XB-ART-39737Biophys J May 20, 2009; 96 (10): 4319-25.
Influence of lamin A on the mechanical properties of amphibian oocyte nuclei measured by atomic force microscopy.
The nuclear lamina is part of the nuclear envelope (NE). Lamin filaments provide the nucleus with mechanical stability and are involved in many nuclear activities. The functional importance of these proteins is highlighted by mutations in lamin genes, which cause a variety of human diseases (laminopathies). Here we describe a method that allows one to quantify the contribution of lamin A protein to the mechanical properties of the NE. Lamin A is ectopically expressed in Xenopus oocytes, where it is incorporated into the NE of the oocyte nucleus, giving rise to a prominent lamina layer at the inner nuclear membrane. Nuclei are then isolated and probed by atomic force microscopy. From the resulting force curves, stiffness values are calculated and compared with those of control nuclei. Expression of lamin A significantly increases the stiffness of oocyte nuclei in a concentration-dependent manner. Since chromatin adds negligibly to nuclear mechanics in these giant nuclei, this method allows one to measure the contribution of individual NE components to nuclear mechanics.
PubMed ID: 19450502
PMC ID: PMC2712154
Article link: Biophys J
Genes referenced: lmna
GO keywords: nuclear envelope
Disease Ontology terms: cardiomyopathy
OMIMs: CARDIOMYOPATHY, DILATED, 1A; CMD1A
Article Images: [+] show captions
|Figure 1 TEM of the NEs of lamin A-expressing oocytes. Prelamin A was expressed in oocytes by nuclear injection of plasmid. TEM sections of isolated oocyte nuclei are shown. The lamina (NL) (brackets in B and C) forms a thick electron-dense layer in oocytes expressing lamin A (B and C), which leaves the NPCs (arrows) free. The lamina in noninjected control oocytes is hardly discernible (A). NC: nuclear content; arrows point to NPCs; open triangles in B point to adjacent lamina layers of different thickness. Images were taken at the same magnification.|
|Figure 2 Typical force curve on a NE. The deflection (i.e., loading force) is proportional to the sample height z, as expected from the mechanical response of a thin elastic shell. (A) Force curves are shown for control nuclei (no DNA injected) and nuclei with DNA injected at 37 μg/mL and 110 μg/mL DNA, respectively. Depending on the amount of DNA injected, lamin A is expressed and the nucleus stiffens. The lamin A nuclei mostly exhibit a linear force curve, which shows that the mechanical response is mainly determined by the NE. However, in the softer samples (control), the nonlinearity of the force curve indicates that the underlying nucleoplasm, including its actin cytoskeleton, also contributes to sample stiffness. In B the softest force curve of A (control) has been fitted to a line (shell model) or to the Hertz fit. As can be seen from these two fits, the Hertz fit does not adequately describe our data.|
|Figure 3 (A) Calculated stiffnesses from AFM force curves on isolated nuclei. The data are grouped for each nucleus. The color code refers to the concentration of injected DNA. Red (asterisk) corresponds to control (no DNA injected), green (circles) to 37 μg/mL, and blue (diamonds) to 110 μg/mL of injected DNA, respectively. (B) Although there is some variability for subsequent measurements of one nucleus, the differences between the different DNA concentrations are obvious and become more pronounced when all measurements of each individual nucleus are averaged. Here the error bars correspond to the SD of each nucleus. There is still some variability between different experiments corresponding to different batches of oocytes. Oocytes within one experiment are all from the same frog. Nevertheless, the degree of expression of lamin A seems to vary to some degree from experiment to experiment and within experiments.|
|Figure 4 Average stiffness from all measurements as a function of concentration of injected DNA. The difference in stiffness at the three concentrations is highly significant between control and injected samples, and significant between low and high concentrations of injected DNA (see Table 1 for data).|
References [+] :