Effect of myosin IIC antisense oligonucleotides on the phenotype of Neuro-2A cells. DIC images of Neuro-2A cells treated with oligonucleotides for 96 h that targeted myosin IIC. Whereas control cells (a) exhibited typical profiles of neurite outgrowth, cells treated with antisense oligonucleotides showed a substantial increase in cell body diameter with profuse vacuolization (b). This aberrant phenotype was reversed after a further 72-h recovery period in the absence of oligonucleotide (c). Bar, 60 μm.

Localization of myosins IIA, IIB, and IIC in Neuro-2A cells as seen in projection images of confocal stacks. The normal distribution of myosin IIC in Neuro-2A cells was determined relative to F-actin and myosin IIA (A) or myosin IIB (B) through immunofluorescence by using confocal laser scanning microscopy. In A and B, myosin IIC immunofluorescence is green (FITC-labeled secondary) (b) and rhodamine-phalloidin F-actin, red (c). Both myosin IIA (A) and myosin IIB (B) are violet (Alexa Fluor 633-labeled secondary) (a). Merged images are seen in d. Scale bars are shown in d.

Effect of oligonucleotides targeting myosins IIA, IIB, and IIC on neurite outgrowth when supplied to Neuro-2A cells at the time of plating. Cells were plated for 96 h in the presence of isoform-specific oligonucleotides before replenishment with oligonucleotide-free media for a further 72 h. Data shown include sense (S), antisense (AS), and control (CTL) (none) time courses for myosin IIA, myosin IIB, and myosin IIC oligonucleotides. Scrambled data, similar to those obtained with sense oligonucleotides, were omitted for clarity. Symbols used are identified within the key (sidebar). Error bars are SEs of the mean from four experiments (>100 cells/time point). Arrow indicates removal of oligonucleotides at 96 h.

Effect of oligonucleotides targeting myosins IIA, IIB and IIC on the mean cell body area. Cells were plated for 96 h in the presence of isoform-specific oligonucleotides before replenishment with oligonucleotide-free media for a further 72 h. Data shown are measurements of the mean cell body area of Neuro-2A cells subsequent to sense (S), antisense (AS), and control (CTL) (none) oligonucleotide regimens targeting myosin IIA, myosin IIB or myosin IIC. Scrambled data, similar to those obtained with sense oligonucleotides, were omitted for clarity. Symbols used are identified within the key (sidebar). Error bars are SEs of the mean from four experiments (>100 cells/time point). Arrow indicates removal of oligonucleotides at 96 h.

Effect of oligonucleotides targeting myosins IIA, IIB, and IIC on neurite outgrowth when supplied to Neuro-2A cells 96 h after initiation of outgrowth. Cells were plated for 96 h in the absence of isoform-specific oligonucleotides before their application for a further 72 h. Data shown include neurite length measurements from sense (S), antisense (AS), and control (CTL) (none) oligonucleotide regimens targeting myosin IIA, myosin IIB, or myosin IIC. Scrambled data, similar to those obtained with sense oligonucleotides, were omitted for clarity. Symbols used are identified within the key (sidebar). Error bars are SEs of the mean from four experiments (>100 cells/time point). Arrow indicates addition of oligonucleotides at 96 h.

Visualization of the effect of antisense oligonucleotides targeting myosins IIA, IIB, and IIC subsequent to LPA-induced neurite retraction. Neuro-2A cells were treated with a pulse of 1 μM LPA subsequent to 48-h incubation with antisense oligonucleotides targeting myosin IIA, myosin IIB, or myosin IIC sequence. DIC images were obtained at the time of LPA application (LHS) or 30 min later (RHS). Arrows indicate examples of cells with processes that do not seem to have changed in length during the 30-min period. Double arrowheads indicate examples of cells with processes that have undergone a considerable decrease in length during the 30-min period. Examples of each extreme exist side by side following when cells are preincubated with antisense targeting myosin IIC. Bar, 50 μm.

Cartoon illustrating the separate roles of the three conventional myosin isoforms in neuronal dynamics, and the consequences of isoform-specific knockdown. In all diagrams, green arrows indicate process outgrowth, blue arrows indicate process retraction, and red arrows illustrate adhesion at cellular “feet.” Bold, solid arrows indicate normal action; thin, dotted arrows indicate diminished action through the loss of either myosin IIC (ii), myosin IIA (iii), or myosin IIB (iv). i, unhindered growth. A neuron with associated dendrites and a single main process terminating in a branch-point with two growth cones is drawn as seen from above (top cartoon) and in profile (bottom cartoon). The actions of myosins IIA, IIB, and IIC are indicated. ii, antisense targeting myosin IIC. The effect of myosin IIC knockdown is illustrated here. Continuous cell profile shows the consequence of myosin IIC knockdown; myosins IIA and IIB remain active and their roles are indicated. Normal cell profile, as seen in i, is illustrated in dotted outline. iii, antisense targeting myosin IIA. The effect of myosin IIA knockdown is illustrated here. Continuous cell profile shows the consequence of myosin IIA knockdown; myosins IIB and IIC remain active and their roles are indicated. Normal cell profile, as seen in i, is illustrated in dotted outline. iv, antisense targeting myosin IIB. The effect of myosin IIB knockdown is illustrated here. Continuous cell profile shows the consequence of myosin IIB knockdown; myosins IIA and IIC remain active and their roles are indicated. Normal cell profile, as seen in i, is illustrated in dotted outline.

