2-5: Millard Flashcards
Describe the technique used to visualise Actin Filaments in cells
AND the technique for visualising Actin Dynamics in Migration
PHALLOIDIN binds F-actin but not G-actin -> fluorescently labelled Phalloidin can be used to stain F-actin in cells
GFP-ACTIN fusion protein shows actin dynamics
Are actin filaments NECESSARY for cell migration? How can this be demonstrated?
YES they are - if actin polymerisation is blocked by Cytochalasin D, lamellipodium collapses and cell can’t migrate in that direction
Describe the significance of photobleaching in our understanding of actin dynamics in lamellipodia
When GFP actin in a region near the front of the lamellipodium is photobleached, we see fluorescence recovery at FRONT of lamellipodium as new, non-bleached actin is incorporated (only at front) - all replaced within a minute
If the region BEHIND the lamellipodium is bleached, a wave of dark, bleached monomers appears at the front of the lamellipodium (reinforces first result)
Describe the dynamics of actin polymerisation observed in vitro (e.g., speed, concentrations etc)
Nucleation: occurs spontaneously, but slowly as it is kinetically unfavourable (formation of dimers/trimers/oligomers is slowest part of filament formation)
Elongation: rate of growth proportional to monomer concentration (occurs at both ends)
As monomers are incorporated into filaments, the monomer concentration falls; at Critical Concentration (around 0.1µM in phys. conditions) monomers are added/dissociate at same rate - equilibrium
Describe the dynamics of actin polymerisation observed in CELLS (e.g., speed, concentrations etc) and state why this is different from in vitro
Nucleation: rapid once triggered, but NOT spontaneous
Elongation: rapid but ONLY ONE END; branched network in lamellipodia
Monomer concentration is ~100µM so system NOT IN EQUILIBRIUM (hence rapid growth)
-> ACTIN BINDING PROTEINS control AF assembly/disassembly
Describe the role of profilin and why this explains the different actin dynamic in vivo vs in vitro
Profilin is an Actin Binding Protein that binds monomers, but NOT filaments (most G-actin in cell is bound to it)
Once bound, profilin changes the behaviour of actin:
1. It cannot bind to minus ends of AFs, can only incorporate into PLUS end
2. Readily exchange ADP for ATP
3. Cannot spontaneously nucleate new filaments
If profilin prevents spontaneous nucleation of new filaments in the cell, how does nucleation occur?
The Arp2/3 complex (in its active state) forms a filament “seed” into which profilin-bound actin monomers can be incorporated
Describe how Arp2/3 nucleation occurs
- Arp2 and Arp3 are activated (by various activators - see later lectures) and come together to form a filament “seed”
- Profilin-bound monomers can be incorporated into this seed
- Arp2/3 remains bound to the minus end, while the filament grows at the plus end
Note: Arp2/3 also binds to the sides of existing filaments (70 degree angle) leading to the branched networks seen in lamellipodia
What stops actin filaments from growing indefinitely once nucleated?
Capping protein (CP, or CapZ)! It binds to filament plus ends (usually within 1 second of nucleation)
How can polymerisation at the leading edge and depolymerisation at the minus end be experimentally visualised?
Photobleaching experiments:
-Use a strong laser to destroy fluorescence of GFP-actin at front of lamellipodium
-> newly incorporated fluorescent actin repopulates the entire lamellipodium within a minute
If AFs are capped at the plus end by capping protein, and at the minus end by Arp2/3, how can they be depolymerised?
ADF/Cofilin family (ADF = Actin Depolymerising Factor) promote depolymerisation of older actin
Describe the function of ADF/Cofilin proteins
(Exact mechanism unclear but thought to include the following)
- ADF/Cofilins are localised to cellular regions with high actin turnover, e.g. the leading edge of migrating cells
- They sever AFs and also increase the rate at which actin monomers “fall off” the pointed end
- ADF remains bound to monomers to prevent immediate reincorporation
How can ADF/Cofilins determine which sections of the actin network are “old” and need to be depolymerised?
ATP Hydrolysis acts as a molecular timer:
A few seconds after incorporation, ATP is hydrolysed to ADP, so sections of the network containing lots of ADP are older sections (and these are the sections ADF binds to)
If all dissociated monomers from AFs are bound to ADP, how can they be reincorporated at the plus end?
They bind to profilin, which catalyses the exchange of ADP for ATP
What are filopodia (structure and cellular function)?
Long, thin actin protrusions which act as “sensors” for the cell to explore its environment; they contain unbranched actin filaments, tightly bundled together
How do filopodia form?
The TIP COMPLEX (a group of many actin regulating proteins) associates with the barbed ends of some actin filaments, and prevents them from being capped
-> These filaments converge with each other, and are cross-linked by actin-bundling proteins e.g., FASCIN (shown by knockdown to be essential for filopodia stability)
- Filopodia grow until the tip complex dissociates
What are Actin Bundling Proteins? (And name an example found in each type of actin network)
They are proteins which cross-link and stabilise actin filaments in a network - they have at least 2 F-actin binding sites to allow this
alpha-actinin (in contractile bundles/stress fibres)
filamin (in gel-like networks in the cell cortex)
fascin (in tight parallel bundles in the filopodia)
Which proteins are found in the filopodia tip complex, and what do they do?
