Activation of CaMKII in single dendritic spines during long term potentiation

In this paper, what was the purpose of constumizing the original CamuiA to Green-Camuia

To measure real time CaMKII activation in single dendritic spines using a FRET-Based biosensor 

How does green-Camuia FRET sensor work?

CaMKII activation changes its confromation → FRET decreases → increases fluorescence lifetime of mEGFP

What fluorophores make up green-CamuiA

Donor: mEGFP

Acceptor: REACh

Why did they use 2-photon FLIM instead of measuring fluorescence brightness?

FLIM measures lifetime which is what thyere interested in, not brightness

What CaMKII states can green-Camuia detect?

Both T286 autophosphorylation and Ca/Calmodulin binding activation states 

After two photon glutamate uncaging, what happened to spine volume?

It increased rapidly by 376% and then stabilized at 104% above abseline for more than 30 mins

How was CaMKII activation related to spine structural plasticity

CaMKII activation (green-CamuiA signal) increased in the stimulated spine and was roughly proportional to the amount of spine enlargment

What happened when NMDA receptors were blocked with AP5

Both spine growth and CaMKII activation were stopped. Proving that NMDAR mediated Ca influx is required

What is KN62 and what are the effects of this molecule on spine plasticity?

KN62 = CaMKII inhibitor

Partially blocked the sustained phase of structural plasticity but not the initial transient enlargment

What role does autophphorylation at T286 play in spine structural plasticity?

T286A mutant decreased sustained spine enlargment, meaning it is needed for maintaining structural changes

Explain CaMKII’s activation after stimulation

CaMKII activity instantly rose after stimulation and only happened at the stimulated spine and not neighbouring spines 

What does the T286A mutant of CaMKII prevent, and what effect did it have?

iT PREVENTS AUTOPHOSPHORYLATION at T286A, and caused reduced CaMKII activation in spines

What does the T305D mutant of CaMKII prevent, and what effect did it have?

T305D blocks calmodulin binding, also reduced CaMKII activation in spines

What do the results from T286A and T305D mutants together suggest?

That both calmodulin binding and T286 autophosphorylation are NEEDED for full CaMKII activation during spine plasticity 

CaMKII activation reaches its peak 6 seconds after glutamte uncaging (stimulaiton), but what are the phases of CamKII inactivation after sitmulation?

A fast decay = 6 seconds 

A slower decay = 45 seconds 

What does biphasic inactivaiton of CaMKII tell us

CaMKII is not permanently active but rather shuts off quickly

How did the T286A mutant affect CaMKII activation

It showed much less activated and decayed in 2 seconds instead of the normal 6 + 45 sec phases

What is the role of T286 autophosphorylation do for CaMKII activaiton 

It prolongs activation, allowing CaMKII to accumulate during repetitive stimulaiton

What does changing the number of uncaging pulses (1 vs 45) do to CaMKII decay time constants?

Nothing, the decay constants for CaMKII dont change no matter how many pulses are given

What tool did the researchers use to measure CaMKII mobility?

Photoactivatable GFP (paGFP) attached to CaMKII 

What did photoactivation experiments reveal about CaMKII mobility, and why is this important

CaMKII diffuses out o spines very slowly (1-20 mins), even during plasticity. Since CaMKII inactivates in sconds, its activity ends before it can spread, keeping activation ONLY on the stimulated spine

How does pairing depolarization with glutamate uncaging induce LTP, and what happens to CaMKII?

• Depolarization (via VSCC): CaMKII is activated more in dendrites than in spines

• Glutamate uncaging (via NMDARs): CaMKII is activated more in stimulated spines, but not in neighbouring spines

• Outcome: Both EPSC and spine volume increase, showing LTP, while CaMKII activation remains brief and compartmentalized

What did blocking L-type VSCCs show about CaMKII activation?

Blocking L-type VSCCs stopped spine CaMKII activation, showing that CaMKII depends on the local Ca nanodomains that these VSCCs create. 

What is the key concept about CaMKII activation from this experiment?

CaMKII activation is channel-specific and location dependent.

It relies on LOCAL Ca from L-TYPE VSCCS nanodomains and not global Ca

Whats the difference between EGTA and BAPTA as Ca chelators?

BAPTA = binds faster; removes nanodomain + global Ca

EGTA = binds slower; mainly removes global Ca and spares nanodomains

What did BAPTA vs EGTA show for depolarization-induced CaMKII activation (VSCC; dendrites)

• BAPTA blocked activation of CaMKII = nanodomains are gone

• EGTA didnt block activation of CaMKII = nanodaomain Ca alone activates CaMKII during depolarization

How did EGTA increase CaMKII activation during depolarization

EGTA reduces GLOBAL Ca, so whats left is Nanodomain Ca which is the one that activates CaMKII

What did EGTA and BAPTA both do to uncaging induced CaMKII activation (NMDAR; spines) 

Both EGTA and BAPTA reduced activation → showing nanodoamin Ca alone is not sufficient; uncaging requires BOTH nanodomain and global Ca spread 

Whats the key difference in CaMKII activation between depolarization (VSCC) and uncaging (NMDAR)

Depolarization (dendrites) = nanodomain Ca from VSCCs is sufficient

Uncaging (spines) needs both Nanodomain + global Ca