Changes in native sarcomeric α-actinin during mitosis of NRVM.

(A) Immunofluorescence staining of sarcomeric α-actinin (red) in NRVM during different mitotic stages (interphase to telophase, i–vi, as indicated below each panel). DNA is stained with DAPI (blue), and this is shown alone and in close-up to the right of each panel. An ordered sarcomeric striation pattern of α-actinin staining is observed in interphase. Sarcomere disassembly begins in prophase as seen by a loss in striations and an increase in a more diffuse and higher intensity fluorescence throughout the cytoplasm. The disassembly of α-actinin peaks in metaphase, continues in anaphase, and is still observable during telophase. Reassembly starts in late telophase/cytokinesis in which the striated pattern of sarcomeric α-actinin starts to re-appear from the margin of cells. (B) Changes in the α-actinin disassembly index during NRVM mitosis. Immunofluorescence staining of α-actinin in NRVM at different mitotic stages were recorded with Volocity 6.1.1 via confocal microscopy. For each sub-phase of mitosis, 20 mitotic cells randomly chosen from our collected image library (from three independent cell isolations) were compared with 20 neighboring, interphase cells to determine the α-actinin disassembly index. The α-actinin disassembly index was calculated as mean AlexaFluor 568 intensity of the mitotic cell divided by that of an interphase cell inside the same field of view and presented as the median in a box and whisker plot. One way ANOVA followed by Tukey's multiple comparison test was performed to analyze differences between each mitotic phase. * p < 0.01 compared to interphase. (C) Time lapse live-cell images show the rapid progress of α-actinin disassembly during early prophase. NRVM were transduced with a lentiviral vector harboring C-terminal HaloTag fused α-actinin. The cells were stained with cell permeable TMRDirect halo ligand (red) 48 hr after transduction. Nuclear DNA was stained with Vybrant DyeCycle Violet (blue). The cell was observed in a live cell imaging chamber (37°C) supplied with 5% CO2 in room air and imaged using spinning disk confocal microscopy. Photos selected at the indicated times are displayed. Original video available in the supplementary video.