[21,555 characters not including Abstract, Methods and ...Feb 05, 2020 · nematodes. Nematodes were placed in . 75. between two membranes, stretched equiaxially and nucleus deformation

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Measuring nucleus mechanics within a living multicellular organism: 3

Physical decoupling and attenuated recovery rate are physiological 4

protective mechanisms of the cell nucleus under high mechanical load 5

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Noam Zuela-Sopilniak,1 Daniel Bar-Sela,2 Chayki Charar,1 Oren Wintner,2 Yosef Gruenbaum,1,* 8 and Amnon Buxboim.2,3* 9

1Departments of Genetics and 2Cell and Developmental Biology, The Alexander Silberman 10 Institute of Life Sciences. 11

3Alexander Grass Center for Bioengineering, The Rachel and Selim Benin School of Computer 12 Science and Engineering. 13

The Hebrew University of Jerusalem, Jerusalem 9190416, Israel. 14

*Corresponding authors. E-mail: [email protected], [email protected] 15

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[21,555 characters not including Abstract, Methods and references] 18

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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 6, 2020. ; https://doi.org/10.1101/2020.02.05.935395doi: bioRxiv preprint

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Abstract [185 words] 20

Nuclei within cells are constantly subjected to compressive, tensile and shear forces, which 21

regulate nucleoskeletal and cytoskeletal remodeling, activate signaling pathways and direct cell- 22

fate decisions. Multiple rheological methods have been adapted for characterizing the response 23

to applied forces of isolated nuclei and nuclei within intact cells. However, in vitro measurements 24

fail to capture the viscoelastic modulation of nuclear stress-strain relationships by the 25

physiological tethering to the surrounding cytoskeleton, extracellular matrix and cells, and tissue- 26

level architectures. Using an equiaxial stretching apparatus, we applied a step stress and 27

measured nucleus deformation dynamics within living C. elegans nematodes. Nuclei deformed 28

non-monotonically under constant load. Non-monotonic deformation was conserved across 29

tissues and robust to nucleoskeletal and cytoskeletal perturbations, but it required intact Linker 30

of Nucleoskeleton and Cytoskeleton (LINC) complex attachments. The transition from creep to 31

strain recovery fits a tensile-compressive linear viscoelastic model that is indicative of 32

nucleoskeletal-cytoskeletal decoupling under high load. Ce-lamin (lmn-1) knockdown softened 33

the nucleus whereas nematode ageing stiffened equilibrium elasticity and decreased 34

deformation recovery rate. Recovery lasted minutes due to physiological damping of the released 35

mechanical energy thus protecting nuclear integrity and preventing chromatin damage. 36

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Introduction 38

The viscoelastic properties of the nucleus are required to protect the genetic material from 39

applied forces(Furusawa et al., 2015) while permitting flexibility to allow constricted cell 40

migration(Denais et al., 2016; Raab et al., 2016; Irianto et al., 2017) and 41

mechanosensitivity.(Athirasala et al., 2017) Externally applied and cell-generated forces 42

propagate along cytoskeletal filaments and are transmitted to the nuclear lamina across Linker 43

of Nucleoskeleton and Cytoskeleton (LINC) complexes. In response to tensile forces that are 44

transmitted to the nuclear lamina across LINC attachments, the nucleus becomes stiffer 45

concomitantly with tyrosine phosphorylation of the nuclear envelope protein Emerin.(Guilluy et 46

al., 2014) Forces pulling on the nuclear lamina are transduced into biochemical cues via tension- 47

suppressed phosphorylation-mediated disassembly and turnover of lamin-A filaments, (Buxboim 48

et al., 2014) which scale with tissue stiffness.(Swift et al., 2013b) Applied forces were also shown 49

to upregulate transcription rate by stretching chromatin, thus acting directly independent of 50

molecular relays. (Tajik et al., 2016) 51

Multiple complementary rheological methods have been employed for measuring the stress- 52

strain relationship of amphibian and mammalian cell nuclei over a range of deformation length 53

scales and dynamic profiles.(Stephens et al., 2018) Indentation experiments of isolated 54

nuclei(Lherbette et al., 2017) and nuclei within adhering cells (Krause et al., 2013) highlighted the 55

stiffness rendered by condensed chromatin, with contributions from linker DNA and inter- 56

nucleosomal interaction.(Shimamoto et al., 2017) Micromanipulation techniques that typically 57

apply intermediate strains separate between small deformation regime dominated by chromatin 58

and large deformations that are resisted by strain-stiffening of the lamina.(Stephens et al., 2017) 59

Compared with indentation and micromanipulation techniques, micropipette aspiration induces 60

large deformations.(Hochmuth, 2000; Gonzalez-Bermudez et al., 2019) Nuclei responded to 61

applied suction as a viscoelastic solid(Guilak et al., 2000; Rowat et al., 2005) spanning over a 62

broad spectrum of timescales(Dahl et al., 2005) showing decreased deformability with cell 63

differentiation(Pajerowski et al., 2007). Viscous and elastic contributions were affiliated with A- 64

and B-type lamins, respectively.(Swift et al., 2013a) 65


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In vitro mechanical characterization of isolated nuclei and nuclei within cells has made 66

fundamental contributions. However, these studies cannot account for the modulating effects of 67

the multiscale physiological surroundings that consists of the cytoskeleton, the cell cortex and 68

membrane, the extracellular environment of cells and matrix, and tissue architectures. Already 69

within intact cells, nucleus stiffening was shown to increase cytoskeletal strain by serving as a 70

stress concentrator.(Heo et al., 2016). In turn, the physical tethering with the cytoskeleton 71

effectively stiffens the nucleus and slows down the release rate of the stored mechanical 72

energy.(Wang et al., 2018) 73

To measure the strain-stress relationship of the nucleus within its physiological multicellular 74

settings, we performed creep test of living C. elegans nematodes. Nematodes were placed in 75

between two membranes, stretched equiaxially and nucleus deformation dynamics was 76

recorded. Counter to the whole nematodes and cells within tissue, nuclei exhibited a non- 77

monotonic response under constant load. Once critical strain was reached, the nucleus 78

transitioned from creep to strain recovery. This anomalous response was conserved across 79

tissues, cytoskeletal perturbations and nuclear perturbations, yet it required intact LINC 80

attachments. We propose a linear viscoelastic model that superimposes apical compression and 81

tensile stretching which is relaxed due to nuclear decoupling via LINC complex detachment at 82

high load. Lmn-1 knockdown increased dissipative recovery time and softened nucleus resistance 83

to continuous stresses but had no effect on instantaneous elasticity. Despite maintaining 84

constant Ce-lamin protein level, nuclei became stiffer and deformation recovery became slower 85

with ageing. Nuclei responded within minutes – much slower than in vitro recovery times scales 86

over seconds. Hence, the physiological surroundings of the cytoskeleton, extracellular matrix and 87

cells, and tissue architectures attenuate the release rate of the mechanical energy stored within 88

the nucleus thus protecting the nuclear envelope and the genetic material from mechanical 89

damage. 90

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Results 94

Creep test of live nematodes reveals non-monotonic nuclear response to applied stresses 95

