Signalling Pathways

Question 1

What are the 4 methods which cells can signal by?

Answer 1

• Direct contact- either through gap junctions on touching cells or via cell surface proteins fitting into receptors on other cells
• Autocrine system- when molecules are produced that affect the cell that produces them
• Paracrine system- when molecules are produced that act on local cells over a short distance
• Endocrine system- when molecules act on cells over a long distance (via bloodstream)

Question 2

What is the term for when a ligand binds to a receptor?

Answer 2

When the ligand binds to the receptor this is called a signal perception

Question 3

What is signal transduction?

Answer 3

When the protein bound to the receptor starts to change shape or catalysing a reaction.

Question 4

Describe the general mechanisms of cell signalling

Answer 4

• There will always be a signalling molecule (probably coming from extracellular environment)
• This will bind to a receptor (Probably on the plasma membrane).
- A lot of receptors are transmembrane proteins- they will have a part of the receptor which is extracellular and a part intracellular
• Upon interaction of the signalling molecule to the receptor, the receptor could change conformation, become phosphorylated, can start attaching to other cytoplasmic molecules and this leads to the transduction of the signal inside the cell
• These transduction pathways can be extremely complex in some, simple in others. Can have varying numbers of relay molecules.
• The cellular response can have various effects, including transcriptional repression/activation, metabolic responses, changes in cell behaviour.

Question 5

What are the 4 steps of cell signalling?

Answer 5

So the four main steps are reception, transduction, response, and feedback (positive/negative).

Question 6

Give some examples of paracrine signals: morphogens

Answer 6

Paracrine signals: morphogens
•	Hedgehog
•	Wnts
•	Transforming Growth Factor b superfamily
•	Receptor Tyrosine Kinases family

Question 7

Give some examples of cell-cell contact signals

Answer 7

Cell-cell contact
• Eph/ephrins
• Semaphorins
• Notch

Question 8

Give some example of extracellular matrix signals

Answer 8

Extracellular matrix

• Integrins

Question 9

What genes are expressed in the ICM and TE?

Answer 9

• Oct3/4, Sox2, Sall4, Nanog expressed in ICM

* Cdx2, Gata4 expressed in TE

Question 10

Summarise the Hippo pathway

Answer 10

On image

Question 11

How was the Hippo pathway first identified?

Answer 11

• Hippo identified as a tumor suppressor in Drosophila
• Mutation causes overgrowth (tumor formation)
• Mouse homologues known as Mst1 and 2 {mutation again leads to overgrowth and tumor formation}

Loss of function -> overgrowth

Question 12

Which cells are polar and apolar?

Answer 12

• Position determines whether cells are polar or apolar
• Cells that are located outside have a free surface so they become polar
• Cells on the inside (ICM) are completely surrounded and bind tightly to other cells and remain apolar

Question 13

Describe the Hippo pathway

Answer 13

• Difference in polarity determines Hippo activity
• In apolar cells a complex of kinases becomes activated – one of them is Mst (the hippo gene). These kinases phosphorylate Yap (a co-transcription factor that normally binds to Tead4). But when it is phosphorylated it cannot translocate into the nucleus so there is no transcription// tead cannot activate transcription. So hippo pathway is active
• In TE, the complex of kinases does not become activated, Yap is not phosphorylated. So Yap binds with Tead activating TFs. Hippo protein is inactive.

Question 14

How does the hippo pathway become activated?

Answer 14

• Its still not fully understood how the hippo pathway becomes activated
• Recent studies have shown us that the presence of an apical surface on the polar cells are important to inactivate the hippo pathway. Molecules that are located on this apical surface will sequester proteins (kinases) which will inactivate the pathway so Yap can enter the nucleus and bind Tead 4
• In the ICM, this does not happen as the cells do not have an apical surface

Question 15

Describe Eph/ephrin signalling pathway

What family are the EPH receptors part of?

What happens downstream following activation?

What happens downstream following signal perception?

What are EPHs classified?

Answer 15

EPH receptors are trans-membrane proteins from the Receptor Tyrosine Kinase family. Downstream transduction of the signal involves the oligomerisation and cytoplasmic cross-phosphorylation of the receptors.

