Intracellular single molecule microscopy reveals two distinct pathways for microRNA assembly

“Microinjected, singly-fluorophore labeled, functional miRNAs were tracked within diffusing particles, a majority of which contained single such miRNA molecules.” Page 8: “...While close to 50% of all particles contained single fluorophore labeled miRNAs 2 h after microinjection, a significant fraction held up to seven labeled miRNAs.” As for the suggested controls: 1) We know that these experiments are concentration sensitive and titration of labeled miRNAs, especially to lower concentrations, will merely further move the distribution to single photobleaching steps, as the probability of encountering a particle with two or more fluorescent probes will decrease. Again, since we do not claim to count the number of ALL, but only that of fluorophore labeled miRNAs, there is really no point to this experiment in the context of the current manuscript. The number of microinjected let-7 miRNAs is similar to that already present endogeneously, hence one would expect about equal probability to encounter a labeled and unlabeled let-7 miRNA molecule in any given complex. Since all our conclusions only depend on the changes over time of the relative (not absolute) numbers of a given miRNA assembled into RNPs and PBs, no absolute quantification of ALL miRNAs in such complexes (which would be very challenging at best) is necessary. 2) As suggested, we have now added new data (Supplementary Fig. 2B, discussed on page 7) showing colocalization of the mutant miRNA with the mutant target sequence, signifying functional assembly of microinjected miRNAs on their cognate mRNA targets. 2. Controls using mutant let-7 will be important to establish that the observed fluorescent spots are actually related to the miRNA function as proposed in Fig. 4. If their interpretation is correct and the observed spots are indeed functional important complexes for miRNA-based repression, dynamics and assembly states of the spots should be dramatically different for the mutant let-7. Essentially as suggested by the reviewer, we have now added control experiments with the artificial and particularly target-poor cxcr4 miRNA, in lieu of the mutant let-7 miRNA. As expected, the assembly states of cxcr4 miRNA are indeed dramatically different from those of wild-type let-7, favoring monomers (Fig. 3E). Going even further, we observe a rescue of multimeric miRNA assembly when specific mRNA targets of the cxcr4 miRNA are co-injected with the miRNA (Supplementary Fig 7), strongly suggesting that the observed spots are indeed functionally important for miRNA mediated mRNA repression. These new data are discussed on pages 10/11. Interestingly, the distribution of diffusion constants of cxcr4-miRNA containing particles is only mildly different from that of let-7 miRNA (Supplementary Fig. EMBO reports Peer Review Process File EMBOR-2011-35549 © European Molecular Biology Organization 1C, D, E). This is a consequence of the fact that our particle tracking can only report diffusion coefficients of a fraction of all diffusing particles, i.e., those diffusing slowly enough to be visible in the focal plane for over 9 imaging frames (0.9 s). Please note that we added a brief discussion of our time resolution on page 7 of the revised manuscript (see also referee 3 below). 3. Fast vs. slow diffusion and single vs. multiple miRNAs. SPT reveals that the miRNA containing particles have two distinct diffusion constants, which closely resemble those of messenger RNPs and processing bodies (PBs), respectively. In the photobleaching step counting experiment, miRNA containing particles are categorized into the ones with single miRNA and with multiple miRNAs. The time-dependent change of relative populations of these two classes suggests the existence of two kinetic processes, which by the authors, interpreted as the assembly of miRNA and/or miRNA RISC (containing single miRNA) into functional RNPs (including both messenger RNPs and PBs, and containing multiple miRNAs) and the disassembly of RNPs after mRNA degradation. Since the latter experiment has been performed in fixed cell, single miRNA or miRNA RISC can be well detected, while they are not resolvable in SPT experiment because of their fast diffusive property and the time resolution. In this scenario, a much larger fraction of particles containing multiple miRNAs in SPT experiment should be expected compared to the photobleaching step counting experiment. However, the authors have observed a strong overlap in the distribution of fluorescence intensities (an indication