Lecture 10 Flashcards
What are the main distinguishing features of the growth cone
Filopodia – long projections lamellae – web-like fanning between projections
What is the difference in actin arrangement in the structures of the growth cone
F-actin is bundled together in a polarised fashion in filopodium whereas in the lamellae they are cross-linked to form a net
Describe the different structures of the central peripheral and transitional domains of the growth cone
The central zone or base is a microtubule rich domain. The regional furthest from central domain is the F-actin rich peripheral domain that possesses the filopodia. The transitional zone in the middle of central and peripheral domains
Explain the actin treadmilling that is seen in the resting growth cone
F-actin subunits are added at the peripheral zone move through the microfilament and are removed at the central zone. Tubulin is sporadically dragged into the filopodia
Growth cones can turn T or F
F – they don’t turn they reorganise
What happens when the growth cone comes into contact with an attractive cue
F-actin treadmilling slows down and F-actin begins to accumulate which stabilises the filopodia. A molecular clutch engages the extension over the substrate and an actin-tubulin link pulls the microtubules into the wake of the extending filopodium
When a growth promoting cue is encountered two key components lead to filopodial extension what are these
A molecular clutch is engaged and rearward actin treadmilling slows down. Next an actin-tubulin links pulls the microtubules into the wake of the extending filopodium
Filopodia are not necessarily attached to the substrate but the central domain is T or F
T
Attachment of the growth cone to a substrate is enough to drive forward movement T or F
F – need a stimulus or a cue to trigger rearrangement of the cytoskeleton
Describe the evidence for a molecular clutch in growth cone extension
Micropatterned substrate containing dots of N-cadherin was generated. Neurons were then transfected with an N-cadherin-GFP fusion protein and allowed to move over the substrate. N-cadherin in the cells will bind homophilically to the N-cadherin dots on the substrate. It was observed the following extension of the growth cone the labelled GFP-N-cadherin fusion proteins become localised to the regions of the substrate where the N-cadherin dots were indicating a link between the extracellular environment and the cytoskeleton
Describe the effects of actin cross linking on engagement of the molecular clutch
The clutch could also be controlled by actin cross-linking. Uncrosslinked F-actin has little strength with no net movement resulting in treadmilling. In contrast branched and crosslinked F-actin has strength to push membrane forward
Which family of GTPases are responsible for the coordination of actin cytoskeletal organisation and the control of neuronal morphology movement and polarity
Rho GTPases
What two factors are important in the regulation of GTPases
GAPs and GEFs
In an inactive state GTPases are bound to GDP what is required to activate signalling
Displacement of the GDP by GTP activates the GTPase and initiates signalling
What is the result of the intrinsic nature of GTPases to hydrolyse GTP
Hydrolysis of the bound GTP by the GTPase releases a phosphate and switches it back to an inactive state
Nucleotide-free GTPases are extremely energetically favourable T or F
F – its extremely unfavourable
What are GEFs and what is the role of these proteins in the cyclic nature of GTPase activity
Guanine nucleotide exchange factors stabilise GTPases in a transition state so that GTP can then bind after GDP release
GTPase activating proteins are responsible for catalysing the hydrolysis of the GTP bound to GTPases thus do they act as positive or negative regulators of GTPase signalling
GAPs are negative regulators of GTPase signalling as they promote the catalyses of GTP hydrolysis to the inactive GDP-bound form
What is the role of guanine nucleotide dissociation inhibitors
GDIs effectively pull the GDP bound GTPases out of the cycle and hold it in the cytoplasm to create a pool of inactive GTPases
What changes happen at the molecular level as a result of GTP nucleotide binding to GTPases
This causes a very small conformational change dictated by the presence of a final phosphate that changes the orientation of the switch 1 and switch 2 domains. This leads to an activation of signalling
What are the three members of the Rho family of GTPases
RhoA Rac1 and Cdc42
What is the role of Cdc42
Cdc42 is a RhoGTPase that controls the polymerisation of actin filaments and the formation of actin spikes or filopodia
What is the role of Rac1
Rac1 controls the organisation of new actin filaments particularly branched actin into dynamic ruffling structures or lamellipodia
What is the role of RhoA
RhoA stabilises and consolidates actin filaments into a more rigid skeletal framework known as stress fibres
Describe a loss of function approach that can be used to elucidate the precise function of GTPases
Create a dominant negative mutant GTPase with a point mutation in the nucleotide-binding site. This will result in a GTPase that is always off and inhibitory due to never binding to GTP. The dominant negative effect of this mutant is due to its binding to and mopping up of active GEFs to prevent their action on functioning GTPases. By binding to these inhibitory mutant GTPases the GEFs are no longer available to activate other functions wild type GTPases.
