The timing of Start is determined primarily by increased synthesis of the Cln3 activator rather than dilution of the Whi5 inhibitor
2022; American Society for Cell Biology; Volume: 33; Issue: 5 Linguagem: Inglês
10.1091/mbc.e21-07-0349
ISSN1939-4586
AutoresAthanasios Litsios, Pooja Goswami, Hanna M. Terpstra, Carleton H. Coffin, Luc-Alban Vuillemenot, Mattia Rovetta, Ghada Ghazal, Paolo Guerra, Katarzyna Buczak, Alexander Schmidt, Sylvain Tollis, Mike Tyers, Catherine A. Royer, Andreas Milias‐Argeitis, Matthias Heinemann,
Tópico(s)RNA and protein synthesis mechanisms
ResumoMolecular Biology of the CellVol. 33, No. 5 ResponseFree AccessThe timing of Start is determined primarily by increased synthesis of the Cln3 activator rather than dilution of the Whi5 inhibitorAthanasios Litsios, Pooja Goswami, Hanna M. Terpstra, Carleton Coffin, Luc-Alban Vuillemenot, Mattia Rovetta, Ghada Ghazal, Paolo Guerra, Katarzyna Buczak, Alexander Schmidt, Sylvain Tollis, Mike Tyers, Catherine A. Royer, Andreas Milias-Argeitis, and Matthias HeinemannAthanasios LitsiosMolecular Systems Biology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9747 AG Groningen, NetherlandsSearch for more papers by this author, Pooja GoswamiBiological Sciences, Rensselaer Polytechnic Institute, Troy, NY 12180Search for more papers by this author, Hanna M. TerpstraMolecular Systems Biology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9747 AG Groningen, NetherlandsSearch for more papers by this author, Carleton CoffinBiological Sciences, Rensselaer Polytechnic Institute, Troy, NY 12180Search for more papers by this author, Luc-Alban VuillemenotMolecular Systems Biology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9747 AG Groningen, NetherlandsSearch for more papers by this author, Mattia RovettaMolecular Systems Biology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9747 AG Groningen, NetherlandsSearch for more papers by this author, Ghada GhazalInstitute for Research in Immunology and Cancer, University of Montréal, Montréal, H3T 1J4 QC, CanadaSearch for more papers by this author, Paolo GuerraMolecular Systems Biology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9747 AG Groningen, NetherlandsSearch for more papers by this author, Katarzyna BuczakProteomics Core Facility, Biozentrum, University of Basel, 4056 Basel, SwitzerlandSearch for more papers by this author, Alexander SchmidtProteomics Core Facility, Biozentrum, University of Basel, 4056 Basel, SwitzerlandSearch for more papers by this author, Sylvain Tollis*Address correspondence to: Sylvain Tollis (E-mail Address: [email protected]); Mike Tyers (E-mail Address: [email protected]); Catherine A. Royer (E-mail Address: [email protected]); Andreas Milias-Argeitis (E-mail Address: [email protected]); Matthias Heinemann (E-mail Address: [email protected]).Institute for Research in Immunology and Cancer, University of Montréal, Montréal, H3T 1J4 QC, CanadaInstitute of Biomedicine, University of Eastern Finland, FI-70210 Kuopio, FinlandSearch for more papers by this author, Mike Tyers*Address correspondence to: Sylvain Tollis (E-mail Address: [email protected]); Mike Tyers (E-mail Address: [email protected]); Catherine A. Royer (E-mail Address: [email protected]); Andreas Milias-Argeitis (E-mail Address: [email protected]); Matthias Heinemann (E-mail Address: [email protected]).Institute for Research in Immunology and Cancer, University of Montréal, Montréal, H3T 1J4 QC, CanadaSearch for more papers by this author, Catherine A. Royer*Address correspondence to: Sylvain Tollis (E-mail Address: [email protected]); Mike Tyers (E-mail Address: [email protected]); Catherine A. Royer (E-mail Address: [email protected]); Andreas Milias-Argeitis (E-mail Address: [email protected]); Matthias Heinemann (E-mail Address: [email protected]).Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY 12180Search for more papers by this author, Andreas Milias-Argeitis*Address correspondence to: Sylvain Tollis (E-mail Address: [email protected]); Mike Tyers (E-mail Address: [email protected]); Catherine A. Royer (E-mail Address: [email protected]); Andreas Milias-Argeitis (E-mail Address: [email protected]); Matthias Heinemann (E-mail Address: [email protected]).Molecular Systems Biology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9747 AG Groningen, NetherlandsSearch for more papers by this author, and Matthias Heinemann*Address correspondence to: Sylvain Tollis (E-mail Address: [email protected]); Mike Tyers (E-mail Address: [email protected]); Catherine A. Royer (E-mail Address: [email protected]); Andreas Milias-Argeitis (E-mail Address: [email protected]); Matthias Heinemann (E-mail Address: [email protected]).Molecular Systems Biology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9747 AG Groningen, NetherlandsSearch for more papers by this authorSophie G Martin, Monitoring EditorPublished Online:28 Apr 2022https://doi.org/10.1091/mbc.E21-07-0349AboutSectionsView articleSupplemental MaterialView PDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail View articleINTRODUCTIONHow cells convert shallow-input gradients into all-or-none decisions is a fundamental problem in biology. Commitment to cell division is thought to require a threshold level of cell growth, but how incremental changes in growth are measured by the cell is unknown. Various hypotheses have been proposed to explain growth-dependent cell cycle commitment in late G1 phase in budding yeast (called Start), including two recent models. One model (Schmoller et al., 2015) posits that as the nucleus grows during G1 phase, the nuclear concentration of the G1/S transcriptional inhibitor Whi5 slowly decreases, thereby increasing the probability of the Start transition. This model of passive dilution is predicated on estimates of Whi5 nuclear concentration as assessed by wide-field epifluorescence microscopy as a function of time in G1 phase daughter cells. An alternative model (Litsios et al., 2019) posits that an increase in protein synthesis toward the end of G1 rapidly drives up the concentration of the highly unstable G1 cyclin Cln3, which activates Cdc28 (Cdk1), thereby triggering the phosphorylation-mediated dissociation of Whi5 from the SBF G1/S transcription factor complex and activation of the G1/S program. This model of active control of Start is supported by measurements of protein synthetic rate and Cln3 concentration as a function of time. Notably, based on wide-field epifluorescence detection of a Whi5-fluorescent protein (FP) fusion and mass spectrometry, Litsios et al. (2019) concluded that there is only “a small or no change in Whi5 concentration” throughout G1, consistent with earlier reported results (Dorsey et al., 2018) using scanning Number and Brightness (sN&B) microscopy, a method that allows for determination of absolute protein concentrations.In their Letter to Molecular Biology of the Cell, Schmoller et al. (2022) raise questions about the results and conclusions presented in our published studies (Dorsey et al., 2018; Litsios et al., 2019). Here, we respond to the criticisms of Schmoller et al. and demonstrate how wide-field fluorescence microscopy experiments to determine nuclear Whi5 concentration dynamics can be confounded by uncontrolled effects, which include photobleaching, partial confocal effects, and nuclear-to-cytoplasmic volume scaling. Further, we provide additional experimental evidence demonstrating that nuclear Whi5 concentration is essentially constant as cells grow in G1 phase and that Cln3 and protein synthesis dynamics occur as reported in Litsios et al. (2019). These results suggest that instead of being triggered by dilution of the stable inhibitor Whi5, Start is rather primarily controlled by the increase in protein synthesis rate in G1 and the concomitant production of the unstable activator Cln3.RESULTS AND DISCUSSIONThere is little or no change in Whi5 concentration during G1It was originally proposed by Schmoller et al. (2015) that Whi5 concentration decreases during G1 and thereby triggers Start. This model is based on estimates of Whi5 concentration during G1 obtained by time-lapse wide-field fluorescence microscopy. In their Letter to Molecular Biology of the Cell, Schmoller et al. (2015) argue that normalization and alignment in time of single-cell Whi5 traces can mask the extent to which Whi5 is diluted. Plotting the data aligned for either birth (as done by Schmoller et al., 2015) or for Start (as done by Litsios et al., 2019) can indeed lead to different visual impressions (Supplemental Figure S1, A and B, respectively). Aligning the data for birth