- Ena/VASP proteins are thought to prevent Capping Protein from binding plus-ends, and promote filament elongation
- Formins (e.g., dDia2, mDia2) nucleate unbranched actin filaments by binding to the PLUS END; they also promote elongation
What role do Ena/VASP proteins have OUTSIDE of the filopodia?
They regulate filament length in lamellipodial networks:
High Ena = longer filaments with weaker pushing force but faster advance
Low Ena = shorter filaments with stronger force but slower advance
Need the right balance of Ena vs Capping Protein for the environment
If filopodia form via convergence of existing actin filaments - but they can form in the absence of Arp2/3. How?
They can also be nucleated by FORMINS! E.g., DAAM
Knocking down BOTH Arp2/3 and DAAM prevents Filopodia, showing that at least one of these two is necessary
Describe the structure and components of contractile stress fibres
Long, unbranched actin filaments (nucleated by formins)
and the following actin-binding proteins:
- Myosin II (motor protein)
- a-actinin (cross-links filaments to stabilise)
- Tropomyosin (wraps around and prevents AFs from depolymerising)
How does profilin ensure that actin monomers can only incorporate into PLUS ends?
Profilin binds to the opposite face of the monomer from where the ATP-binding cleft is - thus blocking the side that could associate with minus ends, while leaving the plus-end-binding side exposed
Cell migration requries co-ordination of actin dynamics throughout the cell. How does this co-ordination happen?
The Rho-GTPase family are master regulators (Rho, Rac and Cdc42) as they regulate MANY Actin Binding Proteins
What roles for each of the RhoGTPase family proteins were suggested by Alan Hall (and what type of experiments revealed this)?
Injected constitutively active forms of each one into fibroblasts and observed:
Rho -> Stress Fibres
Rac -> Lamellipodia
Cdc42 -> Filopodia
What technique can be used to visualise where GTPases are ACTIVE in the cell (not just where they happen to be)?
Fluorescence Resonance Energy Transfer (FRET):
GFP - Protein that binds to active Rac - Rac - RFP (All in Fusion Protein)
Shine blue light: When Rac is active, brings all 4 proteins close enough together for GFP to activate RFP via resonant energy transfer, so red light emitted
How was it discovered that Rac is responsible for Arp2/3 activation?
It was discovered indirectly via studies on Listeria:
- Listeria can move within host cells by triggering actin polymerisation (“actin comets”)
- This requires ActA, an Arp2/3 activator expressed by Listeria
- ActA contains a Pro-rich domain (binds Ena), a WH2 domain (binds G-actin) and an acidic domain (binds Arp2/3)
- (!)The WH2 and Acidic domains are similar to those found in the N-WASP and WAVE/Scar proteins in humans(!)
How does Rac cause Arp2/3 activation?
Rac activates WAVE, by binding to its inhibitory complex to release it
WAVE then activates Arp2/3 by a similar mechanism to ActA:
- The Acidic domain binds Arp2/3 and induces conformational change to activate it
- The WH2 domain brings a G-actin to begin polymerisation
Besides activating Arp2/3, what other role does Rac have in regulating actin dynamics (and describe the pathway)?
It INactivates ADF/cofilin (via an enzyme pathway) to reduce depolymerisation
Rac activates p21-Activated Kinase (PAK)
-> PAK activates (via Phos) LIM Kinase
-> LIMK inactivates (via Phos) ADF/Cofilin
State the two ways Cdc42 can promote Filopodia formation?
Filopodia formation can be Arp2/3-dependent OR Formin-dependent
Cdc42 can activate BOTH of these!
How does Cdc42 cause Filopodia formation (via Arp2/3)?
Cdc42 binds to a binding site on N-WASP, inducing a conformational change and allowing it to activate Arp2/3
(unclear exactly why Arp2/3 promotes Filo not Lam here, but probably to do with specific ABPs involved and size of area of activation)
How does Cdc42 cause Filopodia formation (via Formins)?
It binds to a binding site on mDia (a formin) inducing a conformational change to expose the nucleation domain
Besides promoting nucleation, what other role of Cdc42 promotes Filopodia formation?
It INactivates ADF/Cofilin (similar to Rac):
Cdc42 activates p21-activated Kinase (PAK)
-> PAK activates (via Phos) LIM Kinase
-> LIMK inactivates (via Phos) ADF/Cofilin
How does Rho promote stress fibre formation?
It activates Myosin II
Rho activates RhoKinase (ROCK), which activates (via Phos) Myosin II (motor activity)
How does Rho promote AF nucleation?
