DAG study I am doing recently


基本的に、あまり進んでいない。2006年のうちらの論文および2010年のNewton ACの論文によるそれぞれの膜においてのシグナルのcompartmentalizeが、cell biologyのDAGに関する新しい発見である。これは、imaging技術の発展なしにしてはわからなかったことである。

I found this paper with the search with the word, SMase and diacylglycerol.

Methylmercury-induced toxicity is mediated by enhanced intracellular calcium through activation of phosphatidylcholine-specific phospholipase C


These are the papers related to that DAG is cruical for membrane funsion.

1. Biochemistry. 1989 May 2;28(9):3703-9.

Physiological levels of diacylglycerols in phospholipid membranes induce membrane fusion and stabilize inverted phases.

Siegel DP, Banschbach J, Alford D, Ellens H, Lis LJ, Quinn PJ, Yeagle PL, Bentz J.
Procter and Gamble Company, Miami Valley Laboratories, Cincinnati, Ohio 45239-8707.

2.J Cell Biol. 2004 Dec 20;167(6):1087-98.

Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles.

Fratti RA, Jun Y, Merz AJ, Margolis N, Wickner W.
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

In this paper, they say DAG has inhibitory effect on vesicle fusion.

3.J Biol Chem. 2004 Dec 17;279(51):53186-95. Epub 2004 Oct 12.

Diacylglycerol and its formation by phospholipase C regulate Rab- and SNARE-dependent yeast vacuole fusion.

Jun Y, Fratti RA, Wickner W.
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755-3844, USA.

In this paper, they say that DAG is masked by C1B domain, vacuola fusion is inhibited.

4. Biochem Biophys Res Commun. 2010 Apr 9;394(3):733-6. Epub 2010 Mar 15.

A novel complete reconstitution system for membrane integration of the simplest membrane protein.

Nishiyama K, Maeda M, Abe M, Kanamori T, Shimamoto K, Kusumoto S, Ueda T, Tokuda H.
Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. nishiyam@iwate-u.ac.jp

5.Biochim Biophys Acta. 2010 Jan;1798(1):59-64. Epub 2009 Nov 3.

End-products diacylglycerol and ceramide modulate membrane fusion induced by a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa.

Ibarguren M, Bomans PH, Frederik PM, Stonehouse M, Vasil AI, Vasil ML, Alonso A, Goñi FM.
Unidad de Biofísica (Centro Mixto CSIS-UPV/EHU), y Departamento de Bioquímica, Universidad del País Vasco, Apto. 644, 48080 Bilbao, Spain.

6.Biophys J. 2009 May 6;96(9):3638-47.

A comparison of the membrane binding properties of C1B domains of PKCgamma, PKCdelta, and PKCepsilon.

Sánchez-Bautista S, Corbalán-García S, Pérez-Lara A, Gómez-Fernández JC.
Departamento de Bioquímica y Biología Molecular A, Facultad de Veterinaria, Universidad de Murcia, E-30080-Murcia, Spain.

7.Biochim Biophys Acta. 2009 Mar;1788(3):701-7. Epub 2008 Dec 3.

Calcium inhibits diacylglycerol uptake by serum albumin.

Ahyayauch H, Arana G, Sot J, Alonso A, Goñi FM.
Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU), and Departamento de Bioquímica, Universidad del País Vasco, Aptdo. 644, 48080 Bilbao, Spain.

Progress in Lipid Research
Volume 38, Issue 1, January 1999, Pages 1-48

Structure and functional properties of diacylglycerols in membranes

Félix M. Goñi* and Alicia Alonso
Grupo Biomembranas (Unidad Asociada al CSIC), Departamento de Bioquímica, Universidad del País Vasco, Aptdo. 644, 48080 Bilbao, Spain

About the experiment for biological significance of outer DAG.
Previously I did experiment about the difference in cell growth between the cells with or without DAG probe.
At the time, it seems that the flowting cells exist more in the cells incubated with DAG probe. This means that cell growth is suppressed with DAG probe. But MDCK cells's growth is very fast.
I am gonna reduce the % of serum and make growth rate slow down.

I need to finish this paper now around.
But there are several things I have to do...

