Distinct roles of nonmuscle myosin II isoforms for establishing tension and elasticity during cell morphodynamics

Figure 1.. Phenotypes of NM IIA, NM IIB, and NM IIC-KO cell lines on homogenously coated substrates are very distinct. (A) U2OS WT cells show a polarized phenotype with prominent dorsal stress fibers (dSF) (1), transverse arcs (tA) (2), and ventral stress fibers (vSF) (3). Mature focal adhesions (FA) are visualized by elongated paxillin clusters that localize at the distal ends of dSF or both ends of vSF. (B) The NM IIA-KO leads to drastic morphological changes and the loss of most SFs and mature FAs. The overall actin structure resembles a dense meshwork of fine actin filaments (1). At the trailing edge, long cell extensions remain (2) and the only bundled actin fibers resemble vSF (3). (C) NM IIB-KO cells reveal slight changes in SF organization and FA structure. dSF (1), tA (2) and vSF (3) are present but their distinct localization pattern is disturbed. (D) The phenotype of NM IIC-KO cells is comparable to the WT. dSF (1), tA (2) and vSF (3) localize in a distinct pattern along the cell axis of polarized cells. (E) The mean FA area per cell is reduced for NM IIA-KO and NM IIB-KO cells, whereas FA density is only reduced in NM IIA-KO cells (F). Scale bars represent 10 µm for overviews…

Figure 1—figure supplement 1.. Knockout of NMHC IIA, NMHC IIB and NMHC IIC via CRISPR/Cas9. NM II-KO cell lines were generated via CRISPR/Cas9 and the loss of protein expression was confirmed via western blot (A) and immunofluorescent labeling (B). Deletions in the coding sequences of the first coding exons (exon 2) of NMHC IIA (C) and NMHC IIB (D) lead to frame shifts and premature stop codons in corresponding protein sequences (marked by asteriscs). Scale bar represents 20 µm.

Figure 1—figure supplement 2.. Quantification of FA number and cell spreading area. Quantifications showing the number of focal adhesions (A), their size frequency (B), and the cell area (C) of WT and the respective NM II-KO cell lines. (A) NM IIA-KO cells show a reduced number of mature focal adhesions (larger than 0.25 µm²), while no difference was observed for NM IIB-KO and NM IIC-KO cells. (B) The frequency distribution of FAs is shifted to smaller sizes in NM IIA-KO and NM IIB-KO cells as compared to WT cells. No difference was observed for NM IIC-KO cells. (C) The cell spreading area is not affected by the loss of NM IIA, NM IIB, or NM IIC.

Figure 1—figure supplement 3.. Paralog localization in NM II-KO cells. Immunfluorescent labeling was used to analyze the localization and intensity of the NM II paralogs in U2OS WT or the respective NM II-KO cells. (A) In polarized WT cells, all isoforms co-localize with actin fibers throughout the cell body (NM IIA and NM IIC) or in the cell center (NM IIB). (B) In NM IIA-KO cells, the remaining paralogs show an altered localization pattern: NM IIB and NM IIC minifilaments are clustered along the remaining SFs in the cell center. (C) In NM IIB-KO cells, NM IIA and NM IIC minifilaments still localize throughout the cell body, comparable to WT cells. (D) The loss of NM IIC did not change the localization of the remaining paralogs. NM IIA is homogenously distrubuted and NM IIB accumulates in the cell center. (E) The mean fluorescence intensities of NM IIA-C was measured along segmented actin fibers and the fold change to the WT was calculated. All scenarios resulted in only mild intensity increases and no significant differences were observed. Scale bars represent 10 µm in (A)-(D).

Figure 1—figure supplement 4.. Intensity quantification of NM II-paralogs. Segmented actin fibers were used to determine the fluorescence intensities of NM IIA-C in WT and the respective NM II-KO cells. Shown are the absolute intensity values, used to calculate the fold changes in Figure 1—figure supplement 3E.

Figure 2.. Overexpression of NM II B cannot compensate for the loss of NM II A. (A) GFP intensities were measured along segmented actin fibers to compare the ratios of NM IIA and NM IIB in WT and NM IIB overexpressing NM IIA-KO cells. When expressed under the endogenous promoter, the mean GFP intensity of NM IIB is 4.5 times lower compared to NM IIA. To increase the total amount of NM IIB without the interference of NM IIA, NM IIA-KO cells were transiently transfected with exogenous GFP-NM IIB under the CMV promoter. Even in NM IIA-KO cells with high NM IIB expression, NM IIB filament distribution is less homogenous compared to NM IIA in WT cells. (B) Quantitative comparison of endogenous NM IIA and NM IIB in WT cells, and exogenous NM IIB in NM IIA-KO cells. (C) PRLC signal intensity was used as a marker for active NM II filaments. The quantitative comparison of WT, NM IIA-KO, and NM IIB overexpressing NM IIA-KO cells shows a substantial reduction of pRLC intensity in NM IIA-KO cells, which is not restored in NM IIB overexpressing NM IIA-KO cells. (D) NM IIB overexpression does not phenocopy the WT situation. Immunfluorescent labeling of the actin cytoskeleton (yellow) and the FA marker…

