Neurobio Test2: Recitation 4

Question 1

What is the main question addressed in this paper?

Answer 1

Do vertebrates have a synapse forming protein with:

• extracellular Ig domains
• intracellular PDZ domain-binding domain like invertebrates?

Question 2

Why was the main question asked?

Answer 2

What is the missing protein to explain synapse formation?

Question 3

What characteristics of the Ig domain proteins were the authors looking for in the sequence databank search? Why?

Answer 3

• extracellular IG domains
• intracellular PDZ-domain protein interaction sequence Proteins like that found in invertebrates which perform functions in synaptic cell adhesion.

Question 4

Structural properties of SynCAM?

Answer 4

3 extracellular Ig domains
1 TM sequence
PDZ-domain protein interaction sequence

Question 5

Point of 1b? How are the presence of SynCAM mRNA but absence of protein justified?

Answer 5

Expression of SynCAM limited to brain.

Posttranscriptional mechanisms restrict SynCAM expression to brain.

Question 6

Principle behind western blotting?

Answer 6

• separate proteins based on weight through gel electrophoresis
• visualize with tagged antibodies.

Question 7

Why multiple bands in western blot?

Answer 7

Different levels of glycosylation

Question 8

What is the significance of this statement: “co-expression of SynCAM with CASK, recruited CASK from the cytosol to the membrane”

Answer 8

CASK has PDZ-binding domain

Question 9

What questions are addressed in figure 2?

Answer 9

Will SynCAM bind SynCAM? Is it a homophilic protein?

Question 10

Affinity chromatography

Answer 10

Substance eluted through column containing immobilized ligand

Question 11

Describe four different methods the authors use to show that SynCAM is localized to synapses.

Answer 11

DISQ

1. Immunoelectron microscopy
2. Colocalization of synaptic vesicle protein and SynCAM revealed by fluorescence
3. Enriched in plasma membranes also containing synaptic proteins
4. Quantified distribution of signals obtained with SynCAM antiserum and control sera to rule out artifacts of immunoelectron microscopy

Question 12

How do the authors determine if SynCAM is required for synapse formation, synaptic transmission, or both?

Answer 12

1. Recorded spontaneous mini synaptic currents from hippocamplus neurons expressing full-length SynCAM, SynCAM w/o Ig binding domain, and vector alone.
2. Put in SynCAM and just the cytoplasmic tail of SynCAM
3. FM dyes in membrane taken up in endocytosis

Question 13

Does expression of SynCAM in non-neuronal cells induce synapse formation? How do the authors test this?

Answer 13

Yes: coculuture embryonic kidney cells with nerve cells and look for evidence of synapse formation and function

Question 14

What experimental evidences support the role of SynCAM in glutamatergic synaptic transmission? Identify some possible caveats.

Answer 14

• 293 cells needed to have both glutamate receptor and SynCAM to produce current.
• Current in post-synaptic cell decreased with addition of tetrodotoxin (blocks sodium channel, so indicates current was generated by AP)
• Glutamate receptors usually opposite presynaptic terminal

Question 15

The function of SynCAM is Ca2+ independent. What are the experimental evidences that support this statement?

Answer 15

affinity chromatography assay worked equally well with and without calcium.

Question 16

What are the experimental evidences that suggest that the IgG domains of SynCAM protein are important for trans-synaptic interactions?

Answer 16

MADE

1. Minis: stimulation of minis in neurons transfected with full-length SynCAM
2. Adhesion: cell adhesion in S2 cells transfected with full-length SynCAM
3. Dye: Dye in membrane reveals induction of quantitatively normal release properties of nerve terminals only seen in cells with full-length SyCAM
4. Embryonic cells: Synapse formation by embryonic kidney cells

Question 17

What is the significance of Calcium and salt in Figure 2?

Answer 17

Looking to see if SynCAM Ca++ independent since synaptic cell adhesion is Ca++ independent.

Question 18

Over-expression of SynCAM led to an increased frequency of miniature synaptic currents (post-synaptic), but not amplitude. How did the authors explain this?

Answer 18

Amplitude relates to # of post-synaptic receptors

Frequency relates to # of synapses/ release probability
“freqINcy - sINapse”

Question 19

How did the authors select SynCAM as the candidate for synapse formation?

Answer 19

Has extraceeular Ig-binding domain and intracellular PDZ-domain protein-interaction sequence like invertebrate protein involved in synapse formation.

Question 20

What is the significance of using a dominant negative fragment of SynCAM on presynaptic terminals (Figure 4)? How is this experiment used to describe the synaptic function of SynCAM?

Answer 20

Does PDZ-binding matter? If it does, its function may be more than structural.

Question 21

What is the function of synaptophysin? And what is the co-localization of synaptophysin with the SynCAM telling us?

Answer 21

Synaptic vesicle protein - SynCAM localized at synpases

Question 22

Define “recycling pool” and explain how it teaches us about the function of the synapse.

Answer 22

proximate to cell membrane. Size of recycling pool indicates not kinetics of exocytosis, but amplitude.

Question 23

What are the requirements for a functional synapse (non-neuronal cells)?

Answer 23

full-length SynCAM and glutamate receptor in post-synaptic cell.

Question 24

Neuroglin-neurexin cannot explain synapses b/c

Answer 24

They require calcium and cell adhesion does not.

Question 25

Ephrines cannot explain synapses b/c

Answer 25

Ephrines are not adhesion molecules.

Question 26

Cadherins cannot explain synapses b/c

Answer 26

Are either not evolutionarily conserved or are not present in established junctions.