Editor's evaluation: Probe-free optical chromatin deformation and measurement of differential mechanical properties in the nucleus
Year: 2022
Type: peer-review
Abstract: Full text Figures and data Peer review Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Article and author information Metrics 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. Editor's evaluation Seelbinder et al. describe a valuable new method for perturbing chromatin in living cells by strongly localized temperature gradients. Solid analysis reveals that chromatin shows both elastic and viscous properties at the timescales of seconds, with heterochromatin showing solid-like properties. While some details of the nuclear response to local heating remain to be elucidated, the ability of the method to reveal local mechanics in vivo makes the approach likely to be of broad interest to both the cell biophysics and cell biology communities. https://doi.org/10.7554/eLife.76421.sa0 Decision letter Reviews on Sciety eLife's review process Introduction It is widely believed that the spatial organization of the nucleus is supported by and makes functional use of distinct material properties to support homeostatic function (Cremer et al., 2020; Falk et al., 2019; Mirny and Dekker, 2022), and that may be temporally adapted to facilitate cell cycle dynamics and differentiation (Mittasch et al., 2020; Strom et al., 2021; Sun et al., 2018) or cell migration through confined spaces (Denais et al., 2016; Irianto et al., 2017; Pfeifer et al., 2018; Shah et al., 2021). Starting at the nanometer scale, molecular interactions are thought to give rise to the spatial organization of nuclear constituents that become visible at the micrometer scale. For example, the nucleus features membraneless organelles, such as the nucleolus, Cajal bodies, nuclear speckles, PML bodies, and others, which are comprised of RNA and proteins and are considered to be formed by liquid-liquid phase separation (LLPS) (Zidovska, 2020a; Feric et al., 2016). Most of the nucleus is occupied by chromatin, however, which is hierarchically organized into different compartments: (i) a few nucleosomes (5-20) loosely assembled into clutches, which further assemble into chromatin nanodomains, (ii) nanodomains are further grouped into local continuous gene clusters called topologically associated domains (TADs), and (iii) TADs from different loci group together to form two main compartments: active A-compartments and inactive B-compartments (Jerkovic´ and Cavalli, 2021; Mirny and Dekker, 2022; Misteli, 2020). Using data from chromatin conformation capturing assays (Hi-C), computer simulations suggest that strong interactions between B constituents together with weak interactions of A constituents drive the separation of compartments (Falk et al., 2019; MacPherson et al., 2018; MacPherson et al., 2020). Furthermore, condensed chromatin is thought to solidify with increasing strength of molecular interactions, yielding elastic rather than viscous responses (Hansen et al., 2021) and increase in euchromatin leads to softening of the nucleus (Stephens et al., 2018). Recent studies demonstrated that chromatin compaction by HP1 proteins results in phase-separated liquid condensates (Sanulli et al., 2019; Keenen et al., 2021). Hence, differences in interactions within compartments should also be directly measurable as a reflection of different material properties. Experimental characterization of the material properties of nuclear compartments will be crucial to understand spatial nuclear organization and its role in nuclear information processing. Despite great progress, an integrated physical picture of the nucleus is still missing. A recent point of discussion has been whether chromatin behaves like a liquid or a solid (Strickfaden et al., 2020; Zidovska, 2020b). For chromatin it has been suggested that its material properties are predominantly liquid in line with the view that heterochromatin domains form through LLPS, facilitated through the binding to scaffold proteins, similar to nuclear organelles (Gibson et al., 2019; Larson et al., 2017; Strom et al., 2017). However, FRAP experiments in interphase nuclei that harbored fluorescently labeled chromatin observed that bleached hetero- or euchromatin regions did not recover their intensity after bleaching at the minute to hour scale (Strickfaden et al., 2020). Contrary to the earlier view, this indicates that chromatin cannot move freely and therefore behaves more like a solid. To reconcile the observed discrepancies, it has been suggested that chromatin, like other polymers, shows a more complex behavior that can be viscous, elastic, or viscoelastic depending on the time and length scales that are being probed and the energy-driven enzymatic activity in the environment (Zidovska, 2020b; Zidovska et al., 2013). For example, the liquid-like behavior of chromatin observed at the nanoscale (Nozaki et al., 2017) likely emerges from enzymatically driven