Single-cell monitoring of dry mass and dry mass density reveals exocytosis of cellular dry contents in mitosis
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Evaluation Summary:
The authors measure dry mass and its density in growing and proliferating cells at high temporal resolution and with high precision. Using this method to study mitotic cells, the authors show that some cell types lose dry mass early in mitosis by a mechanism involving exocytosis. This work improves upon the authors' method to measure the mass of single cells and its thought-provoking conclusion is that dividing cells 'clean out' their contents to give the daughter cells a clean start.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Cell mass and composition change with cell cycle progression. Our previous work characterized buoyant mass dynamics in mitosis (Miettinen et al., 2019), but how dry mass and cell composition change in mitosis has remained unclear. To better understand mitotic cell growth and compositional changes, we develop a single-cell approach for monitoring dry mass and the density of that dry mass every ~75 s with 1.3% and 0.3% measurement precision, respectively. We find that suspension grown mammalian cells lose dry mass and increase dry mass density following mitotic entry. These changes display large, non-genetic cell-to-cell variability, and the changes are reversed at metaphase-anaphase transition, after which dry mass continues accumulating. The change in dry mass density causes buoyant and dry mass to differ specifically in early mitosis, thus reconciling existing literature on mitotic cell growth. Mechanistically, cells in early mitosis increase lysosomal exocytosis, and inhibition of lysosomal exocytosis decreases the dry mass loss and dry mass density increase in mitosis. Overall, our work provides a new approach for monitoring single-cell dry mass and dry mass density, and reveals that mitosis is coupled to extensive exocytosis-mediated secretion of cellular contents.
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Author Response
Reviewer #1 (Public Review):
In this article, Miettinen and colleagues exploit the suspended microchannel resonator developed in their lab and optimize the method to be able to record single live mammalian cells for very long periods of times, across several cell division cycles, while performing a double measure of their buoyant mass in media of different densities (H2O and D2O). Because water exchanges fast enough inside the cell, it allows them to define a dry mass and a dry volume, and thus a density of dry material for single cells along the entire cell division cycle. These measures lead them to confirm and clarify some points from previous studies from their lab and others, such as exponential growth also in dry mass and the fact that buoyant mass and this new dry mass are the same thing in interphase cells. …
Author Response
Reviewer #1 (Public Review):
In this article, Miettinen and colleagues exploit the suspended microchannel resonator developed in their lab and optimize the method to be able to record single live mammalian cells for very long periods of times, across several cell division cycles, while performing a double measure of their buoyant mass in media of different densities (H2O and D2O). Because water exchanges fast enough inside the cell, it allows them to define a dry mass and a dry volume, and thus a density of dry material for single cells along the entire cell division cycle. These measures lead them to confirm and clarify some points from previous studies from their lab and others, such as exponential growth also in dry mass and the fact that buoyant mass and this new dry mass are the same thing in interphase cells. They then find that this is not true during mitosis, mostly because dry mass density increases in early mitosis (dry mass decreases and dry volume decreases even more, suggesting that there is a loss of material of density lower then the average dry mass density). The authors rule out a number of potential mechanisms and give evidence for a role of exocytosis, more precisely exocytosis of lysosomal content. Blocking this phenomenon prevents the change in dry mass density but does not affect cell division. They propose some potential function for this phenomenon, including the interesting hypothesis that this helps cleaning the lysosomal content which might contain some toxic components, so that daughter cells are born with 'clean' lysosomes. Cool idea! It is also quite amazing that the precision of their method allows them to detect this event.
The main question I have concerns the definition of dry mass and dry volume. The authors should discuss in more details what it represents physically. Technically, this is defined by their equation 1, which relates their measure of buoyant mass to a dry mass and a volume of water as parameters to fit from the buoyant mass data. One gets to this equation by writing the definition of buoyant mass as the mass of the cell minus the mass of the equivalent volume of the surrounding medium. But then, to get what the authors find, one has to write that the cell mass is the sum of the dry mass and the mass of water contained in the cell (which makes the dry mass easy to understand) and then to write that the cell volume is the sum of a volume of water and of a volume of dry material. This then defines a dry volume, as the difference between the volume of the cell and the volume of the water contained in the cell (which is the parameter Vwater in the equation 1). At least this is how I got to this equation. The question I asked myself then is: what is this dry volume? Is it really the volume occupied by the dry mass in the cell? This is probably not the case, since dry mass is solvated in the cell. One can estimate this solvated volume using the van't Hoff/Ponder relation, which can be found changing the osmolarity of the external medium. It defines an excluded volume, which is the total volume excluded by macromolecules (like for a van der Waals gas) - it is usually between 25 and 30% of the cell volume. This volume contains the dry mass plus a certain fraction of the water, so it is not exactly the dry mass volume as defined here by the authors. I am worried that this dry mass volume, which is mathematically defined here and calculated from the fit of the equation, is not a standard physical quantity and so it is not easy to relate it to standard biophysical theories (e.g. equations of state), and its behavior could be very unintuitive even for simple systems. This makes the variation in this quantity not easy to interpret, and thus also the variation in dry mass density is not easy to interpret in physical terms.
