Generation of stress fibers through myosin-driven reorganization of the actin cortex
- HiLIFE Institute of Biotechnology, University of Helsinki, Finland
- Section of Pathology, Department of Veterinary Biosciences, Faculty of Veterinary Medicine, University of Helsinki, Finland
Figures
Figure 1 with 2 supplements
Stress fiber architecture of migrating cells.
3D-structured illumination microscopy (SIM) maximum intensity projections (MIPs) of the actomyosin networks in cells migrating on fibronectin. (A) Human osteosarcoma (U2OS) and mouse embryonic …
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Figure 1—source data 1
Combined data of RMS traction forces.
- https://cdn.elifesciences.org/articles/60710/elife-60710-fig1-data1-v3.xlsx
Figure 1—figure supplement 1
Cortical stress fibers display periodic non-muscle myosin II (NMII) — α-actinin pattern and assemble on different ECMs.
(A) Human osteosarcoma (U2OS) and mouse embryonic fibroblast (MEF) cells (related to Figure 1A), where vinculin and NMII were detected by specific antibodies, and F-actin and nucleus by fluorescent …
Figure 1—figure supplement 2
Cortical stress fibers are not linked to fibrillar adhesions.
(A) 3D-structured illumination microscopy (SIM) maximum intensity projections (MIPs) of human osteosarcoma (U2OS) cells and mouse embryonic fibroblasts (MEFs) cultured on uncoated high precision …
Figure 2 with 1 supplement
Cortical stress fibers assemble de novo from the actin cortex.
TIRF time-lapse imaging of a migrating human osteosarcoma (U2OS) (A) and mouse embryonic fibroblast (MEF) cell (B) expressing LifeAct-TagGFP2 (gray) and vinculin-mApple (magenta). Selected …
Figure 2—figure supplement 1
Cortical stress fibers emerge from the ventral actin cortex through different intermediate assembly states.
(A) Photoactivation of PA-GFP-actin (gray) at both cortical and dorsal stress fibers in human osteosarcoma (U2OS) cells that additionally expressed Lifeact-mKate1.31 (red) and miRFP670-paxillin …
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Figure 2—figure supplement 1—source data 1
PA-actin intensity profiles from photoactivated foci.
- https://cdn.elifesciences.org/articles/60710/elife-60710-fig2-figsupp1-data1-v3.xlsx
Figure 3 with 1 supplement
Non-muscle myosin II (NMII)A pulse-mediated cortical stress fibers assembly occurs predominantly underneath the nucleus in migrating cells.
(A) TIRF time-lapse imaging of a migrating human osteosarcoma (U2OS) cell. The selected time-lapse frames (from the yellow boxed area) illustrate the assembly of a cortical stress fiber (exemplified …
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Figure 3—source data 1
Analysis of de novo forming cortical SFs.
- https://cdn.elifesciences.org/articles/60710/elife-60710-fig3-data1-v3.xlsx
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Figure 3—source data 2
Analysis of NMIIA pulse dynamics.
- https://cdn.elifesciences.org/articles/60710/elife-60710-fig3-data2-v3.xlsx
Figure 3—figure supplement 1
Myosin pulses are associated with cortical stress fibers assembly underneath the nucleus, and morphologically similar adhesion-attached actin filament bundles also exist in cells migrating in 3D matrix.
(A) TIRF time-lapse imaging of a migrating human osteosarcoma (U2OS) cell expressing histone H2B-eGFP (gray), mApple-NMIIA (green), and miRFP670-paxillin (focal adhesions, magenta). Selected …
Figure 4 with 2 supplements
Cortical non-muscle myosin II (NMII) pulses orchestrate F-actin bundling for the assembly of cortical stress fibers.
(A) Formation of a cortical stress fiber from the ventral cortex of a migrating human osteosarcoma (U2OS) cell as studied by time-lapse TIRF microscopy. The entire cell is shown on the right, and …
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Figure 4—source data 1
Cortical SF number quantification after pharmacological inhibitions.
- https://cdn.elifesciences.org/articles/60710/elife-60710-fig4-data1-v3.xlsx
Figure 4—figure supplement 1
Different ways to assemble cortical stress fibers from the ventral actomyosin cortex.
(A) Example of a formation of cortical stress fiber in a migrating human osteosarcoma (U2OS) cell, where one end of the stress fiber connects to a focal adhesion and the other one to a cortical …
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Figure 4—figure supplement 1—source data 1
Cortical SF number quantification after formin inhibitions.