EPHs are classified in type-A and type-B, according to the Ephrin ligand they bind to: Ephrin-A ligands are attached to the membrane by a lipid modification, while Ephrin-B ligands are transmembrane proteins. EPH receptors can only bind to either Ephrin-A or Ephrin-B ligands, with one exception: EPHA4, which can bind to both types of ligands.

The interaction between Ephrin ligands and EPH receptors leads to a bidirectional response – in both the receiving and the signalling cell.

Question 16

What happens when the notch-delta signalling pathway is inactive and active?

Answer 16

Inactive
• When notch is inactive (nothing is bound to its extracellular domain), CSL in the nucleus is complexed with repressor proteins, meaning the expression of target genes is prevented.
• Notch consists of an extracellular domain and an intracellular domain (found in the cytoplasm).
Active
• The notch protein is activated by a ligand binding to its extracellular domain which is then cleaved from the rest of the protein (first cleavage)
• This leads to the cleavage of the notch intracellular domain by enzymes of the Presenilin complex (second cleavage) and is then released
• The intracellular domain will translocate to the nucleus
• The intracellular domain will bind to the CSL complex activating it, causing the repressor to be released
• The co-activator protein mastermind is recruited, alongside other co-activators and they bind to the complex
• The target genes are then expressed

Question 17

Describe lateral inhibition

Answer 17

• This pathway is used to make two types of cell from a homogenous group of cell types
• An example of when this happens is during neurogenesis during embryonic development
• This pathway will single out cells that will eventually form the correct cell types, such as neurones during neurogenesis

1 All cells express notch and delta (the receptor for notch)
2 Delta will bind to notch and vice versa in the two cells present, so both cells can signal to each other
3 When notch is activated it will inhibit the activity of delta
4 Levels of delta are not identical between cells, so the pathway is not turned off.
5 This difference is amplified
6 Cells with slightly more delta will activate more notch and lead to repression of delta in adjacent cells
7 This results in cells with lots of delta and little notch and cells with high levels of notch and no delta
8 The cells with high levels of delta are the cells that will form the neurones
9 This process is called lateral inhibition

Question 18

What process is notch involved in?

Answer 18

Lateral inhibition by Notch is used during neurogenesis

Here we have the loss of purple cells to single out into neurones

No notch activity leads to lateral inhibition

So lots of cells express delta leading to neuron formation

Question 19

What is a morphogen?

Answer 19

In the embryo, signalling molecules are often produced in a specific region, and disperse throughout the tissue generating concentration gradients. Cells will respond differently depending on the levels of signal they detect - often acquiring different fates. Signalling molecules that confer different fates at different concentrations are called morphogens.

Question 20

What produces morphogens?

Answer 20

• Morphogens are secreted by discrete groups of cells in developing embryos called signaling centers

Question 21

How do cells respond to morphogens?

Answer 21

• Can provide positional information within the embryo
• Cells will respond to different levels of morphogen produced from the source, so they will acquire different identifies or behave in different ways

Question 22

Give an example of a morphogen that is not a diffusible molecule (Drosophila)

Answer 22

• Bicoid (transcription factor that binds to DNA sequences) and AP (anterior posterior axis) patterning in Drosophila embryo
• An early embryo of Drosophila consists of many nuclei produced by mitosis
• Bicoid Mrna is deposited in the anterior cortex of the embryo (blue stain), it is concentrated at one end of the embryo
• The protein will diffuse through the embryo forming a concentration gradient
• This gradient will instruct the nuclei to different fates, more bicoid leads to more transcription of genes
• This forms the anterior posterior axis in the embryo

Question 23

Describe the FGF pathway, RTK signalling pathway

Answer 23

• The FGF molecule binds to its receptor resulting in dimerization (or stabilization of dimers) of the receptor molecule
• The intracellular tyrosine kinase domains are then activated, and phosphorylate each other
• The phosphorylated receptor tails then recruit two adaptor proteins – Grb and Sos
• These two molecules then recruit and activate Ras at the plasma membrane
• This results in the activation of the first serine/threonine kinase (raf in mammals)
• Raf phosphorylates and activates the next kinase in the cascade – MAPKK
• MAPKK then phosphorylates and activates MAPK which then phosphorylates other kinases
• MAPK can enter the nucleus and phosphorylate transcription factors
• This activates gene expression

Question 24

Describe the structure of RTK

Answer 24

Above shows various types of RTKs. Extremely diverse extracellular domains. Intracellular side mainly characterised by the tyrosine kinase domain (red box). Downstream effects can include cell proliferation, survival, metabolism, cell migration etc. Can do this during embryogenesis and in adult tissues.

