1
Q
What are the basic components of the cytoskeleton?
A
Actin Filaments - two stranded polymer
Microtubules - alpha and beta tubulin
Intermediate filaments - family of related proteins
2
Q
What is the general function of the cytoskeleton?
A
It is involved in a variety of celll processes such as muscular movement and transport of molecules within a cell
3
Q
What are the three groups of actin isoforms?
A
Aplha actin - contractile structures
Beta actin - cell cortex and motile cells
gamma actin - stress fibres
4
Q
Describe actin filaments
A
They are a globular monomer that have a polarity and act as an ATPase. They also contain magnesium ions.
5
Q
How do cells use actin?
A
They use them in structural roles and also use their contractile mechanisms to move vesicles around a cell
6
Q
Describe the structure of G-actin
A
It has two lobes and a deep cleft where the ATPase fold resides
7
Q
Describe the structure of F-actin
A
Helical structure that is polarised with the ATP biinding cleft at the (-) end
8
Q
What do actin microfilaments need to polymerise?
A
The presence of a nucleus made up of 3 combined actin microfilaments.
For the nucleus to form a mimimum concentration of actin monomers must be reached known as the critical concentration.
nucleating proteins such as the formin protein family and arp2/3 nucleate actin under signal transduction pathways
9
Q
What are the actin binding proteins?
A
Profilin binds ATP-actin to the + end
Cofilin binds ADP-actin to the - end
Thymosin beta-4 prevents binding of actin to either end
10
Q
What are the two actin cappin proteins?
A
CapZ for the + end
Tropomodulin for the - end
11
Q
What do formins do?
A
They can form FH2 domains on the + end of actin filaments to prevent capping
They also form FH1 domains adjecent to recruit profilin-ATP-G-actin complexes to the + end
12
Q
What do ARP 2/3 complexes do?
A
they assemble branched filaments with a 70 degree angle but is a poor nucleator unless activated by a nucleation promoting factor (NPF)
13
Q
Examples of F-acting crosslinking proteins (not as important)
A
Fimbrin
Filamin
14
Q
What does dystrophin do?
A
links muscle actin network to membrane protein complex of cells
15
Q
What is Duchenne muscular dystrophy?
A
Genetic disorder that revents dystophin production.
16
Q
what is immunolabelling?
A
A sample is prepared and a primary antibody is washed over and unattatched antibodies are washed away
then flourecent antbodies that attatch to the primary antibody are attatched in the same way so the presence of the primary antibody can be observed
17
Q
What is confocal microscope used for?
A
It allows optical sectioning of a thick sample without physical sectioning (cutting)
18
Q
What is advanced light microscopy?
A
light microscopy that allows observation of living transparent cells via phase contrast and differential interference contrast
19
Q
What are the two families of microtuble based proteins and which direction do they each move cargo?
A
Kinesins move towards (+) end
Dyneins move towards (-) end
20
Q
What do the different domains of kinesin do and what are they?
A
Head domain binds ATP and microtubles
Linker domain is critical for forward movement
Stalk domain involved in dimerisation
Tail domain binds to receptors on vesicles containing cargo
21
Q
How does kinesin-1 use atp to walk down a microtubule?
A
The leading and trailing heads take turns using ATP to swing the other ahead of itself and binding the furthest one before repeating
22
Q
Are the kinesin protein family or dynein protein family more diverse?
A
The kinesin is more diverse
22
Q
What is the structure of cytoplasmic dynein?
A
Stem attatched to linker region attatched to two heads that have a stalk on the end of which is the microtubule binding site
23
Q
How does dynein walk across the microtubule?
A
Dynein head binds to ATP causing a movement which detatches the other half allowing twisting movement that can be repeated.
24
What types of kinesin are there?
Kinesis 1 -identical light and heavy chains
Kinesis 2 -two different heavy chains and third polypeptide to bind cargo to tail
Kinesin 5 - 4 heavy chains with heads on both sides
Kinesin 13 - no motor activity but enhance deoplymerisation of microtubules
25
How is ciliary and flagellar bending mediated?