1. see if DAG comes out to the outer plasma membrane in response to ATP.
2. biological significance of outer DAG.

The day after tomorrow, I gonna do the following expreiments.

1. Time course of outer DAG production in response to ATP in MDCK cells.
2. inhibitor of R59949 and U73122 at the time point of 10 min
3. mGluR expressed MDCK cells

1. SMnaseBcPLC, SMnase, ATP, ctrl.

1. ctrl(no D609),ctrl(D609), 1min(D609,ATP) 4min, 7min,10min.
2. ctrl(no D609), ctrl(D609), ctrl(D609ATP)D609ATPU73122, D609ATPR59949,

biological significance:
1. cell growth
2. incorporation of LDL or something.


I am gonna do the following experiments.

1. Incubate with 1 microM ATP and 10 U SMase and 12.5 unit/ml Bc-PLC for 4 min and 10 min and 10 min respectively. Do nothing about ctrl.
2. Fix cells with 3 % PFA for 15 min.
3. Add NH4Cl for 5 min.

Preincubation with D609 for 5 hour.
1. Preincubation of the cells with 15micro/L U73122 for 10 min shortly before starting experiments.
2. ATP stimulation and after 10 min, incubate with EFYP-C1AB for 2 min.
3. Fix cells with 3 % PFA for 15 min.
4. Add NH4Cl for 5 min.


Transfection of DsRED2 and EYFP-C1AB into MDCK cells.
What I want to do is that examination of DAG in stady state in ctrl SMase, Bc-PLC and ATP treated MDCK cell.


I am gonna do the experiment for the paper.
1. incubation with D609 for 0, 1, 3, 6 and 12 hrs.
2. ATP stimulation after incubation with D609 for 6 hrs.
3. the effect of SMase, ATP and BcPLC on the inner DAG.
4. the comparison between Bc-PLC and ctrl.

12:33 12 hour incubation finish
17:06 6 hour incubation finish
12:12 1 hour incubation finish
14:12 3 hour incubation finish.


The things I am gonna do from now is as follows.
1. At first, it is very weird that DAG flip should occured in stady state upon addition of SMase or incubation with ISP1.
So,I am gonna observe DAG in inner leaflet of plasma membrane.
RFP and YFP-C1AB are expressed in MDCK cells.
Fix this cell.
If there are some making a difference in fluorescence between RFP and YFP,
YFP-C1AB can detect inner DAG.
After identifing this, I am gonna do SMase addition to MDCK cells.
I gonna compare the fluorescence pattern with or without SMase.


Today I am gonna do the experiment of staining of lymphocyte with DAG probe.

The way to do is something like that.

MDCK, SMS-+ and Jurkat cells.
(Actually I picked up 5×105 lympho cells: 50microl)
I want to difference in staing between MDCK and other lympho cells.
1. trypsinize
2. rip off the cells.
3. spin down
4. replace medium with HBSS.
5. incubate with DG probe for 2 min
6. fixed with 3%PFA

And then, I want to see the staining pattern of Jurkat cells which have aggrigation.
1. pich up several Jurkat cells on the glass base dish.
2. replace the medium with HBSS carefully in the glass base dish.
3. 2 min incubation with DG probe
4. wash with HBSS 3 times carefully
5. fixed with 3% PFA.

I examined the effect of D609 on DAG on the outer leaflet of plasma membrane several days ago. Previously I thought that DAG is gonna dissapear after 6hour. However I found out that DAG dissapear more quickly within 3 hour. Basically within 1 hour, almost of DAG dissapears.So I am gonna do the following experiment next.
1. Incubate with D609 for 3 hour. At this time, I believe that internalization of recepters does not occur. So I can examine DAG flop that is drived from DAG producing in response to ATP stimulation.
2. 100microM ATP stimulation and stain the outer leaflet of DAG at 0,2,4,7,10 min.


I did following experiments.
MDCK cells were incubated with 50microg/ml D609 for 6 hour.
ripped off the cells with trypsine.
spread MDCK cells 24 well dish with D609.
After 1 hrs, wash out the flouting cells 3 times, fixed and counted the number of cells.

In control cells without D609, cells attached the bottom of dish well.
However in the presence of D609, cells hardly attached the dish.