processes such as transcription and loop extrusion (Fudenberg et al., 2016; Golfier et al., 2020). A high molecular mobility on the nanoscale might still be locally constrained and prevent large-scale rearrangements of chromatin. Hence, observing liquid-like behavior at the nanoscale does not have to be in contradiction with solid-like behavior at the micron scale. One functional adaptation of nuclear architecture is the central compaction of heterochromatin in rod cells, termed nuclear inversion (Solovei et al., 2009). Nuclear inversion is triggered by LBR downregulation (Solovei et al., 2013) and describes the successive fusion of chromocenters during terminal stages of retinal development, which leads to improved contrast sensitivity under low light conditions (Subramanian et al., 2021). Motivated by these findings, it has been suggested that heterochromatin cohesion drives nuclear inversion, as well as the separation of hetero- and euchromatin in conventional interphase nuclei (Falk et al., 2019). Yet, it remains an open question which material properties compacted heterochromatin adapts, how more heterochromatin is mechanically integrated into the nucleoplasm, and how chromatin interfaces with other nuclear compartments. To better understand chromatin organization across scales, new experimental approaches are needed to measure material properties in living cells, ideally spatially resolved and dynamically. A wide range of different complementary techniques have been proposed to infer compartment interactions or nuclear material properties. At the nanoscale, Hi-C-based methods have been used to map the spatial interactions between different chromatin compartments (Belaghzal et al., 2021; Falk et al., 2019; Lieberman-Aiden et al., 2009) and reconstitute the genomic 3D organization in silico (Stevens et al., 2017). While Hi-C reports on genome interactions and allows to infer the spatial organization to a very good degree, Hi-C methods themselves do not capture dynamic processes or facilitate making predictions about the material properties, without being complemented by other techniques. To acquire dynamic data of chromatin motion at the mesoscale (0.01–1 µm), passive micro-rheology approaches can be used to infer material properties from the spatio-temporal dynamics of discernable features using video microscopy in conjunction with tracking algorithms (Armiger et al., 2018; Eshghi et al., 2021; Herráez-Aguilar et al., 2020; Nozaki et al., 2017; Zidovska et al., 2013). These methods are successful in gaining insights into apparent material properties non-invasively; however, their use is limited to short time scales during which thermal fluctuation dominates over active processes. At larger time scales, motion appears to be largely driven by active ATP-dependent processes and material properties cannot be quantified anymore by assuming thermal motion as the driver (Guo et al., 2014; Zidovska et al., 2013). For example, chromatin shows ATP-dependent coherent motion on time scales above 1 s (Zidovska et al., 2013). To overcome the limitations of passive micro-rheology in energy-consuming materials, active micro-rheology approaches have been developed that use an external stimulus to drive materials out of their mechanical equilibrium. Methods to test nuclear material properties include AFM (Sheng et al., 2014), micropipette aspiration (Dahl et al., 2004; Dahl et al., 2005; Davidson et al., 2019; Pajerowski et al., 2007), micromanipulation (Stephens et al., 2017; Stephens et al., 2018), non-invasive techniques using natural and artificial probes (Caragine et al., 2018; Caragine et al., 2021; Lee et al., 2021; Shin et al., 2018), magnetic beads (Guilluy et al., 2014; Keizer et al., 2022), and membrane stretch devices (Schürmann et al., 2016; Seelbinder et al., 2020). A difficulty, however, is that the nuclear lamina, an outer stiff shell that surrounds and protects the nucleus, is believed to be at least 10× stiffer than chromatin and would therefore mask the internal material properties when probed from the outside (Harada et al., 2014; Isermann and Lammerding, 2013), while chromatin governs response to small extensions (<3 μm) and the lamina to larger extensions (Stephens et al., 2017). These methods provide a good understanding of the mechanics of the nucleus as an integrated whole, but lack spatial resolution. Recently, the injection of magnetic beads into live nuclei allowed for the estimation of local material properties inside the nucleus, but spatial control of the bead location is still limited (Keizer et al., 2022). Spatially resolved maps of material properties can be obtained by Brillouin microscopy (BM) (Brillouin, 1922; Prevedel et al., 2019; Scarcelli and Yun, 2008), by quantifying Raman shifts of scattered photons. BM has been used to map mechanical properties in zebrafish (Sánchez-Iranzo et al., 2020) and living cell nuclei (Zhang et al., 2020; Zhang et al., 2017). While it is a powerful method, BM