Opinions of referees of the Journal 
"Cellular and Molecular Life Sciences"

Before publication in "Physiol. Chem. Phys. Med. NMR", the manuscript of my article "Evidence of a new type of protein-protein interaction: desensitized actomyosin blocks Ca²⁺-sensitivity of the natural one. A possible model for an intracellular signalling system related to actin filaments" was proposed to the Journal "Cellular and Molecular Life Sciences". See below: (i) the letter of the editor; (ii) reviewer responses about the manuscript; (iii) my answers to reviewer remarks.

Editor-in-chif letter

Basel, 8 May 2000 

CMLS 00 68 
Evidence for fast diffusion of troponin-tropomyosin complex in actomyosin gel. A possible model for an intracellular signaling system related to actin filaments. 
V. V. Matveev 

Dear Dr. Matveev, 
We are sorry to tell you that according to the referee reports, which are enclosed for your information, your paper has not been recommended for publication in CMLS. We very much regret not being able to accept your paper and thank you nonetheless for your interest in our journal. 

Prof. Dr. P. Jolles Editor-in-chief 

Yours sincerely, 
Prof. Dr. P. Jollès 

REFEREES

Referee a) The experiments described in this paper are simply incredible. The conclusion from an immensely long discussion section (7 1/2 pages) is that tropomyosin and troponin can move from one set of actin filaments to another at a rate faster than free diffusion in water (by two orders of magnitude!). Surely the author must have considered that this was unlikely for two tightly-binding proteins (in my experience the dissociation of tropomyosin from actin filaments has a half life of many minutes, reference 19). 

A clue to what's going on can be guessed from the methods section. Desensitized actomyosin has a different - more open - gel structure than natural actomyosin. It is like that under centrifugal force the natural actomyosin ended up at the bottom of the tube and the desensitized actomyosin, being less dense was on top. The author should label actin or myosin in the desensitized actomyosin (radioactive of fluorescent) to track where it moved to (if it moved). I do not think any journal should publish this manuscript. 

Referee b) The author has overlayered a gel of actomyosin rich in the troponin/tropomyosin complex (TT-complex) over a gel of actomyosin lacking this complex. Then the distribution of the TT-complex was analyzed in the two layers after a specified time of incubation. The author observed that the upper gel layer is depleted of the TT-complex which can be recovered in the lower layer. The diffusion coefficient of the TT-complex into the lower layer was determined and was found to be about three orders of magnitude higher than values for protein diffusion in water. The molecular mechanism of this 'facilitated diffusion" process is unclear. Experiments with control proteins are lacking, and no evidence is provided for the possible biological relevance of this process. 

Specific points: 
   - The manuscript is very unclear in parts (for example page 7, lines 13-15; page 16, lines 3-5 etc) and should be revised by an English-speaking colleague. 
   - The author states in the text (without showing a figure) that the TT complex is lost from the upper layer and can be recovered in the lower layer (page 10). However experiments are lacking on whether calcium-sensitivity is regained in the lower layer or not, as a consequence of 'movement" of TT into the lower layer. The author should provide data on behaviour of control proteins (BSA, myoglobin etc), as compared to the TT complex, in the two layer system. 
   - The Discussion is much too long (7 pages) and should be shortened and focused. 
   - The reference list is too long (66 references). 
   - The word 'bilayer" is used in the manuscript for the two gel phase system. This word is used normally in another context (lipid bilayer) and should be replaced for example by 'two layer system" 'Migration" should be replaced by 'diffusion into". 

