Willin

Question 1

When does myelination occur?

Answer 1

In late embreyonic gestation and post-natal development in a caudal-rostral gradient in the CNS before developing in the PNs

Question 2

What is an internode made from?

Answer 2

An oligodendrocyte (CNS) or Schwann cell (PNS)

Question 3

What is located in the internode, paranode and node?

Answer 3

Intenode - K+ chanels
Paranode - CASPR and Coactin (where myelin adheres)
Node - Neurofascin p186 and Ankyrin G

Question 4

When comparing the mRNA of schwann/oligodendrocytes from pre and post myelination what was found?

Answer 4

2 different types of Neurofascin (NF)
NF155 - found in glial cells
NF186 - found in neuronal cells

Question 5

What was found when NF155 was immunolabelled?

Answer 5

• Found in cell bodies of oligodendrocytes
• Concentrated at the paranode
• co-localised with CASPR at neuron-glial junction
• Responsible for myelin adhesion to neuron

Question 6

How is NF155 proposed to adhere myelin to neurons?

Answer 6

• Anchored in the glial paranodal loop

- Binds with CASPR and contactin which are anchored in the axon membrane

Question 7

What method has shown the binding of neurodascin?

Answer 7

• Use yeast two hybrid with Gal4-UAS system expressing cytplasmic tail of neurofascin (bait)
• optic and sciatic nerve yeast-two libraries (prey)

Question 8

What does the cytoplasmic tail of neruofascin bind to?

Answer 8

• Ankyrin isoforms
• Ezrin
• A novel protein (willin)

Question 9

What is ezrin?

Answer 9

• Part of 4.1 superfamily
• Contains FERM domain which can bind phospholipids and proteins, coil-coil and tail
• Can link transmembrane proteins to the cytoskeleton (actin)
• Controlled by signalling pathways

Question 10

Where do neurofascin and ezrin co-localise?

Answer 10

At interdigitating Schwann cells

Question 11

What are the properties of Willin?

Answer 11

• Contains FERM domain
• Binds phospholipids
• Seen in cell-cell interactions
• Stimulated by growth factors

Question 12

What are the roles of merlin and expanded?

Answer 12

• Have a role in cell adhesion and structure and control activation of the hippo complex
• Regulate cell growth and proliferation
• When knocked down cause uncontrolled proliferation

Question 13

What is the hippo pathway?

Answer 13

• Kinase cascade first identified in Drosophila
• Mutants have wing hyperplasia and eye defects
• Components have different names but process is conserved in humans

Question 14

What are the steps of the hippo pathway?

Answer 14

1. Receptor ‘fat’ activates merlin/expanded
2. This phosphorylates Hpo/Sav which phosphorylates wts/mats
3. Phosphorylation of yorki transcription factor then stays in cytoplasm and signals apoptosis, if not enters nucleus and regulates survival

Question 15

How is the hippo pathway linked to cancer?

Answer 15

1. YAP (human yorki homolog) is an oncoprotein

2. Has a role in organ size homeostasis which is inhibited by hippo pathway

Question 16

What is the TRex method?

Answer 16

• Express TetR (tetracycline repressor) in cells, these bond to TetO2 (operator) sequences repressing transcription of gene of interest
• Addition of tetracycline binds to TeR allowing transcription of gene of interest

Question 17

How have Willin and the hippo pathway been linked?

Answer 17

Using TRex method (Willin is very toxic)

• Upon aplication of Tet there is activation of hippo components
• Decrease in ammounts of nuclear YAP

Question 18

Does willin behave similarly to expanded?

Answer 18

• When expressed in drosophila goes to the same place

- However slightly different effect on wing structure, but not suprising as it is a smaller protein

Question 19

What are MCF10A cells?

Answer 19

• Breast cancer line used as they show signalling components of epithelial mesenchymal transition

Question 20

What is epithelial mesenchymal transition?

Answer 20

• Epithelial cells are usually stationary, show apical-basal polarity, tight junction and expression of cell-cell adhesions (cadherin)
• When undergo EMT loose polarity and adhesion, gain migratory and invasive properties to become mesenchymal stem cells (metastatic cancers)
• Caused by YAP expression

Question 21

What is the role of Willin in MCF10A cells?

Answer 21

• Willin knockout leads to loss of epithelial markers (E-cadherin and occludin) and gain of mesenchymal markers (N-cadherin and vimentin)
• However dependent on YAP phosphorylation (effect removed if YAP phosphorylation site is removed)
• Only willin FERM domain needed to reduce YAP induced genes