L-glutamine, Neurobasal medium, B-27 supplement (50×, minus antioxidants), soybean trypsin inhibitor, Dulbecco’s modified Eagle’s medium (DMEM), Opti-modified Eagle’s medium^® (Opti-MEM^®), Lipofectamine^® RNAiMAX, Lipofectamine^® 3000, Alexa Fluor^® 488 donkey anti-rabbit antibody and Alexa Fluor^® 568 phalloidin were obtained from Thermo Fisher Scientific (San Jose, CA, USA). Fetal bovine serum was from ScienCell (San Diego, CA, USA). Penicillin, streptomycin and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Amresco (Solon, OH, USA). DNase, cytosine arabinoside, poly-L-lysine, N-acetyl-L-cysteine (NAC), blebbistatin, cytochalasin D, ML-7 and H₂O₂ and were from Sigma-Aldrich (St. Louis, MO, USA). Y27632, z-VAD-fmk and z-DEVD-fmk were from Selleck Chemicals (Houston, TX, USA). Anti-MLC, anti-P-MLC (Ser19), anti-caspase-3, anti-microtubule-associated protein 2 (MAP2) antibodies were from Cell Signaling Technology (Danvers, MA, USA). Anti-myosin IIA and anti-actin antibodies were from Abcam (Cambridge, UK). Anti-ROCK1 and anti-ROCK2 antibodies, Protein A/G PLUS-Agarose, normal rabbit IgG and normal mouse IgG were from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-GAPDH antibody was purchased from KangCheng (Shanghai, China). HRP-conjugated secondary antibodies were from Boster (Wuhan, China). DAPI was from Beyotime Biotech (Haimen, China). Protease inhibitor cocktail, RIPA buffer and ECL were purchased from Vazyme Biotech (Nanjing, China). Dimethyl sulfoxide (DMSO) was obtained from SunShineBio (Nanjing, China).

Highly differentiated PC12 (rat adrenal pheochromocytoma) cells were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. According to the protocol from Shanghai Institute of Cell Biology, PC12 cells were differentiated in differentiation medium (DMEM with 100 ng/ml nerve growth factor) for 5–7 days to induce neurite formation and differentiation. The media was changed with new differentiation medium every 2–3 days. The differentiated PC12 cells were cultured in DMEM, supplemented with 10% fetal bovine serum, 100 I.U./mL penicillin and 100 μg/mL streptomycin, in a humidified cell culture incubator in 5% CO₂ atmosphere at 37°C.

Primary cultures of cortical neurons were obtained from Sprague-Dawley rats on embryonic day 16–17 and processed as described (Cao et al., 2015). The pregnant Sprague-Dawley rats were provided by the Reference Animal Research Centre of Yangzhou University (Yangzhou, China; certificate no SCXK 2014-0004). The pregnant rats were euthanized by CO₂ asphyxiation, and embryos were removed under sterile conditions. Neocortices were collected, removed of their meninges, minced by trituration with a Pasteur pipette, and digested in trypsin at 37°C for 25 min. The cells were further dissociated by two successive trituration and sedimentation steps in isolation buffer containing soybean trypsin inhibitor and DNase. The cells were then centrifuged and resuspended in Eagle’s minimal essential medium with Earle’s salt (MEM), supplemented with 2 mM L-glutamine, 10% fetal bovine serum, 100 I.U./mL penicillin and 100 μg/mL streptomycin, pH 7.4. The dissociated cells were plated onto poly-L-lysine-coated 96-well culture plates or 6-well culture plates at a density of 1 × 10⁶ cells/mL. For the immune-fluorescent experiments, cells were plated onto poly-L-lysine-coated 35-mm confocal dishes (Glass Bottom Dish) at a density of 2 × 10⁵ cells/mL. Cells were cultured at 37°C in a 5% CO₂ and 95% humidity atmosphere. Cells were treated with Cytosine arabinoside (10 μM) after 24–48 h post plating to prevent proliferation of non-neuronal cells. The culture medium was changed every 3 days using Neurobasal medium supplemented with B-27 supplement (minus antioxidants), 2 mM L-glutamine, 100 I.U./mL penicillin and 100 μg/mL streptomycin, pH 7.4. The cultures were used for experiments between 7 and 9 DIV (days in vitro). This study was carried out in accordance with the National Institutes of Health Guide for the care and use of laboratory animals. Animal experiments and surgical procedures were approved by the Animal Ethics Committee of the School of Chinese Materia Medica, China Pharmaceutical University.

