Abstract
Nonmuscle myosin II (NM II) powers myriad developmental and cellular processes, including embryogenesis, cell migration, and cytokinesis [1]. To exert its functions, monomers of NM II assemble into bipolar filaments that produce a contractile force on the actin cytoskeleton. Mammalian cells express up to three isoforms of NM II (NM IIA, IIB, and IIC), each of which possesses distinct biophysical properties and supports unique as well as redundant cellular functions [2-8]. Despite previous efforts [9-13], it remains unclear whether NM II isoforms assemble in living cells to produce mixed (heterotypic) bipolar filaments or whether filaments consist entirely of a single isoform (homotypic). We addressed this question using fluorescently tagged versions of NM IIA, IIB, and IIC, isoform-specific immunostaining of the endogenous proteins, and two-color total internal reflection fluorescence structured-illumination microscopy, or TIRF-SIM, to visualize individual myosin II bipolar filaments inside cells. We show that NM II isoforms coassemble into heterotypic filaments in a variety of settings, including various types of stress fibers, individual filaments throughout the cell, and the contractile ring. We also show that the differential distribution of NM IIA and NM IIB typically seen in confocal micrographs of well-polarized cells is reflected in the composition of individual bipolar filaments. Interestingly, this differential distribution is less pronounced in freshly spread cells, arguing for the existence of a sorting mechanism acting over time. Together, our work argues that individual NM II isoforms are potentially performing both isoform-specific and isoform-redundant functions while coassembled with other NM II isoforms.
🔬 Techniques
🔭 Microscopes
💻 Software
✨ Fluorophores
🧪 Sample Preparation
🔬 Cell Lines
🏭 Microscope Brands
📷 Detectors
🔎 Objectives
💻 Software Details
💾 Data Repositories
🏛️ Research Organizations (ROR)
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📋 Methods
Microscopy
Confocal images were acquired using a Zeiss LSM 780 laser scanning confocal microscope equipped with a Zeiss 63x/1.4 NA oil objective (Zeiss, Thornwood, NY). TIRF-SIM imaging was performed using a Zeiss Axio Observer.Z1 inverted microscope outfitted with an apparatus enabling structured illumination [ 30 ]. The key component in this structured-illumination apparatus is a spatial light modulator (SLM) that functions as a phase grating with sub-millisecond pattern switching speed [ 14 , 30 ]. Two-color TIRF-SIM imaging requires that the excitation beams of the two different wavelengths be confined within the TIRF annulus at the objective’s back focal plane. The SLM is capable of this because it can change the diffraction angles for different excitation wavelengths by loading patterns of different periods. An Olympus 100x 1.49 NA (Olympus, Tokyo, Japan) objective was used instead of the Zeiss 1.45 NA objective because the slightly larger NA of the Olympus objective gives higher tolerance for placing the excitation beams inside the TIRF annulus. The high-NA TIRF objective also provides an increase in resolution over standard 3D-SIM. Reconstructed TIRF-SIM images were generated from the raw data as previously described [ 14 ]. For two-color TIRF-SIM, each channel was captured on a separate sCMOS camera and image registration was performed on reconstructed images using the alignment function in Priism (UCSF, San Francisco, CA). The 3-D SIM imaging was performed on the DeltaVision OMX 3-D SIM Imaging System (Applied Precision, Issaqua, WA) equipped with an Olympus 60x 1.42 NA objective. Raw images were reconstructed using Softworx software (Applied Precision, Issaqua, WA). Linear adjustments were made to images using ImageJ. Quantitation of Relative NM IIA/NM IIB in Individual Filaments For Figure 4F , TIRF-SIM images of well-polarized MDA-MB-231 cells that had been immuno-stained for both NM IIA and NM IIB were used for analysis. For Figure S4A , TIRF-SIM images of U2OS cells expressing EGFP-NM IIB and NM IIA-mApple that had been allowed to polarize (“Polarized”), or were trypsin-treated, re-plated on coverslips, and imaged between 20 and 40 minutes post-plating (“Spreading”), were used for analysis. Integrated pixel intensities in both the red and green channels were measured inside a 230 nm circle drawn in ImageJ around discrete red and/or green puncta for Figure 4F , and inside a 450 nm circle around discrete NM IIA-mApple puncta for Figure S4A . The integrated pixel intensities for individual filaments were then divided by the integrated pixel intensity in each channel for the entire image to provide the percentage of the total intensity in each channel that is present within each individual filament. The percentage of the total for NM IIA was then divided by the percentage of the total for NM IIB to give the relative NM IIA to NM IIB ratio in each individual filament. The distance of each punctum from the leading edge was also determined. For “Spreading” cells, the leading edge was considered the nearest cell edge. The ratios were then grouped into 2 μm bins based on distance from the leading edge. Because the final numbers are ratios, calculating mean values would skew the data in favor of the numerator. Therefore, we plotted the data on a log scale as geometric means with 95% confidence intervals. Image analysis was performed with ImageJ and the data was plotted using GraphPad Prism (GraphPad Software, Sand Diego, CA). We note that while some of the filaments in this quantitative analysis are probably homotypic, the unequivocal scoring of filaments as homotypic is problematic because of the presence of endogenous proteins, the potential for misfolded fluorophores, and the technical limitations in trying to identify one or a few fluorophores. It is also important to note that the ratios reported in Figures 4F and S4A are relative values and do not necessarily represent that actual ratio of NM IIA to NM IIB in individual filaments due to differences in expression and assembly levels.