Axonemal dynenin attatched to the A tubule and B tubule in these components are responsile for the bending of the tubules due to them being bound from moving by nexin
26
What are intermediate filaments?
A filament that is intermediate in diameter (10nm) between actin micro filaments and myosin thick filaments.
That create polymers like nails and hair with great tensile strength and are not intrinsically polar
27
What are the 5 classes of intermediate filaments?
Acidic keratins
Basica keratins
Desmin (muscle)
neurofilaments
Lamins (nucleus)
28
What is the difference between hard and soft keratins?
Hard keratins such as hair and nails are rich in cysteine and become oxidized to form disulfide bridges making them strong
Soft keratins such as skin cells associate with desmosomes linking adjacent cells together.
29
What is epidermolysis bullosa simplex?
A rare disorder where K14 keratin cant form protofilaments and basal cells are easily damage causing skin to delaminate and blister.
30
What does the nuclear lamin mesh do?
it maintains the integrity of the nuelcus and Lamin A wich makes a part of it is regulated to deform nuclei when needed such as in neutrophils
31
What is the structure of myosin
They have a consistent head made of heavychains with an actin inding site connected to light chains (essential and regulatory) and then a variable tail made of heavy chains
32
How many types and classes of myosin in humans?
40 different myosins ad 20 classes
33
What are 3 common classes of myosin and their functions?
Class 1 - single head domain
Class 2 - 2 head and 2 light chains assemble into bipolar filaments
Class 5 - 2 head 6 light chains per neck and transport organelle with specific receptors
34
How does myosin move?
ATP binds to it causing a conformational change (amplified by neck domain) as it is hydrolysed. Then it binds to actin and when Pi is released it moves along the actin and repeats
35
How does neck domain length relate to myosin step size?
The longer the neck domain size the larger the step size
36
How does step size relate to myosin 5 walking?
The step size is double due to having 2 heads moving hand-over-hand
37
What are the states of myosin 5
Active where it is bound to cargo
Inactive where the tail domain inhibits activity
38
What holds myosin and actin filaments in place in muscle sarcomere?
Titin holds myosin filaments in place
Nebulin holds actin filaments in place
39
How does an increase in Ca2+ cause a contraction
Causes comformational change in tropomoysin and troponin that expose actin binding sites
40
What actin myosin contractile bundles are there besides mucles
Adherens belt in epithelial cells
Stress fibres in migrating cells
Contractile ring in cell division
41
How is myosin light chains regulated in smooth muscle contaction?
It is done by phospohrylation at certain levels of calcium ions.
Low Ca2+ causes relaxation and vice versa in smooth muscle
42
What is the nernst potential?
The transmemrane voltage where transmembrane ion movement is at equilibrium
43
How does membrane voltage come about?
Selective permeability and ion concentration gradients create it
44
What classes of transport system are there?
Primary pumps (use primary energy source such as ATP) and are electrogenic.
Carriers either symporters or antiporters or facilitators (facilitated diffusion)
Channels (passive electochemical gradients)
45
What are animal plasma membranes centered around?
Na+ ions are controlled with pumps carriers and channels along with Cl- channels for osmoregulation and K+ for membrane voltage
46
What are plant and fungal plasma membranes centered around
Centered around H+ pumps and carriers using H+ as a driver for nurtients and sodium
47
What are bacterial plasma membranes centered around?
Mainly pumping H+ out but also a hint of Na+
48
What is the relative turnover for pumps carriers and channels?
Pumps have lowest 10^2
Carriers middle 10^3
Channels most 10^8
49
What are the 3 groups of ion channels and what do they do?
Voltage-gated - detect a change in voltage and open/close to ions allowing them to reach equilibrium
Neurotransmitter gated channels - receptors on the channel for neurotransmitters open/close/inhibit the channel when the chemical signal is recieved.
