Nuclear mechanical resilience but not stiffness is modulated by αII-spectrin

Abstract

Spectrins are multi-domain, elastic proteins that provide elasticity to the plasma membrane of erythrocytes and select nucleated cells. Spectrins have also been found in the nucleus of non-erythrocytes, but their function remains to be uncovered. It has been hypothesized that a spring-like spectrin network exists within the lamina nucleoskeleton, however, experiments testing a spectrin network׳s mechanical impact on the nucleus are lacking. Here, we knock-down levels of nuclear αII-spectrin with the goal of disrupting this nucleoskeletal spectrin network. We mechanically test live cells with intranuclear particle tracking and compression assays to probe changes in nuclear mechanics with decreases in αII-spectrin. We show no changes in chromatin mechanics or in the stiffness of nuclei under compression. However, we do observe a reduction in the ability of nuclei with decreased αII-spectrin to recover after compression. These results establish spectrin as a nucleoskeletal component that specifically contributes to elastic recovery after compression.

Keywords: Lamina; Mechanobiology; Nucleoskeleton mechanics; Spectrins.

Fig. 1

Western blot showing αII-spectrin protein level decrease in whole cells, and isolated nuclei. Bands below the primary band in whole cells demonstrate αII-spectrin isoforms may be present in the cytoskeleton which are not seen in the nucleus.

Fig. 2

Immunofluorescence (IF) and quantification of fluorescence of KDSp and KDC cells. (A) Shows IF of scramble control (KDC – green) cells which express control levels of αII-Sp. Green arrows show locations in the αII-Sp channel of cells which express the GFP-reporter, and thus have taken up the given vector. (B) Shows IF images of αII-Sp which demonstrates decreased αII-Sp levels in cells which have taken up the knock-down vector (KDSp – green). (C) Shows quantification of intensity of αII-spectrin channel from IF images. Average intensity of the αII-Sp channel was calculated for cells which had taken up a given vector, and divided by the average intensity of the αII-Sp channel of non-transfected cells in each field of view (n=12 fields of view for each condition). Error bars are standard deviation between fields of view. Asterisk denotes significant difference (p<0.05). (D) Shows a comparison of initial nuclear area (i.e. no compression) between KDC and KDSp cells. No significant difference in nuclear area was detected in these cells as measured by Student’s t-test. n=19 KDSp nuclei, and n=28 KDC nuclei. Error bars represent standard deviation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Fig. 3

Intranuclear particle tracking analysis. (A) Shows an example of a cell nucleus (yellow outline) expressing RFP-TRF1. This telomeric protein appears as punctate fluorescent speckles in the nucleus which are tracked at 3-minute time intervals for 1 hour. (B) Shows tracks (blue overlay) of these chromatin-bound proteins demonstrate Brownian motion. (C) Shows a plot of mean squared displacement (MSD) vs. lag time for αII-spectrin knock-down cells compared to wild type (non-treated) cells. Interestingly, no significant difference is seen in MSD between groups for any lag time, indicating no changes in intranuclear movement when spectrin levels are decreased. n=12 WT cells, with 37 total points tracked and n=17 KDSp cells, with 65 total points tracked.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Fig. 4

Example of nuclear area increase, and subsequent decrease in wild type control cells after compression, and release of compression. Nuclei are stained with Hoechst. Propidium iodide (PI) was added to media for compression assays to assure cell membrane remained intact for the entire experiment. In the release frame, a PI stain is seen in one cell, indicated by a red arrow. This cell would not be used for analysis. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Fig. 5

Images of live cell compression assay comparing KDSp to KDC. Images are taken before, during, and after application of a 50 g static weight, and nuclear area was calculated. The GFP reporter is expressed in cells which have taken up the given vector, thus only these cells were used for analysis. Control cells which did not take up the knock-down vector are designated as side by side cells in Fig. 6. PI stain was added and only cells with intact membranes, which persisted for the entire assay were used for analysis. Nuclei are stained with Hoechst 33342.

Fig. 6

Quantification of nuclear area for the various treatment groups after compression assay. (A) KDSp cells (red) fail to return to their initial nuclear area, in contrast to the various control groups (KDC, side-by-side, WT) which all return to their initial nuclear area upon removal of the static weight. Side-by-side cells are cells which did not take up the spectrin knock-down vector, but were in the same field of view as the KDSp cells. This control demonstrates that the failure to return to initial area was not an artifact of experimental variability. WT cells are control cells which did not receive any transfection treatment. Error bars show standard error of the mean. Asterisk denotes significant different (p<0.01). (B) Individual data points of each nucleus are shown for each sample comparing strains under compression (x-axis) to the strain of release (y-axis) normalized to the original size. In each case, high compression strain maps with larger strain after removal of the compressive force. In many control cases, removal of force in nuclei under small strains resulted in a shrinking of the nucleus (blue and green points). Compression of KDSp cells also resulted in the largest nuclear dilation. Regression analysis of the data (inset box) shows similar slopes for all samples, similar intercepts for transfected samples and largely divergent offset for KDSp cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).