質問対策
だいたいこんなもの?
こんなかんじ。
1. In this presentation, you don't mention SHIP protein.
How do you think SHIP protein is involved in this pathway?
Thank you very much for good question.
Exactly, SHIP is the protein that degrades PIP3 to PI(3,4)P2.
I did not examine the effect of SHIP on spinule formation.
Some paper shows that SHIP is not expressed in HP.
Anyway it is very interesting to know the effect of SHIP on spinule formation.
1.0
How did you analyze spinule formation?
It is pretty difficult that as you know spinule is very fine structure. So it is pretty difficult to observed.
In our study we used the hamahoto H7422 Gasp detector.
Spinules that protruded from the spine head were counted. Each synapticspinule, regardness of its size and orientation, was scored as in previous studies(Tao-Cheng et al., 2009)
And we did analyze the circularity. The effect of PTEN inhibitor, which increses PIP3, reduced the circularity.
In the case of PI3K inhibitor, which decreases PIP3, there was the tendency that circularity increases but no significant difference.
1. 1.
PIP3 or PIP2?
very good question.
This study is in the middle of revise.
Reviewer pointed out about this point.
I am looking for the protein of downstream of PIP3 such as Akt protein.
And basically PIP2 is 100 times more than PIP3. even though PIP3 increase or decreases,
PIP2 level is not reflected.
So I think that PIP3 is the factor to regulate spinule formation.
1. 2.
The relationship between schizophronia and PIP3
Inositol metabolism is deeply related to the depression and bipolar disorder. Because old days drug lithium is well known to work this pathway. currently it seems that lithium effects the GSK3beta. And PTEN mutant causes coden disease, which is accompanied by sometime mental reterdation. So PIP3 and inositol metabolism is related to the disease like these. But I think there is mainly two cause inositol metabolism regulates such a diseases. One is that PIP3 regulates the neuronal development in develop brains. The other is synapse function after neuron grown up. In the present study, I examined the effect of PIP3 on spine morphology. In near future, I want to examine the effect of PIP3 on neuron development. In some part, Dr. sheng reveal that PIP3 regulates the dendritic arborization though.
1.3
How about the mobility of PIP3
側方拡散lateral diffusion is roughly 1 microm/sec.
But Dr. Kusumi shows that there is some barria mechanism which prevent the lateral diffusion of lipids.
The molecules is surrounded by the fenses the size is 30 to 200 nm.
So we cannot say that PIP3 is easily do the lateral diffusion.
1.4
PIP3 and raft
Basically PIP3 fatty acid composition is steroyl and arachidonyl.
1.5
Did you try RNAi for PI3K and PTEN
I tried PTEN RNAi. But I don't analyzed yet.
1.6
Did you check the decrease in PIP3 in the presence of PI3K inihibitor?
Yes, I did it.
I observed PIP3 reduction and increase in the presense of PI3K and PTEN inhibitor respectively in this figure.
In the presence of PI3K inhibitor, PIP3 decrease more in spine than dendritic shaft.
In the presence of PTEN inihibitor, PIP3 increase more in dendritic shaft than spines.
Previously several paper shows that PTEN is localize in the dendritic shaft.
Also some paper showed that PI3K is localized in spines and dendrites, but PI3K directly binds to AMPA receptors, leadting to PI3K activation.
This data answers that why PIP3 is accumulated in the spines.
2. In the middle of glutamate uncaging, is PTEN related to this decrease?
At first, I expect that PIP3 decrease during glutamate ungaging is due to PTEN.
But I examined the effect of PTEN inhibitor on PIP3 decrease during glutatate uncaging.
There is no obvious difference with or without PTEN inhibitor about PIP3 decrease.
So I think this decrease of PIP3 is mainly due to menbrane insertion.
3. PTEN is dual phosphatase? Did you remove the possiblility PTEN phosphatase activity works in this pathway?
Exactly PTEN has dual phosphatase activities. burabura.
4. PIP3 is involved in the functional plasticity? and structural plasticity?
As I showd in introduction, there is no doubt that PIP3 regulates functional plasticity.
5. Does PIP3 regulate spinule length?
Actually PIP3 regulates the number of spinule. But the length of spinule is not regulated by PIP3.
6. Explain the FLIM more.
7. What is the strength of FLIM compared to FRET?
Previously I developed the ratiometric FRET probes including PIP3 and DAG probes.
In the case of ratiometric-based FRET probe, in the thick tissues like hippocampal slice, wave-dependent fluorescence absorabance is gonna happen. We cannot measure appropriate FRET efficiecy. So I newly developed the florescence lifetime-based probe. Fluorescence lifetime never change even though thick tissues. That is the strength of FLIM.
8. PI3K works in the middle of glutamate uncaging?
I did the experiment to see the effect of PI3K inhibitor on PIP3 concentration during glutamate uncaging.
But there is no significant difference between with or without PI3K inhibitor.
This means that enzymatic induced PIP3 is extremely little compared to dilution of PIP3 which is caused by membrane insertion.
But if we want to observe more detail PIP3 dynamics which is caused by the PI3K or PTEN during glutamate uncaging, I plan to do this experiment as follows. At first I make the situation that spine size doesn't change even during glutamate uncaging using cytokarasin and catrunculin, which is inhibitor for actin polymerization. In this situation I am gonna measure the PIP3 increase or decerase. So I can observe real PIP3 change not induced by membrane insertion.
9. What is the down stream pathway of spinule formation?
10. What is the most difficult point for this work? How did you take over this problem?
11. What number of PIP3 is there in spine?
Rough caluculation is 10-100 number of PIP3.
How about the number and length of spinule ?
raft and PIP3.
my probe is subjected with palmitoyl and farnesyl modification.
Recently raft proteins clustering regulate the localization of N-Ras.
At least I get the response of FLIMPA3 in culture and neurons.
The important proteins including PI3K is localized in raft. So PIP3 also localized in raft.
But it is very interesting to discriminate the PIP3 production or reduction in raft or non-raft.
このサイトはreCAPTCHAによって保護されており、Googleの プライバシーポリシー と 利用規約 が適用されます。
1文字以上入力してください
本文は少なくとも1文字以上必要です。
1文字以上入力してください。