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A BMS 381 - Developmental neurobiology > Rat sensory growth cones in vitro > Flashcards

Rat sensory growth cones in vitro Flashcards

1
Q

What is the anatomy of the growth cone?

A

3 domains:

  • Central
  • Transitional
  • Peripheral
  • Lamellapodium
  • Filopodium
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2
Q

What is the difference between lamellapodia and filopodia?

A

Made of different kinds of F-actin:

  • Lamella - actin bundles are CROSSLINKED into a NET
  • Filopodia - actin bundles are POLARISED to form larger bundles
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3
Q

What happens to actin in a resting growth cone?

A

Actin is added at the ENDS of the filopodia and ‘treadmills’ back towards the central domain (actin is severed in the central domain)

Tubulin (from the central domain) is dragged sporadically into the filopodia

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4
Q

What is microtubule capture?

A

Sporadic movement of tubuliin into the filopodia

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5
Q

When does microtubule capture happen more dramatically?

A

When the growth cone comes into contact with an ATTRACTIVE CUE

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6
Q

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

A

It REORGANISES itself to establish a new direction:

1) F-actin treadmilling SLOWS
2) F-actin ACCUMULATION

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7
Q

What does F-actin accumulation cause?

A

Filopodium to be STABILISED

Microtubule capture

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8
Q

What part of the growth cone is/isn’t attached to the substrate?

A

Is attached by:
The central domain (palm)

Not attached:
Filopodia

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9
Q

Is attachment of the growth cone to the substrate enough to drive the growth cone forwards?

A

NO - need stimulus of cue to rearrange cytoskeleton

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10
Q

What 2 things lead to reorientation of microtubules and filopodia extension in the growth cone?

A

1) MOLECULAR clutch

2) Actomyosin-based actin-tubilin link

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11
Q

What does the molecular clutch do?

A

Slows rearward actin treadmillling
Forward movement of filopodium

Microtubules extend up the back of the filopodium

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12
Q

What does the actomyosin-based actin-tubulin link do?

A

Captures microtubules in the wake of the extending filopodium - to reorganise them

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13
Q

What is the evidence for the molecular clutch?

What does this show?

A

1) N-cadherin and cytophobic agent in a dish and put neurons over the top

N-cadherin-GFP in the neuron

Where neuron sticks to Ncad in the dish - bright colour

2) Labelled actin in the cell - allows to track what happens to actin over the point of contact with the substrate

Shows:
- Actin treadmills back but SLOWS DOWN/STATIONARY where there is Ncad

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14
Q

What is Ncad?

A

An adhesion molecule (cadherin)

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15
Q

Does the clutch require F-actin to be joined directly to the substrate?

A

Not necessarily

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16
Q

Describe uncrosslinked F-actin

A

In the lamella:
Little strength
No net movement
Treadmilling occurs

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17
Q

What happens when encounter an attractive cue in the lamellopodia?

A
  • Drives a signal
  • Activates intracellular CROSS-LINKING protiens via SECONDARY MESSENGERS
  • Cross-linking stops the treadmilling of the fibres - pushes the membrane out –> membrane moves FORWARDS
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18
Q

What is the crosslinking protein in the growth cone?

A

Likely to be a myosin

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19
Q

What are the 2 alternative ideas for how the clutch works?

A

1) Physical cross-linking of the membrane receptor to the actin cytoskeleton
2) Secondary messengers and cross-linking agents

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20
Q

What is sempahorin?

A

A NEGATIVE cue that causes growth cone collapse (destabilisation of the F-actin)

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21
Q

What are Rho GTPases?

What are they important in?

A

MOLECULAR SWITCHES that respond to EXTRACELLULAR signals

Important in regulating the actin cytoskeleton

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22
Q

What is the ON state of a Rho GTPase?

What is the OFF state?

A

ON - bound to GTP

OFF - bound to GDP

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23
Q

What molecule turns GTPases ON?

A

GEF (Guanine nucleotide exchange factor)

Donates a phosphate –> converts bound GDP to GTP

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24
Q

What molecule turns GTPases OFF?

A

GTPase activating protein!! (GAP)

(GTPase function is to convert GTP–>GDP, so if activated, GTP is turned to GDP, GTPase bound to GDP is OFF)

(activates the cleavage of bound GTP –> GDP)

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25
Q

What effects do RhoGTPases have on the actin cytoskeleton?

