Rac1-Dependent Phosphorylation and Focal Adhesion Recruitment of Myosin IIA Regulates Migration and Mechanosensing

Published on Jan 19, 2015in Current Biology9.601
· DOI :10.1016/J.CUB.2014.11.043
Ana Maria Pasapera11
Estimated H-index: 11
(NIH: National Institutes of Health),
Sergey V. Plotnikov16
Estimated H-index: 16
(U of T: University of Toronto)
+ 3 AuthorsClare M. Waterman39
Estimated H-index: 39
(NIH: National Institutes of Health)
Summary Background Cell migration requires coordinated formation of focal adhesions (FAs) and assembly and contraction of the actin cytoskeleton. Nonmuscle myosin II (MII) is a critical mediator of contractility and FA dynamics in cell migration. Signaling downstream of the small GTPase Rac1 also regulates FA and actin dynamics, but its role in regulation of MII during migration is less clear. Results We found that Rac1 promotes association of MIIA with FA. Live-cell imaging showed that, whereas most MIIA at the leading edge assembled into dorsal contractile arcs, a substantial subset assembled in or was captured within maturing FA, and this behavior was promoted by active Rac1. Protein kinase C (PKC) activation was necessary and sufficient for integrin- and Rac1-dependent phosphorylation of MIIA heavy chain (HC) on serine1916 (S1916) and recruitment to FA. S1916 phosphorylation of MIIA HC and localization in FA was enhanced during cell spreading and ECM stiffness mechanosensing, suggesting upregulation of this pathway during physiological Rac1 activation. Phosphomimic and nonphosphorylatable MIIA HC mutants demonstrated that S1916 phosphorylation was necessary and sufficient for the capture and assembly of MIIA minifilaments in FA. S1916 phosphorylation was also sufficient to promote the rapid assembly of FAs to enhance cell migration and for the modulation of traction force, spreading, and migration by ECM stiffness. Conclusions Our study reveals for the first time that Rac1 and integrin activation regulates MIIA HC phosphorylation through a PKC-dependent mechanism that promotes MIIA association with FAs and acts as a critical modulator of cell migration and mechanosensing.
📖 Papers frequently viewed together
1,888 Citations
773 Citations
133 Citations
#1Yongho Bae (UPenn: University of Pennsylvania)H-Index: 13
#2Keeley L. Mui (UPenn: University of Pennsylvania)H-Index: 8
Last. Richard K. Assoian (UPenn: University of Pennsylvania)H-Index: 60
view all 9 authors...
Tissue and extracellular matrix (ECM) stiffness is transduced into intracellular stiffness, signaling, and changes in cellular behavior. Integrins and several of their associated focal adhesion proteins have been implicated in sensing ECM stiffness. We investigated how an initial sensing event is translated into intracellular stiffness and a biologically interpretable signal. We found that a pathway consisting of focal adhesion kinase (FAK), the adaptor protein p130Cas (Cas), and the guanosine t...
115 CitationsSource
#1Dylan T. Burnette (NIH: National Institutes of Health)H-Index: 21
#2Lin Shao (HHMI: Howard Hughes Medical Institute)H-Index: 51
Last. Jennifer Lippincott-Schwartz (NIH: National Institutes of Health)H-Index: 119
view all 12 authors...
How adherent and contractile systems coordinate to promote cell shape changes is unclear. Here, we define a counterbalanced adhesion/contraction model for cell shape control. Live-cell microscopy data showed a crucial role for a contractile meshwork at the top of the cell, which is composed of actin arcs and myosin IIA filaments. The contractile actin meshwork is organized like muscle sarcomeres, with repeating myosin II filaments separated by the actin bundling protein α-actinin, and is mechani...
129 CitationsSource
#1Neil BillingtonH-Index: 21
#2Aibing Wang (NIH: National Institutes of Health)H-Index: 8
Last. James R. SellersH-Index: 73
view all 5 authors...