Second messenger-gated - these channels bind chemicals (second messengers) in the cytosol. such as cyclic nucleotide-gated channels like cAMP and cGMP in the retinal and olfactory (eyes and smells into action potential)
50
What is the basic strucute if voltage-gated channels?
6TMS domains (S1-6)
1 Pore domain (pore open is the activates state and if it is closed or blocked then it is deactivated) Changes in the P domain conserved amino acid sequence determines what type of ion passes through
S4 is a voltage-sensor
Long N terminus forms ball and chain involved in inactivation
51
What is the mechanism of ion selectivity and transport in K+ and Na+ channels?
Ions in water are dehydrated before entering water by replacing the water in oxygen with carbonyl groups on the amino acid allowing for easy transport.
K+ channels:
Smaller ions such as sodium are more charge dense and therefore require energy to dehydrate and move through and that energy isn't there
Larger ions are unable to fit through the pore so only K+ ions can make it through
Na+ channels:
Na+ passes through partially hydrated as energy of total dehydration is too great
K+ is too large to get close to allow dehydration and therefore can't move through
52
How do voltage gated ion channels sense a change in voltage?
The S4 domain has a helix with arginine residues that moves in response to changes in voltage forcing confomational changes in the shape opening the gate.
53
What is the ball and chain model in voltage gated channels?
The N terminal residues swing around and block the pore inactivating the channel in response to (ion channel lecture 6)
54
What different types of ATP powered pums are there?
P-type ATPases
ABC superfamily
V-type proton pumps
F-type proton pumps
55
What are P-type ATPases and what do they do?
They are pumps for cations made of large alpha catalytic subunits
They are all inhibited by micromolar orthovanadate
atp donates gamma-phoshpate to conserved aspartate during atp hydrolysis
They have a conserved structure across different functions suggesting conserved transport mechanism
Resonsible for housekeeping functions such as maintaining K+ and Na+ electrochemical gradients across plasma membranes. (inhibited by ouabain)
also responsible for housekeeping in plant and fungal cells regulating H+ levels
adittionally responsible for Ca2+ levels in sarcoplasmic reticulum and plasma membrane across animals plants and fungi
Finally in the membrane of gastric epithelium cells and secretes H+ (electroneutral due to taking in K+ simultaneously)
56
What is the structure of P-type ATPases?
N terminus with 4 TMS respulting in 4 stalk regions
C terminus with 6 TMS and a 5th stalk
majority of the pump is in the cytoplasm
Regions B C and J
B induces confomational changes in ion binding sites
C phosphorylates nucleotide
J is the hinge to allow cytosolic regions to interact
57
What is the E1-E2 model of P-type pump-mediated ion transport?
Makes confomational changes between E1 state with binding sites on one side and E2 state with them on the other.
this change induces changes in cation binding allowing to bind ions at low concentration and sissociate them at high concentration.
58
What are CPx pumps and what do they do?
They tansport toxic and nutrient metals.
If they misfunction diseases such as menkes (copper deficiency) and wilsons (copper accumulation in liver) diseases occur
quite similar to P-type pumps with the main differences being a lomger N terminus, shorter C terminus and a CPx motif
The similar structures suggest similar mechanisms of transport and suggest E1-E2 transport
59
What are V-type pumps and what do they do?
They almost exclusively operate as H+ ATPases on intracellular membrane in eukaryotes
They undergo rotational catalysis which rotates c subunits which contain protons.
Main point is it is a completely different mechanism to E1-E2
60
What are ABC transporters?
ATP Binding Cassette transporters transport a wide variety of solutes in or out of cells.
They are of high clinical importance due to them affecting cystic fibrosis and MDR transporters (pathogen/cancer drug resistance)
They use the flippase model in membrranes to solutes disolved in the leaflet of plasma membrane to the other side of the membrane
61
What are the 4 main steps of cell signalling?