exaggerates the extremes and does not reflect the average population behavior. This is because the rightmost part of the average Whi5 concentration profile is dominated by the few cells that spend an unusually long time in G1 before Start. These cells grow more and thus show a larger apparent Whi5 dilution than most of the population. Because every cell passes Start at a different time point, in Litsios et al. (2019), we aligned the single-cell traces at Start, so that the average dilution of Whi5 at Start can be directly read off the plot (Supplemental Figure S1B).However, it is important to note that to determine the Whi5 dilution factor from single-cell Whi5 data, one does not need to align the data at all. The dilution factor is a number that can be calculated for each cell using the Whi5 fluorescence intensity values and cell volume at birth and at Start, as shown in Supplemental Figure S1C. When calculating the Whi5 dilution factor using the integrated cell fluorescence divided by total cell volume as a proxy for protein concentration (as Schmoller et al., 2015), various literature sources find the following values: Using Whi5-mCitrine, Schmoller et al. (2015) reported an average decrease in Whi5 concentration of 25% between birth and Start in daughters grown on synthetic complete (SC) medium with 2% glycerol and 1% ethanol as the carbon source (Figure 1G of Schmoller et al., 2015), as measured in a bck2Δ strain that is partially defective for Start and 50% larger than the wild type. An average Whi5-mCherry decrease of 18% was observed for daughters grown in 2% glucose minimal medium (Figure 5A of Litsios et al., 2019; Supplemental Figure S1B, C) using the same wide-field technique and concentration proxy as Schmoller et al. (2015). Decreases of similar magnitude (i.e., 12–25%) have also been reported in Qu et al. (2019) as well as in Litsios et al. (2019) for daughter cells grown on different carbon sources. Thus, using wide-field microscopy and the proxy of Schmoller et al. to estimate protein concentration consistently results in Whi5 dilution factors in the range of 12–25%.FIGURE 1: Determination of Whi5 concentration with wide-field fluorescence microscopy may be confounded by photobleaching. Photobleaching of sfGFP (A) and mCherry (B) expressed from the TEF1 promoter. Cellular fluorescence was quantified as average fluorescence intensity within the cell mask and normalized to the value at the first time point. Cells were imaged over the course of 250 s for sfGFP and 270 s for mCherry to generate 49 images (“frames”) with approximately 5 s between subsequent exposures. A change in fluorophore concentration due to protein synthesis, dilution, and degradation over the short time course of the experiment can therefore be neglected. Comparing fluorescence between wild-type cells and cells expressing the fluorophores from the TEF1 promoter shows that cellular autofluorescence is negligible compared with the signal of the fluorescent proteins (inserts).As these are relatively small changes in apparent Whi5 concentration, it is critical 1) to assess any potential confounding effects arising from the use of time-lapse wide-field fluorescence microscopy and 2) to estimate the time dependence of Whi5 concentration with alternative experimental methods.Variables affecting Whi5 concentration quantification by wide-field time-lapse fluorescence microscopyPhotobleaching is an inevitable confounding factor in time-lapse quantitative fluorescence microscopy (Cranfill et al., 2016; Fadero et al., 2018), and from our experience, it is extremely difficult, if not impossible, to fully avoid photobleaching when the same cells are imaged repeatedly over time. Here, we assessed to what extent our own published Whi5 experiments (i.e., Litsios et al., 2019), obtained using wide-field fluorescence microscopy, were affected by photobleaching.To quantitatively determine the extent to which bleaching can contribute to a decrease in Whi5 signal, we determined the bleaching rate of sfGFP and mCherry with imaging settings that closely resemble (for sfGFP) or are identical to (for mCherry) those that we used in Litsios et al. (2019), with which we had found 12% and 18% drops in Whi5 concentration, respectively. For these new photobleaching control experiments, we used strains