Nonmuscle myosin IIs (NM IIs) are a group of molecular motors involved in a wide variety of cellular processes including cytokinesis, migration, and control of cell morphology. There are three paralogs of the NM II heavy chain in humans (IIA, IIB, and IIC), each encoded by a separate gene. These paralogs are expressed at different levels according to cell type and have different roles and intracellular distributions in vivo. Most previous studies on NM II used tissue-purified protein or expresse...
124 CitationsSource
#2Anne R. BresnickH-Index: 33
Nonmuscle myosin-II is an actin-based motor that converts chemical energy into force and movement, and thus functions as a key regulator of the eukaryotic cytoskeleton. Although it is established that phosphorylation on the regulatory light chain increases the actin-activated MgATPase activity of the motor and promotes myosin-II filament assembly, studies have begun to characterize alternative mechanisms that regulate filament assembly and disassembly. These investigations have revealed that all...
50 CitationsSource
#1Seham Ebrahim (NIH: National Institutes of Health)H-Index: 10
#2Tomoki Fujita (Kyoto Prefectural University of Medicine)H-Index: 1
Last. Bechara Kachar (NIH: National Institutes of Health)H-Index: 81
view all 14 authors...
Summary Nonmuscle myosin II (NMII) is thought to be the master integrator of force within epithelial apical junctions, mediating epithelial tissue morphogenesis and tensional homeostasis [1–3]. Mutations in NMII are associated with a number of diseases due to failures in cell-cell adhesion [4–8]. However, the organization and the precise mechanism by which NMII generates and responds to tension along the intercellular junctional line are still not known. We discovered that periodic assemblies of...
105 CitationsSource
#1Miguel Vicente-Manzanares (UAM: Autonomous University of Madrid)H-Index: 44
Summary A new study reveals that non-muscle myosin II plays a central role in the durotaxis of mesenchymal stem cells, with the two major isoforms, II-A and II-B, being cooperatively required for this cell movement, and serine phosphorylation of the II-A isoform playing a negative role.
3 CitationsSource
#1Sergey V. Plotnikov (NIH: National Institutes of Health)H-Index: 16
#2Ana Maria Pasapera (NIH: National Institutes of Health)H-Index: 11
Last. Clare M. Waterman (NIH: National Institutes of Health)H-Index: 39
view all 4 authors...
Summary Cell migration toward areas of higher extracellular matrix (ECM) rigidity via a process called "durotaxis" is thought to contribute to development, immune response, and cancer metastasis. To understand how cells sample ECM rigidity to guide durotaxis, we characterized cell-generated forces on the nanoscale within single mature integrin-based focal adhesions (FAs). We found that individual FAs act autonomously, exhibiting either stable or dynamically fluctuating ("tugging") traction. We s...
503 CitationsSource
#1Tamar Geiger (TAU: Tel Aviv University)H-Index: 37
#2Ronen Zaidel-Bar (NUS: National University of Singapore)H-Index: 27
Cell biologists studying cell adhesion have already figured out that cell–extracellular matrix connections, mediated by integrin receptors, are diverse and extremely complex structures. Dozens of adaptors — linking integrins with the cytoskeleton, and numerous enzymes and signaling proteins — regulating adhesion site dynamics, collectively referred to as the integrin adhesome, cooperate in mediating adhesion and activating specific signaling networks. Recent proteomic studies indicate that the k...
79 CitationsSource
#1Maria S. Shutova (UPenn: University of Pennsylvania)H-Index: 7
#2Changsong Yang (UPenn: University of Pennsylvania)H-Index: 22
Last. Tatyana Svitkina (UPenn: University of Pennsylvania)H-Index: 51
view all 4 authors...
The contractile system of nonmuscle cells consists of interconnected actomyosin networks and bundles anchored to focal adhesions. The initiation of the contractile system assembly is poorly understood structurally and mechanistically, whereas system’s maturation heavily depends on nonmuscle myosin II (NMII). Using platinum replica electron microscopy in combination with fluorescence microscopy, we characterized the structural mechanisms of the contractile system assembly and roles of NMII at ear...