1) signal recieved from the extracellular mediator
2) Perception where a cell surface-receptor is activated by the signal molecule
3) Transduction where the signal is passed along signal transduction proteins and second messnegers
4) response which is either in the cytoplasm modifuing metabolism, function or movement OR nuclear modification of gene expression or development
62
What are the 3 main types of signalling?
Endocrine - long range blood hormones
Paracrine - short range among adjacent cells
Autocrine - same cell (signals still leave cell)
63
What are receptors involved in signal transduction?
G protein coupled receptors - short-term changes in cell function (metabolism, movement)
Enzyme-coupled receptors with INTRINSIC enzyme activity - receptors like kinases (they use phosphorylated regions for energy to pass on the singal down the pathway)
Enzyme-coupled receptors ASSOCIATED with an enzyme like cytokine receptors that are associated with kinases rather than being them
Ion channel receptors (ligand-gated ion channels) - e.g Transient receptor potential channels (cation channels)
64
What is the criteria for a second messenger?
1) must be a small molecule
2) Must have a mechanism for inducing rapted concentration alterations
3) controlled by extracellular stimuli
4) must regulate enzyme activity or protein function
5) must involve highly specific interactions
6) must have amplification in the system
65
What are some examples of second mesengers?
Cyclic nucleotides like cAMP (activates protein kinase A (PKA) to breakdown lipids and synthesise glucose) and cGMP (activates PKG to open cation channels in rod cells)
Lipid-derived messangers like DAG (1,2Diacylglycerol) which activates PKC which decreases glucose synthesis and increases transcription AND IP3 which opens Ca2+ channels in ER which are also second messengers
66
What is amplification in cell signalling?
Its where a small signal is multiplied (like a pyramid scheme) as it travels through the transduction pathway to the response.
67
How is the G-protein coupled receptor bound to the membrane?
At the C4 end, H8 is covalently bound to palmitic acid which is embedded in the cell membrane
68
What are the 5 peincipal groups of G protein-coupled receptors in humans?
Rhodopsin (659)
Secretin (15)
Glutamate (22) - perception of ligand is done by the Venus fly trap (VFD) domains
Adhesion (33)
Frizzled (11)
69
What is epinephrine and what is its pathway?
Epinephrine is the "fight or flight" hormone that stimulates glycogen breakdown and release of fatty acid.
It is detected by beta-adrenegernic receptor which is a G-protein coupled receptor in a barrel shape in the membrane.
Epinephrine interacts with 15 amino acids on 4 transmembrane alpha helices
This passes a signal onto a heterotrimeric G-protein which is bound to GDP/GTP depending on 'activation' state and this also serves as a timer for how long GTP lasts. (GTP replaces GDP it is not phosphorylated)
This GTP bound G-protein (active) activates adenylate cyclase which has 2 catalytic domains that are activated upon G-protein binding allowing it to convert ATP to cyclic AMP (cAMP)
4 cAMP activate protein kinase a by binding to cyclic nucleotide-binding domains which cause the regulatory dommains to detach from the binding sites activating the kinase.
70
What are methods of signal termination from G-protein coupled receptors?
1) dissociation as ligand binding reactions can be reversible.
2) G-protein coupled receptor kinases phosphorylates the receptors tail allowing for the binding of arrestin which removes the receptor via endocytosis.
A seperate hormone receptor can send an inhibitory G protein to inhibit the assoicated enzyme
71
What are receptor tyrosine kinases?
Very large family of kinases involved in:
Insulin receptors
Vascular endothelial growth factor receptors
Platelet derived growth factor receptors
Epidermal growth factor receptor/
They possess intrinsinc tyrosine protein kinase activity.
72
How does the activation of the tyrosine kinase occur?