Measuring nucleus mechanics within live nematodes was performed using a custom made device 96

constructed based on a former cell stretcher design (Lammerding and Lee, 2009) and mounted 97

onto an inverted epifluorescence microscope (Fig. 1a). Live nematodes were placed in between 98

two parallel silicon membranes and immersed in M9 buffer. Pulling the two parallel membranes 99

downwards against a static cylinder generated an in-plane uniform and homogenous equibiaxial 100

tensile strain as calculated by tracking the relative displacements of distinct defects. The 101

deformation dynamics of the nematodes was recorded before and during membrane stretching 102

by time-lapse phase contrast and fluorescence imaging (Fig. 1b). Similarly, the deformation 103

dynamics of nuclei within different tissues was evaluated by time-lapse recording of a GFP-fused 104

Ce-lamin fluorescence signal (Fig. 1c). 105

We characterized the mechanical response of worms, cells and nuclei to applied stresses using 106

creep test rheology. Specifically, we pulled the membranes at ~70% equibiaxial strain and 107

maintained stretching for 24 min (Fig. 1d-i). As a result, the nematodes were stretched 108

longitudinally but not perpendicular to the main axis of the worm due to their thin cylindrical 109

geometry (Fig. 1d-ii). The nematodes responded elastically to applied stress: instantaneous 110

deformation at t=0, which was maintained as long as the membranes were held stretched. To 111

evaluate the axial deformation of cells, we estimated the distances between the centers of 112

consecutive pairs of segmented nuclei within the obliquely striated myofibrillar lattice of the 113

body wall muscle tissue (Gettner et al., 1995). Nuclear areal strain dynamics was also evaluated 114

using the segmented GFP-signal regions of interest (ROI’s). Both cell and nucleus strains were 115

normalized by worm strains to compensate for differences in the forces that were applied within 116

different nematodes. We found that cells responded elastically to applied stresses just like the 117

nematodes (Fig. 1d-iii). However, nuclei exhibited a complex dynamic response that was marked 118

by a non-monotonic deformation under constant load (Fig. 1d-iv). 119

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Figure-1 | Mechanical creep test of cell nuclei within live nematodes reveals anomalous response to applied stresses. a, Live C. elegans nematodes are placed between two elastic silicon membranes on a stretching apparatus mounted onto an inverted microscope. b, Fluorescent time-lapse images of the nematodes are recorded as the membranes are being radially stretched. c, Zoom-in images (red frames in b) of GFP-conjugated Ce-lamin nuclei show induced deformation along the primary axis of the nematode. d, Creep test is performed by maintaining a 70% radial strain of the membranes (left). Average dynamic strain profiles of the nematodes (n=3), cells (estimated as the distance between proximal nuclei, n=7) and nuclei (n=12) are evaluated based on time lapse imaging. Unlike nematodes and cells that exhibit a constant elastic axial deformation, nuclei respond non-monotonically to applied stress (right).

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Nucleus mechanics fit a tensile-compressive linear viscoelastic model 122

The irregular deformation dynamics of nuclei was observed both in muscle and in hypodermis, 123

indicating that it is an invariant property across tissues (Figs. 3a-i,ii), which is a property of 124

individual nuclei in both tissues (Fig. S1). It consists of a creep phase between t=0 and a critical 125

time 𝑡𝑡𝑐𝑐𝑐𝑐, where strain increases with time, and a recovery phase at 𝑡𝑡 > 𝑡𝑡𝑐𝑐𝑐𝑐, where strain decreases 126

(Fig. 2a). To obtain insight into the mechanisms that give rise to the creep-recovery response to 127

load, we tested the effects of the LINC complex on this response. LINC complexes are required 128

for mediating the transmission of forces that are propagated along cytoskeletal filaments to the 129

nuclear matrix of lamins and chromatin and back.(Buxboim et al., 2010) To this end, we 130

performed creep test as described above on Unc-84-null nematodes that lack integral LINC 131


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attachments.(Malone et al., 1999; Bone et al., 2014) Remarkably, the irregular deformation 132

dynamics that was conserved across tissues vanished and instead Unc-84-null nuclei responded 133

elastically to applied forces (Fig. 3b). 134

Based on our nematode stretching experiments, we hypothesize that the LINC complexes 135

undergo physical detachment once a critical strain is reached, leading to nucleoskeletal- 136

cytoskeletal decoupling (NCD). Importantly, even if only a few LINC complexes break, the average 137

loading per attachment will increase on the remaining LINC complexes, thus leading to rapid 138

avalanching detachment of all LINC complexes as reflected here by the sudden decrease in 139

nuclear strain. Despite the fact that NCD occurred at different times in muscle and hypodermis 140

nuclei, it was observed at similar levels of critical strain (Fig. 3a-i,ii). NCD is a protective 141

mechanism of the chromatin from physical tears and breaks due to high tensile strain as was 142

recently reported for a culture model of mammalian cells (Gilbert et al., 2019). In addition to 143

tensile stresses, our membrane stretching apparatus also applies compressive forces on the 144

nuclei as the membranes are pulled downwards and against each other (Fig. 2b). Unlike tensile 145

stresses, compressive load is applied to the nucleus independent of the integrity of the LINC 146

complexes and is maintained constant independent of NCD. 147

We employ the Kelvin representation standard linear solid (SLS) to model nucleus response to 148

tensile stress 𝜎𝜎𝑡𝑡 and compressive stress 𝜎𝜎𝑐𝑐 in living nematodes (Fig. 2c). Nucleus resistance to 149

tensile stresses is modeled by a solid-like Kelvin-Voigt viscoelastic module, which is composed of 150

a spring and dashpot connected in parallel (𝑘𝑘𝑡𝑡 and 𝜇𝜇𝑡𝑡). Nucleus resistance to compressive stresses 151

is modeled using a spring (𝑘𝑘𝑐𝑐). During creep phase, the LINC complexes remain intact and tensile 152

and compressive deformations are superimposed. The strain-stress relationship is: 153