That being said, it is still clear that whatever this is, it changes in early mitosis, and it seems to be related to exocytosis, so I am not saying that the authors are wrong here. They potentially indeed detect this increase of exocytosis. But they should discuss more what they think this quantity is, either in the methods or in the discussion of the article. In particular, the sentence at the bottom of page 5, line 104, is not clear ('We are not aware of any other single cell methods capable of quantifying this biophysical feature of a cell'), since this measure is not really clearly a biophysical feature of a cell, but is defined a bit artificially from the equation which defines the dry mass volume from the measures of buoyant mass.
Thank you for the detailed and very constructive feedback. As stated above in the Essential Revisions section, we have now clarified the terminology we use and made the terminology more consistent with existing literature. We have also better defined the concept behind our method. Our updated Measurement Method section now states (page 3) that: “In our approach, we consider the buoyant mass of a cell to be dependent on two distinct physical “sections” of the cell, the dry content and the water content. To measure the cell’s dry content independently of the water content, we measure the cell’s buoyant mass in H2O and D2O-based solutions. Under these conditions, the influence of the water content on buoyant mass can be excluded, because the intracellular water is exchanged with extracellular water, making the intracellular water content neutrally buoyant with extracellular solution. This allows us to detect the cell’s dry mass (i.e. total mass – water mass), dry volume (i.e. total volume – water volume) and dry mass density (i.e. dry mass / dry volume).”
The reviewer is also correct that our method measures a dry volume which is, by our model’s definition, the volume occupied by the dry mass independently of water. In other words, our method & measurement model assumes that the intracellular water exchange is 100% complete. The reviewer is correct that some water may be retained, and we cannot directly measure the amount of H2O left inside the cell after immersion in D2O-based media. However, our results indicate that our dry volume measurements are not limited by the water exchange time that the cell experiences (Figure 1–figure supplement 2). In other words, in our measurements, cells exchange all the water they can exchange, be that 100% or 98%. This is further supported by our new estimations of the time needed to transport all water in and out of the cell (see above, other comments section #1, and our updated manuscript page 5). Note that, as our method only exchanges H2O to D2O instead of removing all water from the cell, dry mass will always remain solvated in either H2O or D2O, which makes it plausible that 100% of the water content is exchanged.
As the reviewer keenly points out, our measured dry volume is biophysically distinct from the more classically measured excluded cell volume (or dehydrated cell volume), which still includes some water in the excluded cell volume quantifications. Consistently, our method measures dry volumes that are smaller (~15%) than what the excluded volumes typically are (~25-30%). We do not consider this a limitation of our method, but rather an opportunity for new measurements. That being said, we completely agree with the reviewer that this may cause confusion in the readers. To address this point, our Measurement Method section now states (page 4) that: “Importantly, our approach assumes that all water within the cell is exchangeable between H2O and D2O. Accordingly, our dry volume measurement is distinct from the excluded cell volume detected by measuring cell volume following strong hyperosmotic shocks, which does not remove all water from the intracellular space.”
Finally, we have also changed the sentence “We are not aware of any other single cell methods capable of quantifying this biophysical feature of a cell” (page 5) so that it only refers to a metric, which hasn’t been quantified before on a single-cell level. We believe that this minor change will avoid the suggestion that dry volume is of biophysical importance on its own.
Reviewer #2 (Public Review):
The new suspended microchannel resonator (SMR)-based method described in this paper enables high precision and high temporal resolution single-cell measurements of key physical properties: cell dry mass and the density of cell dry mass, which depends on the macromolecular composition of the cell. The validity of the method is rigorously tested with several convincing control experiments. This method will be useful for future studies investigating cell size and growth regulation and the coordination of mass, volume and density in animal cells.