94 CitationsSource
#1Patrick W. Oakes (U of C: University of Chicago)H-Index: 21
#2Yvonne Beckham (U of C: University of Chicago)H-Index: 7
Last. Margaret L. Gardel (U of C: University of Chicago)H-Index: 53
view all 4 authors...
Focal adhesion composition and size are modulated in a myosin II–dependent maturation process that controls adhesion, migration, and matrix remodeling. As myosin II activity drives stress fiber assembly and enhanced tension at adhesions simultaneously, the extent to which adhesion maturation is driven by tension or altered actin architecture is unknown. We show that perturbations to formin and α-actinin 1 activity selectively inhibited stress fiber assembly at adhesions but retained a contractil...
233 CitationsSource
Cited By62
#1Robert S. Fischer (NIH: National Institutes of Health)H-Index: 30
#2Xiaoyu Sun (UMD: University of Maryland, College Park)H-Index: 5
Last. Clare M. Waterman (NIH: National Institutes of Health)H-Index: 39
view all 11 authors...
Contact guidance is a powerful topographical cue that induces persistent directional cell migration. Healthy tissue stroma is characterized by a meshwork of wavy extracellular matrix (ECM) fiber bundles, whereas metastasis-prone stroma exhibit less wavy, more linear fibers. The latter topography correlates with poor prognosis, whereas more wavy bundles correlate with benign tumors. We designed nanotopographic ECM-coated substrates that mimic collagen fibril waveforms seen in tumors and healthy t...
#1Joseph G. Duman (BCM: Baylor College of Medicine)H-Index: 10
#2Francisco A. Blanco (BCM: Baylor College of Medicine)H-Index: 2
Last. Kimberley F. Tolias (BCM: Baylor College of Medicine)H-Index: 21
view all 9 authors...
Synaptic connections between neurons are essential for every facet of human cognition and are thus regulated with extreme precision. Rho-family GTPases, molecular switches that cycle between an active GTP-bound state and an inactive GDP-bound state, comprise a critical feature of synaptic regulation. Rho-GTPases are exquisitely controlled by an extensive suite of activators (GEFs) and inhibitors (GAPs and GDIs) and interact with many different signalling pathways to fulfill their roles in orches...
#1Shefali Talwar (UPenn: University of Pennsylvania)H-Index: 5
#2Aayush Kant (UPenn: University of Pennsylvania)
Last. Richard K. Assoian (UPenn: University of Pennsylvania)H-Index: 60
view all 5 authors...
Summary Reversible differentiation of vascular smooth muscle cells (VSMCs) plays a critical role in vascular biology and disease. Changes in VSMC differentiation correlate with stiffness of the arterial extracellular matrix (ECM), but causal relationships remain unclear. We show that VSMC plasticity is mechanosensitive and that both the de-differentiated and differentiated fates are promoted by the same ECM stiffness. Differential equations developed to model this behavior predicted that a null ...
#2Mar Arias-GarciaH-Index: 3
Last. Chris Bakal (ICR: Institute of Cancer Research)H-Index: 26
view all 5 authors...
YAP and TAZ are transcriptional co-activators that are often constitutively active in triple negative breast cancer (TNBC) cells driving proliferation, invasion, and drug resistance. Through multiplexed quantitative genetic screens for YAP/TAZ localisation and cell shape, we found that the RhoGEF DOCK5 is essential for YAP/TAZ activation in metastatic cells and is required for the maintenance of polarity during migration. DOCK5 regulates cell shape and thus YAP/TAZ through different genetic inte...
#2Mar Arias-GarciaH-Index: 3
Last. Chris Bakal (ICR: Institute of Cancer Research)H-Index: 26
view all 5 authors...
