One of the most challenging endeavors in the biological sciences is linking the microscopic details of cellular components to the macro-scale physiology of the organism. This formidable task has been met repeatedly in the recent history of biology, especially in the era of DNA sequencing and single molecule biochemistry. For example, the scientific community has been able to connect sickle-cell anemia to a single amino acid substitution in Hemoglobin which promotes precipitation under a change in O\(_2\) partial pressure (24–26). Others have assembled a physical model that quantitatively describes chemosensation in bacteria (27) in which the arbiter of sensory adaptation is the repeated methylation of chemoreceptors (28–31). In the past ~50 years alone, numerous biological and physical models of the many facets of the central dogma have been assembled that give us a sense of the interplay between the genome and physiology. For example, the combination of biochemical experimentation and biophysical models have given us a picture of how gene dosage affects furrow positioning in Drosophila (32), how recombination of V(D)J gene segments generates an extraordinarily diverse antibody repertoire (33–35), and how telomere shortening through DNA replication is intrinsically tied to cell senescence (36, 37), to name just a few of many such examples.

     By no means are we ``finished” with any of these topics. Rather, it's quite the opposite in the sense that having a handle on the biophysical knobs that tune the behavior opens the door to a litany of new scientific questions. In the case of mechanosenstaion and osmoregulation, we have only recently been able to determine some of the basic facts that allow us to approach this fascinating biological phenomenon biophysically. The dependence of survival on mechanosensitive channel abundance is a key quantity that is missing from our collection of critical facts. To our knowledge, this work represents the first attempt to quantitatively control the abundance of a single species of mechanosensitive channel and examine the physiological consequences in terms of survival probability at single-cell resolution. Our results reveal two notable quantities. First, out of the several hundred single-cell measurements, we never observed a cell which had less than approximately 100 channels per cell and survived an osmotic shock, irrespective of the shock rate. The second is that between 500 and 700 channels per cell are needed to provide \(\geq 80\%\) survival, depending on the shock rate.

    Only recently has the relationship between the MscL copy number and the probability of survival been approached experimentally. In van den Berg et al. (2016), the authors examined the contribution of MscL to survival in a genetic background where all other known mechanosensitive channels had been deleted from the chromosome and plasmid-borne expression of an MscL-mEos3.2 fusion was tuned through an IPTG inducible promoter (8). In this work, they measured the single-cell channel abundance through super-resolution microscopy and queried survival through bulk assays. They report a nearly linear relationship between survival and copy number, with approximately 100 channels per cell conveying 100% survival. This number is significantly smaller than our observation of approximately 100 channels as the minimum number needed to convey any observable degree of survival.

     The disagreement between the numbers reported in this work and in van den Berg et al. may partially arise from subtle differences in the experimental approach. The primary practical difference is the rate and magnitude of the osmotic shock. van den Berg et al. applied an approximately 600 mOsm downshock in bulk at an undetermined rate whereas we applied a 1 Osm downshock at controlled rates varying from 0.02 Hz to 2.2 Hz. In their work, survival was measured through plating assays which represent the population average rather than the distribution of survival probability. While this approach provides valuable information regarding the response of a population to an osmotic shock, the high survival rate may stem from a wide distribution of channel copies per cell in the population coupled with bulk-scale measurement of survival. As has been shown in this work, the expression of MscL from a chromosomal integration is noisy with a single strain exhibiting MscL copy numbers spanning an order of magnitude or more. In van den Berg et al., this variance is exacerbated by expression of MscL from an inducible plasmid as fluctuations in the gene copy number from plasmid replication and segregation influence the expression level. Connecting such a wide and complex distribution of copy numbers to single-cell physiology requires the consideration of moments beyond the mean which is a nontrivial task. Rather than trying to make such a connection, we queried survival at single-cell resolution at the expense of a lower experimental throughput.

    Despite these experimental differences, the results of this work and van den Berg et al., are in agreement that MscL must be present at the level of 100 or more channels per cell in wild-type cells to convey appreciable survival. As both of these works were performed in a strain in which the only mechanosensitive channel was MscL, it remains unknown how the presence of the other channel species would alter the number of MscL needed for complete survival. In our experiments, we observed a maximum survival probability of approximately 80% even with close to 1000 MscL channels per cell. It is possible that the combined effort of the six other mechanosensitive channels would make up for some if not all of the remaining 20%. To explore the contribution of another channel to survival, van den Berg et al. also queried the contribution of MscS, another mechanosensitive channel, to survival in the absence of any other species of mechansensitive channel. It was found that over the explored range of MscS channel copy numbers, the maximum survival rate was approximately 50%, suggesting that different mechanosensitive channels have an upper limit to how much protection they can confer. Both van den Berg et al. and our work show that there is still much to be learned with respect to the interplay between the various species of mechanosensitive channel as well as their regulation.

     Recent work has shown that both magnitude and the rate of osmotic down shock are important factors in determining cell survival (4). In this work, we show that this finding holds true for a single species of mechanosensitive channel, even at high levels of expression. One might naïvely expect that this rate-dependent effect would disappear once a certain threshold of channels had been met. Our experiments, however, show that even at nearly 1000 channels per cell the predicted survival curves for a slow (\(< 1.0\) Hz) and fast (\(\geq 1.0\) Hz) are shifted relative to each other with the fast shock predicting lower rates of survival. This suggests either we have not reached this threshold in our experiments or there is more to understand about the relationship between abundance, channel species, and the shock rate.

     Some experimental and theoretical treatments suggest that only a few copies of MscL or MscS should be necessary for 100% protection given our knowledge of the conductance and the maximal water flux through the channel in its open state (11, 38). However, recent proteomic studies have revealed average MscL copy numbers to be in the range of several hundred per cell, depending on the condition, as can be seen in Table 1 (15, 16, 39). Studies focusing solely on MscL have shown similar counts through quantitative Western blotting and fluorescence microscopy (3). Electrophysiology studies have told another story with copy number estimates ranging between 4 and 100 channels per cell (17, 40). These measurements, however, measure the active number of channels. The factors regulating channel activity in these experiments could be due to perturbations during the sample preparation or reflect some unknown mechanism of regulation, such as the presence or absence of interacting cofactors (41). The work described here, on the other hand, measures the maximum number of channels that could be active and may be able to explain why the channel abundance is higher than estimated by theoretical means. There remains much more to be leared about the regulation of activity in these systems. As the in vivo measurement of protein copy number becomes accessible through novel single-cell and single-molecule methods, we will continue to collect more facts about this fascinating system and hopefully connect the molecular details of mechanosensation with perhaps the most important physiological response -- life or death.