Using their method, the authors report two important results. First, they confirm that buoyant mass measurement is a valid proxy for cell mass in interphase, an important finding given that SMR measurements have been one of the best and most productive approaches to investigating cell mass growth regulation. Second, they provide evidence that some cell types lose dry mass during metaphase by a mechanism that involves exocytosis, emphasizing how mass, volume, and density dynamics are more complex than during the rest of the cell cycle.
While this paper presents very interesting results, it would benefit significantly from two main improvements. First, the different physical variables studied here (dry mass, dry density, dry mass density, dry volume) should be better defined, and the terminology revised to provide a more straightforward and intuitive description of their biological meaning. Several sections of the paper (especially the introduction and the discussion of Fig. 2-4) should be re-written to help the reader understand the message. Second, some of the drug treatments require more replicates to provide more conclusive answers.
Thank you for this constructive feedback. As stated above in the Essential Revisions section, we have now changed our terminology to increase clarity. Our new density measurement in this manuscript (dry mass divided by dry volume) is now defined as ‘dry mass density’. This change has been applied throughout our manuscript, including our manuscript title. In addition, we have added clearer definitions of each term to our Introduction and Measurement Method sections. Furthermore, we have minimized the use of the term ‘dry composition’ throughout our manuscript, as we now realize this may cause confusion to some readers.
More specifically, our introduction (page 3) now states: “Here, we introduce a new approach for monitoring single cell’s dry mass (i.e. total mass – water mass), dry volume (i.e. total volume – water volume), and density of the dry mass (i.e. dry mass / dry volume), which we will refer to as dry mass density.” These definitions are also repeated in our Measurement Method section (page 4), as many readers may look for the definitions in that section. We have also done many other minor modifications to our main text throughout the manuscript to help the readers understand our message.
In addition, as detailed above in the Essential Revisions section 3, we have adjusted the writing of our manuscript to avoid overly strong claims where our replicate numbers are insufficient. More specifically, we now avoid conclusions where we claim that inhibition of cytokinesis has no influence on dry mass and dry mass density changes in mitosis.
Reviewer #3 (Public Review):
In this manuscript, the authors extend the Manalis lab's vibrating cantilever approach by adding the ability to rapidly exchange media with heavy water. This allows the authors to measure dry mass and its density in growth and proliferating cells. This resolves a previous discrepancy of the cantilever approach and quantitative phase imaging and shows that cells in early mitosis likely increase lysosomal exocytosis. This is an interesting piece of work.
The authors report that: "On average, the FUCCI L1210 cells lost ~4% of dry mass and increased dry density by ~2.5%, and these changes took place in approximately 15 minutes (Figure 3C). In extreme cases, cells lost ~8% of their dry mass while increasing dry density by ~4%". Although these changes may sound small, I believe they would require significant changes to the cell composition. I.e., to increase the overall dry mass density by 4% while losing 8% of the cell's dry mass, the cell would need to lose almost exclusively low-density components, which may not be typical for exocytosis. Moreover, even if all of those lost 8% of cell dry mass are exclusively lipids (or other low-density components), it is not intuitively obvious that such a loss would be sufficient to cause a 4% change to the dry density. To make this more convincing, the authors should provide a simple mathematical model that would roughly estimate how the cell composition (e.g., the contents of lipids vs proteins) needs to change and what the composition of the lost (secreted) components needs to be to provide the observed changes to the dry mass and density, given the existing information on average cell composition and the densities of different biomolecules (lipids, sugars, proteins, etc).
Thank you for this comment. The reviewer is correct that significant changes to the cell composition are needed to explain the phenotypes we observe. As stated above in the Essential Revisions section, we fully agree that such calculations could be very useful in interpreting our results. Our manuscript now contains a new paragraph (discussion section, page 13), where we state: “The magnitude of dry mass density increase in mitosis was large. We have previously observed similar magnitude changes in dry mass density when perturbing proliferation in mammalian cell (Feijo Delgado et al., 2013). To provide some rough estimates of what kind of compositional changes would be required to achieve the dry mass loss and dry mass density increase, we carried out a back-of-the-envelope calculations. Assuming a typical mammalian cell composition and typical macromolecule dry mass densities (Alberts, 2008; Feijo Delgado et al., 2013), we calculated the degree of lipid loss needed to increase dry mass density by 2.5%. This suggested that cells would have to secrete ~1/3 of their lipid content in early mitosis. This could be achieved via lysosomal exocytosis of lipids. Lipid droplets, the main lipid storages inside cells, are frequently trafficked into and degraded in lysosomes (Singh et al., 2009), and lipid droplets can also be secreted via lysosomal exocytosis (Minami et al., 2022). However, it seems likely that the mitotic dry mass density increase also involves secretion of other low dry mass density components (e.g. lipoproteins, specific metabolites) and/or a minor, transient increase in high dry mass density components (e.g. RNAs, specific proteins) in early mitosis. Indeed, CDK1 activity has been suggested to drive a transient increase in protein and RNA content in early mitosis (Asfaha et al., 2022; Clemm von Hohenberg et al., 2022; Miettinen et al., 2019; Shuda et al., 2015).”
