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Question 1

What molecule makes up lemallapodiu and filopodium

Answer 1

Different types of F-actin

Question 2

How is the F-actin structured in lamella

Answer 2

Actin bundles are cross linked into a net

Question 3

How is F-actin structured in filopodia

Answer 3

Actin bundles are polarised to form larger bundles

Question 4

What process does F-actin undergo in the resting growth cone

Answer 4

Treadmilling - tubulin is dragged sporadically into the filopodia, this happens much more dramatically when the growth cone comes into contact with an attractive cue

Question 5

What 2 things happen when a growth cone comes into contact with an attractive cue

Answer 5

1) F-actin treadmilling slows (attenuated) this results in the accumulation of F-actin
2) F-actin accumulation stabilises the filopodium and drags microtubules into the back of the filopodium (microtubule capture)

Question 6

What is the model for how control of actin treadmilling may control filopodia extension

Answer 6

Attachment of the GC to a substrate is not enough to drive forward movement need stimulus of cue to rearrange cytoskeleton
As growth cone promoting cue is encountered two things happen
1) A molecular clutch is engaged and rearward actin treadmilling slows, this results in forward movement of the filopodium
2) An actomyosin-based tubulin link captures MTs in the wake of the extending filopodium

Question 7

What is the evidence for the clutch

Answer 7

N-cadherin-FC micropatterned
N-Cadherin-GFP transfected neurons are added
You can see over the dots evidence for N-cadherin binding as expected.
OR
Tracking of individual actin molecules. Where N-cadherin is placed there is arresting of some actin molecules. Where the actin is not over N-cadherin, the speed in which they’re moving is much faster and toward the C terminus.

Question 8

What else could be controlling the clutch

Answer 8

Actin cross linking
Clutch may not require F-actin joining to the substrate especially in the lamella
Uncrosslinked F-actin has little strength: No not movement = treadmilling
Branched and crosslinked F-actin has strength to push the membrane forward = less retrograde flow
Crosslinking is myosin mediated, could be relevant to lamella while substrate crosslinking to filopodia

Question 9

What are Rho GTPases

Answer 9

Molecular switches that respond to extracellular signals

Question 10

What is a GAP

Answer 10

GTPase activating protein

Question 11

What is a GEF

Answer 11

Guanine exchange factor

Question 12

What is the role of GAP

Answer 12

puts RhoGTPase into an off state by activating cleavage of bound GTP to GDP

Question 13

What is the role of GEF’s

Answer 13

Puts RhoGTPase into an on state by donating a phosphate to convert bound GDP to GTP

Question 14

What effect does Rho GTPase have on actin based structures

Answer 14

When constitutively active in fibroblasts.
RhoA induces stress fibres (structures associated with cell attachment)
Rac induces lamellipodia
Cdc42 induces filopodium
Dominant negative version suppresses formation of these structures

Question 15

What is the result of a dominant negative RhoGTPase

Answer 15

Always off and compete with normal RhoGTPases effectively turning them off too.

Question 16

What is the general model for axon growth controlled by Rac, Cdc42 and RhoA

Answer 16

When activated, Rac and Cdc42 seem to be positive regulators of axon growth, whereas activated RhoA is a negative regulator.

Question 17

What experiments showed the model for Rac, Cdc42 and RhoA action

Answer 17

Expression of constitutively active RhoA causes neurite retraction, whereas as a dominant negative blocks the collapse response
DN-Cdc42 blocks formation of both dendrites and axons
DN-Rac effects only axonal, not dendrite growth
Interestingly, CA Rac or Cdc42 blocks axonal growth

Question 18

Why do models with CA Rac or Cdc42 prevent axon growth

Answer 18

Structures whose formation they promote must also be able to be disassembled in order for growth.

Question 19

How were RhoGTPases shown to be instructive

Answer 19

Factors that collapse growth cones activate RhoA and downregulate Rac.

Question 20

How do guidance factor receptors interact with RhoGTPases

Answer 20

either 1) Bind and modulate GTPases directly

OR 2) Bind to GEFs or GAPs which regulate RhoGTPases.

Question 21

What are plexins

Answer 21

Receptors for semaphorins

Question 22

What is the role of semaphorins in flies

Answer 22

Semaphorins are expressed in certain muscles, to guide innervation of motor neurons. Changing relative levels of Plexin B, Rac and RhoA changes the sensitivity of motor axons to the Semas.

Question 23

What is the result of more plexin

Answer 23

More sensitive

Question 24

What is the result of less Rac

Answer 24

More sensitive

Question 25

What is the result of less RhoA

Answer 25

Less sensitive

Question 26

What is the current model for sema signalling considering direct binding of Plexin B to Rac

Answer 26

After addition of semaphorin. Plexin B sequesters Rac, this leads to an increase in free RhoA and leads to growth cone collapse. Compared to when there is no semaphorin where free Rac outweighs free RhoA

Question 27

Why would regulation of growth via GEFs and GAPs afford greater specificity

Answer 27

The Rho GTPase family is relatively small (20 in human) compared to GEF and GAP families which contain many more.

Question 28

Which GEF do ephrins signal through

Answer 28

Signal via the GEF, ephexin, which simultaneously regulates RhoA, Rac and Cdc42.

Question 29

What molecules transient flux can turn growth cones

Answer 29

Localised calcium flux

Ryanodine induced release of calcium stores can attract growth cones

Question 30

What is the action of calcium on the growth cone

Answer 30

Activates Rac and Cdc42, whilst inhibiting RhoA

Question 31

How do RhoGTPases modulate the actin cytoskeleton

Answer 31

1) Filament disassembly activated by cofilin
2) Branching initiated by Arp2/3 in response to activation by WASP proteins
3) Termination of branch extensions by capping proteins
4) Filament assembly regulated by profilin and thymosin
5) Actomyosin contractility essentially crosslinking between branched structures

Question 32

What did a bead experiment suggest about MTs role in growth cone extension

Answer 32

MTs were seen to invade the contact site before the onset of cessation of retrograde flow. Suggests that first MTs must uncouple from F-actin
Cue contact thought to stimulate uncoupling of MTs followed by filopodia invasion by MTs
Invading Mts bring in important signal transduction elements. Some RhoGEFs are known to associate with MTs at plus end tracking proteins (TIPS)

Question 33

What is the role of CSPG and where is it found as a result

Answer 33

chondroitin sulphate proteoglycans inhibit laminin induced outgrowth of cultured cells. CSPGs are also expressed in the roof plate of the neural tube and inhibit the crossing of axons as a result. After CNS lesions CSPGs are upregulated in these areas limiting axon regeneration after damage.