Describe a gain of function approach that can be used to elucidate the precise function of GTPases
Create a constitutively active GTPase mutant that is always on and remains in the GTP-bound form. This will perturb GTP hydrolysis and creates an always active GTPase
What is the result of microinjection of constitutively active RhoA into quiescent cells
Leads to the formation of stress fibres
What is the results of microinjection of dominant negative RhoA into active cells
Leads to the loss of stress fibres
What is the result of microinjection of constitutively active Rac or Cdc42 into cells
Leads to the formation of membrane ruffles or filopodia respectively
What is the specific role of Rac Cdc42 and RhoA in the regulation of axon growth
Activated Rac and Cdc42 are positive regulators of axon growth whereas activated RhoA is a negative regulator
Describe the effects of dominant negative and constitutive active versions of Rho GTPases on neurites
The expression of constitutively active RhoA causes neurite retraction whilst dominant negative RhoA blocks collapse response. By expression a dominant negative form of Cdc42 you can block formation of both dendrites and axons. Dominant negative Rac affects only reduces axonal not dendrite growth.
Why is it thought that constitutively active Rac or Cdc42 also blocks axon growth
It is like that this is because the disassembly of the cytoskeleton is prevented but this is actually vital to assemble new elements and form axons.
Why is it thought that Rho GTPases act as instructive signals in neuronal outgrowth
Factors that collapse growth cones activate RhoA and downregulate Rac. In addition several guidance factor receptors either bind and modulate Rho GTPases directly or bind to GEFs/GAPs which regulate Rho GTPases.
What are the receptors for semaphorins
Plexins
Explain how semaphorins guide the growth cone
Normally when there isn’t semaphorins around the amount of Rac exceeds the amount of RhoA resulting in growth cone extension. Semaphorins bind to their receptor causing dimerization that sequesters a proportion of the Rac leading to growth cone collapse due to increased proportion of RhoA
Below is a table listing changes to the relative levels of Plexin B Rac and RhoA. Fill in the table to describe the changes in sensitivity of motor axons to semaphorins
See completed table below
What is the overall effect of Rac on growth cone sensitivity
Rac is acting to desensitise the growth cone to semaphorins
Explain why guanine exchange factor regulation of growth cone extension is better suited than the regulation of the Rho GTPases themselves
The Rho GTPase family is relatively small and members are broadly expressed. In contrast GEF and GAP families are much larger and more restricted in their expression. This suggests that regulation via GEFs and GAPs of these would afford much greater specificity
Give an example of the GEF regulation of growth cone extension
The inhibitory guidance cues ephrins effect the growth cone by signalling thought the GEF ephexin. This turns of RhoA and turns Rac and Cdc42 off hence leading to growth cone collapse
Describe the role of Ca2+ in turning growth cones
Growth cones exhibit transient fluxes in intracellular Ca2+ as they navigate in vivo and in vitro. The induction of local Ca2+ transient currents can also turn a growth cone. Ryanodine-induced release of Ca2+ from intracellular stores can attract growth cones by activating Rac/cdc42 and suppressing RhoA
What are the five ways in which RhoGTPases can modulate the cytoskeleton in order to influence axon guidance?
- Filament disassembly – activated by Cofilin
- Branching - initiated by Arp2/3 in response to activation by WASp proteins
- Termination of branch extension by capping proteins
- Filament assembly – regulated by Profilin and Thymosin
- Actomyosin contractility – essentially crosslinking between branched structures