Diamerization of the receptor with the ligand binding said ligand causes the TK to hydrolyse ATP which phosphorylates the tyrosine side chain activating the TK
TK phosphorylates additional tyrosine residues (that don't phosphorylate further) to the RTK cytoplasmic domain creating binding sites for more signalling proteins
73
Describe the role of Ras as a molecular switch
Ras is a G protein (small monomeric G protein with one subunit)
If GDP is bound then Ras in inactive and all singnalling downstream of Ras is incactive
If GDP is exchanged for GTP then it becomes active and allows for the the rest of the signalling pathway to take place
74
Outline the molecular mechanisms of Ras-Raf-MAPK signalling
75
What is the pathway of Ras activation?
(Outline the molecular mechanisms of Ras-Raf-MAPK signalling)
Autophosphorylation of RTK creates a binding site for SH2 domain via GRB2
GRB2 contains SH3 domain which binds to Sos allowing for Sos to bind to Ras and this allows Ras to exchange GDP for GTP and vice versa by causing a conformational change to open the GDP binding site.
Active Ras dissociates from Sos and activates downstream signalling
76
How does Ras activate Ras?
Ras.GTP binds to N-terminal regulatory domain of Raf
This dephosphorylates one of two serines that bind Raf to 14-3-3 releasing Raf
Hydrolysis of Ras (GTP to GDP) release Raf from Ras
Raf is fully acitvated by dimerization and phosphorylation of serine/threonin residues
77
How is the Ras-Raf-MAPK pathway an example of enzyme cascades in signal transduction?
After Raf is activated it activates MEK (via phosphorylation) which in turn activates (via phosphorylation) MAPK (mitogen activated protein kinase)
Essentially its a 3 long phosphorylation cascade (kinases of kinases of kinases)
Active MAPK phosphorylates PK p90 which both migrate to the nucleus and activate TCF (Ternary complex factor) and SRF (serum response factor) which bind to SRE (serum response element)
78
What is the difference between the cell cycle in normal animal cells and budding yeast?
Budding yeast forms a bud in G1 phase that expands through the cell cycle and can be used to identify the stages through growth and DNA replication.
79
What are the cell fusion experiments?
Cells in different phases of cell division are fused and the characteristics (phase) of the cell is obsereved.
E.g M phase fused with any other phase of the cell cycle (besides G0) results in the fused nuclei moving to M phase. Thus M promoting factor affects all these phases.
E.g 2 S phase fused with G1 triggers S phase in the G1 nuclei but S phase does nothing to G2. Thus S promoting factor does nothing to G2 phase nuclei
80
Describe the oocyte maturation process?
G2-arrested oocytes mature in virto in the presence of progesterone and are arrested in meiois 2 (metaphase)
MPF is responsible for arresting the egg in metaphase 2 and wont progess until MPF levels drop afterwards fertilization can take place
81
what is the experimental evidence that lead to the defintion and charactirzation of MPF
Sea urchins were used to identify proteins using radioactive isotopes. This showed cyclin rising and falling soon after fetilization.
Frog oocytes were tested with sperm chromatin which identified that sperm chromatin allows the oocytes to undergo mitosis in vitro
Cyclin B levels were found to parallel MPF activity (MPF component) and RNase treated extracts were found to no longer undergo mitosis. After further testing it was identified that deleting a portion of the cyclin B mRNA (ubiquitin ligase binding site) would prevent degredation. and when this cyclin B was added the cell remained stuck in mitotic arrest (DNA was still uncondensed and tubulin was still present)
82
83
84
How are fission yeast cells used to identify cell cycle arrests differently to mamalian cells?
The stage of division is directly proportional to the length of the fission yeast cell so the length can be used to identify arrests.
85
How was it found that cdc28 homologues were conserved across yeast species? And how does this relate to humans?