Show full methods section
Microscopy
Confocal images were acquired using a Zeiss LSM 780 laser scanning confocal microscope equipped with a Zeiss 63x/1.4 NA oil objective (Zeiss, Thornwood, NY). TIRF-SIM imaging was performed using a Zeiss Axio Observer.Z1 inverted microscope outfitted with an apparatus enabling structured illumination [ 30 ]. The key component in this structured-illumination apparatus is a spatial light modulator (SLM) that functions as a phase grating with sub-millisecond pattern switching speed [ 14 , 30 ]. Two-color TIRF-SIM imaging requires that the excitation beams of the two different wavelengths be confined within the TIRF annulus at the objective’s back focal plane. The SLM is capable of this because it can change the diffraction angles for different excitation wavelengths by loading patterns of different periods. An Olympus 100x 1.49 NA (Olympus, Tokyo, Japan) objective was used instead of the Zeiss 1.45 NA objective because the slightly larger NA of the Olympus objective gives higher tolerance for placing the excitation beams inside the TIRF annulus. The high-NA TIRF objective also provides an increase in resolution over standard 3D-SIM. Reconstructed TIRF-SIM images were generated from the raw data as previously described [ 14 ]. For two-color TIRF-SIM, each channel was captured on a separate sCMOS camera and image registration was performed on reconstructed images using the alignment function in Priism (UCSF, San Francisco, CA). The 3-D SIM imaging was performed on the DeltaVision OMX 3-D SIM Imaging System (Applied Precision, Issaqua, WA) equipped with an Olympus 60x 1.42 NA objective. Raw images were reconstructed using Softworx software (Applied Precision, Issaqua, WA). Linear adjustments were made to images using ImageJ. Quantitation of Relative NM IIA/NM IIB in Individual Filaments For Figure 4F , TIRF-SIM images of well-polarized MDA-MB-231 cells that had been immuno-stained for both NM IIA and NM IIB were used for analysis. For Figure S4A , TIRF-SIM images of U2OS cells expressing EGFP-NM IIB and NM IIA-mApple that had been allowed to polarize (“Polarized”), or were trypsin-treated, re-plated on coverslips, and imaged between 20 and 40 minutes post-plating (“Spreading”), were used for analysis. Integrated pixel intensities in both the red and green channels were measured inside a 230 nm circle drawn in ImageJ around discrete red and/or green puncta for Figure 4F , and inside a 450 nm circle around discrete NM IIA-mApple puncta for Figure S4A . The integrated pixel intensities for individual filaments were then divided by the integrated pixel intensity in each channel for the entire image to provide the percentage of the total intensity in each channel that is present within each individual filament. The percentage of the total for NM IIA was then divided by the percentage of the total for NM IIB to give the relative NM IIA to NM IIB ratio in each individual filament. The distance of each punctum from the leading edge was also determined. For “Spreading” cells, the leading edge was considered the nearest cell edge. The ratios were then grouped into 2 μm bins based on distance from the leading edge. Because the final numbers are ratios, calculating mean values would skew the data in favor of the numerator. Therefore, we plotted the data on a log scale as geometric means with 95% confidence intervals. Image analysis was performed with ImageJ and the data was plotted using GraphPad Prism (GraphPad Software, Sand Diego, CA). We note that while some of the filaments in this quantitative analysis are probably homotypic, the unequivocal scoring of filaments as homotypic is problematic because of the presence of endogenous proteins, the potential for misfolded fluorophores, and the technical limitations in trying to identify one or a few fluorophores. It is also important to note that the ratios reported in Figures 4F and S4A are relative values and do not necessarily represent that actual ratio of NM IIA to NM IIB in individual filaments due to differences in expression and assembly levels.
Supplementary Material 01 02 03 04
📊 Figures
Figure 1
TIRF-SIM of cells expressing NM IIA with N- and C-terminal fluorescent tags allows identification of individual NM IIA bipolar filaments. (A) Cartoon of NM II alone, with an N-terminal EGFP reporter, ...
Figure 2
NM IIA and NM IIB form heterotypic filaments in live cells. U2OS cells co-expressing NM IIA-mApple and EGFP-NM IIB were imaged using either confocal microscopy (A) or TIRF-SIM (B-E). The white arrow i...
Figure 3
NM IIA and NM IIB form heterotypic filaments in the contractile ring of LLC-Pk1 cells. LLC-Pk1cells expressing NM IIA-mApple and EGFP-NM IIB were imaged during anaphase using TIRF-SIM. The two images ...
Figure 4
Endogenous NM IIA and NM IIB form heterotypic filaments in live cells. (A) Cartoon depicting the co-assembly of EGFP-NM IIA with endogenous NM IIB. When NM IIB is localized using an antibody that reco...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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