that expressed the fluorescent proteins from a strong promoter (TEF1), such that the signal from the fluorescent protein was substantially greater than cellular autofluorescence (Figure 1, A and B, inserts) and hence the cellular autofluorescence can be neglected. To obtain the photobleaching curve, we then imaged 49 times the same cells (same fields of view [FOVs]) over ∼4.5 min. Through this approach, which has been used in other studies to evaluate the rate of photobleaching (Fadero et al., 2018), the acquired fluorescence data are only minimally affected by synthesis of the fluorescent protein during the imaging period. The amount of light per exposure (i.e., light intensity times exposure duration) that we used for these bleaching tests was equal to (for mCherry) or very closely resembled (for sfGFP) the amount of light we used previously (Litsios et al., 2019) to measure mCherry-Whi5 and Whi5-sfGFP. Specifically, for mCherry we used a light intensity of 50% (amounting to 5.1 nW/µm2 measured at the specimen) and a 600 ms exposure and for sfGFP, 6% (9.8 nW/µm2 measured at the specimen) and 100 ms. Even at this low-light intensity, we found that the fluorescence signal of sfGFP dropped by 3% over the course of the first four exposures and then subsequently by 0.067% with each additional exposure (Figure 1A) while each exposure reduced the mCherry fluorescent signal by 0.29% (Figure 1B).While these bleaching rates appear low, with a G1 daughter length of 60 min and a 3-min sampling interval, the determined bleaching rate of mCherry would yield a signal drop of about 6% (i.e., [1-0.0029]^20 = 0.9435) in our wide-field microscopy experiments with Whi5-mCherry, assuming minimal synthesis of Whi5 during G1 (Litsios et al., 2019). This photobleaching effect would thus account for approximately one third of the observed Whi5-mCherry signal decrease of 18% in our published data (Supplemental Figure S1B, C). With the more photostable sfGFP a lower drop in Whi5 signal would be expected, and consistently we previously found only an about 12% decrease in the signal of Whi5-sfGFP during G1 (Litsios et al., 2019). These controls indicate that photobleaching occurs even with high-numerical-aperture objectives and low exposure settings and that it can contribute to an apparent decrease in Whi5 concentration observed with wide-field epifluorescence microscopy.Another potential artifact connected with the determination of protein levels via wide-field microscopy can arise by the so-called “partial confocal effect” (Gordon et al., 2007; Joglekar et al., 2008; Verdaasdonk et al., 2014). Light emerging from different depths of the specimen does not equally contribute to the overall signal that is measured at a pixel. In other words, while light coming from the focal plane is entirely detected and measured, slightly out-of-focus light is only partially accounted for. This effect, which has been observed for yeast cell imaging with high-numerical-aperture objectives (Gordon et al., 2007; Joglekar et al., 2008; Verdaasdonk et al., 2014), could skew the concentration estimates as obtained by Schmoller and colleagues. Specifically, Schmoller and colleagues assume that the sum of pixel fluorescence intensities of a given cell is proportional to the amount of fluorescent protein in that cell. They then divide this sum by the cell volume to obtain a proxy for protein concentration. However, given the fact that the fraction of the fluorescence intensity attributable to out-of-focus light increases in larger cells (Gordon et al., 2007), the concentration proxy used by Schmoller et al. (2015) could decrease as a cell grows during G1, even if the actual protein concentration stays constant. The contribution of this phenomenon to the quantification of large (e.g., severalfold) changes in protein concentration can be neglected but becomes potentially relevant for small changes in protein concentration such as those reported for Whi5. While we cannot quantitatively determine the degree of underestimation of the protein concentration due to this effect, when we alternatively use the average fluorescence intensity in a cell as another proxy of cellular protein concentration (a common practice in the field [Lo et al., 2015], which we also implemented in Litsios et al. [2019] together