Furley 1

Question 1

What is the characteristic structure of axons compared to dendrites

Answer 1

Axons have highly polarised microtubules compared to dendrites. Also have a set of microtubule associated proteins that cross link microtubules so they are all highly polarised in their direction. Dendrites have microtubules, however they are much less ordered and have mixed orientations.

Question 2

What is the function of MAP2

Answer 2

MAP2 is a microtubule associated protein the induces cross linking and stabilisation of MTs found more specifically in dendrites

Question 3

What is the function of Tau-1

Answer 3

Same as MAP2 but found mainly in the developing axon

Question 4

What is L1 and how can it be used experimentally

Answer 4

Cell surface adhesion molecule that is restricted to axons. Added to the axon at the growth cone. Used as a marker for the developing axon

Question 5

What is GluR1 and how can it be used experimentally

Answer 5

glutamate receptor component that is restricted to the cell body and dendrites - used in conjunction with L1 to show that there is compartmentalisation in neurons.

Question 6

What is the experiment used to show restriction of molecules across the compartmentalised boundaries shown by L1/GluR1

Answer 6

Beads coated with antibodies to L1/GluR1 placed either on axonal or somatodendritic domains and then dragged using optical tweezers. See how easy it is to move/if it is capable of moving across a boundary. L1 beads cannot be easily dragged into the cell body whereas GluR1 beads can be dragged into dendrites from the cell body

Question 7

How was initial neuronal polarity observed

Answer 7

Stages in neurite outgrowth and polarisation observed in rat embryonic hippocampal cells.

Question 8

How can the developing axon be marked for

Answer 8

Because microtubules are polarised in axons, GFP labelled kinesins (plus end directed) mark where the axon is forming

Question 9

What is characteristic of a tyrosinated growth cone

Answer 9

Contains dynamic MTs

Question 10

What is characteristic of acetylated growth cones

Answer 10

Stabilised MTs, are found in the newly polarised axon

Question 11

What is taxol and how is used experimentally

Answer 11

Taxol is a MT stabilising drug. Used to show that MT stabilisation was sufficient to induce its selection as the future axon.

Question 12

What happens if the newly polarised axon is cut

Answer 12

A new axon is specified.

Question 13

What is the evidence that a positive feedback mechanism is required for axon selectivity

Answer 13

Overexpression of HRas or PI3K activation results in multiple axons
HRas induction of multiple axons is blocked by a PI3K inhibitor (HRas works through PI3K).
HRas accumulates in growth cones so symmetry is broken (once the axon is specified) in a PI3K dependent manner
HRas is depleted from non-axonal regions and shipped toward the growth cone of the developing axon.

Question 14

What are the downstream targets of PI3K that induce axon selection

Answer 14

PI3K converts PIP2 to PIP3. PIP3 activates Akt - there is evidence that phosphorylated Akt is found in nascent axons but not naive neurites.
Akt and PIP3 inhibit GSK3B action which is to prevent microtubule stabilisation by modulating MAP activities.

Question 15

What happens if you experimentally inhibit the action of GSK3b

Answer 15

Results in multiple axons

Question 16

What is the role of the Par 3 complex

Answer 16

required to establish polarity after sperm entry, partition the C elegans giving asymmetrical cell divisions.
Initiates polarity through the mutually antagonistic interactions with Par1/2. This affects microtubule organisation which deferentially localises components of the cell. Found at the growth cone

Question 17

What are SAD kinases

Answer 17

Related to PAR kinases
A loss of SAD kinase activity results in failure to acetylated tubulin to predominate tyrosinated tubulin leading to the loss of axons

Question 18

What is LKB1

Answer 18

A kinase which when lost, also results in failure to make axons

Question 19

Why are studies of hippocampal neurons not completely accurate

Answer 19

All done in vitro - neurons are born in a complex environment with ECM and other cells

Question 20

What is the role of LKB1 in axon initiation in vivo

Answer 20

LKB1 is phosphorylated by PKA - when active it in turn activates SAD kinases which affect microtubule associated protein stability and axon initiation.

Question 21

What is the role of Par-3 complex in axon initiation in vivo

Answer 21

Par-3 complex and PI3K inhibit the inhibition of axon formation by GSK3B, also affecting MAPs.

Question 22

What effect does non phosphorylated LKB1 have on the Par-3 complex

Answer 22

Inhibits the action of the Par-3 complex

Loss of LTB1 in the cortex therefore sees a loss in axon initiation (done by in utero electroporation of siRNA in mice.

Question 23

What is the role of TGFB

Answer 23

expressed in the ventricular zone of the developing cortex. TGFB can initiate axons in vitro, Knock downs do not develop axons in vitro or vivo
Extracellular initiator of polarity

Question 24

What is the role of the extracellular cue - Sema3A

Answer 24

Expressed in a gradient from basal to apical and attracts dendrites basally. It also promotes dendrite formation at the expense of axons in vitro,
Sema3A increases cGMP and suppresses cAMP, thus inhibiting PKA phosphorhylation of LKB1 and GSK3B. Manipulation of cAMP and cGMP have opposing effects on axon and dendrite formation.