The first is that you can tell us all the info respectly the reactives, the study done and the experiment that you have done. What are you looking for at? Why did you inyect that compound in the mitochondrion? What are the conditions of the assay? If you give that information, maybe we can help you. Greetings.

Seconding the questions of Felipe Stambuk I would like to stress that interpretations of "ultrastructural textures" whatever they may be or from whatever tissue they derive CAN NOT be evaluated or commented on seriously if the conditions of how they were generated are not known. If it has to remain a secret for us, ok - one can describe the structures as you - dear Omer Faruk Kuzu - have done briefly ...but you can't expect reading out something about physiological alterations within the specs. With regard to your figs./micrographs 10,15,17 I would not interpret these roundish profile structures to be "myelin-" or "myelin-like" figures. There IS definively a change in ultrastructural appearance of mitochondrial matrix, the "lyosomal" changes shown in Figs.12&13 IMHO do represent degenerative changes of former mitochondria. If this is a consequence of applying some "chemical stuff" or "physical" treatment to the (supposed to be) cultured cells one can not say if not knowing about the conditions of the whole procedure.

I greatly should acknowledge more information on your treatment and processing conditions (in this ResearchGate forum or by personal message, if you like to keep confidency),

best regards and wishes

Wolfgang MUSS, PhD, SALZBURG-AUSTRIA

This is a very interesting question of actuality, I have a look to your EM and come back to you.

Effectively, their is a new article concerning cholesterol and lysosomes... I will send you copy.

Patrice

do you authorize me to transmit the EM data to colleagues?

for explanation?

[email protected]

Yes, But I will be happy to hear your interpretations also...

In my opinion you do NOT have significant mitochondrial damage and you certainly do not have typical myelin figures but you may have some membranous abnormalities probably resulting from malfunction of the Golgi apparatus.The aggregations of membranes indicate some defect in membrane turnover. The fine dark granules are glycogen. You may also have some autophagocytosis as seen by vacuoles containing cytoplasmic organelles. I attach a paper of mine showing typical myelin bodies as seen in Niemann Pick disease. I hope this information is helpful.

This an answer elaborated with eeva-liisa.eskelinen (finland) which is an electron microscopy expert.

Some comments to the EM images:

-The membrane accumulations in the treated cells do not look like myelin figures.

I have never seen such structures before, so I do not have definite answer what these huge membrane accumulation are.

This could be also due to preparation of teh EM and/or problematic concerning the product you use (artefactula situation).

However, If notI can suggest that they could be derived from endoplasmic reticulum (or possibly Golgi complex). You could test this by doing an immunofluorescence staining with an endoplasmic reticulum marker protein (like protein disulphide isomerase), and a Golgi marker protein (like mannosidase II).

The structures should be readily visible in immunofluorescence microscopy, if indeed they are endoplasmic reticulum or Golgi -derived.

I do not see signs of severe mitochondrial damage in the treated cells.

The structures suggested to be swollen lysosomes in one of the images are not easy to identify. I do not want to guess what they are.

PXP: clearly the mitochondrial compartment is not affected

Good luk since teh ER/lysosomes pathway are at teh top interest at the moment together with Cholesterol

Dear Prof. Dr. Jones, dear Prof. Dr. Petit, dear Omer,

at this point of the discussion on “lamellar” or “laminated” structures found and the used terminology in the replies (the structures shown in the JONES et al. 1990 paper concerning do NOT convince me*) and also to let other (less experienced) colleagues know what we are talking about I suggest to go back to the roots of terminology and what has been written and shown with excellent figure tables by the (former?) "pope" of Ultrastructural Pathology, Feroze N. Ghadially (cf. Ultrastructural Pathology of the Cell and Matrix, Fourth Ed, Butterworth-Heinemann, Boston, Oxford...., 1997ISBN 0-7506-9635-4=set of II vols). Not without reason there has been written and imaged so much (7 pages Text, 7 TEM-image plates) about those "myelin"-, - , , , , , , also and so on. And I guess these terms are not the only one for “membrane stacks” of more or less electron dense osmiophilic laminated membranous structures which ever have been published. To provide publicly the reference I mentioned before I enclose a pdf out of F. N. Ghadially’s work (text page always a left one, images on the page on the right).

NB: *) discussion about this would be rather longish, starting with the possibility of “lamella”-formation due to primary fixation with FA. Also with reference to “PXP(Professional Experience?): clearly the mitochondrial compartment is not affected” I cannot agree with Prof. Petit’s opinion, having seen a lot of cristolysis, swelling of inner mitochondrial matrix exhibiting at that places also some less osmiophilic lipid-“like” -substrate (cf. Drop-box-file, figs.8-10 and others) and having seen such alterations in several conditions of diseased muscular tissues in case or suspicion of mitochondrial myopathies which CAN be ischaemic changes due to culture or even fixation conditions but also could picture definite alterations due to a metabolic action of unknown nature yet …. and, yes, I know such might be a completely different situation as compared to cell culture conditions… BTW: do we know the type of cells used for culturing?

Best regards and have a nice weekend, Wolfgang MUSS, Salzburg, Austria

I don't really understand why is the opinion of Wolfgang Muss so agressive.

