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Referee Comments: Referee 1

Posted by PLOS_ONE_Group on 17 Mar 2008 at 21:48 GMT

Referee 1's Review:

For a long time, it has been accepted that Merkel cells with associated nerve terminals may behave as mechanoreceptors although the transduction mechanism has not yet been elucidated. In this study, Haeberle et al. expose Merkel cells to hyposmotic solutions to trigger cell swelling and detect the delayed activation of cytoplasmic Ca signals by fura-2 imaging. Haeberle et al. show that these Ca signals are abolished in Ca-free solutions and inhibited by voltage-activated Ca channel blockers, supporting the conclusion that they arise via activation of cationic channels. Finally, the authors detect the transcripts for several cation channels that may or may not be involved in these events. The experiments have been carefully carried out and analyzed in an appropriate manner. The findings are important because they extend our knowledge of Merkel cell biology and may lay the ground-work to explore the ionic basis of the Ca signals activated by hyposmotic solutions. However, the study has several weaknesses that will need to be addressed prior to publication.

The precise sequence of events underlying the activation of the channels involved in Ca2+ influx is unclear from the paper. If I understand the logic correctly, hyposmotic solutions trigger cell swelling due to water uptake, and this expansion of the cell membrane triggers the activation of unknown mechano-sensitive channels. If this is in fact the case, the model needs to be stated more clearly than is currently presented in the paper.

There is also one issue that seems puzzling. In the majority of vertebrate cells and even most unicellular organisms, swelling is followed by a process termed regulatory volume decrease (RVD) which enables the cell to shrink over a period of minutes to their former volume even though the cell remains in a hypotonic environment. This process is mainly mediated by the loss of K+ and Cl- and a variety of K+ and Cl- channels (even Ca2+-activated channels such as BK channels) have been implicated. Because this process is so widespread, I am perplexed by the fact that this phenomenon is not observed in Merkel cells in this study. The issue is important, because if the Ca2+ elevations reported here are related to RVD, then the conclusions relating them to the activation of mechano-sensitive channels are not valid. Please clarify.

Specific comments:

1) page 8, middle para: authors state that there were 7-28 responding cells/experiment. A more meaningful measure is the fraction of cells that respond/experiment. Please provide this measure.

2) page 11. 1st sentence and page 15 (Discussion). The authors conclusion that localized [Ca] elevations in the processes implies that mechanotransduction occurs at these sites seems overly speculative and I do not follow the logic here. No evidence directly supporting this conclusion is presented in the paper and much more will need to be done to arrive at this conclusion. Merely the fact that Ca2+ elevations in the processes are larger or are detected before the global rises does not support the conclusion that the channels are predominantly localized at these sites: Ca2+ influx may occur in all regions of the cell, but may appear larger or slightly faster because of restricted diffusion in small compartments, differences in Ca2+ buffering, or clearance. I found this section overly speculative and it will need to be appropriately modified.

3) Figure 5C and page 11, last para. Why does thapsigargin not empty the stores directly? In virtually all vertebrate cells, the ER has a Ca leak pathway that promotes store depletion. Figure 5C shows that ionomycin applied following TG fails to elicit a Ca response, but how does this compare to a control ionomycin response applied in the absence of TG? This is important to resolve because it may be that these cells have a very small capacity ER Ca store. A related point is that the conclusion for CICR does not directly follow from the TG experiment and is overly speculative, especially in light of the fact that no data is presented showing that the ER contributes to detectable Ca2+ signals in these cells.

4) page 12. Ruthenium Red inhibited the osmotic response by 71%. What was exactly being measured here? If this refers to the cell volume, it needs to be explicitly stated.

5) Finally, the obvious set of experiments seem to be to directly measure and characterize the cationic currents activated by hyposmotic stimuli. Do the authors have any data in this regard? That would greatly strengthen the paper.

N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.