13 - Signaling Mechanisms Regulating Cytoskeletal Dynamics I

Question 1

What mediates the morphological changes of cells during cell polarization?

Answer 1

The cell cytoskeleton

Question 2

What makes up the cytoskeleton?

Answer 2

Microtubules and microfilaments

Question 3

How is the actin cytoskeleton formed?

Answer 3

By monomers of actin forming large oligomers

Question 4

What is needed for actin monomers to bind together?

Answer 4

ATP

Question 5

What is the structure of actin filaments?

Answer 5

A double helix

Question 6

True or false: actin filaments have directionality

Answer 6

True: they have a plus end and a minus end

Question 7

What happens at the plus end of actin?

Answer 7

More monomers of actin are added

Question 8

What higher order structures are possible with actin filaments?

Answer 8

Branching, binding together, etc.

Question 9

True or false: the actin cytoskeleton is a static structure

Answer 9

False: it is highly dynamic

Question 10

How is the actin cytoskeleton dynamic?

Answer 10

It can polymerize or degrade based on the needs of the cell

Question 11

What is the significance of the actin cytoskeleton being dynamic?

Answer 11

Allows for large morphological changes in the cell

Question 12

What happens at the minus end of actin?

Answer 12

The actin filaments get degraded by enzymes

Question 13

What does aPKC do?

Answer 13

1. Phosphorylates and activates crucial downstream targets for polarization
2. Facilitates formation of actin cytoskeleton rearrangement

Question 14

How does PAR-3 regulate cytoskeletal remodeling?

Answer 14

By regulating Rac-1 and recruiting a ring of F-actin

Question 15

How does PAR-3 regulate tight-junction assembly?

Answer 15

By localizing to the site, and recruiting other proteins

Question 16

What will be the phenotype of epithelial cells if PAR-3 and PAR-6 were deleted?

Answer 16

A mix of basal and apical features, thus destroying any unique features

Question 17

How is PAR-3 and PAR-6 localized in the neurons during development?

Answer 17

All around the neurites in stage 2, but only in the axon in stage 3

Question 18

What is the significance of PAR-3 and PAR-6 being localized in the axon in stage 3?

Answer 18

They play a later role in axon development (compared to LKB1)

Question 19

What happens when PAR-3 is overexpressed in neurons?

Answer 19

They develop multiple axons

Question 20

What happens if aPKC is deleted from neurons?

Answer 20

They do not develop axons

Question 21

What does PAR-6 interact with?

Answer 21

aPKC, PAR-3, and Cdc42-GTP

Question 22

What GTPases are found in the positive feedback loop of axon development?

Answer 22

Cdc42 and Rac-1

Question 23

What are GTPases?

Answer 23

GTP-hydrolyzing enzymes, that depend on GTP hydrolysis for their function

Question 24

What functions do GTPases regulate?

Answer 24

Migration, development, trafficking, and regulation of cytoskeletal dynamics

Question 25

When is a GTPase inactive?

Answer 25

When bound to GDP

Question 26

When is GTPase active?

Answer 26

When bound to BTP

Question 27

What helps activate a GTPase?

Answer 27

GEF

Question 28

What helps inactivate a GTPase?

Answer 28

GAP

Question 29

How do GEFs work?

Answer 29

Exchange bound GDP for GTP (activate)

Question 30

How do GAPs work?

Answer 30

Increase rate of GTP hydrolysis of GTPase (inactive)

Question 31

True or false: PAR-6 is always bound to Cdc42

Answer 31

False: it is only bound when Cdc-42 is active (GTP bound)

Question 32

What does Cdc-42 regulate?

Answer 32

The localization of the scaffold proteins (PAR-3 and PAR-6) to the apical domain

Question 33

When happens if Cdc-42 is inactivated?

Answer 33

Delocalization of scaffold proteins (PAR-3 and PAR-6)

Question 34

How does Cdc-42 mediates apical domain polarization?

Answer 34

Through remodeling of the actin cytoskeleton and tight junctions

Question 35

What is TIAM-1?

Answer 35

The GEF for Rac-1

Question 36

What is the GEF for Rac-1?

Answer 36

TIAM-1

Question 37

Where does TIAM-1 bind to?

Answer 37

PAR-3

Question 38

When does PAR-3 and PAR-6 mediate cytoskeletal remodeling?

Answer 38

When PAR-3 is activated by Rac-1

Question 39

What does Rac-1 do?

Answer 39

Recruits F-actin for cytoskeletal remodeling via activation of PAR-3

Question 40

What happens when TIAM-1 is inactivated?

Answer 40

Collapse of apical domain architecture

Question 41

What is the "crux" of the positive feedback loop in neuronal development?

Answer 41

PI3K

Question 42

What does PI3K stand for?

Answer 42

Phosphoinositide 3-kinase

Question 43

What type of protein is PI3K?

Answer 43

A serine/threonine kinase

Question 44

How can PI3K be activated?

Answer 44

1. Association with phosphorylated RTKs 2. Binding to active, GTP-bound Ras 3. Binding to other GTPases such as Rac-1

Question 45

What pathways does PI3K act in?

Answer 45

Cell growth, cell survival, translation, organelle biogenesis, organelle localization, and cytoskeleton regulation

Question 46

Where does PI3K act?

Answer 46

At the cell surface

Question 47

Why is PI3K a critical regulator of the cell surface?

Answer 47

This is where it is localized when it is activated

Question 48

What is the structure of PI3K?

Answer 48

Regulatory domain (p85) and catalytic domain (p110)

Question 49

What is the p85 domain of PI3K made up of?