These picture comes from cells culture.. The measurement of mitochondrial sisfunction could be done... If needed. So, the author of the question has to go back to bench... Since he has now a lot of good expert discussing its topic on which he did not tell all the informations needed for a proper scientific discussion. _We have done our best... so.. Let things going...

If their is mitochondrial changes they have to be measured.. Far from electron microscopy.. Hi!

No salution...

Hi, I am really thankful for all of the interpretations. I am learning a lot. I just wanted to give few more information that I got after asking this question.

Now the data that I have clearly shows that compound is a lysosomotropic compound (probably vacuoles are late endosome originated) and inhibits autophagic flux. I did western blot analyzes and saw that LC3B and p62 is accumulating which states inhibition of autophagic flux. With very high probability lysosomes are not functioning and both endocytotic and autophagic flux is dimnished. I am going to check endocytotic pathways this week. On the other hand these cells are human melanoma cells. I am not %100 sure but these membrane structures might have Golgi origin. I did immunofluorescence staining for PKC members. Interstingly I realized that PKCu immunofluorescence shows circular big spot like structures in treated samples. Interstingly PKCu is reported to be specifically located in Golgi. And these membrane structures might be associated with these spots. However I need to verify this data. I will replicate the experiment this week. I will try to do some immunofluorescence specific for E.R and Golgi.

Moreover, I checked the mitochondrial membrane potential(MMP) through TMRE assay/flow cytometry. The positive control FCCP decreased the TMRE fluorescence to the half with just 30 min of incubation. For the experimental compound; treatment of 3uM (IC50 for 24hr) decreased the TMRE fluorescence just 10-12%. But I treated cells for 6 hours. Probably only 20-30% of the cells were dead at this time. And I am not sure whether I should do this experiment for 24 hours?

In literature it is suggested that, lysosomotropic compounds may induce accumulation of non functional mitochondria due to the inhibition of autophagic flux. However I am not sure whether MMP would reflect this accumulation..

Moreover, compound is inducing caspase independent and protein synthesis independent cell death. I thought it might be related to the lysosomal membrane permeabilization (LMP). However Calpain and cathepsin inhibitors ALLM, ALLN, Leupeptin or Pepstatin A did not inhibited the cell death. However, one professor working on autophagy said to me that no body is able to replicate the reported inhibition of LMP through these compounds. :( I don't know what to believe... However, I will try to do Cathepsin D/B immunofluorescence .

Again thanks to everybody for your interpretations and suggestions...

Good, you are really doing well. If you have a lysosomotropic compounds... You have a high probability to induce cell death also... Lysosomal destabilization may lead io the well known cathepsins activation but also chemotrypsin, both are able to interfere with caspase-8 and Bid to give rise to truncated Bid, mitochondrial destabilization and apoptosis. So, be carefull with you compound... Since this is not very easy to analyse if cell death and autophagy take place at teh same time... a double staining of mitochondria with DiOc6(3), mitoSOXred together with To-PRO-3 for viability will greatly improve your understanding of te processes.

Of course you have to do a time study 4, 8, 16, 24 and 48 h at least...

Also try to synchronized your cells and look at them during the cell cycle progression, you may also measure the sub-G0G1 peak.. for late apoptosis.

I also have a suggestion concerning the ER compartment... If calcium is relased then the PTP is very sensitive and in parrallel of your lysosomes. You can have a classical apoptosis through the mitochondrial compartment and being a 6 h you are just at the start of the process... (autophagic changes, modification of fluxes) before apoptosis will be without return.

Dear Prof. Dr. Petit,

let me say only that I am a little bit surprised that you qualify my answer to the post of Mr. Omer Kuzu as “so aggressive”. This was not at all my intention. Since we are not acquainted I respect your title and honors in Science. If you felt anyhow concerned by my writing style, I apologize.

The communication with Mr. Kuzu off -list focused initially and later on the appearance of lysosomes/endosomes and mitochondria and there was given some information on their appearance and possible reasons for those changes. The huge “tubulus/-ar-like” profile accumulations (in the Drop-Box-file indicated as to be perhaps “myelin figures”, cf. image 28,33,35, and appearing as more loosely profiles within cytoplasm, cf. img.44 guessing altered, dilated ER profiles / ribosomal elements and perhaps an earlier stage of what we see e.g. in image 28,33&35) are not to be termed “myelin figures” and if Omer today tells us that he found striking IF staining for PKC µ (mu) imposing as bigger spots within the treated cells this fits really well with an analog EM-structure of “tubulus-,-ar-like profiles” originating from either ER or better the Golgi apparatus (involvement of trans-golgi network and interrupted transport of substrate to the cell surface). Unfortunately the images (Treated) in the ppt file don't show much/many "classical" Golgi profiles, I have localized one or two images with some Golgi cisternae, at one location with quite well preserved stacks and one with dilated tubules.

For Omer: I wish you great success for the special replication/repetition of your experiment.

Hello again,

Again I want to thank for your helps and suggestions which definitely helps to me a lot.

I will try to follow your suggestions. I want to add couple more notes (and questions).

