by initiating degradation of Cdc6.
- inhibits DNA re-replication.
- activates M-Phase.
allows passage thru mid-mitotic checkpoint.
Describe cell division activation via mitogen to G1-CdK in animal cells.
- Mitogen binds to mitogen receptor which activates Ras.
- Ras activates MAP kinase pathway.
- Myc is produced which leads to expression of cyclin genes including G1-CdK.
Describe cell division activation from G1-CdK in animal cells.
- G1-CdK inactivates Rb which activates E2F
- E2F leads to S-phase gene transcription including G1/S-cyclin and S-cyclin.
- G1/S-cyclin and S-cyclin lead to active S-CdK which leads to DNA synthesis.
In cell division activation for animal cells, what provides positive feedback?
- E2F provides positive feedback for itself.
- G1/S-CdK and S-CdK further phosphorylate Rb providing pos feedback for E2F.
Rb protein ___ is required for ___.
- cells to enter the S-phase
- Tumor suppressor protein.
- Trans-acting repressor that inhibits transcription of genes for S-phase.
- Becomes deactivated by phosphorylation by G1-CdK.
- Forms heterodimer with E2F protein.
What is Skp2?
What does Rb-E2F do to Skp2?
- Represses transcription.
- Stimulates Skp2 removal at G0 resulting no S-phase.
In mid-late G1-phase, what happens with Rb and Skp2?
- Rb is phosphorylated.
- Skp2 transcription increases.
- Skp2 proteolysis decreases resulting in cell division.
In cells that will divide, what is present at ori sites?
When S-CdK is activated, what happens at ori sites?
- Formation of pre-initiation complex and initiation.
- Replication forks.
When M-CdK is activated, what is the result?
- Chromosome segregation.
At the end of mitosis, what is activated, inactivated, and assembled?
- APC/C activation.
- CdK inactivation.
- Assembly of new pre-replicative complexes at origins.
What proteins are used in replication?
- 1. Gyrase - unwinds supercoils.
- 2. Helicase - unwinds dsDNA.
- 3. Polymerase - adds nucleotides.
- 4. Primase - adds primers (attachment points).
- 5. Ligase
Figure 17-23 - Control of initiation of DNA replication.
Describe the control of the initiation of DNA replication.
- Cdc6 and Cdt1 recruit 6 proteins and form the pre-Replicative Comples (pre-RC).
- S-Cdk stimulates assembly of the pre-initiation complex.
- DNA polymerase et. al are recruited to the origin.
- Mcm protein rings are activated as DNA helicases.
- DNA unwinds.
- Replication begins.
What is geminim?
An APC/C target which inactivates Cdt1.
Describe the processes that prevent re-replication.
- S-Cdk triggers destruction of Cdc6 and inactivation of ORC.
- Cdt1 is inactivated by geminim (an APC/C target).
- Thus, a new pre-RC cannot be formed until end of mitosis.
When is DNA checked for damage and repaired?
- After the pre-replication complex is formed.
- Before S-CdK triggers S-phase.
Polymerase and primase are part of what complex?
The pre-initiation complex.
What do the phosphates do that are attached ORC?
When are the phosphates on the ORC removed?
At the end of M-phase.
What is the first step in replication?
Receiving a signal.
How might replication be prevented?
- Remove receptor of start signal.
- Remove enzymes in signal transduction pathway.
- Remove CdK.
How do differentiation events affect the cell cycle?
- Transcriptional activation of CKIs promotes G0 phase.
- Anaphase promoting complex (APC) and ubiquiting ligase promote G0 arrest.
- Remodeling of the chromatin.
What are examples of differentiation signals that activate CKIs?
Members of the bHLH family including MyoD and Hesl
How does APC promote G0 arrest?
- APC binds to Cdc20 or Cdh1 which activates ubiquiting ligase.
- S cyclins are removed.
- Skp2 is also degraded.
- This results in Rb binding to E2F proteins.
Describe Rb and chromatin remodeling.