The experiments below have been very much done.
So what is the next step?
The paper's theme is something involved in DAG flip-flop.
So it is better focusing on DAG flip-flop.
What I gonna do is as follow.
1. I am gonna check that there is no difference in cytosolic DAG production in response to ATP between with or without D609.
I afraid of Receptors internalization during incubation with D609 as previously shown in some papers.
If I can, I want to fix these cells because I can count cell numbers easily.


MDCK was incubated with some drug as follows.
1. 10% LPDS 24 hrs
2. ISP-1 50 nM 24 hrs
3. FB1 40 microM 24 hrs
4. D609 50 mircog/ml 24 hrs
5. U73122 500 nM 24 hrs.
6. D609 50 microg/ml 24 hrs and U73122 500 nM 24 hrs.
7. D609 50 microg/ml 6 hrs
8. ctrl

MDCK cells were incubated with 200 microM D609 for 12hour.
But cells died perfectly.
So I can not use long incubation with 200 microM D609.
I am gonna try 6hour.

The results of yesterday experiment.
In fact, ISP-1 treated cells give annexin V fluorescence a little bit without Bc-PLC.
After incubation with 1.25units/ml Bc-PLC, in the case of 200 nM ISP-1, fluoresecence of annexin V increases little by little.
Same situation gonna happen in the case of 50 nM ISP-1.
But labelling with annexin V is very weaker than 200 nM.
This is the level I can ignore.
So I put 50nM IPS-1 treated cells in my paper.

Today I gonna do the following experiments.
13/Aut/2010, I did 12.5 unit/ml BcPLC application experiment.
I want to confirm that ISP-1 treated cells do not have cell damages during incubation with 12.5 unit/ml Bc-PLC using anexin V.

And I want to confirm whethere DAG produced by Bc-PLC is same between tISP-1 treated cells and normal cells.

I did several important expriments.
1. whether ZS2, ZS2/SMS1 and ZS2/SMS2 have DAG on the outer leaflet of PM.
Answer: Every cells was stained with DAG probe at the same level.
This shows that PC-PLC activity is different from SMS1 and SMS2.

2. whether D609 diminishes the outer DAG amount in MDCK cells.
Answer: at 6hour DAG diminished however 24 hour incubation recovered the DAG? What happen??
Anyhow I am gonna take reproducibility.

3. Glutamte applies MDCK cells expressing mGluR5.
20 microM glutamate was added to this MDCK cells.
However there is no difference between not expressed cells and expressed cells.
This data shows that DAG does not flop to the outer leaflet of the PM.

4. 1.25 unit/ml Bc-PLC was added to the MDCK cells treated with 50 and 200 nM ISP-1.
Flip occured!


Here is most important paper now for me.

In the experiment of 10/Aug/2010, we could not see difference in fluorescence between D609 24hour incubated MDCK cells and intact MDCK cell.
So I am going to do this reproducibility again.
And then I am gonna start ISP1 incubation again.


I am gonna do ISP-1 treatment expeiment.
I am gonna divide MDCK cells, which exposed 200nM and 50nM ISP-1 for observation and biochemistry expeiments.

So I gonna prepare six 3.5mm well dishs for each.

And I gonna do the experiment in which I want to see the effect of D609 on the receptor signalling.
Because it is known that D609 causes the internalization of receptors from plasma membrane.
So I prepare MDCK cells expressing inner DAG probe in three dishes.

I got mGluR-RFP from Matsuura-kun.
I can express this DNA whatever cells I want.
I am gonna express this DNA CHO cells and MDCK cells.

Today's result.
I looked through the samples I prepared yesterday.
Now I found out that DAG exists in static state.
Based on this result, I need to plan the expriments.
Basically, this expreiment follows the experiment of CHO cells.

I am gonna do the experiment as follows.
1. no probe, probe only, ATP, ATP+R59949, ATP+U73122, PC-PLC.
2. MDCK cell growth with or without DAG probe.

I am doing the experiments in which 10 microM R59949 incubation is done upon 100 microM ATP addition.
I expect that more DAG comes to outer leaflet of PM than without R59949.

10 min R59949 incubation and then ATP stimulation containing R59949.
2 min incubation with DAG probe.