I did an experiment with pan caspase inhibitor zVAD-fmk which shows that Caspases are not involved. Although I used excess amount of zVAD-fmk it did not suppressed the cell death. The positive control (TRAIL treatment) was working fine and zVAD FMK protected the cells from death. (Moreover the caspase-9-specific inhibitor did not protected the cells also). So do you think there is a mitochondrial dependent but caspase independent cell death?

To inhibit a possible lysosomal leakage what should I use other than ALLM, ALLN, Leupeptin and Pepstatin A?

Also another interesting finding was related to a compound called Baicalein. This compound is normally used as lipoxygenase inhibitor. This compound also protects cells from the compound that I am working on. 30-40 uM of Baicalein supresses the cell death completely. However neither of the several other lipoxygenase inhibitors shows the same effect. There is a single report that states Baicalein inhibits lysosomal enzyme release but the mechanism was not clear.So I am not sure?

Moreover I did a protein array analyses and Receptor Tyrosine Kinase array analyses where both suggested alterations in RTK signaling. Especially in IGF1R-IRS1 signaling and ALK, EGFR receptor tyrosine kinases. IGF1R Y phosphorylation increases rapidly. IRS1 (S312), IRS1 (Y1179) also being upregulaated. I was not able to validate these findings through western blot analyzes due to antibody and some western issues (and I am fed up with them :( ). However RTK array is highly specific and sensitive. But what I think more than phosphorylation this is related to accumulation. May be related to receptor-mediated endocytosis and inhibition of receptor recycling?

Thanks for all...

Slide 28: this new structure could be similar to the one recent publication about a novel ER stress

http://www.nature.com/cdd/journal/v19/n12/abs/cdd2012108a.html

or it could be the aggresomes (but it doesn’t look like mitochondrial clusters since I don’t see any cristae in them). Most probably ER or Golgi clusters or ERGIC .... The lack of ribosomes on these clusters rule out the rough endoplasmic reticulum..but it may be smooth ER or Golgi. you need fluorescence markers to test this such as TGN markers or ER residents or aggresome markers such as GFP-250 to see your drug induce such bright spots.

Slide 29: Compared to control I don’t think it has much change in mitochondria.

The fine dark granules could be free ribosomes (I have no idea how glycogen will look like). I don’t think it is an artifact (since it is not in control cells).

PS: Do not post novel data on public domains.

The PKC data indicates it could be Golgi compartments in the aggregation. The p62 data shows either the drug inhibits autophagy or proteasome so that it stimulates unfolded protein response, which will ultimately lead to ER stress and might cause structures similar to http://www.nature.com/cdd/journal/v19/n12/abs/cdd2012108a.html

Agian I would strongly recommend you to confirm cell death by assays similar to our recent publication http://www.nature.com/cdd/journal/vaop/ncurrent/full/cdd2012140a.html This paper shows cells can resurrect even after morphological and biochemical apoptosis. So test the viability after drug treatment carefully to make the statements on cell death. I think your EM data is strikingly different from blebbishields (see the second link pdf+supp data) but not sure whether your melanocytes can resurrect similarly or not...but worth testing it. By the way what assay you use to quantify cell death? See the second link for testing confirmatory cell death. In your case drug wash-out studies will be appropriate. Good luck.

I just ran across this discussion string. Omer, if the matter is resolved and you have moved on to other things, this comment can be ignored. I noticed some regions of treated cells look rather empty. Could be aggregation of one or more membrane classes after loss of organization provided by cytoskeleton. You could check how the melanoma cells look after treatment with cytoskeleton disruptive compounds, like colchicine and cytochalasin. But some regions of the treated cells look fine, with nice mito-ER associations etc. Overall impression is overproduction of some class of membrane that likes to engulf itself and/or other membranes, creating those weird tangled figures. There seem to examples in various phases of growth. (EM tomography would probably be fascinating but prove nothing.) Candidate membranes are mentioned in the string. BTW, I think both control and treated cells do show a bit of mitochondrial disruption, low level PTP activation perhaps, common in cancer and other culture cells not reliant on mitochondria for all their ATP. Re: Jinesh's comment about publically posting new data, if want to preserve IP, don't disclose anything that reveals the identity or chemical nature of the compound you are testing. I think sharing images is OK. But that is moot in this case anyway.

We have noticed such membrane stack inclusions in animal ileum treated with cell-free Salmonella toxins given at my researchGate page https://www.researchgate.net/profile/Rakesh_Yashroy/publications/2?sorting=published

Also these isolated toxic extracts cause apopotosis-like changes in liver cells of the same animals https://www.researchgate.net/profile/Rakesh_Yashroy/publications/2?sorting=published

This toxic preparation also causes degranulation of liver endoplasmic recticulum https://www.researchgate.net/profile/Rakesh_Yashroy/publications/2?sorting=published

Also this toxic preparation causes loss 3-D structure of proteins into random coil https://www.researchgate.net/profile/Rakesh_Yashroy/publications/2?sorting=published

It may be interesting to correlate these changes with initial chain to apoptosis of cells https://www.researchgate.net/profile/Rakesh_Yashroy/publications/1?sorting=published

Thanks!