A

Depends on which Rho GTPase is activated:

  • RhoA
  • Rac
  • Cdc42
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26
Q

Why are Rho GTPases called RHO??

A

Because one of their members is called Rho

But have many different other members that aren’t called Rho

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27
Q

What happens when CA RhoA?

A

Stress fibre formation

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28
Q

What happens when CA Rac?

A

Lamellipodia formation

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29
Q

What happens when CA Cdc42?

A

Filopodia formation

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30
Q

What happens when DN RhoA, Rac or Cdc42?

How?

A

SUPRESS formation of their structures

By COMPETING with the normal Rho-GTPases, turning them OFF

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31
Q

What does modulation of GTPases do?

A

Control axon growth

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32
Q

What RhoGTPase(s) are POSITIVE regulators of axon growth when they are ON?

A

Rac and Cdc42

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33
Q

What RhoGTPase(s) are NEGATIVE regulators of axon growth when they are ON?

A

RhoA

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34
Q

What is a positive regulator of axon growth?

Negative regulator?

A

Positive - STIMULATES axons growth

Negative - INHIBITS axon growth

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35
Q

What does DN RhoA cause?

A

Neurite retraction

36
Q

What does DN Cdc42 cause?

A

BLOCKS formation of BOTH dendrites and axons

37
Q

What does DN Rac cause?

A

BLOCKS formation of AXONAL growth

38
Q

What is unusual about CA Rac/Cdc42?

What does this suggest?

A

Also BLOCKS formation of AXON growth (even though DN block axon growth)

Suggests structures whose formation cdc42 and rac normally promote may need to be disassembled as well as assembled for ordinary growth!!!

39
Q

Where is assembly and dissembly for normal growth seen?

A

1) Actin treadmilling (addition of F-actin at one end, disassembly at the other)
2) During guidance - f-actin is broken down in the direction the growth cone is no longer travelling in and used for growth in the new direction

40
Q

How does DN/CA rac/cdc42 block growth?

A

CA - blocks disassembly

DN - blocks assembly

41
Q

Does evidence indicate that RhoGTPases are involved in guiding axons (instructive) or just allow axons to grow (permissive)

A

INSTRUCTIVE

42
Q

What is the relationship between Rac and RhoA in growth cone turning?

A

BALANCE between the two determined growth cone turning

43
Q

How do factors that COLLAPSE the growth cone affect Rac and RhoA?

A

ACTIVATE RhoA

DOWNREGULATE Rac

44
Q

How do factors that EXTEND the growth cone affect Rac and RhoA?

A

DOWNREGULATE RhoA

ACTIVATE Rac

45
Q

Which 2 ways do guidance factor receptors regulate Rho GTPases?

A

1) Bind DIRECTLY and modulate the GTPase

2) Bind to GEFs and GAPs, which regulate the GTPase

46
Q

What is plexin B?

A

Component of the semaphorin receptor

Regulates Rac by directly binding to it

47
Q

How are semaphorins used in flies?

A

They are expressed by some muscles but not others - to guide innervation by motor neurons

48
Q

What muscles do motor axons AVOID in the flies?

A

Avoid muscles that make the semaphorins

49
Q

What muscles do motor axons INNERVATE in the flies?

A

Muscles that don’t make the semaphorins

50
Q

What happens to the motor neurons when increase plexin on motor neuron axons?

A

Become more sensitive to sema in the environment

Fail to innervate muscle

51
Q

What happens to the motor neurons when DECREASE rac?

A

More sensitive to sema (repulsive cue)

Fail to innervate muscle

52
Q

What happens to the motor neurons when INCREASE rac?

A

LESS sensitive to sema

Innervate muscle

53
Q

What happens to the motor neurons when DECREASE RhoA?

A

Less sensitive to sema

Innervate muscle

54
Q

What does it mean if something is INSTRUCTIVE in guiding axons?

A

They GUIDE axons:
Cues can directly switch them on/off
(downstream of attractive/repulsive cue)

55
Q

What does it mean if something is PERMISSIVE in guiding axons?

A

They allow axon growth in general:

- Upstream of the attractive/repulsive cues

56
Q

What is the model for sema signalling?