- https://cdn.elifesciences.org/articles/60710/elife-60710-fig4-figsupp1-data1-v3.xlsx
Figure 4—figure supplement 2
Cortical stress fiber assembly is dependent on functional non-muscle myosin II (NMII) and integrin-based adhesions.
(A) TIRF time-lapse images of a human osteosarcoma (U2OS) cell expressing mApple-NMIIA (green), miRFP670-paxillin (magenta), and LifeAct-TagGFP2 (gray), after 1 hr incubation with 50 µM para-amino …
Figure 5
Two different mechanisms for generation of contractile stress fibers.
(A) Schematic representation of the de novo assembly of a cortical stress fiber from the ventral actomyosin cortex. 1. Non-muscle myosin II (NMII) pulses occur frequently at the ventral actin …
Author response image 1
Analysis of cortical stress fiber half-life in U2OS cells migrating on a fibronectin and expressing LifeAct-TagGFP2 and mApple-Paxillin.
120 min TIRFM time-lapse videos with 1-minute imaging interval. N = 15 cells; 77 cortical stress fibers. Please note that all stress fibers that were visible already in the beginning of videos …
Videos
Video 1
Cortical stress fiber formation at the ventral actin cortex of a migrating human osteosarcoma (U2OS) cell.
Zoom in of a U2OS cell expressing LifeAct-TagGFP2 (gray) and mApple-vinculin (magenta), migrating on a fibronectin (10 µg/ml)-coated high-precision imaging dish. Related to Figure 2A. Time-lapse …
Video 2
Cortical stress fiber formation at the ventral actin cortex of a migrating mouse embryonic fibroblast (MEF) cell.
Zoom in of a MEF cell expressing LifeAct-TagGFP2 (gray) and mApple-vinculin (magenta), migrating on a fibronectin (10 µg/ml)-coated high-precision imaging dish. Related to Figure 2B. Time-lapse …
Video 3
Cortical stress fiber assembly at the trailing edge of a migrating human osteosarcoma (U2OS) cell.
Zoom in of a U2OS cell expressing LifeAct-TagGFP2 (magenta) and mApple-vinculin (green), migrating on a fibronectin (10 µg/ml)-coated high-precision imaging dish. Related to Figure 2—figure …
Video 4
Cortical stress fiber assembly under the nucleus of migrating human osteosarcoma (U2OS) cell.
Zoom in of a U2OS cell expressing LifeAct-TagGFP2 (gray), histone-H2B-mCherry (blue), and miRFP670-paxillin (magenta), migrating on a fibronectin (10 µg/ml)-coated high-precision imaging dish. …
Video 5
Non-muscle myosin II (NMII)A pulses assemble into cortical stress fibers under the nucleus of migrating human osteosarcoma (U2OS) cell.
Zoom in of a U2OS cell expressing histone H2B-eGFP (gray), mApple-NMIIA (green), and miRFP670-paxillin (magenta), migrating on a fibronectin (10 µg/ml)-coated high-precision imaging dish. Related to …
Video 6
3D-reconstruction of human osteosarcoma (U2OS) cells migrating in a 3D fibrin matrix.
U2OS cells expressing eGFP-paxillin (green), stained further with phalloidin for F-actin (magenta) and DAPI (blue) for visualization of the nuclei. Related to Figure 3—figure supplement 1B. Confocal …
Video 7
Non-muscle myosin II (NMII)A pulses at the trailing edge of a migrating human osteosarcoma (U2OS) cells.
Video 8
Non-muscle myosin II (NMII)A pulses coordinate the cortical stress fiber assembly in a migrating human osteosarcoma (U2OS) cell.
Zoom in of a U2OS cell expressing LifeAct-TagGFP2 (gray), mApple-NMIIA (green), and miRFP670-paxillin (magenta), migrating on a fibronectin (10 µg/ml)-coated high-precision imaging dish. Related to F…
Video 9
NMIIA pulse-coordinated cortical stress fiber assembly in a migrating U2OS cell, where one end of the fiber connects to actomyosin mesh.
Zoom in of a U2OS cell expressing LifeAct-TagGFP2 (gray), mApple-NMIIA (green), and miRFP670-paxillin (magenta), migrating on a fibronectin (10 μg/ml)-coated high-precision imaging dish. Related to F…
Video 10
NMIIA pulse-coordinated cortical stress fiber assembly in a migrating U2OS cell, where one end of the fiber connects to a pre-existing adhesion.