Cells were treated with NAC (500 μM), blebbistatin (1 μM), cytochalasin D (1 μM), ML-7 (5 μM), Y27632 (10 μM), z-VAD-fmk (10 μM) or z-DEVD-fmk (10 μM) for 1 h, followed by co-incubating with 100 μM H₂O₂ for another 12 h. All compounds were dissolved in DMSO and finally diluted in the serum free medium with DMSO at the final concentration of 0.1%. DMSO was added in the control groups.

The siRNA oligonucleotides used to knockdown myosin IIA or IIB were purchased from Biomics Biotech (Nantong, China). The antisense sequence for myosin IIA (Myh9) is 5′-GAGACAAUGGAGGCCAUGA-3′. The antisense sequence for myosin IIB (Myh10) is 5′-CUAUUCAGGACUCAUCUAU-3′. PC12 cells growing on 6-well plates at 50%–60% confluency were transfected with 5 μg of siRNA and 5 μL of Lipofectamine^® RNAiMAX in the Opti-MEM^® medium. The cells were incubated with the medium containing siRNA for 6 h. The transfection medium was then replaced with complete medium without antibiotics. Cells were processed for analysis 48–72 h after transfection.

MYH9 clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) knockout plasmid (Santa Cruz Biotechnology, Dallas, TX, USA) consists of a pool of three plasmids, encoding three guide RNAs (gRNAs) to target different regions of Myh9 gene for maximum knockout efficiency. The gRNAs sequences targeted by Myh9 CRISPR plasmids are 5′-TGGTTCAAAGCCATTCTTGG-3′, 5′-ACCAGCCAGCCTTAAGGAGG-3′ and 5′-TATCTACTCAGAGGAGATCG-3′. Control CRISPR/Cas9 Plasmid does not recognize any DNA sequence used as a negative control (NC). Each plasmid encodes Cas9 nuclease and green fluorescent protein (GFP). PC12 cells were seeded in 6-well plates (at a density of 1 × 10⁶ cells/well) the day before transfection. Cells were transfected with 2 μg of the plasmid using lipofectamine 3000^®. After incubation of 72 h, GFP-positive cells were sorted and grown as single cell in 96-well plates using the BD FACSAria III cell sorter (BD Biosciences, San Jose, CA, USA). Colonies derived from single cells were expanded in media with 20% FBS. After 3 weeks, Myh9 knockout cells were screened by Western blotting using myosin IIA-specific antibodies.

Cell viability was determined by the MTT assay. Cells were seeded in 96-well plates and cultured at 37°C in an atmosphere of 5% CO₂ and 95% relative humidity. Following treatment, the medium was aspirated, and 100 μL of culture medium containing 5 mg/mL of MTT dye was added to each well. Cells were incubated for 4 h. The reaction mixture was carefully taken out and 150 μL of DMSO was added to each well. The 96-well plates were shaken for 10 min and the absorbance was then recorded with dual waves at 570 and 650 nm by a microplate reader (Epoch, BioTek, Winooski, VT, USA). The cell viability was expressed as percentage of vehicle-treated controls.

Caspase-3 activity was evaluated according to the manual of the Caspase-3 Activity Assay kit (Beyotime Shanghai, China). Briefly, the whole cell lysates (WCL) were added into 96-well plates and incubated with 2 mM of caspase-3 substrate (Ac-DEVD-pNA) at 37°C for 4 h. The absorbance was measured at 405 nm using a microplate reader. The protein levels of the samples were measured by the Bradford protein assay (Beyotime, Shanghai, China). Relative caspase-3 activity was expressed as percentage of vehicle-treated controls.

PC12 cells were seeded in 6-well plates at a density of 1 × 10⁵ cells/mL, treated as mentioned above, and observed with a phase contrast microscope (Olympus Corporation, Tokyo, Japan). PC12 cells were considered blebbing when blebs extended and cell bodies contracted. The percent of blebbing cells was determined by counting total and blebbing cells under blinded conditions. Neurite length was measured from the tip of the neurite until the soma of the cell using the ImageJ software (National Institutes of Health, Bethesda, MD, USA). Cell body area was measured using the ImageJ software. Neurite length and cell body area were presented as percentage of controls.