YAP and TAZ are transcriptional co-activators that are often constitutively active in triple negative breast cancer (TNBC) cells promoting proliferation, invasion, and drug resistance. However the mechanisms responsible for YAP/TAZ dysregulation in TNBC remain unclear. Through unbiased systematic RNAi screening we found that the Rho GTP Exchange Factor (RhoGEF) DOCK5 promotes YAP/TAZ nuclear translocation, and is required for maintenance of migration, invasion, cell survival, and resistance to M...
#1Yongsheng Jia (Beckman Research Institute)H-Index: 1
#2Yujun Wang (Beckman Research Institute)H-Index: 1
Last. Mike Yue Chen (Beckman Research Institute)H-Index: 1
view all 4 authors...
Abstract Metastasis is associated with poor prognosis in cancer, and is a multistep process that includes invasion and migration. Several epigenetic factors are involved in this process, including chromobox protein homolog 8 (CBX8). Here, we show that CBX8 is overexpressed in many cancers compared with normal tissues. Functional analyses indicated that CBX8 promoted invasion and migration in glioblastoma, breast cancer, and lung cancer in vitro and in vivo. WNK2 was identified as a target gene o...
#1Eric Guberman (College of Wooster)
#2Hikmet Sherief (College of Wooster)
Last. Erzsébet Ravasz Regan (College of Wooster)H-Index: 13
view all 3 authors...
Abstract Epithelial cells respond to their physical neighborhood with mechano-sensitive behaviors required for development and tissue maintenance. These include anchorage dependence, matrix stiffness-dependent proliferation, contact inhibition of proliferation and migration, and collective migration that balances cell crawling with the maintenance of cell junctions. While required for development and tissue repair, these coordinated responses to the microenvironment also contribute to cancer met...
#2Mar Arias-GarciaH-Index: 3
Last. Chris Bakal (ICR: Institute of Cancer Research)H-Index: 26
view all 5 authors...
YAP and TAZ are transcriptional co-activators that are often constitutively active in triple negative breast cancer (TNBC) cells. However the mechanisms responsible for YAP/TAZ dysregulation in TNBC remain unclear. We hypothesised that RhoGEF activity in TNBC cells underpins YAP/TAZ activation in TNBC cells. Through systematic RNAi screening we found that the Focal Adhesion (FA) associated RhoGEF DOCK5 promotes YAP/TAZ nuclear translocation in proliferating TNBC cells. DOCK5 is required for 2D m...
#1Hawa Racine Thiam (NIH: National Institutes of Health)H-Index: 8
#2Siu Ling Wong (Harvard University)H-Index: 22
Last. Clare M. Waterman (NIH: National Institutes of Health)H-Index: 39
view all 9 authors...
Neutrophil extracellular traps (NETs) are web-like DNA structures decorated with histones and cytotoxic proteins that are released by activated neutrophils to trap and neutralize pathogens during the innate immune response, but also form in and exacerbate sterile inflammation. Peptidylarginine deiminase 4 (PAD4) citrullinates histones and is required for NET formation (NETosis) in mouse neutrophils. While the in vivo impact of NETs is accumulating, the cellular events driving NETosis and the rol...
34 CitationsSource
#1Fei Xie (DUT: Dalian University of Technology)H-Index: 4
#2Shuai Shao (DUT: Dalian University of Technology)H-Index: 5
Last. Bo Liu (DUT: Dalian University of Technology)H-Index: 12
view all 7 authors...
The activity of Rho-specific guanine nucleotide dissociation inhibitor alpha (RhoGDIalpha) is regulated by its own phosphorylation at different amino acid sites. These phosphorylation sites may have a crucial role in local Rho GTPases activation during cell migration. This paper is designed to explore the influence of phosphorylation on shear stress-induced spatial RhoGDIalpha activation. Based on the fluorescence resonance energy transfer biosensor sl-RhoGDIalpha, which was constructed to test ...