𝜀𝜀𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 = −𝜈𝜈𝜎𝜎𝑐𝑐𝑘𝑘𝑐𝑐

+𝜎𝜎𝑡𝑡𝑘𝑘𝑡𝑡�1 − 𝑒𝑒−𝑡𝑡 𝜏𝜏⁄ � 𝐸𝐸𝐸𝐸𝐸𝐸 1 154

where 𝜏𝜏 = 𝜇𝜇𝑙𝑙𝑎𝑎𝑡𝑡 𝑘𝑘𝑙𝑙𝑎𝑎𝑡𝑡⁄ is the Kelvin-Voigt response time and 𝜈𝜈 is the Poisson’s ratio of the nucleus 155

responsible for converting compressive loading to lateral deformation. Upon NCD, only 156

compressive stress remains leading to a gradual recovery of the Kelvin-Voigt module with time. 157

In this case nucleus strain asymptotically approaches the instantaneous deformation as imposed 158

by compressive stress: 159


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𝜀𝜀𝑐𝑐𝑐𝑐𝑐𝑐𝑟𝑟𝑟𝑟 = −𝜈𝜈𝜎𝜎𝑐𝑐𝑘𝑘𝑐𝑐

+𝜎𝜎𝑡𝑡𝑘𝑘𝑡𝑡�1 − 𝑒𝑒−𝑡𝑡𝑐𝑐𝑐𝑐 𝜏𝜏⁄ �𝑒𝑒−(𝑡𝑡−𝑡𝑡𝑐𝑐𝑐𝑐) 𝜏𝜏⁄ 𝐸𝐸𝐸𝐸𝐸𝐸 2 160

The instantaneous elastic modulus 𝐸𝐸0 is evaluated based on creep phase strain at t=0: 161

𝐸𝐸0 =𝑘𝑘𝑐𝑐

−𝜈𝜈 ∙ 𝜎𝜎𝑐𝑐 𝐸𝐸𝐸𝐸𝐸𝐸 3 162

Nuclear response time 𝜏𝜏 and equilibrium elastic modulus 𝐸𝐸𝑐𝑐𝑒𝑒 are evaluated by fitting the recovery 163

phase strain dynamics: 164

𝐸𝐸𝑐𝑐𝑒𝑒 = �−𝜈𝜈𝜎𝜎𝑐𝑐𝑘𝑘𝑐𝑐

+𝜎𝜎𝑡𝑡𝑘𝑘𝑡𝑡�−1

𝐸𝐸𝐸𝐸𝐸𝐸 4 165

𝐸𝐸𝑐𝑐𝑒𝑒 accounts for steady state elastic resistance to applied stresses of the two springs 𝐸𝐸𝑡𝑡 and 𝐸𝐸𝑐𝑐 in 166

series. 167

Figure-2 | Tensile-compressive viscoelastic model of the cell nucleus in live nematodes. a, Nucleus response to constant load consists of an instantaneous elastic response to compressive stresses (𝜀𝜀𝑐𝑐), followed by creep response to tensile stresses and partial deformation recovery (𝜀𝜀𝑐𝑐). The non-monotonic transition from creep to recovery occurs at critical time 𝑡𝑡𝑐𝑐𝑐𝑐 once critical strain 𝜀𝜀𝑐𝑐𝑐𝑐 is reached. b, Equiaxial stretching of the two membranes generates tensile stresses on the nuclei that are mediated via LINC attachments as well as compressive stresses that are independent of intact LINC as the membranes are pulled against each other due to in-plane tension. c, A minimal viscoelastic model of the nucleus consists of a viscoelastic solid-like Kelvin-Voigt module and an elastic spring connected in series. The Kelvin-Voigt module resists tensile stresses whereas the spring models the response to compressive stresses.


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Muscle and hypodermis nuclei have similar stiffness but different viscosities 168

Nuclear viscoelastic properties in muscle and in hypodermis tissues were obtained by fitting the 169

average strain dynamics curves (Fig. 3a-i,ii, solid lines). Fitting quality was scored by R-square 170

0.97 in muscle and 0.98 in hypodermis. Since the nucleus is physically decupled from the 171

surrounding cytoskeleton in Unc-84-/- nematodes, areal strain was fitted using an elastic spring 172

(R-square 0.93, Fig. 3b, solid line). The instantaneous elastic modulus, equilibrium elastic 173

modulus and recovery response time were evaluated for muscle, hypodermis and Unc-84-/- nuclei 174

based on fitting of the normalized nuclear strain curves. The instantaneous and equilibrium 175

elastic moduli, 𝐸𝐸0 and 𝐸𝐸𝑐𝑐𝑒𝑒, were both equal in muscle and hypodermis nuclei (Fig. 3c-i,ii). Since 176

instantaneous deformation in our stretching experiments is dominated by compressive stresses, 177

𝐸𝐸0 is equal in Unc-84 null and LINC-intact nuclei (Fig. 3c-i). However, 𝐸𝐸𝑐𝑐𝑒𝑒 is higher because tensile 178

stresses are disrupted only in Unc-84 null nuclei whereas 𝐸𝐸𝑐𝑐𝑒𝑒 in LINC-intact nuclei is set by 𝑘𝑘0 and 179

𝑘𝑘𝑐𝑐𝑒𝑒 springs in series (Fig. 3c-ii). Response time 𝜏𝜏 was relatively short in muscle nuclei and long in 180

hypodermis nuclei (Fig. 3c-iii). Taken together, our stretching measurements of nuclear 181

mechanics in live nematodes support the proposed linear viscoelastic model that accounts for 182

NCD at critical strain and combines viscoelastic tensile response and elastic compressive 183

response (Fig. 2c). 184

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Chromatin and lamin control the viscoelastic properties of the nucleus 186

To obtain a mechanistic understanding of nuclear mechanics, we studied how nucleus resistance 187

to applied stresses depends on cytoskeletal and nucleoskeletal organization in live nematodes. 188

We perturbed filamentous actin polymerization by treating live nematodes with Latrunculin-A 189

(Lat-A; Fig. 4a-i) and disrupted the LINC complex attachments by feeding the nematodes with 190

RNAi against unc-84 (unc84i; Fig. 4a-ii). In addition, we targeted the nuclear lamina and 191

chromatin by feeding live nematodes with RNAi against lmn-1 (lmn1i, Fig. 4a-iii) and treatment 192

with Trichostatin-A (TSA, 4a-iv), respectively. TSA is a potent Class-I and –II histone deacetylase 193

inhibitor (HDACi) that is broadly used for chromatin de-condensation. This is achieved by rapid 194

induction of a global increase in histone acetylation, thus removing positive charges and relaxing 195


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DNA interactions. Acetylation of histone H4K16 that interferes with the structural compaction of 196

the 30 nm chromatin fibers was also reported.(Shogren-Knaak et al., 2006; Robinson et al., 2008; 197

Chen and Li, 2010) 198

Figure-3 | Non-monotonic nucleus deformation is conserved across tissues and depends on LINC attachments. a, Creep tests of nuclei in (i) muscle tissue (12 nuclei in 2 nematodes) and in (ii) hypodermis tissue (11 nuclei in 2 nematodes) share a non-monotonic response to applied load. b, Nuclei in Unc-84-null nematodes (18 nuclei in 4 nematodes) maintain a constant deformation under a constant load. c, Evaluation of the (i) instantaneous and (ii) equilibrium elastic moduli and the (iii) viscoelastic response time. Muscle and hypodermis parameters were evaluated by curve fitting using the tensile-compressive model. Red lines represents unc-84 null stiffness evaluated for a purely elastic response.

