13 - Signaling Mechanisms Regulating Cytoskeletal Dynamics I Flashcards
What mediates the morphological changes of cells during cell polarization?
The cell cytoskeleton
What makes up the cytoskeleton?
Microtubules and microfilaments
How is the actin cytoskeleton formed?
By monomers of actin forming large oligomers
What is needed for actin monomers to bind together?
ATP
What is the structure of actin filaments?
A double helix
True or false: actin filaments have directionality
True: they have a plus end and a minus end
What happens at the plus end of actin?
More monomers of actin are added
What higher order structures are possible with actin filaments?
Branching, binding together, etc.
True or false: the actin cytoskeleton is a static structure
False: it is highly dynamic
How is the actin cytoskeleton dynamic?
It can polymerize or degrade based on the needs of the cell
What is the significance of the actin cytoskeleton being dynamic?
Allows for large morphological changes in the cell
What happens at the minus end of actin?
The actin filaments get degraded by enzymes
What does aPKC do?
- Phosphorylates and activates crucial downstream targets for polarization
- Facilitates formation of actin cytoskeleton rearrangement
How does PAR-3 regulate cytoskeletal remodeling?
By regulating Rac-1 and recruiting a ring of F-actin
How does PAR-3 regulate tight-junction assembly?
By localizing to the site, and recruiting other proteins
What will be the phenotype of epithelial cells if PAR-3 and PAR-6 were deleted?
A mix of basal and apical features, thus destroying any unique features
How is PAR-3 and PAR-6 localized in the neurons during development?
All around the neurites in stage 2, but only in the axon in stage 3
What is the significance of PAR-3 and PAR-6 being localized in the axon in stage 3?
They play a later role in axon development (compared to LKB1)
What happens when PAR-3 is overexpressed in neurons?
They develop multiple axons
What happens if aPKC is deleted from neurons?
They do not develop axons
What does PAR-6 interact with?
aPKC, PAR-3, and Cdc42-GTP
What GTPases are found in the positive feedback loop of axon development?
Cdc42 and Rac-1
What are GTPases?
GTP-hydrolyzing enzymes, that depend on GTP hydrolysis for their function
What functions do GTPases regulate?
Migration, development, trafficking, and regulation of cytoskeletal dynamics
When is a GTPase inactive?
When bound to GDP
When is GTPase active?
When bound to BTP
What helps activate a GTPase?
GEF
What helps inactivate a GTPase?
GAP
How do GEFs work?
Exchange bound GDP for GTP (activate)
How do GAPs work?
Increase rate of GTP hydrolysis of GTPase (inactive)
True or false: PAR-6 is always bound to Cdc42
False: it is only bound when Cdc-42 is active (GTP bound)
What does Cdc-42 regulate?
The localization of the scaffold proteins (PAR-3 and PAR-6) to the apical domain
When happens if Cdc-42 is inactivated?
Delocalization of scaffold proteins (PAR-3 and PAR-6)
How does Cdc-42 mediates apical domain polarization?
Through remodeling of the actin cytoskeleton and tight junctions
What is TIAM-1?
The GEF for Rac-1
What is the GEF for Rac-1?
TIAM-1
Where does TIAM-1 bind to?
PAR-3
When does PAR-3 and PAR-6 mediate cytoskeletal remodeling?
When PAR-3 is activated by Rac-1
What does Rac-1 do?
Recruits F-actin for cytoskeletal remodeling via activation of PAR-3
What happens when TIAM-1 is inactivated?
Collapse of apical domain architecture
What is the “crux” of the positive feedback loop in neuronal development?
PI3K
What does PI3K stand for?
Phosphoinositide 3-kinase
What type of protein is PI3K?
A serine/threonine kinase
How can PI3K be activated?
- Association with phosphorylated RTKs
- Binding to active, GTP-bound Ras
- Binding to other GTPases such as Rac-1
What pathways does PI3K act in?
Cell growth, cell survival, translation, organelle biogenesis, organelle localization, and cytoskeleton regulation
Where does PI3K act?
At the cell surface
Why is PI3K a critical regulator of the cell surface?
This is where it is localized when it is activated
What is the structure of PI3K?
Regulatory domain (p85) and catalytic domain (p110)
What is the p85 domain of PI3K made up of?