- Rb recruits histone deacetylase (HDAC) complex.
- HDAC associates with SWI/SNF ATP-dependent nucleosome remodeling complex.
- Rb binds to enzyme that methylates H3 histones.
- HP1 is recruited and keeps chromatin in repressive state.
What does HDAC do?
Packs DNA together.
What does the SWI/SNF ATP-dependent nucleosome remodeling complex do?
Repackages DNA tightly for activation of replication.
What does HP1 do?
Blocks access to chromatin especially in areas that contain cyclin and promoter genes.
Describe the pathway to inactivate HP1.
There is no such pathway.
What affect does Rb/chromatin remodeling have on differentiated cells?
Those cells can't divide and can't lead to cancer.
What is an easy method to damage DNA?
When DNA is damaged right before cell division, various ___ are recruited that ___.
- protein kinases
- initiate a signaling pathway that causes cell cycle arrest.
What are the first kinases at the site of DNA damage?
ATM or ATR
What is recruited after ATM or ATR?
What do Chk1/2 do?
Phosphorylates p53 preventing Mdm2 binding resulting in CKI p21 production.
What does p21 do?
Binds to G1/S-CdK & S-CdK and inactivates them arresting cell in G1.
Abnormally high levels of Myc cause ___ even if there is no signal for cell division.
activation of Arf.
What does Arf do?
Binds and inhibits Mdm2 thereby increasing P53 levels.
What does stable/active p53 do?
Cell-cycle arrest or apoptosis depending on the cell.
The telomere model of senescence is not as strong as ___.
Altering the insulin and insulin-like pathways results in ___.
- Increase in life span in C. elegans, Drosophila, and mice.
- Keeps mutant animals healthy with less age-related problems.
Effects of altering the insulin and insulin-like pathways are only detected in ___.
in the adult and not during developmental stages.
Animals with the mutant insulin pathway are generally ___.
The Daf-2 mutant was first discovered in ___.
Age-related genes in C. elegans include ___.
- Age-1 - Phosphoinositide-3-kinase (PI3K)
- Daf-2 - insulin receptor
- Daf-10 - forkhead transcription factor
Age-related genes in flies include ___.
- InR - Insulin receptor
- Chico - fruit fly receptor
Age-related genes in mice include ___.
IRS1 - insulin receptor substrate 1
In neurons, replacing ___ and ___ of ___ and ___ worms ___ wt age of the worms.
- age-1, daf-2
- age-1(-/-), daf-2(-/-)
Mutant with age-1(-/-) and daf-2(-/-) have lifespans that ___.
are twice as long.
In the metabolic model, in order to increase lifespan, ___.
- Insulin-signaling needs to be appropriate.
- Amount of insulin in resto of body (w.r.t. neurons) needs to lowered.
Metabolic rate appears to be ___ to lifespan possibly due to ___ and ___.
- inversely proportional.
- Exposure to oxidative stress.
- DNA damage
In fat tissue, ___ knockouts can ___ lifespan.
In flies, overexpressing ___ or ___ can also extend lifespan.
Data suggests that a ___ signal originating from fat tissue and the ___ pathway regulates longevity.
- Insulin/Insulin-like Signaling (IIS)
Why would pathway changes in fat tissue make a difference?
Related to cardiovascular disease
Germline ___ in worms extends lifespan.
Transplantation of ___ into older mice extends their lifespan.
In mice, mutants have lowered rates of ___, ___, and ___.
- cardiac problems
- Alzheimer's disease
There are relatively high numbers of gene variants coding for ___ in ___ species.
In vertebrates, there is/are ___ gene(s) coding for insulin, but ___ receptor variants.
What is the phenotype for IRS-4 knockout?
mild growth defect in males
What is the phenotype for IRS-2 knockout?
- short lifespan, diabetes (insulin resistance) in males.
- longer lifespan if brain-specific
What is the phenotype for IRS-1 knockout?
extended lifespan in females
Basic areas where lifespan extension mechanisms are unknown include ___ and ___.