measures the mechanics at very short time scales (sub-nanosecond) due to its reliance on acoustic waves in the GHz range. Highly attractive would therefore be a micro-rheology method that permits probe-free, spatially resolved mapping of material properties on physiologically relevant time scales, while leveraging the conceptual advantages of active perturbations. Here, we report on a novel approach that utilizes highly localized temperature gradients to displace and strain chromatin inside the nucleus. Since displacements can reach hundreds of nanometers to few microns, local displacements can be quantified through tracking algorithms. As temperature stimuli pervade the stiff nuclear lamina, chromatin motion occurs without disturbing nuclear size or shape. Using this method, we observed that interphase chromatin displays highly reversible, viscoelastic behavior in response to deformation with a characteristic time of τ~1 s. We find that material properties are spatially distinct for different compartments. The nucleolus, in particular, shows high mechanical resilience to deformation, but significant adhesion to surrounding chromatin. Together, our results showcase the utility of this new approach to actively probe nuclear material properties in a spatially and temporally resolved manner, enabling to assign material responses to biochemical identity of compartments and gain new insights into the mechanics of compartment interfaces. Results Engineered temperature gradients facilitate controlled chromatin deformation in living cells To generate a highly localized temperature gradient, a heating IR laser beam (λ=1455 nm) was focused and rapidly scanned (1 kHz) along a line (Figure 1a). Temperature profiles for various laser intensities (low, medium, high) were measured using the temperature-sensitive dye rhodamine B at a chamber temperature of 36°C (Figure 1b–c, Figure 1—figure supplement 1a). The average temperature increase inside the nucleus could further be confirmed via temperature-sensitive mCherry-H2b, which corresponded well to rhodamine measurements averaged along a typical nuclear length of 20 μm (Figure 1d–e, Figure 1—figure supplement 1b). Unless otherwise indicated, experiments were run at medium laser intensity resulting in an average temperature increase of 2°C inside the nucleus. Figure 1 with 3 supplements see all Download asset Open asset Engineered temperature gradients facilitate controlled chromatin deformation in living cells. (a) Overview of the microscopy setup. An IR laser is scanned along a line to generate a temperature gradient perpendicular to the scanning line. Cells are cultured in temperature chambers that maintain a reservoir temperature of 36°C via Peltier elements. Sapphire, rather than glass, was used due to its high heat transfer coefficient. Shown is the side view of chamber. (b) The temperature-sensitive dye rhodamine B can be used to visualize temperature gradients during laser scanning. The red dotted line indicates the laser scan path and outlines of an average size nucleus are superimposed in white. (c) Temperature profile and gradient were quantified perpendicular to the laser scan path via rhodamine B for three different laser intensities (low, medium, and high). Raw data of temperature measurements were fitted and differentiated to achieve noise-robust estimates of the temperature gradients. See Figure 1—figure supplement 1 for dye calibration. (d) Confirmation of instantaneous temperature changes inside the nucleus, at low laser intensity, via relative quantum efficiency measurements of mCherry-H2b, which reduces ~1.3% for each 1°C heating. See Figure 1—figure supplement 1 for calibration. (e) Comparison of thermometry results measured inside the nucleus via mCherry-H2b and inside the chamber via rhodamine B. Chamber temperature was averaged along 20 μm, reflecting the average size of a nucleus, to compare both measurements directly; n=5, error = STD. (f) Top: Response of NIH-3T3 nuclei to an applied temperature gradient at medium laser intensity. Bottom: Image difference analysis of the same data and controls, indicating mesoscopic, partially reversible chromatin displacements due to temperature stimulation; scale = 5 µm. See also Figure 1—videos 1; 2. (g) Temporal analysis of image differences shows that chromatin rearrangements follow a simple viscoelastic material model by Kelvin-Voigt (inset), and have characteristic times (τc - creep or retardation time, τr - relaxation time) on the time scale of seconds; n=10, error = STD. The Kelvin-Voigt model consists of a purely elastic spring and a purely viscous dashpot arranged in parallel, with uniform distribution of strain. There is no possibility for the spring and dashpot to expand independently leading to a typical creep-recovery response. When placing the heating stimulus in close proximity to the nucleus, we observed chromatin motion down the temperature gradient as visualized by GFP-H2b in NIH-3T3 cells (Figure 