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Evaluation Summary:
The authors measure dry mass and its density in growing and proliferating cells at high temporal resolution and with high precision. Using this method to study mitotic cells, the authors show that some cell types lose dry mass early in mitosis by a mechanism involving exocytosis. This work improves upon the authors' method to measure the mass of single cells and its thought-provoking conclusion is that dividing cells 'clean out' their contents to give the daughter cells a clean start.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this article, Miettinen and colleagues exploit the suspended microchannel resonator developed in their lab and optimize the method to be able to record single live mammalian cells for very long periods of times, across several cell division cycles, while performing a double measure of their buoyant mass in media of different densities (H2O and D2O). Because water exchanges fast enough inside the cell, it allows them to define a dry mass and a dry volume, and thus a density of dry material for single cells along the entire cell division cycle. These measures lead them to confirm and clarify some points from previous studies from their lab and others, such as exponential growth also in dry mass and the fact that buoyant mass and this new dry mass are the same thing in interphase cells. They then find that …
Reviewer #1 (Public Review):
In this article, Miettinen and colleagues exploit the suspended microchannel resonator developed in their lab and optimize the method to be able to record single live mammalian cells for very long periods of times, across several cell division cycles, while performing a double measure of their buoyant mass in media of different densities (H2O and D2O). Because water exchanges fast enough inside the cell, it allows them to define a dry mass and a dry volume, and thus a density of dry material for single cells along the entire cell division cycle. These measures lead them to confirm and clarify some points from previous studies from their lab and others, such as exponential growth also in dry mass and the fact that buoyant mass and this new dry mass are the same thing in interphase cells. They then find that this is not true during mitosis, mostly because dry mass density increases in early mitosis (dry mass decreases and dry volume decreases even more, suggesting that there is a loss of material of density lower then the average dry mass density). The authors rule out a number of potential mechanisms and give evidence for a role of exocytosis, more precisely exocytosis of lysosomal content. Blocking this phenomenon prevents the change in dry mass density but does not affect cell division. They propose some potential function for this phenomenon, including the interesting hypothesis that this helps cleaning the lysosomal content which might contain some toxic components, so that daughter cells are born with 'clean' lysosomes. Cool idea! It is also quite amazing that the precision of their method allows them to detect this event.
The main question I have concerns the definition of dry mass and dry volume. The authors should discuss in more details what it represents physically. Technically, this is defined by their equation 1, which relates their measure of buoyant mass to a dry mass and a volume of water as parameters to fit from the buoyant mass data. One gets to this equation by writing the definition of buoyant mass as the mass of the cell minus the mass of the equivalent volume of the surrounding medium. But then, to get what the authors find, one has to write that the cell mass is the sum of the dry mass and the mass of water contained in the cell (which makes the dry mass easy to understand) and then to write that the cell volume is the sum of a volume of water and of a volume of dry material. This then defines a dry volume, as the difference between the volume of the cell and the volume of the water contained in the cell (which is the parameter Vwater in the equation 1). At least this is how I got to this equation. The question I asked myself then is: what is this dry volume? Is it really the volume occupied by the dry mass in the cell? This is probably not the case, since dry mass is solvated in the cell. One can estimate this solvated volume using the van't Hoff/Ponder relation, which can be found changing the osmolarity of the external medium. It defines an excluded volume, which is the total volume excluded by macromolecules (like for a van der Waals gas) - it is usually between 25 and 30% of the cell volume. This volume contains the dry mass plus a certain fraction of the water, so it is not exactly the dry mass volume as defined here by the authors. I am worried that this dry mass volume, which is mathematically defined here and calculated from the fit of the equation, is not a standard physical quantity and so it is not easy to relate it to standard biophysical theories (e.g. equations of state), and its behavior could be very unintuitive even for simple systems. This makes the variation in this quantity not easy to interpret, and thus also the variation in dry mass density is not easy to interpret in physical terms.
