Probe-free optical chromatin deformation and measurement of differential mechanical properties in the nucleus
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Seelbinder et al. describe a new method for perturbing chromatin in living cells by local heating. Employing this approach, the authors uncover interesting behaviors that underscore the variability in the mechanical response of subnuclear domains and structures. The study is timely, and if some conceptual and technical aspects are improved, it should be of broad interest to both the cell biophysics and cell biology communities, in particular since the method can also be applied to study mechanical relationships of subcellular compartments in other cellular and multicellular systems.
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Abstract
The nucleus is highly organized to facilitate coordinated gene transcription. Measuring the rheological properties of the nucleus and its sub-compartments will be crucial to understand the principles underlying nuclear organization. Here, we show that strongly localized temperature gradients (approaching 1°C/µm) can lead to substantial intra-nuclear chromatin displacements (>1 µm), while nuclear area and lamina shape remain unaffected. Using particle image velocimetry (PIV), intra-nuclear displacement fields can be calculated and converted into spatio-temporally resolved maps of various strain components. Using this approach, we show that chromatin displacements are highly reversible, indicating that elastic contributions are dominant in maintaining nuclear organization on the time scale of seconds. In genetically inverted nuclei, centrally compacted heterochromatin displays high resistance to deformation, giving a rigid, solid-like appearance. Correlating spatially resolved strain maps with fluorescent reporters in conventional interphase nuclei reveals that various nuclear compartments possess distinct mechanical identities. Surprisingly, both densely and loosely packed chromatin showed high resistance to deformation, compared to medium dense chromatin. Equally, nucleoli display particularly high resistance and strong local anchoring to heterochromatin. Our results establish how localized temperature gradients can be used to drive nuclear compartments out of mechanical equilibrium to obtain spatial maps of their material responses.
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eLife assessment
Seelbinder et al. describe a new method for perturbing chromatin in living cells by local heating. Employing this approach, the authors uncover interesting behaviors that underscore the variability in the mechanical response of subnuclear domains and structures. The study is timely, and if some conceptual and technical aspects are improved, it should be of broad interest to both the cell biophysics and cell biology communities, in particular since the method can also be applied to study mechanical relationships of subcellular compartments in other cellular and multicellular systems.
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Reviewer #1 (Public Review):
In this manuscript the authors describe a new method for perturbing chromatin in living cells by delivering a local temperature gradient. Employing this approach, the authors uncover interesting behaviors that underscore the variability in the mechanical response of subnuclear domains and structures. The combination of a new experimental tool that should be accessible to many users and new insights are compelling, although there is the need for some controls and a broader discussion of prior work.
Strengths:
1. There is a need for non-invasive methods for probing the mechanical properties of chromatin, and nuclei and the approach developed by the authors has strong potential to be of broad utility.
2. By and large the authors provide a reasonable characterization of the technical aspects of the method, for …Reviewer #1 (Public Review):
In this manuscript the authors describe a new method for perturbing chromatin in living cells by delivering a local temperature gradient. Employing this approach, the authors uncover interesting behaviors that underscore the variability in the mechanical response of subnuclear domains and structures. The combination of a new experimental tool that should be accessible to many users and new insights are compelling, although there is the need for some controls and a broader discussion of prior work.
Strengths:
1. There is a need for non-invasive methods for probing the mechanical properties of chromatin, and nuclei and the approach developed by the authors has strong potential to be of broad utility.
2. By and large the authors provide a reasonable characterization of the technical aspects of the method, for example how local temperatures rise and the propagation of the temperature gradient relative to the rastering of the IR laser.
3. The findings that different chromatin compartments respond in distinct manners, in ways perhaps that were not intuited previously (for example, the highest level of deformation for "medium dense" chromatin domains regions), is provocative and raises new ideas about how the chromatin polymer and diffusible nuclear constituent molecules in different domains together contribute to the mechanical response.