Ts mutant budding yeast which were only able to grow at 25 degrees were able to grow at 35 with cdc28 added and Ts mutant fission yeast that could only grow at 25 degrees was also able to grow at 35 with cdc28 added
This propmpted the question of if these factors were also conserved in higher eukaryotes and it was found that cdk1 is structually and functionally conserved in eukaryotes.
cdk1 is esentially the same as Cdc28 and Cdc2 so it can be said that they are preserved between yeast and humans
86
How do cdc regulation affect cell division in fission yeast?
Optimal ammounts have standard cell lengths at division
Defficiency results in increased cell growth without mitosis and looooong cells
over expression results in cells enterin gmitosis inappropriately with insucficient cell growth
87
How is the G2/M transition regulated?
It is regulated by the phosphorylation of MPF (phosphorylated cdc2 (cdk1))
It requires the inhibitory phosphorylation (inhibited by Wee1) at Y15 to be removed for full activity
Inhibitory phosphorylation is removed by cdc25 activating CDK which enhances cdc25 which ultimately inactivates Wee1
Excess Wee1/ Cdc25 deficit prevents mitosis (long yeast)
Excess Cdc25 / Wee1 deficit results in inapropriate mitosis (wee cells)
balanced levels ensure regulated mitosis
88
What regulates the cell cycle transitions?
CDKs regulate the cell cycle transition and each cyclin is expressed for a particular phase of the cycle.
Cyclins accumulate in response to growth factors (mitogens) and transcriptional activation
They are degraded after their function is complete.
89
How does CDK regulation happen in the G1/S phase?
Mitogen activates g1-cdk which results in the phosphorylation of Rb protein, freeing the bound E2F protein which stimulates G1/S-cdk in a positive feedback loop
90
How are CDKs regulated?
They are regulated by CDK activating kinases (CAKs)
CDK are catalytically inactive without them being phosphorylated on their T loop by CAK
They are also regulated by CDKI (CDK inhibitors) which bind to and inhibit CDK by mimicing a substrate without being able to be phosphorylated. (preventing S phase entry)
91
How does CDK regulate DNA regulation?
CDK levels increase throughout the cell cycle and are only degraded in mitosis.
There is a licensing phase before S phase that is neccessary for replication
After this point at a higher CDK conc licencing is inhibited so that new DNA can't be licensed.
There is a permissive CDK range where DNA replication can take place that is in the inhibited licensing range.
92
What is ORC and what does it do in DNA?
ORC/Origin recgnition complex associates with DNA in mitosis interacts with Cdc6 and Cdt1 and allows helicase to be recruited (DNA repliaction licensing)
Only functional at low CDK conc as high conc results in it being phosphorylated and dissociating from Cdc6 and Cdt1
MCM complexes are rectuired to origins (2 per origin) when CDK increases late G1 which unwind the helix and allow replication.
MCM is activated by CDK and DDK as well
93
How is ubiquitinylation carried out and why?
Via three coupled enzyme complxes
E1 is the ubiquitin activating enzyme which Ub is conjugated to (ATP used)
E2 conjuating enzyme tranfers Ub to E2
E3: E2 binds to E3 along with the target protein (substrate) and Ub is transfered to the substrate.
Polyubiquitylated substrates are released and degraded by the proteasome
94
How is the G1/S transition regulated by switch from APCchd1 to SCF?
APC/Ccdh1 maintains low CDK activiy in G1 but when CDK increases (due to degredation of p27 the CDK inhibitor) it promotes SCF which degrades APC/CCdh1(mutually degrading)
Cdh1 removal is required to exit G1 as it degrades cyclins when in the APC-Cdh1 complex
Lecture 16 slide 13 summary image
95
How is MPF activated in G2 to initiate mitosis?
MPF is formed by Cyclin B and Cdk1 binding together.
MPF activates cyclin-degrading enzymes
CAK activates MPF via phoshporylation whereas Wee1 inhibits it by phosphorylation
Active MPF has positive feedback for Cdc25 and Wee1
MPF targets factors that ensure chromosome adhesion and inactivates cohesin via APC activation and degredation of Secruin for Separase activation so that sister chromosomes can be seperated.