• Single pas, trans-membrane receptors
• Complex extracellular domains
• Red box = tyrosine kinase domain
• They activate downstream cascades regulating lots of different types of cell process’ such as proliferation, differentiation, survival, migration ect
• This occurs during embryogenesis

Question 25

Describe the general path of RTKs

Answer 25

• First step in activation of RTK receptor is the formation/promotion of the dimerization between the two receptors.
• Often the signals are dimers themselves so they combine the two receptors and then they bring them together.
• The consequence of bringing them together is that the tyrosine kinase domain in the cytoplasmic side of these receptors will phosphorylate a lot of tyrosines in the adjacent cytoplasmic receptor tail
• This leads to the activation of these receptor dimers.
• The phosphorylation of all these tyrosine’s generates docking sites for other proteins, and these will be able to bind to the cytoplasmic domain of these receptors and then subsequently transduce a signal downstream inside the cell.
• One of the main and best understood consequence downstream of RTKs is the activation of the GTPase Ras.

Phospho-tyrosines are docking sites for other proteins
• Once the receptore has dimerized and phosphyrlated. The phosphates act as docking sites for other proteins to bind to.
• The proteins can now bind to the cytoplasmic domain of the receptors which will then transduce a signal

Question 26

Describe the GTP Ras activation

Answer 26

GTPases work by:
• They are usually found inactive bound to GDP. They are activated when this GDP is released and replaced with GTP.
• Activation of a GTPase leads to a downstream response and there are some consequences in the modulation of the activity of other proteins within the cell.
• These molecules also have the ability of removing a phosphate from the GTP that is bound to them and then they become inactive again.
• This transformation from inactive to active and vice versa can be modulated by two other groups of protein- Guanine Exchange Factors (GEF) and the GTPase Activating Proteins (GAP).

• In our case, in the RTK signalling cascade, the activation of the Ras protein is promoted thanks to the recruitment of a Ras GEF next to the membrane next to the activated receptor.
• This occurs when one of the phosphorylated tyrosine regions in the cytoplasmic domain becomes a docking site for the Grb-2 adaptor protein, which in turn binds to Ras GEF, bringing Ras GEF next to the membrane where inactive Ras protein is, which can then promote Ras activation.

Question 27

What happens downstream of RAS?

Answer 27

After this pathway is complete it leads to cell proliferation, cell fate determination ect
• Ras activation leads to the activation of a cascade of phosphorylation.
• The proteins above in the transduction cascade are kinases and all of them phosphorylate other proteins in serines and threonines
• First step is phosphorylation of MAP-kinase-kinase-kinase, which phosphorylates the next and so on.
• The third one will phosphorylate many effector proteins, amongst which we can find transcription factors and many other proteins.
• The final consequence of the activation of this cascade is the modulation of cell proliferation, cell fate determination and many other processes within the cell.
• Activation of Ras and this phosphorylation cascade is not the only consequence of RTK activation.
• The cytoplasmic domain of this RTK is not only a docking site for the adaptor proteins but also docking sites for many other proteins that will lead to the activation or modulation of many other processes within the cell.
• The responses of a cell to the activation of these signalling pathways can be extremely complex:

Question 28

What are the distinctive features of RTK signalling

Answer 28

1 The receptor has kinase activity
2 Upon activation it dimerises and undergoes cross-phosphorylation in tyrosine residues
3 A network of molecules transduces the signal
4 One of the best known transducers: the GTPase Ras

Question 29

Describe the BMP pathway

Answer 29

BMP pathway:
1 The BMP receptor has an extracellular binding site and an intracellular domain
2 The BMP dimer ligand will bind to the receptor
3 The ligand binding to the receptor causes subunit 2 (serine/threonine) to phosphorylate receptor subunit 1 (kinase domain)
4 Receptor subunit then phosphorylates an intracellular Smad protein
5 The phosphorylated Smad protein binds to another type of Smad to form a transcriptional regulatory complex
6 The regulatory complex enters the nucleus and then either activates or represses target genes. Sometimes transcriptional co-factors are also involved
7 This can trigger, for example, the ventralization of mesoderm, formation of bone and cartilage or apoptosis