4. The method provides insights into the viscoelastic properties of different chromatin domains, particularly different time scales of behavior, that have been challenging to access with existing approaches.
5. The authors provide new measurements of the behavior of nucleoli, which leads to insights that will impact our view of the mechanical behavior of such organelles.Weaknesses:
1. Direct or indirect effects of the temperature gradient on the integrity of the DNA needs to be addressed, as this could influence the response particularly given the observation that there is a ~15% of the response that is not reversible (see next point).
2. The authors do not probe the basis for the irreversibility of the chromatin response, which seems to perhaps differ between different chromatin regions. The underlying factors that underlie this need to be further explored.
3. The authors need to acknowledge the time scales of behaviors that can be revealed using the approach and how this influences their observations. For example, they observe the creep behavior on the 1 second timescale, which is an order of magnitude below observations of the behavior of whole nuclei (~15 seconds) for nuclei from mammalian to yeast that has been suggested to reflect chromatin flow.
4. There are numerous studies important for the premise and interpretation of this study that need to be considered/cited. -
Reviewer #2 (Public Review):
In this manuscript, Seelbinder et al, introduce a novel, elegant approach to study the organization of cell nuclei, which complements currently existing technology. The authors employ localized temperature gradients to move chromatin inside the nucleus noninvasively, and they study flow fields and deformations of different nuclear compartments in different experimental settings. The study is timely and should be of broad interest to a wide readership, in particular since the method can also be applied to study mechanical relationships of subcellular compartments in other cellular and extracellular systems.
The non-invasive manipulation of cell organelles in intact cells has been a challenge for decades. The new technique introduced in this study contributes to closing this important gap, enabling experiments …
Reviewer #2 (Public Review):
In this manuscript, Seelbinder et al, introduce a novel, elegant approach to study the organization of cell nuclei, which complements currently existing technology. The authors employ localized temperature gradients to move chromatin inside the nucleus noninvasively, and they study flow fields and deformations of different nuclear compartments in different experimental settings. The study is timely and should be of broad interest to a wide readership, in particular since the method can also be applied to study mechanical relationships of subcellular compartments in other cellular and extracellular systems.
The non-invasive manipulation of cell organelles in intact cells has been a challenge for decades. The new technique introduced in this study contributes to closing this important gap, enabling experiments to better understand spatial and mechanical relationships between different cell compartments. This study is a very nice example of how concepts and approaches from physics can be exploited to better understand biology.
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Reviewer #3 (Public Review):
Seelbinder et al present local heating of the cell nucleus in live cells as a perturbation of the nucleus, which they use to interrogate mechanical properties of the nucleus. The authors use their recently developed technique of generating local heat gradients (Mittasch et al, 2018) and apply it to the cell nucleus, where they then measure the displacements/strains of chromatin as a function of distance from the heat source. They show that during the heat perturbation the nuclear area and shape remain unchanged. They measure spatially resolved strains across the nucleus and find that different parts of the nucleus exhibit different mechanical behavior. Their analysis reveals that chromatin shows both elastic and viscous properties at the timescales of seconds, with heterochromatin showing solid-like …
Reviewer #3 (Public Review):
Seelbinder et al present local heating of the cell nucleus in live cells as a perturbation of the nucleus, which they use to interrogate mechanical properties of the nucleus. The authors use their recently developed technique of generating local heat gradients (Mittasch et al, 2018) and apply it to the cell nucleus, where they then measure the displacements/strains of chromatin as a function of distance from the heat source. They show that during the heat perturbation the nuclear area and shape remain unchanged. They measure spatially resolved strains across the nucleus and find that different parts of the nucleus exhibit different mechanical behavior. Their analysis reveals that chromatin shows both elastic and viscous properties at the timescales of seconds, with heterochromatin showing solid-like properties. In addition, they find that the nucleolus shows high resistance to the heat-induced deformation at the seconds' timescale.
Conceptually, this is an interesting and thought-provoking work allowing for new ways to perturb the cell nucleus and study its internal mechanics.
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