A
  • Sema binds Plexin receptor
  • Receptor gets crosslinked
  • Receptor sequesters Rac in the environment
  • Less free Rac in the environment
  • More RhoA than free Rac
  • Growth cone COLLAPSE
57
Q

When there is more Rac than RhoA what happens to the GC?

A

Extension

58
Q

When there is more RhoA than Rac what happens to the GC?

A

Collapse

59
Q

What determines the outcome of the growth cone?

A

BALANCE between free RhoA and Rac

60
Q

How many members of the Rho GTPase family?

What does this mean?

A

6 in fly
20 in humans

Very small - hard to confer specificity to pathways

61
Q

Why would signalling through GEFs and GAPs allow more regulation and specificity of pathways than just using Rho GTPases?

A

Much larger family

More RESTRICTED expression (GTPases have broad expression)

62
Q

What are ephrins?

A

INHIBITORY cues

63
Q

How do ephrins ultimately regulate RhoA, Rac and cdc42?

A

Signal via GEF called Ephexin, which regulates RhoA, Rac and Cdc42

64
Q

How does Epherin control RhoA, Rac and cdc42?

A

TURNS OFF Rac and cdc42

TURNS ON RhoA

65
Q

What secondary messenger may guidance cues act through?

How?

A

Calcium

Affects the Rac/RhoA balance

66
Q

How does calcium effect growth cones?

A

Localised calcium flux TURNS growth cones

Growth cones turn towards flux of calcium

67
Q

What happens to intracellular calcium as the growth cone navigates in vitro/vivo?

A

Transient fluxes in calcium - happens constantly in response to cues in the environment

68
Q

What visualises calcium in a neuron?

A

Fluo-3 dye

69
Q

What happens when a growth cone is treated with ryanodine?

Why?

A

Growth cone turns towards ryanodine:

  • Ryanodine causes release of calcium from stores
  • Calcium flux occurs
  • Ryanodine activates Rac/cdc42
  • Ryanodine supressed RhoA
70
Q

How guidance cues modulate the actin cytoskeleton? (5 ways)

A

1) Filament disassembly
2) Branching
3) Termination of branch extension
4) Filament assembly
5) Actomyosin contractility

71
Q

What are the known point of input into the actin cytoskeleton for guidance cues?

A

Through RhoGEFs and RhoGAPs which control Rho, RacA and cdc42

72
Q

What is filament disassembly activated by?

A

Cofilin

73
Q

What is branching initiated by?

A

Arp2/3 in response to activation by WAVE/WASP proteins

74
Q

What is termination of branch extension caused by?

A

Capping proteins

75
Q

What is filament assembly regulated by?

A

Profilin and Thymosin

76
Q

What determines actomyosin contractility?

A

Cross-linking between branched structures

77
Q

What is the evidence that MT may be involved earlier than actin in movement of the growth cone?

A

When put guidance cue on growth cone - IMMEDIATELY tubulin accumulates underneath

78
Q

What is the role of MT in growth cone guidance?

A

Bring in important elements (eg. signal transduction elements, factors required for membrane protrusion) into the are of contact with the cue

Bring these elements into the filopodia

Elements important in response of the filopodia to cues

79
Q

What ‘goodies’ are brought into the filopodia by the microtubules?

A

Monomeric actin

Collapsin Response Mediator Protein (CRMP)

Transduction elements

80
Q

What does CRMP do?

A

Promotes MT polymerisation and drives WASP complex proteins into the filopodia

Promotes actin crosslinking

81
Q

What controls the growth cone behaviour?

A

Intimate interaction of pathways that control actin cytoskeleton and pathways that control the MT

82
Q

What is Rac signalling important in?

How?

A

Establishment of polarity

Is involved in the PI3K responses

83
Q

How does cdc42 act in a feedback loop?

A
  • Ras activates PI3K
  • PI3K activates PIP3 levels
  • PIP3 activates Cdc42
  • Cdc42 has effect on the Par3 complex
84
Q

How else does sem3A effect polarity?

A

Via cGMP/cAMP

85
Q

What does the cGMP/cAMP ratio affect?

A

Responses to guidance cues

86
Q

What are axon guidance cues a continuation of?

How?

A

The polarity process

Attraction enhances axonal polarity, repulsion inhibits it

A BMS 381 - Developmental neurobiology (17 decks)

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