Cell suspensions were centrifuged, resuspended in fixative in a Eppendorf tube, spun down again to form a pellet and stored overnight at 4°C. Cells were then postfixed in 1% osmium tetroxide in 0.10 M phosphate buffer, dehydrated in a graded series of ethanol (30, 50, 70, 80, 90 and 100%), infiltrated, and embedded in an “Epon-Araldite” mixture. Seventy-five nanometer sections were stained with uranyl acetate and bismuth subnitrite, and then examined in a transmission electron microscope (JEM-1010, JEOL Ltd, Tokyo, Japan) at 80 Kv accelerating voltage.

PC12 cells and neurons were washed with cold PBS, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS for 30 min at room temperature, blocked with 5% BSA and incubated with rabbit anti-myosin IIA antibody at 1:200 dilution or rabbit anti-MAP2 antibody at 1:200 dilution overnight at 4°C, followed by 1 h incubation with Alexa Fluor^® 488 donkey anti-rabbit antibody at 1:200 dilution at room temperature. Filamentous actin structures were stained with 1:200 dilution of Alexa Fluor^® 568 Phalloidin. Nuclei were visualized with DAPI. Fluorescent images were taken through laser excitation lines 405, 488, 568 nm and Differential Interference Contrast (DIC) with confocal laser scanning microscope (LSM700, Zeiss, Germany). Cells were imaged with the same optical slice thickness in every channel using the Objective Plan-Apochromat 63×/1.40 Oil DIC M27. The resolution of images is 1024 × 1024 and the pinhole is set to one Airy unit. Images were obtained using the ZEN imaging software (Zeiss, Germany). Quantitative co-localization of myosin IIA or IIB with actin filaments was performed using the ImageJ software which can provide the Manders’ coefficients for the overlap of the images. Its values range between 0 and 1.0. A value of 1 represents strong positive correlation and 0 indicates that there is no discernable correlation. Neurite length was quantified from neuronal cultures immunostained with MAP2 antibody. Neurite length was measured from the tip of the neurite until the soma of the neuron using the ImageJ software and was presented as percentage of control. All micrographs include a bar to indicate the scale.

Cells were washed twice with PBS, lysed in RIPA buffer containing complete protease inhibitor cocktail and centrifuged at 12,000 rpm for 10 min at 4°C. Protein concentrations were determined according to BCA protein assay. Equal amounts of proteins (30 μg) were electrophoresed in 10% SDS-PAGE gels, transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore Corporation, Billerica, MA, USA). The targeting proteins were probed with antibodies against myosin IIA (1:1000), myosin IIB (1:1000), ROCK1 (1:300), ROCK2 (1:300), MLC (1:1000), P-MLC (Ser19, 1:1000), caspase-3 (1:1000), GAPDH (1:10,000) and appropriate HRP-conjugated secondary antibodies (1:10,000), followed by visualization with ECL and photographed using the Bio-Rad Gel Imaging System (Bio-Rad, Hercules, CA, USA). Data analysis was done using ImageJ software measuring integrated density of bands after background subtraction.

The Protein A/G PLUS-Agarose was washed with RIPA buffer three times before mixing with antibody. Prior to immunoprecipitation, 2 μg of purified antibodies against myosin IIA, actin filaments or normal IgG were mixed with 30 μL of agarose solution, and incubated at 4°C overnight on a rocker table. The prepared antibody-agarose complex were added to 1 mL of WCL (1 mg/mL) and incubated with rocking for 4 h at 4°C. The agarose-containing lysates were centrifuged and washed three times with RIPA buffer. Agarose-bound proteins were released by adding 2× SDS gel loading buffer and boiling for 5 min. An equal volume of each sample was fractionated by 10% SDS-PAGE and blotted using specific antibodies.

Data are presented as the means ± SD of three independent experiments. Statistical analysis of the data was performed with Student’s t test for two group comparison or one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test for multiple comparisons using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA). P < 0.05 was considered statistically significant.

In the present study, we used highly differentiated PC12 cells and primary cultured rat cortical neurons stimulated by H₂O₂ as the in vitro neuronal oxidative stress models. Caspase-3 activity significantly increased following 100 μM H₂O₂ treatment for 12 h in both PC12 cells and neurons (Figure 1A). The phase-contrast micrographs showed that normal PC12 cells had typical elongated and spreading morphology with extended neurites (Figure 1B, Control). Following H₂O₂ treatment, PC12 cells lost membrane extensions, detached from the culture support and shrunk to a rounded shape with obvious membrane blebbing (Figure 1B, H₂O₂). Transmission Electron Microscopy (TEM) was utilized to obtain fine-detailed photomicrographs of PC12 cells morphology. A typical apoptotic cell was observed with dramatic membrane blebs and chromatin condensation after H₂O₂ treatment (Figure 1C, H₂O₂). In contrast, control PC12 cells did not bleb and their nucleus remained intact (Figure 1C, Control). Consistently, similar results were also observed in neurons (Figure 1D).