Answer 49

SH2, SH3, and proline-rich regions

Question 50

What is the significance of the structure of the p85 domain of PI3K?

Answer 50

Many protein-binding domains for regulation of its activity

Question 51

What does PI3K do once it is activated?

Answer 51

Convert PIP2 into PIP3

Question 52

What is PIP2?

Answer 52

Phosphatidylinositol 4,5-bisphosphate

Question 53

What is PIP3?

Answer 53

Phosphatidylinositol 3,4,5-trisphosphate

Question 54

What does PTEN do?

Answer 54

Converts PIP3 into PIP2

Question 55

What is the role of PTEN?

Answer 55

Negative regulation of PI3K pathway

Question 56

What type of molecules are PIP2 and PIP3?

Answer 56

Lipid second messengers

Question 57

True or false: PIP3 is a stable second messenger

Answer 57

False: it is quickly degraded by PTEN

Question 58

What type of protein is PTEN?

Answer 58

A phosphatase

Question 59

What does PIP3 activate?

Answer 59

Akt, and the rho family of GTPases

Question 60

What is another name for Atk?

Answer 60

PKB (protein kinase B)

Question 61

What do the rho GTPases activate?

Answer 61

PI3K

Question 62

What is the significance of rho GTPases activating PI3K?

Answer 62

This creates a positive feedback loop (PI3K --> PIP3 --> rho GTPases --> PI3K)

Question 63

What is a PH domain?

Answer 63

Pleckstrin homology domain

Question 64

What is the purpose of a PH domain?

Answer 64

Allow binding to PIP3 to regulate activity

Question 65

How does Akt get activated?

Answer 65

By binding to PIP3, being recruited to the cell surface, and being further phosphorylated by PKD

Question 66

How does Akt get phosphorylated by PKD?

Answer 66

PKD is activated and recruited to the cell surface by PIP3

Question 67

What pathways are downstream of Akt?

Answer 67

1. Inhibition of apoptosis and promotion of cell survival 2. Activation of translation (through mTOR) 3. Activation of small GTPases

Question 68

How does PAR-3 get recruited to the cell membrane?

Answer 68

By binding to PIP2

Question 69

Besides PAR-6 and aPKC, what is bound to PAR-3?

Answer 69

TIAM-1, PIP2, and PTEN (and oligomers)

Question 70

Why does PAR-3 bind to PTEN?

Answer 70

PTEN creates more PIP2, leading to stronger anchoring of PAR-3 to the cell membrane

Question 71

How does PAR-3 get recruited to the tight junctions?

Answer 71

By associated with proteins that are critical components of the tight junctions

Question 72

What is the full scaffold complex seen in cell polarization?

Answer 72

PAR-6 binds with aPKC, Cdc-42-GTP, and PAR-3. PAR-3 binds with TIAM-1/Rac-1-GTP, PIP2, PTEN, and PAR-6

Question 73

What is the full, major positive feedback loop in polarization?

Answer 73

PI3K --> Cdc-42 --> aPKC/PAR-3/PAR-6 --> TIAM-1/Rac-1 --> PI3K

Question 74

What are some example of rho GTPases activated by PI3K (and PIP3)?

Answer 74

Rac-1 and Cdc-42

Question 75

What is the significance of PI3K activating rho GTPases?

Answer 75

This creates a positive feedback loop, thus leading to continuous signaling

Question 76

Why does PAR-3 bind to PIP2 and not PIP3?

Answer 76

PIP3 concentrations are kept fairly low, as a means of negative regulation of PI3K

Question 77

True or false: TIAM-1 and Rac-1 are found in physical association when they are both in the scaffold

Answer 77

True: Rac-1 associated with the GEF through a physical association

Question 78

True or false: PAR-6 can oligomerize, similar to PAR-3

Answer 78

True: this leads to robust signaling through recruitment of many scaffolds

Question 79

True or false: many of the signaling determinants in polarization only function in that pathway

Answer 79

False: many are also upstream of cell growth and survival, development, organelle biogenesis, etc.

Question 80

What is needed to regulate positive feedback loops?

Answer 80

Negative feedback

Question 81

How can negative regulation be exerted in the cell?

Answer 81

GAPs, PDEs, and phosphatases

Question 82

What do GAPs negatively regulate?

Answer 82

GTPases

Question 83

What do PDEs negatively regulate?

Answer 83

cAMP (and its downstream activators)

Question 84

What do phosphatases negative regulate?

Answer 84

Kinases (and their products, such as PIP3)

Question 85

In terms of polarization, which regulation is more well understood?

Answer 85

Positive regulation (as opposed to negative regulation)

Question 86

What is the phenotype of neurons with deleted PI3K?

Answer 86

No axons, but also no healthy neurites, smaller cells, not as healthy

Question 87

Why do neurons that have deleted PI3K not look healthy?

Answer 87

PI3K is involved in cell growth and survival as well as neuronal polarization

Question 88

What is the conclusion of the experiments that deleted PI3K from neurons?

Answer 88

Deleting PI3K may not be the best way to look at the question of neuronal development (is it just because the neurons are sick?)

Question 89

Why is deleting PI3K not the best way to study neuronal development?

Answer 89

Not sure if the phenotype is due to the kinase, or just that the neurons are not surviving well

Question 90

Where is aPKC located when it is not bound to PAR-3 and PAR-6?

Answer 90

All over the cell

Question 91

What do the experiments about inhibiting PI3K suggest about its interactions with the neurites?

Answer 91

PI3K may be involved in proper neurite formation