Question 30

Describe the hedgehog pathway

Answer 30

Pathway in absence of hedgehog:
1. The membrane protein Patched inhibits the membrane protein Smoothened
2. Inhibition of smoothened results in the transcription factor Cubitus interruptus (Ci) being held in the cytoplasm
3. Ci is held in two protein complexes. One is associated with Smoothened and the other is associated with the protein suppressor of fused (Su(fu))
4. Ci in the smoothened complex is phosphorylated by three protein kinases: protein kinase A, GSK-3 (glycogen synthase kinase) and CK1 (casein kinase 1)
5. Phosphylation of Ci results in cleavage of Ci. This forms the truncated protein CiRep
6. CiRep enters the nucleus and represses a target Hedgehog target gene
Pathway in presence of hedgehog:
1. Hedgehog binds to Patched membrane protein
2. This lifts the inhibition of Smoothened and blocks the production of CiRep
3. Smoothened is phosphorylated by PKA and CK1
4. Ci is released from both complexes in the cytoplasm
5. Ci enters the nucleus and acts as a gene activator
6. Genes activated include wingless (wg), decapentaplegic (dpp) and engrailed (en)
So overall, in absence of Hedgehog Ci is converted to a repressor of Hedgehog target genes. However in the presence of Hedgehog Ci acts as a gene activator of Hedgehog target genes.

Question 31

What are the types of Wnt signalling pathways?

Answer 31

Canonical Wnt pathway: Frizzled and Beta-catenin dependent

Non-canonical Wnt Pathway: PCP (Planar Cell Polarity) pathway and the Ca2+ pathway.

Question 32

Describe active and inactive Canonical Wnt pathway: Frizzled and Beta-catenin dependent

Answer 32

Pathway in absence of Wnt:
1. When Wnt is absent, the protein beta-catenin is bound by a destruction complex which includes protein kinase CK1-gamma and GSK-3beta
2. The protein kinase phosphorylates B-catenin, targeting it for ubiquitination and degradation in the proteasome, meaning there is no B-catenin free to move into the nucleus
3. In the absence of B-catenin, transcriptional co-repressors bind to TCF transcription factors
4. This prevents the expression of certain genes
Pathway in the presence of Wnt:
1. Wnt binds to the transmembrane receptor Frizzled
2. This causes a signal to be transmitted across the membrane by Frizzled and LRP, activating them
3. Activation of Frizzled and LRP causes protein kinases CK1-gamma and GSK-3Beta (the same as those seen in the destruction complex) to associate with the membrane
4. The protein kinases then phosphorylate the tail of the activated LRP.
5. Next the intracellular signalling protein dishevelled and the protein Axin are recruited to the cytoplasmic tail of LRP and Frizzled
6. This prevents the formation of the destruction complex, meaning B-catenin accumulates in the cytoplasm
7. B-catenin then moves into the nucleus
8. This binds to TCF, displacing co-repressors
9. This enables target genes to be expressed

Question 33

What does B-catenin do in the nucleus?

Answer 33

In the absence of wnt :
• TCF is bound to groucho which blocks transcription
Presence of wnt:
• B-catenin translocates into the nucleus and displaces groucho
• B-catenin binds to TCF and recruits lots of other proteins to form a large protein complex that induces transcription

• In the absence of Wnt we have a transcription factor TCF that is always bound on regulatory sequences of DNA which is also bound to a cofactor called Groucho.
• This blocks transcription. When beta-catenin accumulates and then translocates into the nucleus it displaces Groucho and binds to TCF, and recruits many other proteins.
• This huge protein complex induces transcription, turning these genes on.

Question 34

Describe the Non-canonical Wnt Pathway: PCP (Planar Cell Polarity) pathway and the Ca2+ pathway.

Answer 34

• The PCP pathway was discovered when looking at the direction/polarity of hairs on a fruitflys wings.
• One of the main differences in the PCP pathway is that downstream of dishevelled instead of involving the destruction protein complex it involves Rho GTPases- similar activity to Ras GTPases but are in the cytoplasm (not membrane bound).
• They can activate various proteins that modulate the cytoskeleton proteins. Can affect the dynamics of the actin-myosin cytoskeleton, shape, polarity of the cells etc.
• The Calcium pathway, downstream of dishevelled are molecules that release calcium into the cytoplasm and this pathway is modulating the activity of various proteins that can bind to calcium and depend on it for their activity. Can lead to transcriptional/ cell activity modification.