with the concentration proxy approach of Schmoller et al., 2015), then the fluorescence intensity drop of Whi5 during G1 is nearly zero (see WF-1 data in Figure 5A of Litsios et al., 2019). Thus, partial confocal effects may also lead to an apparent drop in Whi5 concentration during G1, particularly for larger cells.A third potentially confounding factor in the analysis of the Whi5-mCitrine time courses of Schmoller et al. (2015) (and our Whi5-mCherry and Whi5-sfGFP data shown in Figure 5A of Litsios et al., 2019) lies in the fact that if Whi5 is indeed diluted during G1, then it must be the nuclear Whi5 that gets diluted because Whi5 resides mainly in the nucleus before Start. In this context, it is critical to note that neither Schmoller et al. (2015), nor the other authors of Schmoller et al. (2022), nor Litsios et al. (2019) measured the nuclear Whi5 concentration. Instead, all these authors divided the total Whi5 signal of a cell (which they interpret as the Whi5 amount, as discussed above) by the cell volume to obtain an estimate of Whi5 concentration. If the nuclear volume scales proportionally with cell volume during G1, then this approach could indeed be used to estimate the relative nuclear Whi5 concentration dynamics during G1, as illustrated in Figure 2A. However, it has been reported that although the nucleus of daughter cells grows during G1, it does not grow as fast as the cytoplasm (Jorgensen et al., 2007). We confirmed this previous result using a strain with fluorescently tagged Nup133 (Nup133-yeGFP) to delineate the nuclear membrane and found that the average nucleus-to-whole-cell volume ratio decreases from around 10% in small daughter cells to around 7% in large daughters (Figure 2B). When the ratio of nuclear-to-cytoplasmic volume is plotted against cytoplasmic volume, as published previously (Jorgensen et al., 2007), a highly similar regression line and negative correlation coefficient are obtained (Figure 2C), demonstrating that the nuclear-to-whole-cell volume ratio decreases through G1 phase.FIGURE 2: Determination of nuclear Whi5 concentration by wide-field fluorescence microscopy is confounded by nonproportional scaling of the nuclear-to-cell volume. (A) Schematic illustration of the nuclear-to-cell volume scaling problem. Whi5 is in the nucleus prior to Start. As time passes, cells grow, and so does the nucleus. Calculating nuclear Whi5 concentration by dividing total cell fluorescence by cell volume (as performed by Schmoller et al., 2015) presupposes that the nucleus occupies a constant fraction of the whole-cell volume as cells grow during G1 phase. (B) Ratio of nuclear to whole-cell volume as a function of daughter cell volume. Nuclear volume was determined with Nup133-yeGFP, and cell volume was determined from bright-field images (see Materials and Methods). The regression line is shown in blue, and the confidence band for the regression line is shown in red. (C) Ratio of nuclear-to-cytoplasmic volume as a function of daughter cytoplasmic volume. The cytoplasmic volume was calculated as the difference between whole-cell and nuclear volumes. The regression line and confidence band for our data is shown in green. The respective regression line from Jorgensen et al. (2007) (purple), where this relationship between nuclear and cell volume during G1 was first reported, is reproduced for comparison.To put the above numbers in perspective: given a cell volume increase of around 60% between birth and Start for a daughter cell in glucose medium (e.g., Figure 4 of Soifer and Barkai, 2014; Figure 3 of Litsios et al., 2019), if Whi5 concentration is calculated by using cell volume as a proxy for nuclear volume, then the concentration of Whi5 would reveal a drop of around 10%, even if the nuclear protein concentration was assumed constant. This discrepancy becomes even larger if a cell grows more during G1. This means that an observed apparent Whi5 dilution of 18% based on the division of whole-cell fluorescence with cell volume, in reality corresponds to only a dilution of 8% for nuclear Whi5.FIGURE 3: Mass spectrometry analysis demonstrates that Whi5 concentration is constant during G1. (A) Whi5 amino acid sequence; six peptides identified in our mass spectrometry analysis are indicated by colored boxes; red letters: known