Previous studies have shown that morphological changes in apoptotic cells are dependent on actomyosin cytoskeleton remodeling (Wickman et al., 2013; Turney et al., 2016). To understand which myosin II isoform regulates neuronal apoptosis, we examined the relocalization of the two myosin II isoforms and actin filaments in response to H₂O₂. In normal PC12 cells and neurons, myosin IIA and IIB have distinct cellular localization pattern, while actin showed a similar pronounced peripheral localization. Myosin IIA was distributed throughout the cytoplasm and neuritis (Figures 2A,C), whereas myosin IIB tended to be broadly peripheral and associated more with actin comparing to myosin IIA (Figures 3A,C). H₂O₂ exposure induced dramatic changes of cell morphology, as well as the reorganization of myosin IIA, IIB and F-actin. Myosin IIA and F-actin accumulated to form a dense spherical network. Myosin IIA had a stronger association with actin filaments under oxidative stress than normal conditions (Figures 2B,D). Quantitive analysis by Manders’ overlap coefficients (Bolte and Cordelières, 2006) showed that myosin IIA and actin filaments exhibited statistically significant co-localization upon H₂O₂ treatment in both PC12 cells (Figure 2E) and neurons (Figure 2F). Co-immunoprecipitation analysis also confirmed the increased interaction of myosin IIA and F-actin induced by oxidative stress (Figures 2G,H).

H₂O₂ treatment didn’t significantly increase the interaction of myosin IIB with actin filaments in apoptotic PC12 cells (Figures 3B,E). Co-immunoprecipitation analysis also showed that the interaction between myosin IIB and actin didn’t change in PC12 cells induced by H₂O₂ (Figure 3G). While in neurons, myosin IIB staining became dimmer and punctate with decreased co-localization with actin under H₂O₂ exposure (Figures 3D,F). Western blot analysis showed that H₂O₂ treatment significantly decreased myosin IIB expression in neurons (Figure 3H), while had no effect on the protein level of myosin IIA (Supplementary Figure S1A). H₂O₂ also didn’t change the level of myosin IIA of IIB in PC12 cells (Supplementary Figure S1B). We then examined the expression levels of myosin II isoforms in PC12 cells and neurons. There was higher myosin IIA expression in PC12 cells and similar myosin IIB levels in both kinds of cells (Supplementary Figure S1C).

Although the co-localization of myosin IIB with actin filaments was more striking under normal conditions, myosin IIA tended to bind with more actin filaments under H₂O₂ treatment. The increased interaction between myosin IIA and actin suggested that actin polymerization might also play a role during H₂O₂ exposure. Therefore, we applied actin filament assembly inhibitor, cytochalasin D before H₂O₂ injury. Cytochalasin D inhibited PC12 cells membrane blebbing and caspase-3 activation induced by H₂O₂ (Supplementary Figure S2).

To investigate the specific roles of myosin II isoforms in neuronal apoptosis, we transfected PC12 cells with siRNAs targeting at myosin IIA or IIB. Transfection of PC12 cells with #1 Myh9 siRNA (siMyh9) or 1# Myh10 siRNA (siMyh10) led to attenuated expression of myosin IIA or IIB to 20%–30% of the control values, without affecting the levels of the other isoforms (Supplementary Figure S3). Knockdown of myosin IIA caused remarkable expansion in cell body area and increased neurite length. In contrast, depletion of myosin IIB led to a raised and rounded cell body with dramatic decreases in its size and neurite length, which suggests opposing roles of myosin IIA and IIB in regulating the morphology of PC12 cells (Figure 4D, Control). Knockdown of myosin IIA protected PC12 cells from H₂O₂-induced membrane blebbing, cell contraction and neurite retraction, compared with PC12 cells transfected with NC siRNA. In contrast, knockdown of myosin IIB further increased membrane blebbing, cellular contraction and neurite retraction under H₂O₂ exposure (Figures 4A–D, H₂O₂).