Zoom in of a U2OS cell expressing LifeAct-TagGFP2 (gray), mApple-NMIIA (green), and miRFP670-paxillin, migrating on a fibronectin (10 μg/ml)-coated high-precision imaging dish. Related to Figure …
Video 11
Inhibiting non-muscle myosin II (NMII) activity abolishes cortical stress fiber assembly.
Zoom in of a human osteosarcoma (U2OS) cell expressing LifeAct-TagGFP2 (gray), mApple-NMIIA (green), and miRFP670-paxillin (magenta, not shown in the zoom in) migrating on a fibronectin (10 …
Video 12
Inhibiting integrin-based ECM adhesion obstructs cortical stress fiber formation.
Zoom in of a human osteosarcoma (U2OS) cell expressing LifeAct-TagGFP2 (gray), mApple-NMIIA (green), and miRFP670-paxillin (magenta, not shown in the zoom in) on a poly-D-lysine (10 µg/ml)-coated …
Video 13
CK-666 mediated Arp2/3 inhibition does not prevent cortical stress fiber assembly.
Zoom in of a human osteosarcoma (U2OS) cell expressing LifeAct-TagGFP2 (gray) and miRFP670-paxillin (magenta) migrating on a fibronectin (10 µg/ml)-coated high-precision imaging dish after 4 hr …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | Human osteosarcoma cells (U2OS) | ATCC | Cat# HTB-96, RRID:CVCL_0042 | Authenticated through STR profiling |
| Cell line (Mus musculus) | Mouse embryonic fibroblasts (MEFs) | Jiu et al., 2015 | A gift from John Eriksson (University of Turku, Finland) | |
| Recombinant DNA reagent | eGFP-NMIIA (plasmid) | Addgene, Jacobelli et al., 2009 | RRID:Addgene_38297 | A gift from Mathew Krummel (University of California, San Francisco) |
| Recombinant DNA reagent | PA-GFP-Actin (plasmid) | Addgene | RRID:Addgene_57121 | A gift from Michael Davidson |
| Recombinant DNA reagent | LifeAct-mKate1.31 (plasmid) | Addgene, Shcherbo et al., 2009 | RRID:Addgene_54668 | A gift from Michael Davidson |
| Recombinant DNA reagent | mApple-NMIIA (plasmid) | Addgene, Burnette et al., 2014 | RRID:Addgene_54929 | A gift from Michael Davidson |
| Recombinant DNA reagent | Vinculin-mApple (plasmid) | Addgene | RRID:Addgene_54962 | A gift from Michael Davidson |
| Recombinant DNA reagent | α-actinin1-TagRFP-T (plasmid) | Addgene | RRID:Addgene_58033 | A gift from Michael Davidson |
| Recombinant DNA reagent | LifeAct-TagGFP2 (plasmid) | Stefani et al., 2017 | A gift from Emmanuel Lemichez (Pasteur Institute, France) | |
| Recombinant DNA reagent | Histone H2B-eGFP (plasmid) | This paper | A gift from Maria Vartiainen (University of Helsinki, Finland) | |
| Recombinant DNA reagent | Histone H2B-mCherry (plasmid) | This paper | A gift from Maria Vartiainen (University of Helsinki, Finland) | |
| Recombinant DNA reagent | eGFP-Paxillin (plasmid) | This paper | Gallus gallus paxillin ORF with T132S and M133L unattended mutations (pPL1277) | |
| Recombinant DNA reagent | miRFP670-Paxillin (plasmid) | This paper | eGFP in eGFP-Paxillin replaced with miRFP670-N1 (pPL1514) | |
| Recombinant DNA reagent | miRFP670-N1 (plasmid) | Addgene, Shcherbakova et al., 2016 | RRID:Addgene_79987 | A gift from Vladislav Verkusha (Albet Einstein College of Medicine, NY) |
| Transfected construct (human) | SMARTpoolON-TARGETplus siRNA to mDia1 | Dharmacon | Cat# L-010347-00-0005 | Transfected using GeneSilencer |
| Transfected construct (human) | SMARTpoolON-TARGETplus siRNA to mDia2 | Dharmacon | Cat# L-012029-00-0005 | Transfected using GeneSilencer |
| Transfected construct (human) | negative (non-targeting) control siRNA | Qiagen | Cat# 1027281 | Transfected using GeneSilencer |