1f, Figure 1—videos 1; 2). To exclude the possibility that an absolute temperature increase above 36°C is the driver of the observed chromatin movement, rather than the induced temperature gradient, we repeated our experiments at an ambient temperature of 30°C. As with an ambient temperature of 36°C, we observed chromatin motion upon the heating stimulus down the temperature gradient (Figure 1—figure supplement 1c–e). The laser stimulus did not trigger a substantial stress response (Figure 1—figure supplement 1f–h). Formation of dynamic stress granules (SGs) is a typical and reversable response of cells to mitigate several kinds of stress (Hofmann et al., 2021). The protein G3BP1, for example, is a marker of SGs. Under normal conditions, G3BP1 is distributed in the cytoplasm, but accumulates into granules when a cell experiences a stress (Hofmann et al., 2021), such as short treatment with thapsigargin (Sidrauski et al., 2015; Figure 1—figure supplement 1g). Compared to the SG formation upon treatment with thapsigargin, we observed only minor formation of SGs if at all (Figure 1—figure supplement 1h). Within 15 min after a typical laser stimulus of 10 s, in seven repetitions we observed either no SG formation (three times), the formation of a few SGs (two times) or stronger SG formation (two times), but still to a lower extend compared to the positive control of thapsigargin treatment (Figure 1—figure supplement 1h). We concluded that the laser stimulus alone is not sufficient to trigger a stress response, although, additional stresses caused by the experimental setup, for example, mounting of the cells inside the thin chambers, can in sum lead to SG formation in some cases. Yet, in near half of the cases, our perturbations did not trigger any SG formation. This indicates that the laser-induced chromatin motion does not necessarily evoke a stress response. However, some stress response was observed in some cases. Yet, stress responses are typically mounted within minutes. The mechanical response to the laser stimulus, the chromatin movement, can be seen independent as it is much faster. Therefore, mechanical properties of the chromatin movement are independent of a later stress response. To dissect the occasional stress response, further studies would be needed, which could benefit from decoupling of laser heating and the induction of temperature gradients (Minopoli et al., 2023). To better visualize time-dependent chromatin displacements, we generated image difference stacks by subtracting the first image from the image stack (Figure 1f). Analyzing the average image difference over time, we demonstrated that chromatin movement is instantaneous and largely reversible (Figure 1g). Further, image difference dynamics followed an exponential trend that could be fitted to a simple viscoelastic material model (Kelvin-Voigt), with characteristic times τ on the scale of seconds. In the absence of temperature stimulation, we only observed small changes in the image difference stack that likely reflect the spontaneous coherent motion of chromatin as reported before (Zidovska et al., 2013) and might account for some of the residual differences after perturbations. Strongly localized temperature gradients drive intra-nuclear chromatin displacement without affecting nuclear shape To better characterize the observed chromatin displacement away from an applied temperature gradient, we tracked large intra-nuclear features during the perturbation. Large organelles that exclude chromatin, such as the nucleolus, show up as dark pockets (features) in GFP-H2b images and are well suited to track intra-nuclear movement (Figure 2a). Kymograph analysis of chromatin-void features confirm instantaneous directed motion with amplitudes of hundreds of nanometers to a few microns, with reversible asymptotic exponential dynamics (Figure 2b–c). Furthermore, centroid tracking of prominent features reveal that displacements are larger to the temperature Figure with 1 supplement see all Download asset Open asset of intra-nuclear displacement and absence of changes in nuclear shape during temperature (a) displacement over time during 10 s temperature experiments in NIH-3T3 nuclei scale = 5 µm. view of chromatin Kymograph analysis quantifying time and length scale shows largely reversible motion on the of scale = 5 µm. and tracking of intra-nuclear features show their displacements of up to while nuclear area remains largely See also Figure 1; scale = 5 µm. (f) view of the nuclear of the nucleus in (d) further indicates that the remains during and after temperature stimulation; scale = 5 µm. (g) of the of the nuclear with to the nuclear at s, of the nucleus in (d) reveals that the of the nuclear shape are than during temperature to be to the inside of the nucleus, as the centroid of the nucleus and nuclear area only during temperature (Figure Figure quantifying the in nuclear by the of the nuclear to the nuclear before during and after temperature