-
What makes up the cell membrane?
- Phospolipid bilayer
- Proteins
- Cholesterol (animals)
-
What kinds of proteins make up the cell membrane?
- Peripheral
- 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
- Shape
- Receive stimuli
- Site of ECM attachment
-
Where does glycoslyation occur, and what is the destination?
- Golgi
- 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?
Via vesicle
-
Do proteins for organelles get glycosylated? Why or why not?
- No.
- 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?
20 a.a.'s
-
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
- Channel/gate
- 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?
Aquaporin
-
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?
Increases fluidity
-
What does an increase in temp do?
Increases fluidity
-
What does an increase in saturation do?
Decreases fluidity
-
What does an increase in hydrocarbon tail length do?
Decreases fluidity
-
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
- Metabolism
- Movement
-
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?
Endocrine
-
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?
- Survive
- Grow + Divide
- Differentiate
- Die
-
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 ___.
transcription
-
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 ___.
- longer-lasting
- their duration in the blood
-
Non-soluble signals enter the cytoplasm by ___.
diffusion
-
Non-soluble receptor responses include ___.
- Early - first 30 min
- Delyated
- Depends on type of protein and timing of synthesis
-
Gene expression regulation falls into two general categories:
-
All nuclear receptors bind as either ___ or ___.
Homodimers, heterodimers
-
An inactive receptor protein is bound to ___ proteins.
Inhibitory
-
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?
cAMP, cGMP
-
What second messengers are derived from phospholipids
DAG, IP3
-
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?
No.
-
What are 4 classic downstream targets of G Proteins that regulate different effectors?
- Adenylyl cyclases
- Phospholipases
- 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?
Different isoforms
-
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?
Cell division
-
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 ___.
Delta, ligands
-
Notch is composed of ___.
- NECD - Notch extracellular domain
- transmembrane domain
- NID - Notch intracellular domain
-
___ cleaves the NECD from the ___.
ADAM TM-NICD
-
What is ADAM?
A desintegrin and metalloprotease
-
The NICD makes its way to the ___ and generally results in ___.
- nucleus
- transcription, cell division, differentiation
-
Hh are ___, a ___.
- ligands
- family of secreted proteins
-
Hh generally functions in ___.
development
-
In adult cells Hh can lead to ___.
cancer
-
Hh homologues in vertebrates include ___.
sonic, desert, Indian Hh
-
What is the receptor for Hh?
Patch
-
What are the receptors for Hh in mammals?
-
Besides Patch and Hip, what else is needed for Hh pathway activation?
Smo
-
The ultimate target of Hh in the fruitfly is ___, a ___.
- cubitus interruptus (Ci)
- transcription factor
-
What vertebrate homologue most closely resembles Drosophila Hh?
Desert
-
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?
- Holoprosencephaly
- Greig cephalopolysyndactyly syndrome
- Pallister-Hall Syndrome
- Gorlin's syndrome
-
What are some cancers triggered by malfunctioning Hh?
- Basal cell carcinoma
- Rhabomyosarcoma
- Medulloblastoma
- 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)
-
Describe Patch.
- 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 ___.
-
Describe Smoothened.
Intermembrane protein that when activated relays signals to HSC
-
In vertebrates, Smo is always ___.
at the cell membrane
-
Describe HSC.
- 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 ___.
truncated
-
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?
- Autocleavage
- 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 ___.
- 155
- Weak activator - Ci^act
- Strong activator - Ci*
-
If no Hh is present, Ci__ is formed.
75
-
How is Ci phosphorylated?
- PKA
- Glycogen synthase Kinase 3-beta
- Casein Kinase 1-gamma
-
Upon phosphorylation, Ci is ___ by ___, a ___.
- ubiquitinated
- 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
- cMyc
-
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 ___.
medulloblastoma
-
LOF of Ptc1 leads to ___.
cell nevus carcinoma
-
Heterozygous Ptc mice ___.
develop tumors
-
Blocking Smo blocks ___.
binding of Hh
-
What should be inhibited for cancer treatment?
Smo, Gli
-
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 ___.
ECM
-
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.
-
Describe proteoglycans?
"filler" substance. Traps water. Binds cations.
-
Describe collagen?
Most abundant ECM component. Connective tissue.
-
Describe laminin?
Forms network of weblike structures that resist tensile forces.
-
Describe fibronectin?
Glycoproteins. Maintains cell shape.
-
Describe vitronectin.
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
-
Describe cadherins.
Tissue specific; dimerize; Ca+2 dependent; regulate cell shape and migration.
-
Cancer cells also change ___ expression.
cadherin
-
Cadherins affect cell shape and migration via ___.
Indirect binding
-
Describe selectins.
- 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?
- desmoplakin
- plakoglobin
- plakophilin
-
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
-
Describe integrins.
- 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?
- Receptors.
- 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?
G0
-
Where are the checkpoints?
G1/S and G2/M and in the middle of Mitosis
-
Cells increase in size in the ___ phase.
G1
-
DNA replication occurs during the ___ phase.
S (synthesis)
-
After DNA is synthesized, growth occurs during the ___ phase.
G2
-
Cell division occurs during the ___ phase.
M
-
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?
- Health
- 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.
G1
-
What regulates the cell cycle?
- Available space.
- Organ size.
- Signals/hormones.
- 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 ___.
- high
- 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?
Ubiquination
-
How are the inhibitory phosphates removed?
Cdc25
-
How is cyclin degraded?
Proteolysis by APC/C and cdc20 and ubiquitin
-
What regulates cyclin/CdK activity?
phosphorylation/dephosphorylation
-
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
- G2
- mitosis
-
___, not ___, activates CdK.
- Phosphorylation
- Cyclin concentration
-
Memorize:
Figure 17-21 - overview of cell-cycle control system.
-
Favorable extracellular environment ___.
activates G1-CdK
-
DNA damage ___.
inhibits G1/S-CdK, S-CdK, M-CdK
-
Unrepicated DNA ___.
inhibits M-CdK
-
Chromosome unattached to spindle ___.
inhibits APC/C
-
S-CdK ___.
- Activates S-Phase.
- inhibits DNA re-replication by initiating degradation of Cdc6.
-
M-CdK ___.
- inhibits DNA re-replication.
- activates M-Phase.
-
APC/C ___.
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.
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Rb protein ___ is required for ___.
- inactivation
- cells to enter the S-phase
-
Describe Rb.
- 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?
Ubiquitin ligase.
-
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?
pre-replicative complexes.
-
When S-CdK is activated, what happens at ori sites?
- Formation of pre-initiation complex and initiation.
- Replication forks.
- Elongation.
-
When M-CdK is activated, what is the result?
- Chromosome segregation.
- Mitosis.
-
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
-
Memorize:
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?
Prevent replication.
-
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?
Water
-
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?
Chk1/Chk2
-
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 ___.
metabolic pathway
-
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 ___.
Smaller
-
The Daf-2 mutant was first discovered in ___.
C. elegans
-
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(-/-)
- rescues
-
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.
- secondary
- Insulin/Insulin-like Signaling (IIS)
-
Why would pathway changes in fat tissue make a difference?
Related to cardiovascular disease
-
Germline ___ in worms extends lifespan.
ablation
-
Transplantation of ___ into older mice extends their lifespan.
young ovaries
-
In mice, mutants have lowered rates of ___, ___, and ___.
- cancer
- 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.
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