Figure 2—figure supplement 1.. Generation of GFP-NM IIA and GFP-NM IIB fusion proteins. GFP-NM IIA or GFP-NM IIB reporter cell lines were generated according to the guidelines in Koch et al., 2018. (A) Western blots depicting the insertion of the GFP tag in the knock-in cell lines and heterozygous expression of GFP with NM IIA or NM IIB. (B) Illustration depicting localization of the GFP tag and antibody binding sites in bipolar minifilaments. The GFP sequence was introduced at the 5’-End of the coding sequences, resulting in N-terminally tagged NM IIA or NM IIB fusion proteins. The antibodies for NM IIA or NM IIB bind at the C-terminal region, resulting in the depicted bipolar staining pattern. (C) Exemplary images of bipolar minifilaments in GFP-NM IIA or GFP-NM IIB cells. Scale bar in (C) represents 1 µm.

Figure 3.. Quantitative shape analysis on cross-shaped micropatterns reveals distinct phenotypes for NM IIA-KO and NM IIB-KO cells. (A) U2OS WT cells show prominent invaginated actin arcs along the cell contour, with an invagination radius R and a spanning distance d (see inset). Quantitative image analysis reveals a positive R(d)-correlation (correlation coefficient r given at bottom right). Solid lines denote the bootstrapped mean fit of the dynamic tension-elasticity model (dTEM), black dashed lines denote the geometrically possible minimal radius. (B) U2OS NM IIA-KO cells form invaginated shapes on the cross-patterns despite their strongly perturbed shapes on homogenous substrates. The spanning distance of the arcs is shorter, but the positive correlation between R and d remains. (C) Actin arcs of U2OS NM IIB-KO cells are less invaginated compared to WT cells and their measured R(d)-correlation is very weak. (D) The phenotype of NM IIC-KO cells is comparable to WT and the R(d)-correlation is not affected. Scale bar represents 10 µm for (A) – (D).

Figure 3—figure supplement 1.. NM II-KO phenotypes in collagen gels show distinct contractile behaviors with prominent actin arcs along the cell contour. (A) Cell seeded collagen gels (CSCG’s) were cultivated in suspension and the area was measured at the beginning and the end of the experiment. Red circles indicate the initial area, the blue area shows the CSCG after a total incubation time of 20 hr. WT cells contract the CSCG to 10% of the initial area. NM IIA-KO cells barely contract the gel and 91.5% of the initial size is still present after the incubation time. For NM IIB-KO cells, the contraction was also lower than in the WT case with 20% of the initial area left. No difference was observed for WT and NM IIC-KO cells. (B–E) 3D images of WT and NM II-KO cells, encased in collagen gels that were attached to the coverslip. All cell lines flattened in the collagen meshwork and showed prominent actin arcs along parts of the cell contour. For the sake of clarity, only some of the actin arcs were marked by the fitted circles in red. While WT, NM IIB-KO, and NM IIC-KO show comparable phenotypic features, the phenotype of NM IIA-KO cells is characterized by many lamellipodial protrusions and extensions of…

Figure 3—figure supplement 2.. Circularity of invaginated actin arcs. (A) The root mean squared deviation of the distance from points on the arc to the fitted center relative to the fitted radius is a measure for the circularity of the arc, as demonstrated here for parabolic curves. (B) Cell contours were fitted with the ImageJ/Fiji-plugin JFilament (Smith et al., 2010) and then fit to circular arcs with a custom-written software using the hyper least squares algorithm (Kanatani and Rangarajan, 2011). (C) For WT and all three KOs, circularity errors are relatively small, with typical values around 2% and few outliers above. The largest deviations are observed for NM IIA-KO.

Figure 3—figure supplement 3.. Localization of NM IIA and NM IIB minifilaments along peripheral actin arcs. (A) The amount of actin fibers is quantified by the coherency of the structure tensor measured for the cell interior with a custom-made python script. Only NM IIA-KO cells show a significantly weaker coherency, demonstrating that these cells don’t develop internal SFs. (B) In WT cells, NM IIA, and NM IIB minifilaments localize along peripheral actin arcs and internal SFs. (C) In NM IIA-KO cells, the pRLC signal is almost absent and NM IIB minifilaments appear mainly along the actin arcs. (D) In NM IIB-KO cells, NM IIA minifilaments are homogenously distributed along the actin arcs and the internal SFs. The pRLC staining is comparable to the WT cells. Scale bars represent 10 µm for (A)-(C).

Figure 3—figure supplement 4.. NM IIA or B overexpression on micropatterned substrates show the importance of the different motor qualities. (A) GFP-tagged NM IIB was overexpressed in NM IIA-KO cells and the cells were seeded on micropatterned substrates. Higher quantities of NM IIB did not restore the phenotype in the absence of NM IIA. The cells still spread along the cross bars rather than spanning large passivated areas and the R(d)-correlation is positive, comparable to WT and untransfected NM IIA-KO cells. (B) Overexpressing GFP-tagged NM IIA in NM IIB-KO cells did not restore the R(d)-correlation, the values are comparably low to untransfected NM IIB-KO cells. Scale bars represent 10 µm for (A and B).