stimulation, we further that the in nuclear shape were on the of (Figure similar to displacements of the nuclear The nuclear likely reflect the of the nuclear the of the nucleus our from the nuclear that was observed during heating of nuclei et al., 2017). To our to this we that strongly localized temperature gradients with an absolute temperature increase 2°C displacements of chromatin and chromatin organelles inside the nucleus without the nuclear area or disturbing the nuclear compacted heterochromatin behaves as an solid in a genetically induced nuclear inversion model of chromatin to the nuclear is an that chromatin et al., For example, of chromatin from the nuclear by downregulation of and B leads to the inversion of the conventional chromatin architecture in cells with nuclei a condensed heterochromatin in the (Solovei et al., et al., 2009). This in nuclear organization has functional gene as it to in (Subramanian et al., 2021; et al., 2019). to its characteristic inverted nuclei have become a prominent model to heterochromatin formation and genome organization (Solovei et al., 2009). transcription in NIH-3T3 cells, as before et al., we were to chromatin inversion in some leads to the formation of a large central heterochromatin of the organization of nuclei in (Figure nuclei an model for chromatin with to the material properties associated with heterochromatin formation. We observed a highly reversible displacement of the upon temperature in and (Figure Figure which was further by tracking of the centroid (Figure At the same time, area and shape of the during stimulation, indicating strong resistance to deformation and solid-like behavior (Figure Figure 3 with 1 supplement see all Download asset Open asset of movement and shape changes of centrally compacted chromatin during temperature in a genetically induced nuclear inversion (a) NIH-3T3 cells an inverted chromatin organization after with The resulting central heterochromatin shows a significant displacement during temperature See also Figure 1; scale = 5 µm. (b) Kymograph analysis quantifying time and length scale shows reversible scale motion of the analysis of the centroid that its motion is by a and reversible displacement during temperature However, the appears to deformation as it shows in area and and minor (e) view of chromatin during temperature The on the the of strain. on their length the strain is in (c) on the The dynamic displacement fitted well to a simple viscoelastic Kelvin-Voigt model with characteristic times of s during the creep and s during the relaxation phase (Figure Since the in its and showed deformation, this dynamic likely the properties of chromatin that from the to the nuclear (Figure the length of the at μm) and assuming that the centroid displacement was similar to the of the strain of chromatin was to be up to (Figure In the strain of the was indicating that the nucleus features material properties. Spatially resolved strain maps reveal distinct mechanical properties of nuclear sub-compartments Analyzing the chromatin displacement in inverted nuclei further that the nucleus features spatially distinct nuclear material properties. Therefore, we if we could map intra-nuclear strain more in conventional interphase nuclei in to local material with To this we used particle image velocimetry 2004; Zidovska et al., 2013) to generate spatial displacement maps during temperature using the at s as (Figure Figure supplement To further local intra-nuclear deformation, strain maps were calculated from displacement Shown are local changes and displacements nuclear strain integrated over the nucleus, asymptotic exponential dynamics similar to tracked features before (Figure over the nucleus, positive and can each results in a line close to which is with our that is in nuclear area during temperature Figure with supplements see all Download asset Open asset strain within chromatin compartments during temperature using spatially resolved strain (a) NIH-3T3 cells mCherry-H2b were during temperature Spatially resolved displacement maps were generated from image stacks via particle image velocimetry (PIV), using s as and strain maps were further are as relative map is at half see Figure supplement 1 for and large-scale strain scale = 5 µm. (b) and of strain components. The the of different strain as a measure of local (c) were into seven chromatin compartments of different by mCherry-H2b intensity. were further using live A of away from the nuclear was to exclude low displacements close to the nuclear (d) of absolute of the strain is not positive and are were locally for different chromatin by strain maps with compartment Shown is the averaged absolute strain for each compartment measured at deformation for the with the data range via with (e) analysis of averaged of the strain is considered positive and
Source: eLife
Author: Megan C King
Institution: Yale University
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,500
OpenAlex ID: W4390829499