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Changes to the viscoelastic properties of the nucleus due to the cytoskeletal and nucleoskeletal 200

perturbations were evaluated via creep test and model fitting as described above. Compared 201

with control nuclei (Fig. 4; light gray), both actin de-polymerization and LINC disruption led to an 202

overall decrease in nucleus compliance (Fig. 4a-i,ii). Critical strain for NCD appears to be a 203

conserved property across tissues and invariant to nuclear perturbations. However, both 204

cytoskeletal perturbations decreased critical time and strain compared with untreated muscle 205

control (Fig. 4b-i,ii). Relative to non-perturbed muscle nuclei, Lmn-1 knockdown led to a 206

shallower creep slope and a higher critical strain (Fig. 4a-iii). Model fits of nucleus perturbations 207

indicate that knockdown of Lmn-1, which in the nematode combines properties of both A- and 208

B-type mammalian lamins,(Liu et al., 2000; Lyakhovetsky and Gruenbaum, 2014) softened the 209

equilibrium elastic modulus twofold and prolonged response time sevenfold (Figs. 4c-ii,iii). 210

Increase in 𝜏𝜏 is consistent with a decrease in the restoring force due to softening of 𝑘𝑘𝑡𝑡 (Fig. 2c), 211

in tune with previously reported lamin-A knockdown experiments in mammalian cells 212

(Lammerding et al., 2006) and our unpublished data. Instantaneous elasticity was dominated by 213

chromatin rather than Ce-lamin (Fig. 4c-i). On the other hand, chromatin de-condensation 214

attenuated instantaneous deformation in response to impact (Fig. 4a-iv) in a manner that 215

effectively corresponds to stiffening of 𝐸𝐸0 by > 60% (Fig. 4c-i). However, cytoskeletal effects of 216

TSA should be considered (see discussion). 217

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Ageing alters long term stiffness without significantly decreasing normalized lamin levels 219

C. elegans is an established model organism in the research of ageing and associated 220

laminopathic genetic disorders.(Haithcock et al., 2005) To study how nuclear mechanics changes 221

with ageing, we performed creep test of L4 larvae (D0), two-day old (D2) and four-day old (D4) 222

adult nematodes (Fig. 5a). We first compared Ce-lamin protein levels between D0, D2 and D4 223

nematodes via western blotting of whole nematodes lysates (Fig 5b-i). Since D0 nematodes are 224

significantly smaller despite having an equal number of cells and nuclei as D2 and D4 nematodes 225

(Fig. 5a), we normalized Ce-lamin intensities by (total protein)/(nematode volume). Based on 226

three biological replicas, we excluded significant differences in Ce-lamin protein levels with 227

ageing (Fig. 5b-ii). 228


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Instantaneous strain was comparable between D0, D2 and D4 creep test measurements (Fig. 5c). 229

However, strain dynamics slope during creep phase was high in D0 nematodes and low in aged 230

D2 and D4 nematodes. With ageing, critical strain decreased between D0 and D2 and critical time 231

was shorter in D4 nematodes, thus recapitulating the cytoskeletal effects that we observed 232

following actin de-polymerization and LINC disruption (Fig. 4b-i,ii). Instantaneous stiffness was 233

invariant to nematode age. However equilibrium stiffness was low in D0 nuclei and high in D2 234

and D4 nuclei (Fig. 5c-i,ii) and response time was threefold longer in aged nuclei (Fig. 5c-iii). 235

Collectively, we find that ageing increases the elastic resistance of nuclei to applied stresses and 236

the dissipative response time required for reaching mechanical equilibrium. 237


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Figure-4 | Cytoskeletal and nuclear perturbations modulate nucleus deformation dynamics in accordance with the tensile-compressive viscoelastic model. a, Creep tests of muscle tissue nuclei in nematodes treated with (i) Lat-A (3 nematodes, 14 nuclei), (ii) siUnc-84 (3 nematodes, 14 nuclei), (iii) siLmn-1 (4 nematodes, 16 nuclei) and (iv) TSA (3 nematodes, 10 nuclei) are fitted using the tensile-compressive model. Non-treated nucleus deformation curves are plotted in gray as reference. b, Evaluation of the (i) instantaneous and (ii) the equilibrium elastic moduli and (iii) viscoelastic response time. Lat-A: Latrunculin-A. RNAi: RNA interference. TSA: Trichostatin-A.


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Discussion 238

Here we present, to the best of our knowledge, the first nucleus rheology study in a live 239

multicellular organism. Using a custom designed worm stretching device, we performed a creep 240

test and identified anomalous deformation dynamics of individual nuclei. The non-monotonic 241

transition from creep to recovery is indicative of NCD once critical strain is reached, as 242

experimentally supported by the elastic deformation of SUN-domain Unc-84 knockout nuclei that 243

lack intact LINC complex attachments. Nucleus mechanics is well modeled by a three-elements 244

linear viscoelastic material (SLS model) (Guilak et al., 2000) that superimposes LINC-dependent 245

tensile stresses and LINC-independent compressive stresses. Disruption of filamentous actin 246

polymerization and knockdown of unc-84 do not ablate all LINC complexes but rather decrease 247

their number. Hence, earlier NCD at lower critical strain is likely attributed to the increase in the 248

average load per LINC attachment (Fig. 4b-i,ii). The anomalous response of the nucleus to applied 249

stresses is conserved across tissues and across conditions. LINC complex repair appears to be 250

hindered for approximately 20 minutes following NCD, optionally due to retraction of the 251

cytoskeletal filaments away from the nucleus. Tracking LINC repair during longer time periods 252

following NCD was impeded due to loss of nematode viability during creep test. 253

Chromatin de-condensation by HDACi was reported to either soften nucleus resistance to small 254

deformations (Chalut et al., 2012; Krause et al., 2013; Haase et al., 2016; Shimamoto et al., 2017) 255

or not to stiffen nucleus resistance to large deformations.(Stephens et al., 2017) Counter to these 256

in vitro findings that apply forces directly on the nucleus, here we probe nucleus deformation 257

dynamics while indirectly stretching the nuclei by pulling on the entire nematodes (Fig. 1). Hence, 258

we cannot exclude cytoskeletal effects of HDACi. TSA was shown to inhibit HDAC6, which acts on 259

