Fig 1.
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.
Fig 2.
The turnover rate of α-actinin assessed in a pulse-chase experiment.
NRVM were transduced with lentiviral vector harboring C-terminal HaloTag labeled α-actinin. 48 hr after transduction, cells were stained with excess TMRDirect (red fluorescence), a cell permeable ligand which forms a stable covalent bond with HaloTag α-actinin. At specified time points after TMRDirect staining as indicated, a different Halo-tag ligand (R110Direct, green fluorescence) was used to stain de novo synthesized HaloTag α-actinin. Both red and green fluorescence were assessed simultaneously. Any newly synthesized α-actinin in different time intervals after TMRDirect staining would be labeled with R110Direct. Representative images were taken under identical exposure parameters. Slight variations in background fluorescence between durations could be due to cell-to-cell variation, differing locations on cover slips, slight variation in depth of mounting media, or to changes in focus.
Fig 3.
Pattern of titin disassembly during mitosis in NRVM.
(A) Immunofluorescence staining of titin at both its N terminal (Z-disk, T12 antibody, red) and C terminal (M-line, M8 antibody, green) during mitosis. The clear alternating striation pattern of interphase cells (i) is disrupted starting from the end of prophase (ii) through prometaphase (iii). This is shown by a widening of both Z band and M line titin staining and the disappearance of gaps between the Z bands and M lines. Titin is almost completely disassembled by metaphase (iv) and remains disassembled through anaphase (v) until telophase (vi) when signs of reassembly appear in late cytokinesis. DNA is stained with DAPI (blue), and this is shown alone and in close-up above panels i to vi. (B) Proposed model of titin disassembly based on these observations. (C) Titin immunofluorescence image of a typical metaphase NRVM was shown along with two interphase cells. The drawn white lines show the border between cells. The boxed perinuclear region of titin undergoing disassembly is shown at higher magnification on the right. The transition titin disassembly pattern observed suggests that titin disassembly may start from the perinuclear zone and spread towards the peripheral margins of the cell.
Fig 4.
Disassembly of α-actinin precedes that of titin during mitosis.
To delineate the possible sequential order of disassembly between α-actinin and titin during NRVM mitosis, triple staining of α-actinin (red), titin M8 epitope (green) and nuclear DNA (blue) was performed. (A) A typical prophase cell shows that α-actinin disassembly is well underway while titin remains intact. (B) For each image of mitotic NRVM in different mitotic stages found (screened from about 3000 NRVM per individual isolation over 5 isolations), α-actinin and titin disassembly scores were assessed. Mitotic stage determination was based on chromatin morphology. Disassembly score for both α-actinin and titin in each cell assessed is shown in two dimensional dot plots. Titin disassembly consistently occurs after α-actinin disassembly in timing.
Fig 5.
Protease inhibitors fail to interrupt sarcomere disassembly.
NRVM were cultured in the presence of different protease inhibitors (PMSF, 20 μM; ARP100, 100 μM; GM6001, 5 μM or ALLM, 100 μM) for 4 hr before they were processed for immunofluorescence staining of α-actinin, titin (M8) and DAPI. α-actinin and titin disassembly were evaluated together for each single mitotic cell found in different treatment groups. The extent of α-actinin and titin disassembly were scored [0, intact sarcomeric proteins; 1, low (20–40% of sarcomere area) disassembly; 2, medium (50–70% of sarcomere area); 3, high, disassembly (>80% of sarcomere area)]. Imaging screening area and NRVM culture density were comparable between different treatment groups and a total of 584 mitotic NRVM over 13 independent isolations were found and recorded by confocal fluorescence microscope. The proportional distribution of different disassembly stages was plotted based on different mitotic stages and inhibitor treatments. Fisher's exact test was used to analyze the ranking data. None of the treatments show significant difference to vehicle control (p > 0.05).
Fig 6.
Analysis of cytosolic and sarcomeric α-actinin during mitosis.
(A) Synchronized NRVM using thymidine and Ro3306 reveal increased α-actinin in RIPA buffer cell extracts in association with an increase in mitotic index. NRVM were cultured on 12 mm glass cover slips and synchronized with thymidine and 9 μM Ro3306 as indicated in the timeline. Immunofluorescence staining with α-actinin antibody and DAPI were performed to determine the mitotic index of NRVM at the different time points. A separate synchronization experiment was also performed in parallel on cells cultured in 6-well plates. At the same time points cells were lysed in RIPA buffer to obtain total cellular protein and this was analyzed for α-actinin by Western blot. The average mitotic index data from three independent experiments is shown. (B) Cell cycle analysis based on Vybrant DyeCycle Violet DNA staining was performed on non-synchronized NRVM (top panel) and synchronized NRVM (bottom panel). The high DNA content population (G2M population) increased in the cell cycle synchronized NRVM (4 hr after release from Ro3306) in comparison with non-synchronized NRVM. (C) Cytosolic and sarcomeric fractions of NRVM separated by FACS into different cell cycle stages based on DNA content were analyzed for α-actinin by Western blot analysis. (D) Phosphorylation status of cytosolic and sarcomeric α-actinin of FACS-sorted NRVM was determined using Phos-tag PAGE. GAPDH was used as a cytosolic marker. (E) Sarcomeric α-actinin phosphosites identified in large-scale phosphoproteomic screens. Upper bars show all sites reported in the PhosphoSitePlus database. The height of the bars corresponds to the number of species in which a given site was identified. Lower bars show phosphosites from mouse heart, with the height of the bar corresponding to the number of spectral matches, a semi-quantitative measure of phosphosite abundance. Residues of interest from the text are indicated.
Table 1.
In silico analysis to identify potential kinases of known phosphorylated residues in sarcomeric α-actinin (ACTN2).
Fig 7.
Induced exit from mitosis in NRVM is accompanied by rapid reassembly of α-actinin and titin.
(A) After 16 hr of nocodazole exposure to enrich cells at prometaphase, fresh media alone (control) or with 9 μM Ro3306 replaced nocodazole-containing media. For each time point and treatment condition, 3000 NRVM were counted in the low magnification (10X) images and absolute numbers of cells in each mitotic sub-phase were plotted. (B) Immunofluorescence of a single representative cell chosen from each of: interphase cells after thymidine treatment, nocodazole enriched prometaphase cells, anaphase cells 1 hr after nocodazole release and prophase-like cells (indicated by arrows) after 60 min Ro3306 treatment following nocodazole release, are shown for both titin (M8) and α-actinin. (C) The α-actinin disassembly index was assessed for the different cell groups as shown above in (A) and (B). Analyzed cells were randomly chosen from three independent experiments. Data are presented as the median in a box and whisker plot. One way ANOVA followed by Tukey's multiple comparison test was performed. * p < 0.01 between control and Ro3306 groups is highlighted.