of number of fluorephores) between particles in these two experiments (supplementary fig 8), which seems to be self-contradictory. We appreciate the reviewer’s keen observation. However, estimating the number of fluorophores per particle in live cells based on a particle’s intensity, as the reviewer implicitly does here, is error prone due to the blurring effect caused by the particle’s movement. Movement results in a distribution of fluorescence signal over a larger area (number of pixels) on the camera’s CCD chip, thus lowering the fluorescence intensity detectable from a single particle. This effect does not occur for a fixed particle, as the reviewer points out. Consequently, the strong overlap in fluorescence distribution from individual particles in live and fixed cells is NOT self-contradictory as it is likely aided by a lowering of the fluorescence intensity in both cases, but through different effects – blurring in the case of live cells and higher chances to observe particles with fewer fluorophores in fixed cells. What this discussion shows is, however, that live and fixed cell experiments are nicely complementary and speak to different properties (diffusion and assembly state, respectively) of miRNA containing particles, as we emphasize throughout the manuscript. We also note that we report our comparison of particle intensities at a stage (2 h after microinjection) where the ‘slow’ and ‘fast’ populations in live cells are almost equally distributed and where we observe a maximal change in the assembly of miRNAs (Fig 3E). Thus, it is highly likely that a predominant fraction of microinjected miRNAs at this stage has indeed assembled into large RNPs, slowing down their diffusion and further favoring the observed overlap in intensity distributions between live and fixed cells. To clarify this latter point we have made the following changes to the revised manuscript: Page 9: “In addition, we observed a strong overlap in the distribution of fluorescence intensities between particles in fixed and living cells 2 h after microinjection (Supplementary Fig S5E), when most miRNAs are assembled into RNPs, suggesting that our counting results in fixed cells closely reflect the miRNA assembly states in living cells.” 4. Sensitivity of the method. The time-dependent change in the fraction of single and multiple miRNA containing particles observed in let-7a-1 is not observed in cxcr4 miRNA, and the authors attribute this to the fact that cxcr4 has 10-fold fewer mRNA targets, therefore the change in population in this case will be too small to be detected. This interpretation needs be tested by cutting down the amount of cxcr4 microinjected. In addition, based on the observation that the "sensitivity" of the method seems to be dependent on the target level of a specific miRNA, maybe the authors can comment on what is the lower limit of the target level for a particular miRNA when applying this method to study its assembly into functional states. Decreasing the level of microinjected cxcr4 miRNA will further bias the distribution towards monomeric miRNA particles in our fixed cell photobleaching analysis. Thus, we instead EMBO reports Peer Review Process File EMBOR-2011-35549 © European Molecular Biology Organization performed an equivalent experiment, by co-microinjecting ~3,000 mRNA target molecules, each bearing six cxcr4 binding sites, along with ~18,000 cxcr4 miRNA (Supplementary Fig 7). Accordingly, we observed an increase in the number of particles containing multiple labeled miRNAs as compared to those where no mRNA or a control mRNA bearing no cxcr4 binding sites was co-microinjected. Consequently, we have made the following changes to the main text: Pages 10/11: “Consistent with this model, cells microinjected with the artificial cxcr4 miRNA, which is predicted to find 10-fold fewer mRNA target molecules in a HeLa cell (Supplementary Methods), do not show these time dependent changes in the fractions of single and multiple miRNA containing particles (Fig 3E; since the decrease in monomeric miRNAs is expected to be ~10-fold smaller for cxcr4 than let-7-a1, it becomes indiscernible in our experiments with an estimated standard error of the mean of ~5-10%). In further support, co-microinjecting ~3,000 mRNA molecules targeted by cxcr4-miRNA increased the fraction of multiple miRNA containing particles at the expense of monomers, while co-microinjecting control mRNA molecules did not (Supplementary Fig S7).” Referee #2: This reviewer finds our study “interesting”, but asks for additional comparisons and control experiments, which we provide as follows.