α-tubulin both in vivo and in vitro.(Matsuyama et al., 2002) In this manner, TSA can promote 260

acetylation of α-tubulin, likely at the 𝜀𝜀-amino group of the N-terminus conserved lysine residue 261

(Lys40) (MacRae, 1997; Rosenbaum, 2000), thus contributing to the stabilization of the 262

cytoplasmic microtubule filaments.(Matsuyama et al., 2002) In particular, TSA is expected to 263

stiffen skeletal muscle cells that are rich in microtubule networks.(Robison and Prosser, 2017). 264

We therefore conclude that the decrease in instantaneous nuclear strain due to TSA treatment 265

cannot be definitively attributed to chromatin de-condensation. In fact, it may reflect altered 266


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muscle cell and tissue level mechanics rather than nuclear stiffening due to chromatin de- 267

condensation (Fig. 4b-i). 268

Figure-5 | Ageing alters nucleus elasticity and viscosity. a, C. elegans nematodes grow in size as they age. b, (i) Western blot of C. elegans samples, stained for Ce-lamin (ii) Ce-lamin levels normalized according to total protein count and nematode size. c, Nucleus strain dynamics of Day-0 (3 nematodes, 10 nuclei), Day-2 (3 nematodes, 9 nuclei), and Day-4 (2 nematodes, 5 nuclei) nematodes. d, Evaluation of the (i) instantaneous and (ii) the equilibrium elastic moduli and (iii) viscoelastic response time.


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C. elegans nuclei exhibit ageing-related modifications that include changes to nuclear 269

morphology and loss of peripheral heterochromatin, which are also observed in accelerated 270

ageing disorders.(Haithcock et al., 2005) Here we performed creep test of L4, and D2 and D4 271

adult nematodes (Fig. 5a) to characterize the effects of nematode age on nucleus mechanics (Fig. 272

5c). We found that in aged nematodes NCD was observed earlier and at lower critical strain 273

compared with young nematodes (Fig. 5c). A decrease in critical strain is consistent with 274

equilibrium nucleus stiffening and prolonged response time (Fig. 5d-ii,iii). However, nuclei 275

maintain constant instantaneous elasticity with age, which renders mechanical strength to the 276

nucleus against impact (Fig. 5d-i). Our in vivo measurements reproduce in vitro studies that 277

report stiffening of equilibrium elasticity, which is a hallmark of HGPS as well as normal ageing in 278

amphibian and mammalian cells.(Dahl et al., 2006; Verstraeten et al., 2008; Kaufmann et al., 279

2011; Apte et al., 2017) 280

We derive a dissipative response time 𝜏𝜏 that measures the duration of viscoelastic transition 281

towards equilibrium mechanics (Figs 3c-iii, 4c-iii, 5d-iii). Tethering of the nucleus to the 282

surrounding cytoskeleton was shown to slow down strain relaxation and rate of mechanical 283

energy release owing to cytoskeletal viscosity. (Wang et al., 2018) The response time that we 284

measure in live nematodes (minutes) is significantly longer than the equivalent time scales 285

measured for isolated nuclei (< 1 second)(Wang et al., 2018) and nuclei surrounded by 286

cytoskeleton within intact cells (seconds).(Swift et al., 2013b) Rapid release of mechanical energy 287

increases the risk of structural damage to the nucleus and the encapsulated genetic material. We 288

find here that the actual energy release rate within a live organism is even slower than in vitro 289

estimates. Hence, the physiological environment of cells and tissues provide a dissipative 290

mechanism for protecting the nucleus and chromatin from applied forces. 291

292


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Methods 293

Non-aging strains and culture

We used the following C. elegans strains in this study: PD4810 (lmn-1:GFP, ccIs4810 [pJKL380.4 294

Plmn-1::lmn-1::gfp::lmn-1 3'UTR + pMH86 dpy20(+)]), YG751 (lmn-1-GFP:unc-84 null [pJKL380.4 295

Plmn-1::lmn-1::gfp::lmn-1 3'UTR + pMH86 dpy20(+);unc-84(e1174)]). C. elegans strains were 296

maintained and manipulated under standard conditions as previously described.(Brenner, 1974) 297

Strains were out-crossed at least 3 times to ensure a clean background. 298

299

Nematode culture and stretching

C. elegans strains were bleached in 0.06 M NaOH, 0.5% sodium hypochlorite solution. L1 300

synchronization was performed by incubating the embryos on empty NGM plates for 18 hours at 301

20°C. L1 nematodes were washed in M9 buffer (3g KH2PO4, 6g Na2HPO4, 5g NaCl, 1ml 1M 302

MgSO4, H2O to 1 liter sterilize by autoclaving) and reseeded in plates containing a thin lawn of 303

OP50/RNAi bacteria. Late L4 synchronized nematodes were collected into Eppendorf tubes and 304

washed 3 times. To support adhesion, 0.2% Poly-L-Lysine (Sigma P2636) was added at equal 305

volume and supplemented with 10 µL anti-fade solution (9 parts glycerol, 1 part 10X PBS, 0.1 part 306

20%(w/v) n-propyl gallate (Sigma P3130) in dimethyl sulfoxide). Nematodes were allowed to 307

settle down for a few minute. 308

Nematodes were transferred to the center of a 4" x 4" square silicon sheet (cut from 12" x 12", 309

0.005" NRV G/G 40D silicon membrane, SMI) in twenty separate droplets. The nematodes were 310

then covered by a second silicone sheet and trapped air bubbles were removed. The 311

“sandwiched” nematodes were placed on a stretching device(Zuela et al., 2016) and mounted on 312

top of an inverted microscope (Nikon Eclipse Ti-U inverted microscope, equipped with a Nikon S 313

Plan fluor ELWS 40X/0.60 for PD4810 experiments and a Plan Apo VC 20X/0.75 for YG751 314

experiments). Equiaxial stretching was performed within the conjugated plane of the microscope 315

and time-lapse recording (Andor Zyla 4.2 sCMOS camera controlled with NIS Elements 4.5.00 316


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software) was performed both in bright field phase contrast and fluorescence channels (Nikon 317

Intensilight C-HGFI light source and Chroma ET - EGFP (FITC/Cy2) filter set). 318