MTT assay showed that myosin IIA knockdown didn’t change, while myosin IIB knockdown increased cell viability in PC12 cells. Under H₂O₂ treatment, both myosin IIA and IIB knockdown increased cell viability compared with control (Figure 4E). This might result from the increased cell number following myosin IIB knockdown (Figure 4F). To exclude the effects of cell number, we then subtracted H₂O₂-induced cell viability from form untreated cells, which represents the decreased cell viability. There was a more severe reduction in cell viability of myosin IIB knockdown cells than that of control or myosin IIA knockdown cells (Figure 4G). Consistently, caspase-3 activity assay showed that myosin IIA depletion inhibited H₂O₂-induced caspase-3 activation, while myosin IIB knockdown further increased H₂O₂-induced caspase-3 activation in PC12 cells (Figure 4H). Our data indicated a critical role for myosin IIA in the progressive apoptosis of neuronal cells induced by H₂O₂.

To further confirm the role of myosin IIA in regulating H₂O₂-induced neuronal apoptosis, we knocked out Myh9 gene in PC12 cells with the CRISPR/Cas9 system. Western blot analysis revealed that colony #1, #2 and #3 were completely depleted of myosin IIA protein compared with wild-type (WT) cells, without affecting the level of myosin IIB (Figure 5A). Consistently, Myh9 knockout enlarged cell body area under normal conditions, and inhibited cell contraction and membrane blebbing upon H₂O₂ exposure (Figure 5B). Myh9 knockout also increased cell viability and decreased caspase-3 activation after H₂O₂ treatment compared with WT cells (Figures 5C,D). Our observation demonstrates the critical and specific role of myosin IIA in regulating H₂O₂-induced PC12 cells apoptosis.

ROCK1 contributes to actomyosin contractility and causes cellular blebs to protrude through MLC phosphorylation (P-MLC). Caspase-3 has also been reported to be important in such morphological changes (Sebbagh et al., 2001). Time-course study revealed that H₂O₂ increased the cleavage of caspase-3 as early as 3 h after H₂O₂ exposure (Figure 6A). A small cleavage fragment of Mr 30K ROCK1 was detectable using the C-19 antibody after 6 h of H₂O₂ treatment, while the abundance of the full-length Mr 160K protein decreased reciprocally (Figure 6B). In contrast, no such fragment or reduction of the Mr 160K band was observed using the specific anti-ROCK2 antibody (C-20; Figure 6C). MLC phosphorylation culminated after 12 h of H₂O₂ incubation and decreased thereafter (Figure 6D). Caspase-3, ROCK1, ROCK2 and MLC phosphorylation had similar changes in neurons upon H₂O₂ treatment for 12 h (Supplementary Figure S4). Together, there was a sequential activation of caspase3, ROCK1 and MLC during H₂O₂ exposure in neuronal cells.

We further investigated whether the observed association among caspase, ROCK or myosin II during apoptosis could be blocked by related chemical inhibitors. On the basis of cell viability, 1 μM blebbistatin was applied to reduce any possible toxic and nonspecific effects. Pretreatment with 1 μM blebbistatin (myosin II ATPase inhibitor), 10 μM Y27632 (ROCK inhibitor) or 10 μM z-VAD-fmk (caspase inhibitor) increased cell viability (Figure 7A), decreased caspase-3 activity (Figure 7B), inhibited membrane blebbing and increased neurite length (Figure 7C) after H₂O₂ exposure in both PC12 cells and neurons. Consistent with phase-contrast micrographs, TEM results indicated that pretreatment with blebbistatin, Y27632 and z-VAD-fmk blocked membrane blebbing and nucleus condensation induced by H₂O₂ in PC12 cells (Figure 7D). Statistical analysis showed that these inhibitors significantly decreased percentage of blebbing cells and increased neurite length after H₂O₂ treatment (Figures 7E,F). MAP2, a cytoskeletal phosphoprotein, is reported to degrade after ischemia and other injuries. Loss of MAP2 immunoreactivity is a marker of neuronal damage (Shelton et al., 2015). Blebbistatin, Y27632 and z-VAD-fmk rescued the decrease of MAP2 fluorescence and neurite length in neurons under H₂O₂ induction (Figure 8).

Caspase-3 inhibitor, z-DEVD-fmk inhibited H₂O₂-induced membrane blebbing and caspase-3 activity (Supplementary Figure S5), which indicated that caspase-3 activation played a major role in H₂O₂-induced apoptosis. MLC phosphorylation may occur via ROCK or MLCK. To address the role of MLCK in H₂O₂-induced membrane blebbing, cells were incubated with MLCK inhibitor ML-7 prior to H₂O₂ treatment. ML-7 failed to inhibit membrane blebbing and caspase3 activity (Supplementary Figure S5), which indicated that MLCK was probably not involved in H₂O₂-induced neuronal apoptosis.