phosphosites according to BioGRID (https://thebiogrid.org/34481/protein); turquoise boxes: CDK sites according to Wagner et al. (2009). (B, D, E) Signals from six Whi5 peptides, normalized to the signals from housekeeping proteins (Tdh3, Eno2, Act1). The normalized values of a peptide from an experiment were then divided by the peptide’s median value within this experiment. Data are from three independent cultivations, elutriations, and mass spectrometric analyses. Time point 0 indicates the moment when the elutriated cells were released into the fresh glucose minimal medium. Sampled cells were quenched with 10% TCA to prevent abiotic changes in protein phosphorylation. Note that the protein extraction protocol used in Litsios et al. (2019) was not explicitly designed to preserve protein phosphorylation such that any adventitiously dephosphorylated phosphopeptides would be expected to show a flat temporal behavior. (B) The signals of three Whi5 peptides that contain a CDK site and drop in signal. (D) The signals of two Whi5 peptides that do not contain any phosphosites. (E) The signal of a peptide with a single CDK site. An alternative normalization against all detected peptides in the proteome (instead of only those from the three housekeeping proteins) generated identical results (Supplemental Figure S2). (C) Budding indices as obtained in the three independent experiments.In summary, at least three effects can lead to an overestimation of the drop in Whi5 signal based on wide-field microscopy data. First, our photobleaching tests demonstrate that photobleaching can explain part of the dilution we observed with Whi5-mCherry, given the wide-field fluorescence imaging conditions used in our experiments. While we cannot comment on the imaging settings used previously (Schmoller et al., 2015), these should not deviate much from the settings used here. Second, the partial confocal effect in combination with the method of Schmoller et al. to estimate intracellular protein concentration could lead to an underestimation of protein concentration, particularly as cell volume increases. Third, the incorrect assumption that the ratio of nuclear-to-cell volume remains constant during G1 leads to an overestimation of the decrease in Whi5 concentration in the nucleus. Together, these confounding effects could explain a large fraction of the modest apparent dilution of 12–25% observed by Litsios et al. (2019); Schmoller et al. (2015) and Qu et al. (2019), in a range of different growth conditions. Given these potential confounding effects, we believe that it is absolutely crucial to investigate Whi5 concentration changes during G1 with alternative experimental methods. In the following sections, we describe results from such orthogonal measurement approaches.Assessment of Whi5 concentration by mass spectrometry does not reveal dilutionBy mass spectrometric detection of Whi5 peptides, we did not observe any Whi5 dilution during G1 in cell cultures synchronized by elutriation (Litsios et al., 2019). In their Letter, Schmoller et al. (2022) criticize two aspects of this mass spectrometry data set, namely 1) the potential for interference by foreign peptides from the rich growth medium used and 2) that signals of the measured unphosphorylated Whi5 peptides did not decrease as cells approach Start, which should happen due to CDK-dependent phosphorylation of Whi5 and concomitant signal loss of the unphosphorylated peptides. To demonstrate the validity of our original conclusion that Whi5 concentration does not decrease in G1, we performed additional mass spectrometry experiments. Specifically, we used elutriation to generate synchronized populations of cells grown on minimal synthetic medium, to avoid putative contamination from foreign peptides originating from YPD (yeast extract, bacto peptone, dextrose) medium. Synchronized cell populations obtained by elutriation were then subjected to mass spectrometric proteome analyses at 16 different time points. In this experiment, contrary to the protocol used in Litsios et al. (2019), we preserved protein phosphorylation by quenching cells with 10% trichloroacetic acid (TCA) directly after harvesting (Kanshin et al., 2015).Six Whi5 peptides were identified (Figure 3A), the signals of which were normalized to the summed signals of