319

Image processing, mechanical and statistical analyses

Whole nematodes (bright field channel) and nuclei (EGFP fluorescence channel) were segmented 320

using ImageJ. As described in the main text, projected area, contour length and distances were 321

calculated using ImageJ tools. Viscoelastic analyses and curve fitting were performed as 322

described in detail in the main text using Matlab-2018a (Mathworks). 323

324

Cytoskeletal and nuclear drug perturbations

Trichostatin-A (TSA, Sigma T8852) was diluted in M9 medium at 2.5 mM. Five droplets (150 µl in 325

total) were added onto the OP50 bacteria lawn of NGM plates to reach a complete coverage and 326

left to dry. Similarly, Latrunculin-A (Lat-A, Sigma L5163) was freshly diluted in M9 medium at 5 327

mM. Five droplets (150 µl in total) were added onto the OP50 bacteria lawn of NGM plates to 328

reach full coverage and left to dry. Synchronized L1 nematodes were placed on top of the drug- 329

immersed plates at 20°C for 24 hours until reaching the appropriate developmental stage (late 330

L4). 331

332

RNA interference experiments

RNA interference (RNAi) knockdown of target genes was performed via bacteria-feeding as 333

previously described.(Timmons et al., 2001) E-coli clones were obtained from the Vidal's and 334

Ahringer's RNAi libraries.(Kamath and Ahringer, 2003; Kim et al., 2005) L4440 clone was used as 335

an empty vector control. Lmn-1 and unc-84 knockdown was performed using the DY3.2 and 336

F54B11.3 vectors, respectively. Specifically, synchronization L1 nematodes were transferred to 337

feeding plates at 20°C and experiments were performed at late L4 stage as described above. 338

339


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Aging stretching experiments

Synchronized L1 PD4810 were placed on NGM plates at 20°C for 24 hours, until reaching late L4 340

stage. Late L4 nematodes (D0) were extracted by picking for subsequent experiments. The 341

remaining nematodes were further incubated at 20°C for additional 12 hours until reaching adult 342

stage. Adult nematodes were transferred by picking into new NGM plates and incubated at 20°C. 343

Two-day old adult nematodes (D2) were extracted by picking 36 hours after transfer for 344

subsequent experiments. The remaining nematodes were transferred by picking to new NGM 345

plates and incubated for an additional 48 hours at 20°C. Four-day old adult nematodes (D4) were 346

then extracted by picking for subsequent experiments. Extracted nematodes were transferred 347

into M9 droplets and placed onto an unseeded NGM plate for 10 minutes to remove residual 348

OP50 bacteria. The nematodes were then transferred into a single 1 µL droplet containing 3:1 349

M9:anti-fade mixture and placed at the center of a 4" x 4" square silicon sheet. Nematode 350

stretching was performed by placing a second membrane on top and mounted onto the 351

stretching apparatus as described above. Each age group consisted of 20 nematodes. 352

353

Western Blotting

To eliminate the contribution of embryonic Ce-lamin to the calibration of adult Ce-lamin levels 354

across age groups, we used temperature-sensitive sterile CF512 nematode strain [(b26) II; fem- 355

1(hc17) IV]. Bleached CF512 embryos were synchronized as described above. L1 nematodes were 356

transferred to OP50 containing NGM plates and incubated at 25°C for 24 hours. 150 late L4 357

nematodes (D0) were collected for Western blotting and the remaining nematodes were 358

transferred to new NGM plates and incubated at 25°C (24 hours) followed by 20°C (24 hours). 359

150 two-days old (D2) nematodes were collected for Western blotting. The remaining nematodes 360

were transferred to new NGM plates and incubated at 20°C (48 hours). 150 four-days old (D4) 361

nematodes were collected for Western blotting. 362

Western blotting was performed as previously described.(Towbin et al., 1979; Bank et al., 2011) 363

In short, nematode samples were washed three times in M9 buffer and immersed in 100 µL M9 364

buffer. Nematodes were lyzsed by adding 900 µL mixture of RIPA lysis Buffer (10mM Tris-Cl pH 365


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8.0, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.1% Sodium Deoxycholate, 0.1% SDS, 140mM 366

NaCl), X25 protease inhibitor (cOmplete protease inhibitor cocktail by Sigma Aldrich) and 0.1M 367

PMSF (Sigma P7626) at 95:4:1 volume ratios. Nematode lysate solutions were centrifuged at 368

2,000 rpm for two minutes. Supernatant was removed and pellet was dissolved in 200 µL RIPA- 369

PI-PMSF lysis buffer. The samples underwent four freezing-thawing cycles in liquid nitrogen and 370

sonication (Sonics Vibra Cell sonicator with a model CV33 microtip at 34% amplification 4x10 sec 371

sonication periods). Samples underwent two freeze-thaw cycles and centrifuged 7,500 rpm for 5 372

minutes at 4°C. Supernatant was collected and transferred to a new Eppendorf tube for use. 373

Total protein levels were calibrated using Bicinchoninic Acid (BCA) assay (ThermoFisher Pierce 374

BCA Protein Assay kit, OD measured with BioTek synergy 2 multi-mode multiplate reader). 375

Samples were ran on a 9% SDS polyacrylamide gel, transferred to a nitrocellulose membrane and 376

blotted using anti Ce-lamin antibody (KLH-conjugated C-terminal peptide of Ce-lamin; 377

VEFSESSDPSDPADRC; serum 3932 , bleed 6, dilution of 1:1000). Chemiluminescence images were 378

acquired (Vilber Fusion FX). Ce-lamin levels were quantified by measuring total band intensities 379

normalized to total protein levels. 380


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Supplementary material 381

382

383

Figure-S1 | Individual nuclei respond non-monotonically to constant load. Dynamic areal strain profiles of three representative nuclei within (a) muscle and (b) hypodermis tissues are depicted. The non-monotonic mechanical response shared by all nuclei in both tissues is characterized by an instantaneous elastic deformation, creep and deformation recovery.