We next investigated if blebbistatin, Y27632 or z-VAD-fmk attenuated myosin IIA-actin interaction induced by H₂O₂. In the presence of blebbistatin, Y27632 or z-VAD-fmk, the inhibition of membrane blebbing was associated with a reversion of the cell morphology and myosin IIA-actin reorganization toward control cells, exhibiting decreased myosin IIA-actin interaction (Figures 9A–F). The statistical analysis also revealed that blebbistatin, Y27632 or z-VAD-fmk significantly inhibited the co-localization between myosin IIA and actin filaments (Figure 9G). Similar results were also observed in neurons (Figure 10). These findings demonstrated that caspase-3/ROCK1/MLC activation plays a key role in triggering the downstream myosin IIA-actin hyperactivation upon H₂O₂ exposure.

Interestingly, Western blot analysis showed that myosin IIA-actin interaction inhibition by blebbistatin attenuated H₂O₂-induced activation of caspase-3, ROCK1 and MLC, indicating that myosin IIA might reversely influence caspase-3, ROCK1 and MLC activity. Y27632 and z-VAD-fmk also attenuated H₂O₂-induced cleaved caspase-3, cleaved ROCK1 and MLC phosphorylation (Figure 11). These results demonstrated that H₂O₂-induced caspase-3/ROCK1/MLC activation and myosin IIA-actin interaction cascade might form a positive feedback pathway in H₂O₂-induced neuronal apoptosis (Figure 12). The original uncropped images of western blots are shown in Supplementary Figures S6–S9.

The interaction of myosin II with actin filaments in generating forces during the execution phase of apoptosis has been well documented (Ndozangue-Touriguine et al., 2008). However, little is known about the specific functions of myosin II isoforms in neuronal apoptosis. Normally, myosin IIA was dispersed throughout the cytoplasm, in contrast to the pronounced peripheral localization of myosin IIB and F-actin, which indicated that myosin IIA was better suited for large scale contractile processes than myosin IIB (Wylie and Chantler, 2003). Accordingly, the interaction of myosin IIA and actin increased upon H₂O₂ treatment, whereas myosin IIB and actin interaction showed no obvious change in PC12 cells and decreased in neurons as myosin IIB protein degraded. Why was myosin IIA retained but IIB lost after H₂O₂ exposure in neurons? On one hand, our results have demonstrated the requirement of actin filaments for cell contraction and membrane bleb formation during neuronal apoptosis. On the other hand, the interaction of myosin IIB with actin filaments was more striking compared with that of myosin IIA under normal conditions. Therefore, decreased myosin IIB protein level could facilitate the binding between myosin IIA and actin filaments due to lack of competing with myosin IIB in neurons. Although myosin IIB levels were virtually not affected in PC12 cells, myosin IIB is reported to be less active than myosin IIA (Kelley et al., 1996), and bind with actin more slowly than myosin IIA (Kolega, 2003, 2006). Therefore, the differential localization and expression of myosin IIA and IIB, together with the spatial organization of actin filaments are critical to their actions (Beach and Hammer, 2015).

Cellular activity, like growth and motility, is the resultant of a balance of forces, the mechanism involving two opposing processes: outgrowth and retraction. Each of these processes is regulated by the coordinated actions of one or more molecular motors (Chantler and Wylie, 2003; Hyland et al., 2014). It has reported that myosin IIA drives neurite retraction and maintains tensile adhesion (Wylie and Chantler, 2001), while myosin IIB drives outgrowth (Wylie et al., 1998). Knockdown of myosin IIA or IIB resulted in opposite morphological changes. Thus, the functions of myosin IIA and IIB counteracted each other. Our data indicated that loss of myosin IIA would result in a shift in the balance of myosin IIB-based outgrowth, while loss of myosin IIB resulted in the myosin IIA-mediated retraction. As myosin IIA derived large scale cellular contractile processes under oxidative stress-induced apoptosis, myosin IIB knockdown further increased the interaction of myosin IIA with actin and the related contractile forces generation, which caused cells more sensitive to oxidative stress and easier to undergo apoptosis.