APC then is responsible for MPF degredation in the return to G1
96
What are some common model organisms for developmental biology?
Nematode - caenorhabditis elegans (comprehensive connectivity maps with 302 neurons and ~7000 synapses)
Fruit fly - drosophilia melanogaster (small genome)
Zerbrafish - Danio rerio (small robust, cheap and transparent)
African clawed toad - Xenopus laevis (large oocytes)
Mouse - Mus usculus (similar to human genome and easy to genetically modify + cost efficient)
Chick - gallus gallus domsticus (windowing eggs to image development)
97
What is the general picture of early embryonic development
Cleavage - early cell division in embryo
Split into cleavage 1 and 2 which are perpendicular and equal holoblastic and 3 which is perpendiculat but unequal holoblastic
Gastrulation (movements to produce gut and primary germ layers)
98
Name the 3 primary gemr layers and what structures they form
Endoderm - gut, liver, lungs
Mesoderm - skeleton, kidney, muscle, heart and blood
Ectoderm - skin, nervous system
99
What is induction in development?
Induction is essential cell fates detemined by signals from other cells.
It can be form direct cell-cell contact or diffused over distance.
Induction can be used to create new ectoderm on the eye to induce lens formation seperate to the primative brain
100
What is a morphogen and morphogen gradient
A morphogen is a molecule that diffuses across a tissue which affects cell fate
It mainstains a seady gradient using a morphogen sink where active degredation takes place so it doesn't saturate the tissue.
The gradient can determine which side is the posterior and which is anterior such that if you add morphogen to the opposite side to where it is normally produced you will create a mirror image on the organism (e.g two thumbs one hand)
101
How does two-way induction take place in kindey developement?
Mesenchyme (morphogen) induces bud branching in the kidney epithelium and in the buds (also because of the buds) there is mesenchyme-epithelium transition to form the tubules.
(One way) Mesencyme ssecretes mesenchyme and is recieved by GDNFR and Ret on the bud as if either of these genes are knocked out the kidneys do not form.
(two way) The bud releases what is thought to be LIF which activates the mesenchyme to form tubules.
102
What is the TGF-Beta family?
Very large family of proteins that are important for regulating cell division, altering growth factor synthesis and induction and cell specification.
103
How are mature TGF-beta molecules produced?
They are secreted as inactive preursors and processed in the ER/Golgi and must dimerize to activate
They form a cystine know which is a stable and compact structure (lots of disulphide bonds)
104
What is the TGF-beta signalling pathway?
TGF-B dimer binds to receptor
Receptor phosphorylates the domain of type 1 receptor
Kinase activates
R-Smad/Co-Smad dimer forms and enters nucleus
Smad dimer forms complex with DNA-binding protein which binds near target genes to activate transcription.
105
How is TGF-Beta specificty achieved?
Various receptors, R-Smads and co-Smads
106
Describe mesoderm formation in xenopus
Mesoderm forms from the late blastula after Co-culture induces mesoderm from cap calles. Also signal from vegetal cells to induce mesoderm formation (this is because multiple cell types are being formed so multiple signals are needed)
Mesoderm differentiates from marginal zone cells
107
What is the evidence for existence of dorsal vegetal signal (Nieuwkoob centre), ventral vegetal signal and dorsal magrinal zone (organiser) singal in xenopus?
The blasopore is formed at the Nieukwoob centre and it induces cells in the late blastula to form the spemann organizer
The evidence for this Is that the transplantation of a new organiser induces a new axis for growth.
108
Describe neurulation
The dorsal region becomes the neural plate and neural folds develop which fuse forming the neural tube.
The brain froms at the anterior and the rest forms the spinal chord
The neural crest forms above and different cell types become specialised and migrate from here around the embryo.
109
What are the three signals of TGF-Beta family proteins?