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References 384

Apte, K., Stick, R., and Radmacher, M. (2017). Mechanics in human fibroblasts and progeria: Lamin A 385 mutation E145K results in stiffening of nuclei. J Mol Recognit 30. 386 Athirasala, A., Hirsch, N., and Buxboim, A. (2017). Nuclear mechanotransduction: sensing the force from 387 within. Current opinion in cell biology 46, 119-127. 388 Bank, E.M., Ben-Harush, K., Wiesel-Motiuk, N., Barkan, R., Feinstein, N., Lotan, O., Medalia, O., and 389 Gruenbaum, Y. (2011). A laminopathic mutation disrupting lamin filament assembly causes disease-like 390 phenotypes in Caenorhabditis elegans. Mol Biol Cell 22, 2716-2728. 391 Bone, C.R., Tapley, E.C., Gorjanacz, M., and Starr, D.A. (2014). The Caenorhabditis elegans SUN protein 392 UNC-84 interacts with lamin to transfer forces from the cytoplasm to the nucleoskeleton during nuclear 393 migration. Mol Biol Cell 25, 2853-2865. 394 Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71-94. 395 Buxboim, A., Ivanovska, I.L., and Discher, D.E. (2010). Matrix elasticity, cytoskeletal forces and physics of 396 the nucleus: how deeply do cells 'feel' outside and in? Journal of cell science 123, 297-308. 397 Buxboim, A., Swift, J., Irianto, J., Spinler, K.R., Dingal, P.C., Athirasala, A., Kao, Y.R., Cho, S., Harada, T., 398 Shin, J.W., and Discher, D.E. (2014). Matrix elasticity regulates lamin-A,C phosphorylation and turnover 399 with feedback to actomyosin. Curr Biol 24, 1909-1917. 400 Chalut, K.J., Hopfler, M., Lautenschlager, F., Boyde, L., Chan, C.J., Ekpenyong, A., Martinez-Arias, A., and 401 Guck, J. (2012). Chromatin decondensation and nuclear softening accompany Nanog downregulation in 402 embryonic stem cells. Biophys J 103, 2060-2070. 403 Chen, P., and Li, G. (2010). Dynamics of the higher-order structure of chromatin. Protein & cell 1, 967- 404 971. 405 Dahl, K.N., Engler, A.J., Pajerowski, J.D., and Discher, D.E. (2005). Power-law rheology of isolated nuclei 406 with deformation mapping of nuclear substructures. Biophys J 89, 2855-2864. 407 Dahl, K.N., Scaffidi, P., Islam, M.F., Yodh, A.G., Wilson, K.L., and Misteli, T. (2006). Distinct structural and 408 mechanical properties of the nuclear lamina in Hutchinson-Gilford progeria syndrome. Proc Natl Acad 409 Sci U S A 103, 10271-10276. 410 Denais, C.M., Gilbert, R.M., Isermann, P., McGregor, A.L., te Lindert, M., Weigelin, B., Davidson, P.M., 411 Friedl, P., Wolf, K., and Lammerding, J. (2016). Nuclear envelope rupture and repair during cancer cell 412 migration. Science 352, 353-358. 413 Furusawa, T., Rochman, M., Taher, L., Dimitriadis, E.K., Nagashima, K., Anderson, S., and Bustin, M. 414 (2015). Chromatin decompaction by the nucleosomal binding protein HMGN5 impairs nuclear 415 sturdiness. Nat Commun 6, 6138. 416 Gettner, S.N., Kenyon, C., and Reichardt, L.F. (1995). Characterization of beta pat-3 heterodimers, a 417 family of essential integrin receptors in C. elegans. The Journal of cell biology 129, 1127-1141. 418 Gilbert, H.T.J., Mallikarjun, V., Dobre, O., Jackson, M.R., Pedley, R., Gilmore, A.P., Richardson, S.M., and 419 Swift, J. (2019). Nuclear decoupling is part of a rapid protein-level cellular response to high-intensity 420 mechanical loading. Nat Commun 10, 4149. 421 Gonzalez-Bermudez, B., Guinea, G.V., and Plaza, G.R. (2019). Advances in Micropipette Aspiration: 422 Applications in Cell Biomechanics, Models, and Extended Studies. Biophys J 116, 587-594. 423 Guilak, F., Tedrow, J.R., and Burgkart, R. (2000). Viscoelastic properties of the cell nucleus. Biochemical 424 and biophysical research communications 269, 781-786. 425 Guilluy, C., Osborne, L.D., Van Landeghem, L., Sharek, L., Superfine, R., Garcia-Mata, R., and Burridge, K. 426 (2014). Isolated nuclei adapt to force and reveal a mechanotransduction pathway in the nucleus. Nat 427 Cell Biol 16, 376-381. 428


https://doi.org/10.1101/2020.02.05.935395

23

Haase, K., Macadangdang, J.K., Edrington, C.H., Cuerrier, C.M., Hadjiantoniou, S., Harden, J.L., Skerjanc, 429 I.S., and Pelling, A.E. (2016). Extracellular Forces Cause the Nucleus to Deform in a Highly Controlled 430 Anisotropic Manner. Sci Rep 6, 21300. 431 Haithcock, E., Dayani, Y., Neufeld, E., Zahand, A.J., Feinstein, N., Mattout, A., Gruenbaum, Y., and Liu, J. 432 (2005). Age-related changes of nuclear architecture in Caenorhabditis elegans. Proc Natl Acad Sci U S A 433 102, 16690-16695. 434 Heo, S.J., Driscoll, T.P., Thorpe, S.D., Nerurkar, N.L., Baker, B.M., Yang, M.T., Chen, C.S., Lee, D.A., and 435 Mauck, R.L. (2016). Differentiation alters stem cell nuclear architecture, mechanics, and mechano- 436 sensitivity. Elife 5. 437 Hochmuth, R.M. (2000). Micropipette aspiration of living cells. J Biomech 33, 15-22. 438 Irianto, J., Xia, Y., Pfeifer, C.R., Athirasala, A., Ji, J., Alvey, C., Tewari, M., Bennett, R.R., Harding, S.M., Liu, 439 A.J., Greenberg, R.A., and Discher, D.E. (2017). DNA Damage Follows Repair Factor Depletion and 440 Portends Genome Variation in Cancer Cells after Pore Migration. Curr Biol 27, 210-223. 441 Kamath, R.S., and Ahringer, J. (2003). Genome-wide RNAi screening in Caenorhabditis elegans. Methods 442 30, 313-321. 443 Kaufmann, A., Heinemann, F., Radmacher, M., and Stick, R. (2011). Amphibian oocyte nuclei expressing 444 lamin A with the progeria mutation E145K exhibit an increased elastic modulus. Nucleus 2, 310-319. 445 Kim, J.K., Gabel, H.W., Kamath, R.S., Tewari, M., Pasquinelli, A., Rual, J.F., Kennedy, S., Dybbs, M., Bertin, 446 N., Kaplan, J.M., Vidal, M., and Ruvkun, G. (2005). Functional genomic analysis of RNA interference in C. 447 elegans. Science 308, 1164-1167. 448 Krause, M., Te Riet, J., and Wolf, K. (2013). Probing the compressibility of tumor cell nuclei by combined 449 atomic force-confocal microscopy. Phys Biol 10, 065002. 450 Lammerding, J., Fong, L.G., Ji, J.Y., Reue, K., Stewart, C.L., Young, S.G., and Lee, R.T. (2006). Lamins A and 451 C but not lamin B1 regulate nuclear mechanics. J Biol Chem 281, 25768-25780. 452 Lammerding, J., and Lee, R.T. (2009). Mechanical properties of interphase nuclei probed by cellular 453 strain application. Methods in molecular biology 464, 13-26. 454 Lherbette, M., Dos Santos, A., Hari-Gupta, Y., Fili, N., Toseland, C.P., and Schaap, I.A.T. (2017). Atomic 455 Force Microscopy micro-rheology reveals large structural inhomogeneities in single cell-nuclei. Sci Rep 7, 456 8116. 457 Liu, J., Rolef Ben-Shahar, T., Riemer, D., Treinin, M., Spann, P., Weber, K., Fire, A., and Gruenbaum, Y. 458 (2000). Essential roles for Caenorhabditis elegans lamin gene in nuclear organization, cell cycle 459 progression, and spatial organization of nuclear pore complexes. Mol Biol Cell 11, 3937-3947. 460 Lyakhovetsky, R., and Gruenbaum, Y. (2014). Studying lamins in invertebrate models. Adv Exp Med Biol 461 773, 245-262. 462 MacRae, T.H. (1997). Tubulin post-translational modifications--enzymes and their mechanisms of action. 463 Eur J Biochem 244, 265-278. 464 Malone, C.J., Fixsen, W.D., Horvitz, H.R., and Han, M. (1999). UNC-84 localizes to the nuclear envelope 465 and is required for nuclear migration and anchoring during C. elegans development. Development 126, 466 3171-3181. 467 Matsuyama, A., Shimazu, T., Sumida, Y., Saito, A., Yoshimatsu, Y., Seigneurin-Berny, D., Osada, H., 468 Komatsu, Y., Nishino, N., Khochbin, S., Horinouchi, S., and Yoshida, M. (2002). In vivo destabilization of 469 dynamic microtubules by HDAC6-mediated deacetylation. EMBO J 21, 6820-6831. 470 Pajerowski, J.D., Dahl, K.N., Zhong, F.L., Sammak, P.J., and Discher, D.E. (2007). Physical plasticity of the 471 nucleus in stem cell differentiation. Proc Natl Acad Sci U S A 104, 15619-15624. 472 Raab, M., Gentili, M., de Belly, H., Thiam, H.R., Vargas, P., Jimenez, A.J., Lautenschlaeger, F., Voituriez, 473 R., Lennon-Dumenil, A.M., Manel, N., and Piel, M. (2016). ESCRT III repairs nuclear envelope ruptures 474 during cell migration to limit DNA damage and cell death. Science 352, 359-362. 475


