Lamin A in lamina acts as a “Check valve” in response to stress.

<p>Inside lamina A and B-type lamins respond differently with increasing cellular stress at different stages of differentiation.</p

Strain induced changes in the network of wt LA and mutant protein and DLS measurements.

<p>A) Dependence of elastic modulus <i>G′</i> and viscous modulus <i>G′′</i> of wt LA at 0.85 mg/ml concentration on varying the strain amplitude in the range of 0.01 to 1000%, keeping the angular frequency fixed at 5 rad/s. Green and blue arrow indicates critical strain and yield strain respectively. B) Concentration dependence of the critical strain corresponding to the onset of non-linearity and the yield strain (inset) above which the network starts to flow is shown. Concentrations of 0.6, 0.85 and 2 mg/ml were used for this experiment. C) Dependence of elastic modulus <i>G′</i> and viscous modulus <i>G′′</i> of wt LA and E161K at 0.6 mg/ml concentration on varying the strain amplitude in the range of 0.01 to 1000%, keeping the angular frequency fixed at 5 rad/s. Black and blue arrows indicate critical strain, grey and light blue arrows indicate yield strain of wt LA and E161K respectively. Inset shows a model representing the fate of wt LA and mutant LA network upon shear deformation. D) Number percentage statistics of 0.3 and 3 mg/ml of wt LA protein.</p

Expression, folding and ultrastructure of lamin A.

<p>A) 10% SDS PAGE analysis of pure fractions of wt LA, E161K and R190W from Mono S column; immunoblot of the same fractions using mouse monoclonal anti lamin A+C antibody (JoL2). Numbers corresponding to the bands of the marker in lane M are in kilo Daltons. B) CD spectra of 0.7 mg/ml wt LA in 4 M urea, 2 M urea and assembly buffer respectively at 25°C. C) SEM images of WT, E161K and R190W at concentrations of 0.6 mg/ml. Magnification for WT and mutants are 2000x and 3000x respectively. Scale bars for wt LA and mutants are 20 µm and 10 µm respectively. Arrow and Asterisk marks indicate the cross-linked sites and bundled filaments in the network respectively. D) Mesh size of lamina from EGFP tagged wt LA and mutants transfected in HeLa cells were calculated from confocal images and represented as box plot (n =  200–300, in 10 nuclei).</p

Viscosity of wild type and mutant lamin A networks under shear.

<p>A) Steady shear viscosity (<i>η</i>) as a function of shear-rate denoted by open circles and complex viscosity ((ω)) as a function of angular frequency (<i>ω</i>) denoted by solid circles are shown. The inset shows the flow curve of wt LA (0.7 mg/ml) indicating the variation of shear stress (<i>σ</i>) with shear-rate. B) Steady shear viscosity (<i>η</i>) as a function of shear-rate and C) complex viscosity ((ω)) as a function of angular frequency (<i>ω</i>) of 0.7 mg/ml of wt LA and 0.6 mg/ml of E161K and R190W. D) Flow curve indicating the variation of shear stress (<i>σ</i>) with shear-rate of 0.7 mg/ml of wt LA and 0.6 mg/ml of E161K and R190W.</p

Elastic Behaviour of wt LA and mutant proteins.

<p>A) Increase in storage modulus <i>G′</i> and B) loss modulus <i>G′′</i> of wt LA upon assembly in lamin A assembly buffer with increasing concentrations. <i>G′</i> and <i>G′′</i> are the in-phase and out of phase components respectively, of an oscillatory shear of strain amplitude 1% at an angular frequency of 5 rad/s for 1000 – 3000 s. Protein concentrations used were in the range of 0.28–3.2 mg/ml of wt LA. C) Same measurement as in (A) and (B) with wt LA concentration fixed at 2.2 mg/ml and DOPC concentrations in the range 0 – 10 mg/ml. The decrease in <i>G′</i> with different DOPC concentration at the air/water interface is shown in the inset. The <i>G′</i> values obtained from repeated measurements lie within the experimental error bar. D) Concentration dependent increase in <i>G′</i> of wt LA. Comparison of E) Storage modulus <i>G′</i> and F) Loss modulus <i>G′′</i> of wild type and mutants upon assembly in assembly buffer. 0.6 mg/ml concentration of wt LA, E161K and R190W were used for these measurements. The parameters for (E, F) are identical to (A, B).</p

Differential elastic modulus measured from forced oscillations about a prestress.

<p>A) The differential elastic modulus (<i>K′</i>) as a function of steady shear stress () at 0.6, 0.85 and 2 mg/ml concentrations of wt LA protein. The inset shows the variation of maximum stress for breaking the network () with concentration (c). Open and Solid arrow indicates and respectively. B) <i>K′</i> scaled by differential modulus in the linear region () as a function of. The inset shows <i>K′</i> scaled by concentration as a function of_.</p

Frequency sweep measurements of wt LA and mutant proteins.

<p>Measurements for A) elastic modulus <i>G′</i> (<i>ω</i>) and B) viscous modulus <i>G′′</i> (<i>ω</i>) were carried out for probing the structural relaxation in the gel phase by varying the angular frequency in the range 0.1 to 20 rad/s with the strain amplitude fixed at 1%. C) Master curve of the linear viscoelasticity of the lamin A network. Protein concentrations used were in the range of 0.28–2 mg/ml. The variation of the scaling parameters for <i>G′</i> and <i>G′′</i> are shown in the inset where <b>a</b>- <i>G′</i> and <b>b</b>- <i>G′</i>′. Measurements for D) elastic modulus <i>G′</i> (<i>ω</i>) and E) viscous modulus <i>G′′</i> (<i>ω</i>) of wt LA, E161K and R190W at 0.6 mg/ml concentration were carried out for probing the structural relaxation in the gel phase by varying the angular frequency in the range 0.1 to 10 rad/s with the strain amplitude fixed at 1%.</p