It has been reported that myosin IIA depletion remarkably increases cell survival and cloning efficiency of human/mouse embryonic stem cells (h/mESCs; Walker et al., 2010). Myosin IIB, together with actin, constitute the actomyosin cytoskeleton that mediates contractility during TNFalpha-induced shrinkage, detachment and apoptosis in NIH 3T3 fibroblasts (Solinet and Vitale, 2008). Our results demonstrated that myosin IIA knockdown protected cells from cellular contraction, caspase-3 cleavage and apoptosis. We also showed for the first time that PC12 cells with myosin IIB knockdown had a higher proliferative activity than myosin IIA knockdown or control cells. It has been reported that cytokinesis is facilitated by the constriction of a cortical ring made up of myosin II and actin filaments in animal and fungal cells (Cheffings et al., 2016; Zambon et al., 2017). Actomyosin-drived cellular contractility is recognized as an important promoter for increased tumor number, growth and progression (Samuel et al., 2011). And myosin IIA stack occurs in the cleavage furrow of dividing cells and form the contractile ring to divide cells (Fenix et al., 2016). Therefore, myosin IIB depletion might facilitate the myosin IIA-mediated cellular contractility that is essential for cytokinesis, causing increased PC12 cell number. These results indicated that myosin II regulates cell survival in an isoform and cell type specific manner.

Such an antagonistic relationship between these two motors on the morphology of PC12 cells and responses to oxidative stress may be an important mechanism by which the vectorial balance of forces is regulated during cellular growth and apoptosis. Myosin IIA knockout by CRISPR/Cas9 provides evidence of a central role for myosin IIA in cellular contractility-induced apoptosis under oxidative stress. This work also highlights the use of the CRISPR/Cas9 to study neuroscience and cytoskeletal functions.

Activation of the myosin II motor through ATP hydrolysis and binding to actin filaments are required to initiate actomyosin contractility, which further facilitates cellular apoptosis (Sari-Hassoun et al., 2016). Blebbistatin, a small molecular myosin inhibitor, exhibits high affinity and selectivity toward myosin II (Limouze et al., 2004; Allingham et al., 2005). Kinetic analysis shows that in the concentration range 0.5–5 μM, the inhibitor preferentially binds to the myosin-ADP-Pi complex and slows down phosphate release process. Thus, the inhibitor blocks the myosin II heads to interact with actin (Kovács et al., 2004). Several studies indicate the roles of blebbistatin in protection of damaged mammalian cells. Blebbistatin inhibits TNFα/CHX induced apoptotic nuclear breakdown and membrane blebbing in fibroblasts (Croft et al., 2005). Inhibiting myosin II activity by blebbistatin markedly promotes growth cone invasion and accelerates axon growth rate on inhibitory substrates (Burnette et al., 2007; Hur et al., 2011). Although blebbistatin potently inhibits actin-myosin interaction, very little is known regarding its action in the organized contractile system of oxidative stress-induced neuronal apoptosis. Myosin II uses ATP to move along actin filaments, which forms actomyosin system in various cellular functions (Hartman and Spudich, 2012). Cytochalasin D, a blocker of actin polymerization, disruptes actin filaments dynamics (Nair et al., 2008). Cytochalasin D treatment resists serum starvation-induced caspase-3 activation in NIH 3T3 cells by extracellularly activating gelatinase A, which interacts with integrin aVh3, eliciting survival signals mediated through ERK 1/2, p38 and SAPK/JNK pathways (Ailenberg and Silverman, 2003). Consistently, both blebbistatin and cytochalasin D prevented H₂O₂-induced activation of caspase-3, membrane blebbing and neurite retraction in neuronal cells. Our data demonstrate that the function of activated myosin II during neuronal apoptosis is associated with actin filament formation and actomyosin contractility.

Exogenous H₂O₂ influences the function of various proteins, such as kinases, phospholipases, transcription factors, ion channels, G proteins, et al. However, unlike other second messengers, the structure of H₂O₂ is too small and simple to interact with a specific protein target (Rhee et al., 2003). It has been reported that H₂O₂ induces neuronal apoptosis through various signaling pathway, such as nuclear factor-κB, JNK/ERK (Liu et al., 2017), Cdc42/MLK3/MKK7/JNK3 pathway (Wang et al., 2017), P53, mitochondria-related Bax and Bcl-2 (Sohn et al., 2016), Akt/H2A phosphorylation (Park et al., 2016), et al. In out research, time-course study revealed that H₂O₂ activated caspase-3, ROCK1 and MLC in chronological sequence. Thus, H₂O₂ activated caspase-3 through upstream pathways and then activated caspase-3 mediated ROCK1 and MLC activation. However, the upstream signaling of caspase-3 still needs to be further investigated.