Effect of oligonucleotides targeting myosins IIA, IIB, and IIC on LPA-induced neurite retraction. Neuro-2A cells were treated with a pulse of 1 μM LPA either alone (untreated) or preceded by 48-h incubation with either sense or antisense oligonucleotides targeting myosin IIA, myosin IIB, or myosin IIC sequence. In a fourth treatment set, 25 μM Y27632 was added 30 min before addition of LPA. Over a 30-min period, subsequent to addition of LPA, DIC images were taken of the same set of cells at various intervals (1, 2, 3, 4, 5, 10, 15, 20, and 30 min), allowing quantification of neurite lengths for up to 50 cells from any one treatment.

Effect of oligonucleotides targeting myosins IIA, IIB, and IIC on cell density. Cells were plated for 96 h in the presence of isoform-specific oligonucleotides before replenishment with oligonucleotide-free media for a further 72 h. Plated adherent cells were individually counted using Kontron software at 24-h intervals over the 168-h period. Data shown are determinations of the adherent Neuro-2A cell density after oligonucleotide treatments (sense or antisense regimens that target myosin IIA, myosin IIB, and myosin IIC). Cell densities for each cognate group are expressed as a percentage of the combined density average for all untreated controls (no oligonucleotides). Scrambled data, similar to those obtained with sense oligonucleotides, were omitted for clarity. Cells were counted individually from 6 to 12 fields (20x objective) per well at each time point. Symbols used are identified within the key (sidebar). Error bars are SEs of the mean from four experiments (400–800 cells/time point, except time zero where there were 200 cells/time point). Arrow indicates removal of oligonucleotides at 96 h.

Effect of oligonucleotides targeting myosins IIA, IIB, and IIC on the number of neuritic processes emerging from Neuro-2A cell bodies. Digitized DIC image data sets were evaluated to determine the number of processes arising from each cell body throughout the course of myosin IIA, IIB, and IIC oligonucleotide regimens. The numbers (1 through 4 or more), are tabulated as histograms for each myosin isoform: myosin IIA (A), myosin IIB (B), and myosin IIC (C). Four sets of time point are used: immediately upon plating (0); 48 and 96 h after oligonucleotide administration; 72 h after oligonucleotide removal (168 h in total). For each time point, data are shown for antisense (A), sense (S), or scrambled (Sc) treatments as well as for cells cultured in the absence of oligonucleotide (C). The color coding used is identified within the key (sidebar). Data set comprises 400–800 cells per treatment, except at time zero in which there were 200 cells per treatment. Taken from four experiments in all.

(A) Immunostaining of Neuro-2A cells during treatment and recovery from oligonucleotides targeting myosin IIC. Cells were observed by confocal laser scanning fluorescence microscopy immediately after addition of (at 0 h), incubation with (after 96 h) or 72 h of recovery from (168 h), isoform-specific oligonucleotides targeting myosin IIC sequence. Cells were triple stained using antibodies that recognize myosin IIC (FITC-labeled secondary; green) and myosin IIA (Alexa Fluor 633-labeled secondary; violet) as well as rhodamine-phalloidin to localize F-actin (red). Results from sense and antisense treatments are shown. Untreated cells and cells treated with scrambled oligonucleotides gave results similar to those seen with sense oligonucleotides (data not shown). Merged images contain information from all three channels (myosin IIA, myosin IIC, and actin). Each frame represents a field ∼153.5 μm². (B–E). Localization of myosins IIA, IIB, and IIC within Neuro-2A neuroblastoma cells after treatment with oligonucleotides targeting myosin IIC. The distributions of myosin IIA (B and C) or myosin IIB (D and E) relative to F-actin and myosin IIC were observed in Neuro-2A cells subsequent to 96-h exposure to either antisense (B and D) or sense (C and E) oligonucleotides targeting myosin IIC. All images obtained using confocal laser scanning microscopy and are slices taken within 2 μm of the substratum; this ensures inclusion of processes and actin-containing structures thought to be associated with adhesion. Labeling strategies are described in Materials and Methods. In B–D, myosin IIC is localized using a FITC-labeled secondary (green) (b). In all images, rhodamine-phalloidin F-actin is red (c). In B and C, myosin IIA is localized using an Alexa Fluor 633-labeled secondary (violet) (a). In D, myosin IIB is violet (Alexa Fluor 633-labeled secondary) (a). In E, labels are reversed so myosin IIC is violet (Alexa Fluor 633-labeled secondary) (a), whereas myosin IIB is green (FITC-labeled secondary) (b). Remaining frames shown are for merged (d) and phase (e) images. Scale bars are shown in e.

