Lecture 10

Question 1

What are the main distinguishing features of the growth cone

Answer 1

Filopodia – long projections lamellae – web-like fanning between projections

Question 2

What is the difference in actin arrangement in the structures of the growth cone

Answer 2

F-actin is bundled together in a polarised fashion in filopodium whereas in the lamellae they are cross-linked to form a net

Question 3

Describe the different structures of the central peripheral and transitional domains of the growth cone

Answer 3

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

Question 4

Explain the actin treadmilling that is seen in the resting growth cone

Answer 4

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

Question 5

Growth cones can turn T or F

Answer 5

F – they don’t turn they reorganise

Question 6

What happens when the growth cone comes into contact with an attractive cue

Answer 6

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

Question 7

When a growth promoting cue is encountered two key components lead to filopodial extension what are these

Answer 7

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

Question 8

Filopodia are not necessarily attached to the substrate but the central domain is T or F

Answer 8

T

Question 9

Attachment of the growth cone to a substrate is enough to drive forward movement T or F

Answer 9

F – need a stimulus or a cue to trigger rearrangement of the cytoskeleton

Question 10

Describe the evidence for a molecular clutch in growth cone extension

Answer 10

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

Question 11

Describe the effects of actin cross linking on engagement of the molecular clutch

Answer 11

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

Question 12

Which family of GTPases are responsible for the coordination of actin cytoskeletal organisation and the control of neuronal morphology movement and polarity

Answer 12

Rho GTPases

Question 13

What two factors are important in the regulation of GTPases

Answer 13

GAPs and GEFs

Question 14

In an inactive state GTPases are bound to GDP what is required to activate signalling

Answer 14

Displacement of the GDP by GTP activates the GTPase and initiates signalling

Question 15

What is the result of the intrinsic nature of GTPases to hydrolyse GTP

Answer 15

Hydrolysis of the bound GTP by the GTPase releases a phosphate and switches it back to an inactive state

Question 16

Nucleotide-free GTPases are extremely energetically favourable T or F

Answer 16

F – its extremely unfavourable

Question 17

What are GEFs and what is the role of these proteins in the cyclic nature of GTPase activity

Answer 17

Guanine nucleotide exchange factors stabilise GTPases in a transition state so that GTP can then bind after GDP release

Question 18

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

Answer 18

GAPs are negative regulators of GTPase signalling as they promote the catalyses of GTP hydrolysis to the inactive GDP-bound form

Question 19

What is the role of guanine nucleotide dissociation inhibitors

Answer 19

GDIs effectively pull the GDP bound GTPases out of the cycle and hold it in the cytoplasm to create a pool of inactive GTPases

Question 20

What changes happen at the molecular level as a result of GTP nucleotide binding to GTPases

Answer 20

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

Question 21

What are the three members of the Rho family of GTPases

Answer 21

RhoA Rac1 and Cdc42

Question 22

What is the role of Cdc42

Answer 22

Cdc42 is a RhoGTPase that controls the polymerisation of actin filaments and the formation of actin spikes or filopodia

Question 23

What is the role of Rac1

Answer 23

Rac1 controls the organisation of new actin filaments particularly branched actin into dynamic ruffling structures or lamellipodia

Question 24

What is the role of RhoA

Answer 24

RhoA stabilises and consolidates actin filaments into a more rigid skeletal framework known as stress fibres

Question 25

Describe a loss of function approach that can be used to elucidate the precise function of GTPases

Answer 25

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.

Question 26

Describe a gain of function approach that can be used to elucidate the precise function of GTPases

Answer 26

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

Question 27

What is the result of microinjection of constitutively active RhoA into quiescent cells

Answer 27

Leads to the formation of stress fibres

Question 28

What is the results of microinjection of dominant negative RhoA into active cells

Answer 28

Leads to the loss of stress fibres

Question 29

What is the result of microinjection of constitutively active Rac or Cdc42 into cells

Answer 29

Leads to the formation of membrane ruffles or filopodia respectively

Question 30

What is the specific role of Rac Cdc42 and RhoA in the regulation of axon growth

Answer 30

Activated Rac and Cdc42 are positive regulators of axon growth whereas activated RhoA is a negative regulator

Question 31

Describe the effects of dominant negative and constitutive active versions of Rho GTPases on neurites

Answer 31

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.

Question 32

Why is it thought that constitutively active Rac or Cdc42 also blocks axon growth

Answer 32

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.

Question 33

Why is it thought that Rho GTPases act as instructive signals in neuronal outgrowth

Answer 33

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.

Question 34

What are the receptors for semaphorins

Answer 34

Plexins

Question 35

Explain how semaphorins guide the growth cone

Answer 35

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

Question 36

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

Answer 36

See completed table below

Question 37

What is the overall effect of Rac on growth cone sensitivity

Answer 37

Rac is acting to desensitise the growth cone to semaphorins

Question 38

Explain why guanine exchange factor regulation of growth cone extension is better suited than the regulation of the Rho GTPases themselves

Answer 38

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

Question 39

Give an example of the GEF regulation of growth cone extension

Answer 39

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

Question 40

Describe the role of Ca2+ in turning growth cones

Answer 40

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

Question 41

What are the five ways in which RhoGTPases can modulate the cytoskeleton in order to influence axon guidance?

Answer 41

* **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