That being said, it is still clear that whatever this is, it changes in early mitosis, and it seems to be related to exocytosis, so I am not saying that the authors are wrong here. They potentially indeed detect this increase of exocytosis. But they should discuss more what they think this quantity is, either in the methods or in the discussion of the article. In particular, the sentence at the bottom of page 5, line 104, is not clear ('We are not aware of any other single cell methods capable of quantifying this biophysical feature of a cell'), since this measure is not really clearly a biophysical feature of a cell, but is defined a bit artificially from the equation which defines the dry mass volume from the measures of buoyant mass.
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Reviewer #2 (Public Review):
The new suspended microchannel resonator (SMR)-based method described in this paper enables high precision and high temporal resolution single-cell measurements of key physical properties: cell dry mass and the density of cell dry mass, which depends on the macromolecular composition of the cell. The validity of the method is rigorously tested with several convincing control experiments. This method will be useful for future studies investigating cell size and growth regulation and the coordination of mass, volume and density in animal cells.
Using their method, the authors report two important results. First, they confirm that buoyant mass measurement is a valid proxy for cell mass in interphase, an important finding given that SMR measurements have been one of the best and most productive approaches to …
Reviewer #2 (Public Review):
The new suspended microchannel resonator (SMR)-based method described in this paper enables high precision and high temporal resolution single-cell measurements of key physical properties: cell dry mass and the density of cell dry mass, which depends on the macromolecular composition of the cell. The validity of the method is rigorously tested with several convincing control experiments. This method will be useful for future studies investigating cell size and growth regulation and the coordination of mass, volume and density in animal cells.
Using their method, the authors report two important results. First, they confirm that buoyant mass measurement is a valid proxy for cell mass in interphase, an important finding given that SMR measurements have been one of the best and most productive approaches to investigating cell mass growth regulation. Second, they provide evidence that some cell types lose dry mass during metaphase by a mechanism that involves exocytosis, emphasizing how mass, volume, and density dynamics are more complex than during the rest of the cell cycle.
While this paper presents very interesting results, it would benefit significantly from two main improvements. First, the different physical variables studied here (dry mass, dry density, dry mass density, dry volume) should be better defined, and the terminology revised to provide a more straightforward and intuitive description of their biological meaning. Several sections of the paper (especially the introduction and the discussion of Fig. 2-4) should be re-written to help the reader understand the message. Second, some of the drug treatments require more replicates to provide more conclusive answers.
-
Reviewer #3 (Public Review):
In this manuscript, the authors extend the Manalis lab's vibrating cantilever approach by adding the ability to rapidly exchange media with heavy water. This allows the authors to measure dry mass and its density in growth and proliferating cells. This resolves a previous discrepancy of the cantilever approach and quantitative phase imaging and shows that cells in early mitosis likely increase lysosomal exocytosis. This is an interesting piece of work.
The authors report that: "On average, the FUCCI L1210 cells lost ~4% of dry mass and increased dry density by ~2.5%, and these changes took place in approximately 15 minutes (Figure 3C). In extreme cases, cells lost ~8% of their dry mass while increasing dry density by ~4%". Although these changes may sound small, I believe they would require significant …
Reviewer #3 (Public Review):
In this manuscript, the authors extend the Manalis lab's vibrating cantilever approach by adding the ability to rapidly exchange media with heavy water. This allows the authors to measure dry mass and its density in growth and proliferating cells. This resolves a previous discrepancy of the cantilever approach and quantitative phase imaging and shows that cells in early mitosis likely increase lysosomal exocytosis. This is an interesting piece of work.
The authors report that: "On average, the FUCCI L1210 cells lost ~4% of dry mass and increased dry density by ~2.5%, and these changes took place in approximately 15 minutes (Figure 3C). In extreme cases, cells lost ~8% of their dry mass while increasing dry density by ~4%". Although these changes may sound small, I believe they would require significant changes to the cell composition. I.e., to increase the overall dry mass density by 4% while losing 8% of the cell's dry mass, the cell would need to lose almost exclusively low-density components, which may not be typical for exocytosis. Moreover, even if all of those lost 8% of cell dry mass are exclusively lipids (or other low-density components), it is not intuitively obvious that such a loss would be sufficient to cause a 4% change to the dry density. To make this more convincing, the authors should provide a simple mathematical model that would roughly estimate how the cell composition (e.g., the contents of lipids vs proteins) needs to change and what the composition of the lost (secreted) components needs to be to provide the observed changes to the dry mass and density, given the existing information on average cell composition and the densities of different biomolecules (lipids, sugars, proteins, etc).
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