Question 35

What molecules regulate Wnt

Answer 35

Sfrps – secreted frizzled related protein – this molecule has a very similar structure to frizzled so it can bind Wnt in the extracellular space and sequester it away from frizzled or it can bind straight away to frizzled

Dkk1 – Dickkopf1 – this binds to the LRP5/6 receptor and sequesters it away from frizzled, so went Wnt binds to frizzled it cannot recruit LRP5/6.

Wif-1 – Wnt inhibitory factor – This binds directly to Wnt

Question 36

What family does activin belong to?

Answer 36

Activin belongs to the TGFb superfamily

Question 37

What is activin and what does it do?

Answer 37

• Activin is a morphogen that is involved in determining cell fates in xenopus
• There is an animal pole at the top and a vegetal pole at the bottom, and an equator where the mesoderm will be induced
• Activin will diffuse and determine mesodermal cell fates depending on the levels of activin

Question 38

How is BMP signalling involved in neural ectoderm formation in xenopus?

Answer 38

• This is about the establishment of the neural ectoderm in xenopus which is the precursor of the neural tube and the CNS in the vertebrae embryo later on in development
• The neural ectoderm is derived from the ectodermal side of the embryo which will give rise to two main derivatives: neural ectoderm and the epidermis
• Epidermis cell fate is determines by BMP4 (which is expressed in the whole ectodermal region of the embryo) but it only induces epidermis in the ventral side of the embryo – because the organiser positioned in the dorsal side produces molecules that work as antagonists for BMP4 called chordin nogglin and follistastin
• They are secreted into the extracellular medium which will bind to BMP4 and suppress its activity
• In this way the organiser determines some cell fates to neural tissues and others to epidermis

Question 39

How is hedgehog signalling involved in limb patterning?

Answer 39

• The limb derives from a structure called the limb buds, which is the outgrowth of tissue that comes from flank of the developing embryo
• This tissue is flat and has an anterior portion and posterior portion
• The posterior region has organiser properites known as the zone of polarising activity
• These group of cells released a morphogen that is important to establish the symmetries of the developing limb
• This morphogen is hedgehog

Question 40

How was hedgehog discovered to be a morphogen?

Answer 40

• The development of the limbs was acknowledge well before it was known it was caused by this morphogen
• This was discovered from experiments in chicks
• Researchers took grafts from posterior parts of the limb into other regions of the limb
• The first column shows the normal organisation of the chick limb, with the position of the polarising cells. The concentration of morphogen is shown along the anterior posterior axis which is needed to create differences along this axis
• When grafts were placed in the anterior portion they were able to create mirror images of the developing limb. The new graft is a source of morphogen generating a symmetric concentration gradient
• More small grafts were done and were not forming a complete duplication. This is how they discovered a morphogen is needed to form differences along the anterior posterior axis of the limbs

Question 41

What do Shh,Tgfb and Wnts determine in the neural tube?

Answer 41

• The neural ectoderm will give rise to the neural plate which will fold to form the neural tube
• It will differenitiate to give rise to many different types of neurones and the fate of the neurones is different depending on the location of the dorsal/ventral axis. These fates are determines by the actvity of Shh,Tgfb and Wnts

Question 42

What morphogens induce neurone formation in the neural tube and how do they do this?

Answer 42

• We will look at the ventral portions of the neural tube
• The notochord is a source of morphogen which are important for determining cells fates of the ventral portion of the neural tube
• Grafts were taken and put into the lateral portion of the neural tube causing the cell to differentiate to form ventral fated neurones
• The morphogen responsible for this is hedgehog, the notochord is a source for it
• It will diffuse into the neural tube forming a concentration gradient
• This will form different types of neurones along the dorsal/ventral axis
• BMPs from the TFG-Beta family are expressed in the overlying ectoderm that covers the neural tube that forms a concentration gradient from the dorsal to the ventral portion. This gradient counteracts the hedgehog gradient. This is important for forming different cells fates in the neural tube.