(A) RT-PCR demonstration that the isoform-specific antisense oligonucleotides targeting myosins IIA, IIB, and IIC attenuate their targets specifically, and do not affect expression of other myosin transcripts. RNA samples were harvested from ∼3 × 10⁵ Neuro-2A cells, which had been exposed to oligonucleotides targeting either myosin IIA (a), IIB (b), or IIC (c) for 96 h after plating and then used to synthesize cDNA. In each case, cDNA was amplified using primers specific for myosin IIA, IIB, or IIC. R, scrambled oligonucleotides; C, no oligonucleotides; S, sense oligonucleotides; A, antisense oligonucleotides. Lanes 1–4, samples from myosin IIA, IIB, or IIC (R, C, S, and A) that were amplified using primers for mouse β-actin and G3PDH, to demonstrate consistency of loading. Markers are seen at the extreme ends of each gel. Negative controls are seen in lanes 5 and 6. Lane 5, total RNA as template (i.e., no reverse transcriptase [Moloney murine leukemia virus, MMLV] added to PCR in the presence of mouse G3PDH primers), which also tests for genomic DNA contamination; lane 6, no functional amplification in the presence of cDNA (i.e., no Taq polymerase added to PCR in the presence of mouse G3PDH primers), to test for general levels of contamination). Lane 7 indicates amplification of human placental RNA for G3PDH as positive control (supplied with kit) producing a 983-bp product. (B) Immunoblot demonstrating that attenuation of myosin IIA, IIB, and IIC expression arising from antisense knockdown. Homogenates from Neuro-2A cells, plated for 96 h in the presence of isoform-specific oligonucleotides, were run on 7.5% SDS-acrylamide gels before electrophoretic blotting on to PVDF membranes. Sister lanes from blots of sense- and antisense-treated materials were probed with isoform-specific antibodies recognizing myosins IIA, IIB, and IIC. The corresponding myosin heavy chains (MHCs) are indicated in the figure by short bars. Note that the upper band in the myosin IIC immunoblot is unrelated to myosin (Buxton et al., 2004). Isoform-specific antisense oligonucleotides used here attenuate expression of all targeted peptides. See text for details.

Effect of oligonucleotides targeting myosins IIA, IIB, and IIC on the localization of paxillin phosphorylated on Tyr118. The distribution of paxillin phosphorylated on Tyr118 was observed relative to F-actin and myosin IIA (A and B), myosin IIB (C and D), or myosin IIC (E and F) in Neuro-2A cells subsequent to 96-h exposure to either sense (A, C, and E) or antisense (B, D, and F) oligonucleotides targeting myosin IIA (A and B), myosin IIB (C and D), or myosin IIC (E and F). All images were obtained by confocal laser scanning microscopy and are from confocal slices taken within 2 μm of the substratum; this ensures inclusion of all structures associated with adhesion. Labeling strategies are described in Materials and Methods. In all images, myosins are violet (Alexa Fluor 633-labeled secondary) (a), paxillin phosphorylated on Tyr118 is green (FITC-labeled secondary) (b), and rhodamine-phalloidin F-actin is red (c) The merged image and bars are shown in d. Companion 40× Fluar N.A. 1.3 images to the 63× confocal images seen in A–F were used to quantify changes in fluorescence signal intensities. The resultant histograms of summed total intensities (see Materials and Methods for details), expressed as intensity per pixel, are seen in G. An ∼90% knockdown of myosin II immunofluorescence after 96-h exposure to the corresponding antisense oligonucleotide led also to attenuation of paxillin-phospho-Tyr118 immunofluorescence by 57% (myosin IIA antisense) and 68% (myosin IIC antisense), but this did not occur after myosin IIB antisense treatment. Although the error bars shown, generated automatically by our statistical software, are indicative, the intensity data were found not to be normally distributed; consequently, a Wilcoxon signed rank test was used to assess significance, as follows. Pairings displaying significant difference: myosin IIA (sense vs. myosin IIA antisense) W(z) = 2.86, n = 30, p = 0.0042 (2-tailed); myosin IIB (sense vs. myosin IIB antisense) W(z) = 2.37, n = 30, p = 0.0178 (2-tailed); myosin IIC (sense vs. myosin IIC antisense) W(z) = 3.77, n = 30, p = 0.002 (2-tailed); paxillin-phospho-Tyr118 (sense vs. myosin IIA antisense) W(z) = 3.25, n = 30, p = 0.0012 (2-tailed); and paxillin-phospho-Tyr118 (sense vs. myosin IIC antisense) W(z) = 2.93, n = 30, p = 0.0034 (2-tailed). Pairings displaying no significant difference: paxillin-phospho-Tyr118 (sense vs. myosin IIB antisense) W(z) = 0.83, n = 30, p = 0.41 (2-tailed); F-actin (sense vs. myosin IIA antisense) W(z) = 1.14, n = 30, p = 0.254 (2-tailed); F-actin (sense vs. myosin IIB antisense) W(z) = 0.68, n = 30, p = 0.497 (2-tailed); and F-actin (sense vs. myosin IIC antisense) W(z) = 0.03, n = 30, p = 0.976 (2-tailed).