- Downstream mechanisms (e.g. oxidative stress).
- Multi-gene effects.
What are the four types of cell death?
Describe programmed cell death.
- Requires gene activation of specific genes
- Prevention of auto-immune disease
- Uncontrolled cell death.
- Protein precipitation.
- Leads to inflammation in tissue.
- ATP loss.
- Swelling of cells and organelles (burst).
Describe lysosomal/typeII/autophagic cell death.
- Formation of autophagic vessicles.
- "intermediate" step in some cases.
Cleavage of ___ can ___ apoptotic pathways.
When both pro- and anti-apoptotic signals are present ___.
Stronger signalling "wins"
What are some characteristics of apoptotis/cell death?
- Cell shrinkage
- Membrane blebbing
- Chromatin fragmentation
- Leaky mitochondria (Cyt C)
- Nuclear condensation
- Flipping of phosphatidyl serine to outside.
- Formation of apoptotic bodies.
What are the two basic apoptotic pathways?
- Extrinsic - death receptor
- Intrinsic - mitochondrial
Describe extrinsic pathway.
- Ligand binds to death receptor (dimer).
- Cytoplasmic side binds to FADD
- Procaspase 8/10 is cut to caspase 8/10 (active form).
- Caspase 8/10 activates procaspase 3 -> apoptosis,
- and Bid (which can lead to intrinsic).
Describe intrinsic pathway.
- Caspase 8 cuts BID.
- Bid sends Bax to mitochondial membrane to dimerize with Bak.
- Mitochondia is damaged/dimerized(Bax/Bak) - releases Cyt C
- Cyt C cuts procaspase 9 to caspase 9 (active form).
- Caspase 9 and Apaf-1 bind to from Apoptosome (7-er)
- Apoptosome activates caspase3 -> apoptosis.
- Cut to Flips - survival.
- Cut to FlipL - high concentration apoptotic.
- low concentration survival.
Pathways are dependent on ___.
The BCL2 family including ___, can be ___ or ___.
- Bax, Bak, Bid, etc.
Integrin binding to ECM leads to ___ including what three proteins?
- Survival pathway.
- JNK, AKT, ERK
Pro-caspases are activated by ___.
Study of apoptosis has led to therapies that might be helpful in treating ___.
What is anoikis?
Apoptotic cell death caused when ECM-bound cells detach.
How is anoikis activated?
By integrin receptors.
Is anoikis intrinsic or extrinsic?
Why does anoikis occur?
To remove cells that may threaten structure of multicellular organisms.
The ___ family of proteins can be both ___ and ___.
Give examples of pro-apoptotic proteins.
- Bax, Bak, Mtd
- BH3-only (e.g. Bid, etc)
Give examples of anti-apoptotic proteins.
Pro-apoptotic proteins are usually synthesized in ___ form and are ___ by ___.
In normal cells, ___ is found in the cytosol and ___ is found in the mitochondria.
What might result in the upregulation of BH3-only proteins?
DNA damage, starvation, or cell detachment
When ___ and ___ dimerize in the mitochondrial membrane, ___ is released.
___ binding to the ECM and ___ activate pro-survival signals.
Name three growth factors that activate pro-survival signals.
ERK, JNK, AKT
___ binding between neighboring cells can ___ apoptosis by blocking ___.
- BH3-only proteins
___ is a transcription factor that is present in ___ and is ___-apoptotic.
- every cell type
Detachment from the ECM results in: ___.
- Pro-survival proteins are not activated.
- Inhibition of apoptotic pathways is removed.
- Increased expression of Fas receptors on membrane.
Describe the two classes of cells for the extrinsic pathway.
- Class 1 - cas8 activation is sufficient to activate cas3
- Class 2 - cas8 insuff for cas3. Activates intrinsic pathway - Bid, CytC.
What are two basic strategies for anoikis resistance?
- Stimulation of survival signals.