peptides of housekeeping proteins (Tdh3, Eno2, Act1) whose abundances reflect total protein content. The concentration of three Whi5 peptides (SPPTAAR, SEVFLSPSPR, NGFGTPSPPSPPGITK) declined (Figure 3B) until the budding index (Figure 3C) reached a value of ∼0.5, at which point Start had occurred in most of the cells. These three peptides contain CDK phosphosites and are therefore expected to be phosphorylated as cells approach Start (Costanzo et al., 2004; De Bruin et al., 2004; Wagner et al., 2009). As our mass spectrometry analysis detected only the unphosphorylated form of these peptides, the concentration of these forms should decrease over time, which is exactly what the data show (Figure 3B). As a control, the samples were treated with phosphatase to allow the entire pool of Whi5 peptides to be detected. In this test, we found that the concentration of two phosphatase-treated peptides (one with two documented phosphosites, SEVFLSPSPR, and as a control one without a phosphosite, LNYALVK) remained constant throughout G1 (Supplemental Figure S3), supporting the notion that the signal reduction for these peptides is due to increasing phosphorylation as cells approach Start, and contradicting Whi5 dilution. Furthermore, two other Whi5 peptides that do not bear CDK sites (LNYALVK, LQNGWTDK) and another peptide that contains a single CDK site (TLPELETELAPAVQTPPR) remained unchanged over the entire 300 min observation period (Figure 3, D and E). It should be noted that the presence of the CDK consensus site on the latter peptide does not imply that it is phosphorylated as cells grow; our data suggest that the phosphorylation status of this peptide does not change during G1. Collectively, these new mass spectrometry results suggest that Whi5 concentration is constant during G1, consistent with the conclusion drawn previously (Litsios et al., 2019).Assessment of Whi5 concentration by scanning 2-photon microscopy reveals invariant Whi5 concentration in G1 cellsTime-lapse wide-field fluorescence microscopy is subject to photobleaching and partial confocal effects and furthermore cannot directly measure nuclear protein concentrations, as demonstrated above. Mass spectrometry, like immunoblot detection, is a population-level analysis method that also cannot resolve nuclear Whi5 concentration. Alternative experimental methods are therefore required to substantiate conclusions on nuclear Whi5 dynamics. Such alternatives are offered by microscopy methods that can directly measure nuclear Whi5 concentration.In Dorsey et al. (2018), we used 2-photon (2p) scanning number and brightness (2psN&B) to show that nuclear Whi5 concentration is constant as a function of cell size during G1. In their Letter, Schmoller et al. (2022) stated that they are not familiar with 2psN&B and thus cannot judge the approach. 2psN&B is a well-established and widely used fluorescence fluctuation–based method for quantitative single-cell imaging (e.g., Nagy et al., 2010; Hellriegel et al., 2011; Moutin et al., 2014; Bourges et al., 2017; Cutrale et al., 2019; Zamai et al., 2019). 2psN&B can provide absolute protein concentrations, protein complex stoichiometry, and/or dynamic information on diffusion rates and has several advantages over wide-field fluorescence imaging. First, 2p imaging eliminates contributions from out-of-focus light because the 2p excitation volume is <1 fl and thus it is possible to gather light that emanates only from the yeast nucleus. Hence, 2psN&B is immune to partial confocal effects and nucleus-to-whole-cell volume ratio scaling effects. Second, because 2p microscopy excites in the infrared range (1000 nm), it diminishes the contribution of autofluorescence to the overall signal. Using this approach for GFP protein fusions at concentrations above 100 nM (such as nuclear Whi5-GFP), the contribution of autofluorescence is negligible (Dorsey et al., 2018). Third, the lower excitation energy (1000 nm compared with 488 nm) causes less phototoxicity than visible light laser scanning confocal microscopy.Using these advantages of 2psN&B microscopy, Dorsey et al. (2018) implemented an experimental approach that completely avoids photobleaching while determining nuclear Whi5 concentration during G1. Specifically, individual
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