| Antibody | anti-vinculin (mouse monoclonal) | Sigma Aldrich | Cat# V9131, RRID:AB_477629 | 1:400 (IF) |
| Antibody | anti-NMIIA (C-termini) (rabbit polyclonal) | Biolegend | Cat# 909801, RRID:AB_2565100 | 1:800 (IF) |
| Antibody | anti-fibronectin (rabbit polyclonal) | Sigma Aldrich | Cat# F3648, RRID:AB_476976 | 1:400 (IF) a gift from Johanna Ivaska (University of Turku, Finland) |
| Antibody | anti-Y118-paxillin (rabbit monoclonal) | Cell Signaling Technology | Cat# 2541, RRID:AB_2174466 | 1:50 (IF) a gift from Johanna Ivaska (University of Turku, Finland) |
| Antibody | anti-human mDia1 (rabbit monoclonal) | Abcam | Cat# ab129167, RRID:AB_11143749 | 1:1000 (WB) |
| Antibody | anti-human mDia2 (rabbit polyclonal) | Sigma Aldrich | Cat# HPA005647, RRID:AB_1078657 | 1:1000 (WB) |
| Antibody | anti-GAPDH (rabbit polyclonal) | Sigma Aldrich | Cat# G9545, RRID:AB_796208 | 1:10,000 (WB) |
| Antibody | Anti-mouse Alexa Fluor 488 (goat polyclonal) | Thermo Fisher Scientific | Cat# A-11001, RRID:AB_2534069 | 1:200 (IF) |
| Antibody | Anti-mouse Alexa Fluor 568 (goat polyclonal) | Thermo Fisher Scientific | Cat# A-11031, RRID:AB_144696 | 1:200 (IF) |
| Antibody | Anti-rabbit Alexa Fluor Plus 647 (goat polyclonal) | Thermo Fisher Scientific | Cat# A32733, RRID:AB_2633282 | 1:400 (IF) |
| Antibody | HRP-conjugated anti–rabbit (goat polyclonal) | Thermo Fisher Scientific | Cat# 32460, RRID:AB_1185567 | 1:1000 (WB) |
| Commercial assay or kit | Lipofectamine 2000 | Thermo Fisher Scientific | Cat# 11668019 | Plasmid transfection (3.5:1 DNA: Lipofectamine ratio) |
| Commercial assay or kit | GeneSilencer | Genlantis | Cat# T500750 | siRNA transfection |
| Commercial assay or kit | jetPRIME | Polyplus transfection | Cat# 114–01 | Plasmid transfection (TFM) |
| Commercial assay or kit | NEBuilder | New England Biolabs | Cat# E5520S | |
| Chemical compound, drug | Para-amino Blebbistatin | Optopharma | Cat# DR-Am-89 | 50 µM working concentration |
| Chemical compound, drug | SMIFH2 | Sigma Aldrich | Cat# S4826 | 12.5 µM working concentration |
| Chemical compound, drug | CK-666 | Sigma Aldrich | Cat# SML0006 | 100 µM working concentration |
| Chemical compound, drug | DMSO | Sigma Aldrich | Cat# D2650 | Diluent/negative control |
| Software, algorithm | Fiji | Schindelin et al., 2012 | RRID:SCR_002285 | |
| Software, algorithm | OriginPro | OriginLab Corp. | RRID:SCR_014212 | |
| Software, algorithm | NMII pulse quantification (macro) | This paper. Modified from Kim and Davidson, 2011 | Available at: https://github.com/UH-LMU/lmu-users/tree/master/jaakko/NMIIA_pulses | |
| Other | Fibrinogen (from human plasma) | Sigma Aldrich | Cat# 341578 | 2.25 mg/ml working concentration |
| Other | Thrombin (from human plasma) | Sigma Aldrich | Cat# T6884 | 1 U/ml working concentration |
| Other | Fibronectin (human plasma) | Sigma Aldrich | Cat# FIBRP-RO | 10 µg/ml working concentration |
| Other | Laminin | Sigma Aldrich | Cat# L2020 | 25 µg/ml working concentration |
| Other | Collagen | Sigma Aldrich | Cat# C4243 | 500 µg/ml working concentration |
| Other | poly-D-lysine | Sigma Aldrich | Cat# P0899 | 10 µg/ml working concentration |
| Other | DAPI stain | Thermo Fisher Scientific | Cat# D1306 | 0.2 µM working concentration (IF) |
| Other | Alexa Fluor 488-Phalloidin | Thermo Fisher Scientific | Cat# A12379 | 1:200 (IF) |
| Other | Alexa Fluor 568-Phalloidin | Thermo Fisher Scientific | Cat# 12380 | 1:200 (IF) |
| Other | Fluorescent microspheres. 0.2 µm diameter | Invitrogen | Cat# F8848 |