- Inhibition of pro-apoptotic signals.
What types of cells may have anoikis resistance?
- Cells that migrate in early development.
- Cancer cells.
How do cells acquire anoikis resistance for migration?
- Constitutive active expression of AKT, MEK, ERK, NF-KB, etc.
- Change in integrin expression (e.g. melanoma).
- Expression of ROS in cancer cells.
- Low O2 environ inhibits pro-apop (e.g. cells in middle of tumor).
- Upregulation of Fas inhibitory protein (FLICE).
- Activation of snail, twist, NF-KB (transcription factors for survival).
How do normal cells inhibit anoikis?
- Maintain ECM attachment.
- Maintain cell-cell contact (e.g. lymph node binding).
- Temp release from ECM for migration.
- Non-adherent cells (e.g. lymphocytes).
Describe other receptors s.tu. ECM attach -> integrin receptors ->.
- Epidermal growth factor receptor (EGR).
- Platelet-derived GFR.
- Hepatocyte GFR.
- Vascular endothelial GFR.
How do transiently migratory cells avoid anoikis?
Activation of kinases and the ROS pathway.
How do lymphocytes avoid anoikis?
Pro-survival signals from cytokines (IL-2, IL-7, IL-15).
How do cancer cells avoid anoikis?
- Upgregulation of surival signals.
- Inhibition of pro-apoptotic signals.
- Upregulation of GF/Rs (e.g. hijack other cells to release GFs).
What are two "normal" cell characteristics.
- Stable genome
- Ability to regulate cell div and growth
What are extracellular cues for regulating cell division?
- Growth hormones
- Contact inhibition
What are intracellular cues for regulating cell division?
- Available nutrients/energy
- DNA damaged/need repair?
Describe senescence and crisis in normal cells.
- Two proliferation barriers
- Senescence - viable but won't replicate
- Crisis - loss of telomeres -> cell death
What are basic cancer cell characteristics?
- Unstable genome.
- Autocrine signaling of growth factors.
- Anoikis/apoptosis blocked.
- Use of autophagy in hypoxic environments.
- Lack of contact inhibition.
- Ability to migrate/metastasize.
What causes an unstable genome in cancer cells?
Loss of telomeres, chromsomal breakage, joining of with different chromosomes.
Cancer cells use autophagy until ___.
angiogenesis is activated.
Cancer cell migration/metastasis is enabled by ___.
E-cadherin turned off and expression of N-cadherin.
Most cancer cells that enter the blood stream ___.
Describe senescence and crisis in cancer cells.
- Telomere activity
- - Prevents senescence and crisis
- - Increases breakage-fusion-bridge cycles
- - TERT, telomere subunit, inhibits apoptosis by upping Wnt
Describe the late stages of cancer development.
- - uncontrolled cell div
- - angiogenesis
- - cell migration/metastasis
- - Inhibition of apoptosis/anoikis
What are the 6 hallmarks of cancer?
- Sustaining proliferative signaling
- Evading growth supressors
- Activating invasion and metastasis
- Enabling replicative immortality (telomeres)
- Inducing angiogenesis
- Resisting cell death
In normal cells, what controls proliferative signaling?
Growth factors binding to tyrosine kinase receptors
What is regulated when growth factors bind to tyrosine kinase receptors?
Cell division, growth, survival, metabolism
How are growth factors regulated?
- Sequestration in the ECM.
- ECM-degrading enzymes can activate growth factors.
In cancer cells, what controls proliferative signaling?
- Autocrine signaling (GFs)
- Stimulation of stromal cells (GFs)
- Hyper-responsiveness to GFs
- Ligand-independent GFRs (always on)
- GFR independence (always on downstream)
What are two other mechanisms leading to unregulated cell div?
- Mutations on PI3 kinase.
- Inhibition of neg feedback loops (e.g. Ras)
Describe the triggering of senescence/apoptosis in normal cells.