SH2, SH3, and proline-rich regions
What is the significance of the structure of the p85 domain of PI3K?
Many protein-binding domains for regulation of its activity
What does PI3K do once it is activated?
Convert PIP2 into PIP3
What is PIP2?
Phosphatidylinositol 4,5-bisphosphate
What is PIP3?
Phosphatidylinositol 3,4,5-trisphosphate
What does PTEN do?
Converts PIP3 into PIP2
What is the role of PTEN?
Negative regulation of PI3K pathway
What type of molecules are PIP2 and PIP3?
Lipid second messengers
True or false: PIP3 is a stable second messenger
False: it is quickly degraded by PTEN
What type of protein is PTEN?
A phosphatase
What does PIP3 activate?
Akt, and the rho family of GTPases
What is another name for Atk?
PKB (protein kinase B)
What do the rho GTPases activate?
PI3K
What is the significance of rho GTPases activating PI3K?
This creates a positive feedback loop (PI3K –> PIP3 –> rho GTPases –> PI3K)
What is a PH domain?
Pleckstrin homology domain
What is the purpose of a PH domain?
Allow binding to PIP3 to regulate activity
How does Akt get activated?
By binding to PIP3, being recruited to the cell surface, and being further phosphorylated by PKD
How does Akt get phosphorylated by PKD?
PKD is activated and recruited to the cell surface by PIP3
What pathways are downstream of Akt?
- Inhibition of apoptosis and promotion of cell survival
- Activation of translation (through mTOR)
- Activation of small GTPases
How does PAR-3 get recruited to the cell membrane?
By binding to PIP2
Besides PAR-6 and aPKC, what is bound to PAR-3?
TIAM-1, PIP2, and PTEN (and oligomers)
Why does PAR-3 bind to PTEN?
PTEN creates more PIP2, leading to stronger anchoring of PAR-3 to the cell membrane
How does PAR-3 get recruited to the tight junctions?
By associated with proteins that are critical components of the tight junctions
What is the full scaffold complex seen in cell polarization?
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
What is the full, major positive feedback loop in polarization?
PI3K –> Cdc-42 –> aPKC/PAR-3/PAR-6 –> TIAM-1/Rac-1 –> PI3K
What are some example of rho GTPases activated by PI3K (and PIP3)?
Rac-1 and Cdc-42
What is the significance of PI3K activating rho GTPases?
This creates a positive feedback loop, thus leading to continuous signaling
Why does PAR-3 bind to PIP2 and not PIP3?
PIP3 concentrations are kept fairly low, as a means of negative regulation of PI3K
True or false: TIAM-1 and Rac-1 are found in physical association when they are both in the scaffold
True: Rac-1 associated with the GEF through a physical association
True or false: PAR-6 can oligomerize, similar to PAR-3
True: this leads to robust signaling through recruitment of many scaffolds
True or false: many of the signaling determinants in polarization only function in that pathway
False: many are also upstream of cell growth and survival, development, organelle biogenesis, etc.
What is needed to regulate positive feedback loops?
Negative feedback
How can negative regulation be exerted in the cell?
GAPs, PDEs, and phosphatases
What do GAPs negatively regulate?
GTPases
What do PDEs negatively regulate?
cAMP (and its downstream activators)
What do phosphatases negative regulate?
Kinases (and their products, such as PIP3)
In terms of polarization, which regulation is more well understood?
Positive regulation (as opposed to negative regulation)
What is the phenotype of neurons with deleted PI3K?
No axons, but also no healthy neurites, smaller cells, not as healthy
Why do neurons that have deleted PI3K not look healthy?
PI3K is involved in cell growth and survival as well as neuronal polarization
What is the conclusion of the experiments that deleted PI3K from neurons?
Deleting PI3K may not be the best way to look at the question of neuronal development (is it just because the neurons are sick?)
Why is deleting PI3K not the best way to study neuronal development?
Not sure if the phenotype is due to the kinase, or just that the neurons are not surviving well
Where is aPKC located when it is not bound to PAR-3 and PAR-6?
All over the cell
What do the experiments about inhibiting PI3K suggest about its interactions with the neurites?
PI3K may be involved in proper neurite formation