https://doi.org/10.1101/2020.02.05.935395

24

Robinson, P.J., An, W., Routh, A., Martino, F., Chapman, L., Roeder, R.G., and Rhodes, D. (2008). 30 nm 476 chromatin fibre decompaction requires both H4-K16 acetylation and linker histone eviction. Journal of 477 molecular biology 381, 816-825. 478 Robison, P., and Prosser, B.L. (2017). Microtubule mechanics in the working myocyte. J Physiol 595, 479 3931-3937. 480 Rosenbaum, J. (2000). Cytoskeleton: functions for tubulin modifications at last. Curr Biol 10, R801-803. 481 Rowat, A.C., Foster, L.J., Nielsen, M.M., Weiss, M., and Ipsen, J.H. (2005). Characterization of the elastic 482 properties of the nuclear envelope. J R Soc Interface 2, 63-69. 483 Shimamoto, Y., Tamura, S., Masumoto, H., and Maeshima, K. (2017). Nucleosome-nucleosome 484 interactions via histone tails and linker DNA regulate nuclear rigidity. Mol Biol Cell 28, 1580-1589. 485 Shogren-Knaak, M., Ishii, H., Sun, J.M., Pazin, M.J., Davie, J.R., and Peterson, C.L. (2006). Histone H4-K16 486 acetylation controls chromatin structure and protein interactions. Science 311, 844-847. 487 Stephens, A.D., Banigan, E.J., Adam, S.A., Goldman, R.D., and Marko, J.F. (2017). Chromatin and lamin A 488 determine two different mechanical response regimes of the cell nucleus. Mol Biol Cell 28, 1984-1996. 489 Stephens, A.D., Banigan, E.J., and Marko, J.F. (2018). Separate roles for chromatin and lamins in nuclear 490 mechanics. Nucleus 9, 119-124. 491 Swift, J., Harada, T., Buxboim, A., Shin, J.W., Tang, H.Y., Speicher, D.W., and Discher, D.E. (2013a). Label- 492 free mass spectrometry exploits dozens of detected peptides to quantify lamins in wildtype and 493 knockdown cells. Nucleus 4, 450-459. 494 Swift, J., Ivanovska, I.L., Buxboim, A., Harada, T., Dingal, P.C., Pinter, J., Pajerowski, J.D., Spinler, K.R., 495 Shin, J.W., Tewari, M., Rehfeldt, F., Speicher, D.W., and Discher, D.E. (2013b). Nuclear lamin-A scales 496 with tissue stiffness and enhances matrix-directed differentiation. Science 341, 1240104. 497 Tajik, A., Zhang, Y., Wei, F., Sun, J., Jia, Q., Zhou, W., Singh, R., Khanna, N., Belmont, A.S., and Wang, N. 498 (2016). Transcription upregulation via force-induced direct stretching of chromatin. Nat Mater 15, 1287- 499 1296. 500 Timmons, L., Court, D.L., and Fire, A. (2001). Ingestion of bacterially expressed dsRNAs can produce 501 specific and potent genetic interference in Caenorhabditis elegans. Gene 263, 103-112. 502 Towbin, H., Staehelin, T., and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide 503 gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76, 4350-4354. 504 Verstraeten, V.L., Ji, J.Y., Cummings, K.S., Lee, R.T., and Lammerding, J. (2008). Increased 505 mechanosensitivity and nuclear stiffness in Hutchinson-Gilford progeria cells: effects of 506 farnesyltransferase inhibitors. Aging Cell 7, 383-393. 507 Wang, X., Liu, H., Zhu, M., Cao, C., Xu, Z., Tsatskis, Y., Lau, K., Kuok, C., Filleter, T., McNeill, H., Simmons, 508 C.A., Hopyan, S., and Sun, Y. (2018). Mechanical stability of the cell nucleus - roles played by the 509 cytoskeleton in nuclear deformation and strain recovery. Journal of cell science 131. 510 Zuela, N., Zwerger, M., Levin, T., Medalia, O., and Gruenbaum, Y. (2016). Impaired mechanical response 511 of an EDMD mutation leads to motility phenotypes that are repaired by loss of prenylation. Journal of 512 cell science 129, 1781-1791. 513

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[21,555 characters not including Abstract, Methods and ...Feb 05, 2020 · nematodes. Nematodes were placed in . 75. between two membranes, stretched equiaxially and nucleus deformation