Nodal-related signal:
Induces mesoderm
Dorsal mesoderm (high)
Ventral mesoderm (low)
BMP4 induces ventral mesoderm formation and is antagonised by Chordin
Xolloid cleaves chordin allowing BMP4 to bind to it's receptors (antagonist antagonist)
110
What is the process of amphibian axis formation?
Sperm cayses cortical rotation and specifies ventral and dorsal quadrants
Niewkoop forms in dorsal vegetal quadrant
Nodal-related signalling induces BMP4 in margial zone animal cap cells
High noadl induce organiser in dorsal magrinal zone
Organiser secretes chordin and xolloid degrades it.
111
How was segment polarity genes Wg and Hh found in Drosphila?
A group of mutants were identified that afected patterning within the segments and then a mutant that half of segment failed to develop and the larva was identified covered by denticles.
After the mutants were discovered the gene wsa identified and th protein produced was found to be a signalling molecule.
112
Descibe Hh autocatalysis and embedding
Hh is autocatalytic
Cholesterol is added to c-terminus on N-terminal
Cleavage between Gly-257 and Cys-258
Sulfur on Cys forms thioester from peptide bond
Hydroxyl on cholesterol forms ester bond to complete cleavage
Palmitoyl on N-terminus makes bost ends hydrophobic and becomes embeded in membrane by cholesterol (this restricts its activity to adjacent cells)
113
What is detatched?
It is a protein that is responsible for extracting Hh from the membrane (while keeping the cholesterol)
114
What is Patched?
Patched (Ptc) is the Hh receptor that acts more like an ion channel
If knocked out the Hh response is activated.
Smo (smoothened) is important for it but not known how
115
Descibe Hh signalling pathway in absence of Hh ligand
In absence of Shh Ptc sequesters Smo in membrane vesicles and it is degraded
Ci/Cos2/Fu complex is bound to microtubules and Ci is phoshprylated (by PKS)
Slimb recognizes Ci-P and is targeted to the proteosome releasing a fragment (Ci75) which binds to target genes and represses transcription (in absence of Shh
In Hh presence Ptc can't sequester Smo and Cos2/Fu get recruited by Smo
this lets regular Ci (can't be phosphorylated) activate gene expression
116
Where is Shh secreted in development?
In the notocord where it induced the floor plate which also secretes it to specify V1 and V2 interneurons.
117
How does Hh relate to cancer
Hh pathway is often mutated in basal cell carcinoma (BCC) as excess Hh leads to overproliferation of basal cells
118
Descibe Wg signalling pathway in absence of Wg ligand
Similar to Hh pathway
In absence of Wnt GSK3/APC/Axin phosphorylates beta-catenin
completely degrades Beta-catenin so it can't bind to TCF
TCF in absence of Beta-Catenin represses transctiption
In the presence of Beta-Catenin it activates transctiption
In presence of Wnt binds to frizzeld and LRP which prevents GSK3/APC/Axin forming by sequestering Axin
Beta-catenin present binds to TCF and activates transcription.
119
What is the structue of Wnt Proteins?
Small singalling protein similar to Hh
Amalgamation of Wingless and Int
Present in vertebrates
N-terminal signal peptide
Intra-chain disulfide bonds
Hydrophobic modification added by Porcupine
120
What is the parasegment boundary?
The boundary between Hh and Wg (wingless) which induce each other at this boundary and maintains demarcation
121
How was Wnt demonstrated to be inportant for Nieuwkoop centre development
When Beta-catenin (as if Wnt pathway was present) added to both sides of a xenopus embryo a twinned embryo develops with 2 Nieuwkoop centres.
122
How does Wnt relate to cancer?
Over-expression can cause cancer.
Beta-catenin degredation prevention mutations are often found in colon cancer and melanoma cases.
APC breaks down Beta-Catenin and an excess of Wnt results in a loss of APC
Beta-catenin also has mutations that prevent it breaking down with the same effect.
123
BIOL
flashcards
Decks in class (21)
# Cards