It activates the formin mDia by binding to a GTPase binding site
What does Rho inactivate (and what is the pathway by which it does this)?
It inactivates ADF/Cofilin by a DIFFERENT pathway from Rac and Cdc42
It activates ROCK, which activates (via Phos) LIMK, which inactivates (via Phos) ADF/Cofilin
State the three (mentioned) roles of Rho in actin dynamics
- Activates Myosin II (via ROCK)
- Inactivates ADF/Cofilin (via ROCK)
- Activates mDia
Lecture 4 shows Rac, Cdc42 and Rho as part of a complex regulatory network controlling actin dynamics in the cell. But what, in turn, determines the activity of these RhoGTPases?
Extracellular signals! Ligands (e.g., chemoattractants) bind receptors, or focal adhesions are used to determine ECM stiffness
Signals from hundreds of PM receptors are integrated via >60 GEFs and >70 GAPS, which in turn activates/inactivates the 3 key GTPases
What are the three examples in lecture 5 of biological processes in which cell migration is vital?
Wound Healing, Morphogenesis and Immunity (Hemocytes)
Give some examples of morphogenesis processes that require cell motility
- Creating branches/networks (e.g., in the lungs + vessels)
- Lengthening and shortening tissues (by shuffling cells around)
- Folding tissues (e.g., neural tube closure)
- Epithelial closure (joining two tissue sheets together)
What is epithelial closure, and what are some key examples of it?
The joining together of two tissue sheets (e.g., neural tube closure, palate closure, body wall closure in morphogenesis; also Wound Healing!)
What can result if epithelial closure fails?
Birth defects such as spinda bifida (failure to close neural tube) and cleft palate
What is dorsal closure (and what is its relevance here)?
It is an epithelial closure event that occurs in Drosophila development, in which a hole in the epidermis is sealed up
We can use GFP to visualise dorsal closure in Drosophila and use it to learn about Epithelial Closure more generally
When visualising dorsal closure in Drosophila using GFP experiments, which structures (and proteins) appear to be involved?
Filopodia (and Ena proteins at the tips)
Knocking down Ena -> less filopodia + they are shorter
How do Filopodia facilitate dorsal closure in Drosophila?
They “zipper” the two edges together from the edges of the hole to the centre
Besides Drosophila dorsal closure, what other Epithelial Closure Events are Filopodia shown to be involved in?
Neural tube and eyelid closure in Mice
Which experiments show that filopodia are important for ACCURACY of dorsal closure?
Disrupting Ena OR Cdc42 results in INACCURATE dorsal closure 0 segments don’t match up
Actin segments labelled with different colour fluorescent dyes - if filopodia are disrupted, the colours don’t line up
What does the Magenta/Green fluorescent actin experiment show about the role of filopodia in dorsal closure?
Cells use filopodia to “search” for their matching cell on the opposite epithelial edge - shows filopodia DO also have a sensory role in dorsal closure (more familiar role for filopodia)
What is the role of the contractile Actomyosin cable during dorsal closure (and how can this be demonstrated)?
It keeps the epithelial edge Taut and Organised (Myosin II inactivation -> closure is disorganised and incomplete)
What is observed when a laser is used to make a small wound in a Drosophila embryo?
A contractile Actomyosin cable forms around the wound’s edge, then contracts like a “purse-string” to close the wound (Wound Healing)
How can we demonstrate that the Actomyosin cable is NECESSARY for Wound Healing in Drosophila?
Inactivate RHO -> No activation of Myosin II (via ROCK) or mDia, so no contractile actomyosin -> WOUND DOES NOT CLOSE
Why is Cdc42 required for wound healing as well as Rho?
Cdc42 is necessary for Filopodia to form - they “zipper” or seal up the two edges (similar to their role in Dorsal Closure)
Describe the normal role of Hemocytes
They are migratory immune cells in Drosophila:
They protect against invading microbes, clear up debris and secrete ECM (normally patrol through cell seeking anything they need to remove)
What do hemocytes (normally) do when a laser is used to create a wound in a Drosophila embryo?
They all migrate towards it (visualised using magenta FP)
What changes can be seen in migrating hemocytes when each of the RhoGTPases are knocked out?
Rho deficient: hemocytes elongated as no retraction of rear
Rac deficient: small lamellipodia
Cdc42 deficient: multiple lamellipodia (failure to polarise) - Cdc42 has a guiding role
What impact does knocking down each of the RhoGTPases have on the speed and success of hemocyte migration towards a wound?
Rho deficient: fewer reach the wound
Rac deficient: fewer reach the wound
Cdc42 deficient: hemocytes DO reach the wound, but are slower as they take an indirect route
What is the name of the signalling cascade that is essential for all three processes discussed in Lecture 5 (as well as eyelid closure!)?
Jun Kinase (JNK)
Note: also linked to AP-1
What is a possible reason that wound repair as described in Lecture 5 leads to scarring in adults but not in embryos?
It may be due to the inflammatory response in adults, which does not occur in embryos
