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Cellular Signalling > Tomlinson - Tetraspanins > Flashcards

Tomlinson - Tetraspanins Flashcards

1
Q

Knockout of Tspan12 Tetraspanin shows which phenotype and therefore tells us what regarding its function?

A

Tspan12 knockdown shows abnormal retinal vasculature due to defective Wnt signalling. Tspan12 deficient patients develop Familial Exudative Vitreoretinopathy.

Therefore Tspan12 is essential for eye blood vessel development.

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2
Q

How many human tetraspanins are there?

A

33

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3
Q

Discuss the role of CDC151 in regulating Laminin-Binding Integrins

A

CDC151 regulates adhesion of Laminin in the basement membrane to Integrins a3b1 and a6b1. CDC151 also regulates the signalling, survival, internalisation and glycosylation events also.

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4
Q

How many tetraspanins can be found in fish?

A

46 - due to a duplication event

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5
Q

What is the role of CD81 Tetraspanin?

A

CD81 interacts with CD19 which is a signalling molecule. CD81 is essential for CD19 expression on the B cell surface. Without CD81, there is a lack of CD19 on the cell surface hence impaired antibody immunity.

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6
Q

How many tetraspanins can be found in C. Elegans?

A

20

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8
Q

What is Super Resolution Microscopy, and what has been determined from SRM regarding lipid rafts?

A

STED is an SRM used to visualise objects beyond the diffraction limit. In this case it was observed that tetraspanins tend to cluster homogenously with alike tetraspanins

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9
Q

How many tetraspanins are present in insects?

A

36

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10
Q

Knockout of CD9 Tetraspanin has shown which phenotype and therefore tells us what regarding its function?

A

CD9 knockout showed impaired sperm and egg fusion, with shorter, thicker and denser microvilli on the Oocyte. Therefore, it is essential for fertilisation

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11
Q

How can tetraspanins produce a “hierachical network of interactions”?

A

They interact with both non-tetraspanin binding partners and tetraspanins too

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12
Q

Some tetraspanins are specific to certain cell types, while some are ubiquitous. Give two examples of ubiquitous tetraspanins

A

CD9 and CD81

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13
Q

Give four broad classes of tetraspanin binding partners

A

The Immunoglobulin superfamily
Transmembrane proteins
Proteases
Integrins

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14
Q

How many mammalian tetraspanins are there?

A

34

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14
Q

State two difficulties with Tetraspanin research

A

In mice, gene silencing of Tetraspanins often leads to surprisingly subtle phenotypes - despite their heavy importance. For example, single knockdowns of CD9 and CD81 gave similarly healthy lung phenotypes. However, in a double knockdown, a phenotype of diseased lungs were observed. This suggests a compensatory effect and similar functionality of CD9 and CC81.

Secondly, functions of Tetraspanins are difficult to determine. This is because through knockdowns we can’t be sure if this phenotype is due to loss of function or a general disruption of micro domains. Knockdown of CD81, 151, 37 and Tspan32 all showed hyper responsive T cell phenotypes.

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15
Q

How much larger do Tetraspanins protrude over the plasma membrane?

A

3-5nm

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16
Q

Aside from CD81’s role in B-cell receptor signalling, what else is CD81 utilised in? Give two examples

A

CD81 is essential for the entry of malarial parasite Plasmodium Flaciparum into hepatocytes.

It is also an essential receptor for the Envelope protein E2 of the Hepatitis C virus

17
Q

How many domains are present in tetraspanins?

A

4

18
Q

Each cell type can express how many tetraspanins?

A

20

20
Q

Draw a structure of a Tetraspanin

A

-

21
Q

Aside from CDC151 regulating Laminin-binding Integrins, what else has CD151 been implicated to cause?

A

CDC151 is involved in the endocytic uptake of the Human Papilloma Virus (HPV)16 which is an aetiological agent for cervical cancer. It requires interaction with Integrins a6 and palmitoylation for interactions with other Tetraspanins

22
Q

Give four examples of phenotypes observed in non mammalian species following Tetraspanin gene silencing

A

Plants - abnormal morphology
Fungi - abnormal lead penetration
Drosophila - neuronal defects
Worms - epidermal blistering

23
Q

Which four Tetraspanins following knockdown all showed a Hyper responsive T cell phenotype?

A

Tspan 32
CD37
CD151
CD81

24
Q

In knockdown studies of CDC151, what has been the observable phenotype, aside from skin blistering?

A

Kidney failure
Impaired pathological angiogenesis
Impaired thrombus formation

24
Q

What is the function of CDC151?

A

Regulation of Laminin-Binding Integrins

25
Q

What has been observed in patients suffering from CDC151 deficiency?

A

Skin blistering, due to poor epithelial cell attachment to the basement membrane

26
Q

What is Particle Tracking Studies? What has been determined through PTS regarding tetraspanins

A

Particle tracking studies involve selectively labelling tetraspanins with fluorescently-coupled antibodies in very dilute concentrations.

Through PTS it was determined that tetraspanins cluster with binding partners and patrol the plasma membrane. They also tend to cluster to other tetraspanins and their binding partners

27
Q

What is ADAM-10, and what are its functions besides Tetraspanin interaction? Give 6 examples

A

ADAM-10 is a Tetraspanin binding partner, as well as “a molecular scissor”. Functions include:

  • Release of soluble EGF
  • Release of soluble chemokines
  • Neuroprotection against APP in Alzheimer’s
  • Notch signalling for Development
  • Negative regulation of VE-Cadherin
  • Negative regulation of Platelet GPVI
28
Q

What was the protocol to determine the Tetraspanin Binding partners of molecular scissor ADAM10?

A
  1. Make a FLAG epitope tagged Tetraspanin expression construct
  2. Make a HA epitope tagged ADAM10 construct
  3. Transfer each FLAG tagged Tetraspanin into a cell line containing ADAM10
  4. Lyse cells
  5. Immunoprecipitate with FLAG and blot with HA antibodies
29
Q

What were the six Tetraspanin binding partners of ADAM10?

A

Tspan5, 10, 14, 15, 17, 33

30
Q

What group does the Tspans 5, 10, 14, 15, 17, 33 belong to? Why was it named this?

A

The C8 Tetraspanin family.

It the main extracellular loop of the Tspan there are 8 Cysteine residues

31
Q

It was thought the C8 Tspan family may be involved in cellular trafficking of molecular scissor ADAM10. What was the protocol to determine this? What was found?

A
  1. Determine the expression profile of all C8 Tspans
  2. Identify the most readily expressed Tspan
  3. SiRNA mediated knockdown of this Tspan
  4. Observe the sub cellular location of ADAM10

Tspan14 -> Endoplasmic Reticulum exit of ADAM10

Cellular Signalling (5 decks)

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