- High levels of Ras (G-protein)
- High levels of Myc, Raf (transcription factors)
Describe the triggering of senescence in normal cells.
Disabling of Ras, Myc, Raf pathways even at high levels
___ and ___ are tumor suppressor proteins.
RB and P53
RB and p53 have a ___ level of ___.
What does RB respond to?
Signals from outside cell.
What kind of signals does p53 respond to?
- Intracellular signals
- DNA damage
- Metabolic stress
What are two mechanisms of contact inhibition?
- Merlin protein (NF2 gene product)
Describe the merlin protein.
- Strengthens cell adhesion by coupling E-cadherin binding to TKRs.
- Kinase is inactivated in normal cells when in contact.
- Organizes epithelial structure.
- Can suppress Myc's activity.
When do some cancer cells activate autophagy?
- Low nutrient conditions
- Not random.
- Release of IL-1a inducing proliferation.
- Release of pro-inflammation signals.
What induces angiogenesis?
Vascular endothelial growth factor (VEGF)
VEGF in normal cells is ___ and is used in ___.
- Wound healing
- Female reproductive cycle
VEGF in cancer cells is ___ and is used in ___.
- Oncogene signaling
___ can also activate tumor angiogenesis.
Fetal growth factor (FGF)
Describe abnormal blood vessels in tumors.
- Excessive and complex branching.
- Malformed vessels.
- Erratic blood flow/leakiness
- High levels of endothelial apoptosis/cell division.
Describe anti-angiogenic proteins.
- Endostatin, angiostatin, and 12 others.
- Found in healthy mice and humans.
Describe angiogenic promoters.
- Facilitate local invasion.
- Prevent chemo drugs from blocking angio.
- Includes innate immune system.
What immune system components can enhance VEGF?
Macrophages, neuthrophils, mast cells, myeloid precursors
What are two ways to block angiogenesis?
- Anti-VEGF antibodies.
- Soluble VEGFR1 (competitive inhibitor)
What is the only angiogenic factor present thruout tumor's lifecycle?
VEGF is thought to be ___ and unsusceptible to ___.
Metastasis does not occur without ___.
During metastasis, there is a loss of ___.
Describe the metastasis cascade.
- Local invasion (moving, same tissue).
- Intravasation (to blood vessel).
- Transit in blood and lymph vessels.
- Extravasation (exit blood vessel).
- Micrometastasis (still vulnerable).
- Colonization (fairly secure).
What is EMT?
- Mechanism used by embryonic cells to migrate.
What happens during EMT?
- Cells change shape from epithelial to fibroblastic.
- Secrete ECM-degrading enzymes.
- Repress E-cadherin.
- Express N-cadherin.
What activates cell migration?
Twist1 -> RAC1
How do stromal cells aid metastasis?
Mesenchymal stem cells release CCL5/RANTES to stimulate invasion.
Why do cancer cells secrete chemoattractants?
Attract macrophages to increase inflammation.
The release of IL-4 by cancer cells activates ___.
macrophages to release ECM-degrading enzymes.
Cancer cells can also degrade ___.
What are three types of invasion?
- Collective invasion
What are the two steps of colonization?
- Movement of cancer cells to distant tissues.
- Adaptation to other tissue microenvironments.
What are the emergin hallmarks of cancer?
- Avoiding immune destruction.
- Tumor-promoting inflammation.
- Deregulating cellular energetics (glycolysis).
- Genome instability and mutation.
Describe genome instability.
- Abnormal chromosomal rearrangements (B and T cells).
- Mutation rates increase.
- Increased sensitivity to mutagens.
- Inhibition of DNA repair mechanisms.
Describe an unusual metabolic change with some cancer cells.
Switch to glycolysis even in the presence of oxygen.
Some cancer cells secrete TGF-B that ___.
inactivates CTL and Natrual Killer (NK) cells.
What contributes to tumor-promoting inflammation?
- Survival factors
- Pro-angiogenic factors
- EMT-activating signals
- ECM-degrading enzymes