Referee У) This manuscript describes experiments in which actomyosin gels with or without troponin-tropomyosin are layered to form an interface and then the effects of the differential composition of the gels studied. The results are interesting, however they are difficult to interpret due to the confusing methodology. I had a difficult time discerning what samples were gel, and what were sol, and if gel, then how samples were removed. I could not figure out how measurements were made and what was being specifically measured. I do not understand what the nature of the "suspension system" (Fig 2B) is and how it was analyzed. What would have been useful is a step by step description of the experiment and the state of the material at each step. As near as I can figure, they have an actomyosin gel complex with and without TT. These are sequentially centrifuged to form a two layer system in a test tube. After incubation in the cold, a sample is taken from the top. There is no data presented on the composition of this material nor how it was sampled from the gel. Since its TT modulated contraction is examined, I cannot image a reason not to directly determine the mixing of TT into this layer. It is stated that since it is a gel, no mixing is possible makes no sense given the entire phenomenon is apparently due to mixing. I also do not understand how this chunck of gel is assayed for ATP dependent changes given it is presumably a solid gel. They also state that desensitization occurs in 1-3 minutes. If so, then why is mixing done for 15 hours? The authors then determine a diffusion rate by slicing gels and determining the content of TT complex. None of this data is shown. Most importantly, there is no control protein examined. The presumption is that the phenomenon has something to do with actin interaction and not some sort of non-specific mixing. I think it should be possible to incorporate a control protein (BSA?) into the gel layer in sufficient quantitity to be able to measure its diffusion rate by fluorescence or gel cutting as done for TT proteins. If it is no different from TT, then the paper would focus on actin and myosin and rheological concerns and not on TT and a very different conclusion would result. The jump from this line of evidence to the alcohol addition, I found uncompelling. The effects presented seem to be pure speculation given the complex nature of the system. I recommend focusing on the controls for the diffusion and better description of the experiments and dropping the alcohol experiments from this manuscript. Last, I recommend dramatically shortening the discussion. There is a large amount of unsupported speculation that is unnecessary. 

Discussion with referees 

Referee a): 'The experiments described in this paper are simply incredible'.
Matveev: My experiments are very simple and can be tested easily.

Referee a): 'The conclusion: is that tropomyosin and troponin can move from one set of actin filaments to another at a rate faster than free diffusion in water (by two orders of magnitude!). Surely the author must have considered that this was unlikely for two tightly-binding proteins (in my experience the dissociation of tropomyosin from actin filaments has a half life of many minutes, reference 19)'.
Matveev: The mechanism of 'sliding' diffusion remains unclear, so it is impossible to know precisely that dissociation of tropomyosin is a really necessary step: see a possible explanation.

Referee a): ' Desensitized actomyosin has a different - more open - gel structure than natural actomyosin. It is like that under centrifugal force the natural actomyosin ended up at the bottom of the tube and the desensitized actomyosin, being less dense was on top'.
Matveev: I had a control for this possibility which was not described in the manuscript. I marked the surface of the first gel layer by neutral red dye or by colloidal carbon. After a second centrifugation the border between two layers was not deformed.

Referee b): 'The molecular mechanism of this 'facilitated diffusion" process is unclear'.
Matveev: In science there are a lot of mechanisms which are not clear, but this is not an occasion to stop research. If something is clear it is not science anymore; it is praxis.

Referee b): 'Experiments with control proteins are lacking, and no evidence is provided for the possible biological relevance of this process'.
Referee c): 'I think it should be possible to incorporate a control protein (BSA?) into the gel layer in sufficient quantitity to be able to measure its diffusion rate by fluorescence or gel cutting as done for TT proteins. If it is no different from TT, then the paper would focus on actin and myosin and rheological concerns and not on TT and a very different conclusion would result'.
Matveev: OK. We can test some control protein (BSA or hemoglobin). We can expect only one of two possible results: (i) control protein diffuses normally (very slowly) or (ii) the diffusion is also as fast as the investigated complex. Which result denies my findings? The first result will confirm the specific ability of tropomyosin to fast diffusion, and the second result will show that condensedљ actomyosin gel is the unique environment for fast diffusion of any protein. I agree to be the author of both findings.

Referee b): ':experiments are lacking on whether calcium-sensitivity is regained in the lower layer or not, as a consequence of 'movement" of TT into the lower layer'.
Matveev: This is a problem because calcium-sensitivity function is off as a result of interaction of the two type of myosin.

Referee c): 'I had a difficult time discerning what samples were gel, and what were sol, and if gel, then how samples were removed: I do not understand what the nature of the "suspension system".
Matveev: All these doubts and questions testify that the reviewer has no practical operational experience with actomyosin. Actomyosin is insoluble in solutions with low ionic strength (I used these solutions). Therefore, suspension is a colloidal solution of the protein. Actomyosin can be sol only in 0,5 M KCl solution, but I did not use such solutions.

Referee c): ' They also state that desensitization occurs in 1-3 minutes. If so, then why is mixing done for 15 hours?'.
Matveev: My task was to study tropomyosin diffusion in conditions of complete steady-state. A lot of variants of experiments are possible. It is necessary to begin with something.

I am grateful to my reviewers both for their understanding of my and for their mistakes. All these will help me to give to my research a clearer character.

New data about desensitized actomyosin properties
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