It has been previously demonstrated that caspase/ROCK-mediated MLC phosphorylation and the mitochondrial signaling axis regulate cell apoptosis (Liu et al., 2013; Shen et al., 2015b). Our findings extended previous observations by showing that activated caspase-3, ROCK1 and phosphorylated MLC were specifically associated with the myosin IIA-actin cytoskeletal system. Disruption of myosin IIA-actin contraction is the mechanism by which caspase or ROCK inhibitors decreased apoptosis and membrane blebbing of neuronal cells. Interestingly, inhibition of myosin IIA-actin contraction by blebbistatin, myosin IIA knockdown, knockout or cytochalasin D in turn inhibited caspase-3 activation. Blebbistatin also inhibited ROCK1 activation and MLC phosphorylation, which was similar with the effects of Y27632 and z-VAD-fmk. These results indicated that myosin IIA and its interaction with actin filaments could regulate caspase-3/ROCK1/MLC phosphorylation in a positive feedback manner, which has numerous implications. Once activated, this positive feedback loop may be an important mechanism to amplify and sustain actomyosin contractility, signaling cascade activation and ultimate apoptosis. This architecture also explains the observation that disruption of caspase, ROCK, myosin IIA or actin functionally disrupted the entire mechanical system and signaling pathway, resulting in similar phenotypes such as decreased membrane blebbing, apoptosis and neurite retraction.

It has been suggested an existence of a feedback loop between cytoskeletal tension, adhesion maturation and ROCK signaling that contributes to numerous mechanochemical processes (Bhadriraju et al., 2007). Under the treatment of rotenone, PUMA, Bcl-XL and p53 form a positive feedback amplification loop to increase the apoptosis sensitivity (Shi et al., 2014). Additionally, downstream caspase-3 reinforces Bak activation and the release of AIF and endoG through positive loops, which sensitize cancer cells to the treatment with genistein (Guo et al., 2015). The numerous feedback loops between cytoskeletal, apoptotic and mechanical functions of cells and the related signaling pathway highlight the complexity of pathological and physiological mechanisms in cells. Other studies have also reported that myosin IIA knockdown or blebbistatin pretreatment remarkably increase cell survival and cloning efficiency of embryonic stem cells (Walker et al., 2010). Moreover, inhibition of actin polymerization by cytochalasin D inhibits thrombin-induced activation of caspase-3 and caspase-9 in human platelets (Ben Amor et al., 2006). However, how the cellular contractile forces regulate caspase-3 activity has not been fully elucidated. Investigating these mechanisms will be important to us to understand how cells coordinate the mechanical and biochemical events.

As our data support a regulatory loop between myosin IIA and caspase-3/ROCK1/MLC pathway, this raises the question about how the cell specifically regulates myosin IIB activity. It has been shown that myosin IIB is activated upstream by PKC, PAK1 or Rac, which have been shown to regulate the phosphorylation and cellular localization of myosin IIB and induce neurite outgrowth (Even-Faitelson et al., 2005; Solinet and Vitale, 2008). Regulation of myosin IIA and IIB by distinct signaling pathways might be an explanation for myosin II isoforms performing different functions at different places in cells. Clearly, additional work is required to fully understand the specific roles of myosin IIA and IIB, and how the two isoforms are distinctly regulated. In addition, myosin IIA and IIB share 77% identity at the amino acid level, they are believed to have considerable functional overlap in certain circumstances (Bao et al., 2007). Despite our observations that myosin IIA and IIB perform unique cellular functions, it still needs further research on the complementary actions and how the two isoforms influence each other in regulating neuronal function and apoptosis.

In conclusion, we identified the distinct functions of the two highly conserved myosin II motors in oxidative stress-induced neuronal apoptosis. Myosin IIA, rather than myosin IIB, constitutes a continuous mechanical link with actin filaments, and is required to develop contractile forces and membrane blebbing during H₂O₂-induced neuronal apoptosis. Myosin IIA is also involved in a positive feedback loop that links caspase-3/ROCK1/MLC signaling axis. Understanding the cytoskeletal basis of neuronal cells would be helpful to clarify the cellular mechanisms underlying the biochemical, mechanistic and morphological changes induced by oxidative stress, and to develop therapeutics for neuronal diseases.