Structural and functional analysis of LIM domain-dependent recruitment of paxillin to αvβ3 integrin-positive focal adhesions

AbstractThe LIM domain-dependent localization of the adapter protein paxillin to β3 integrin-positive focal adhesions (FAs) is not mechanistically understood. Here, by combining molecular biology, photoactivation and FA-isolation experiments, we demonstrate specific contributions of each LIM domain of paxillin and reveal multiple paxillin interactions in adhesion-complexes. Mutation of β3 integrin at a putative paxillin binding site (β3VE/YA) leads to rapidly inward-sliding FAs, correlating with actin retrograde flow and enhanced paxillin dissociation kinetics. Induced mechanical coupling of paxillin to β3VE/YA integrin arrests the FA-sliding, thereby disclosing an essential structural function of paxillin for the maturation of β3 integrin/talin clusters. Moreover, bimolecular fluorescence complementation unveils the spatial orientation of the paxillin LIM-array, juxtaposing the positive LIM4 to the plasma membrane and the β3 integrin-tail, while in vitro binding assays point to LIM1 and/or LIM2 interaction with talin-head domain. These data provide structural insights into the molecular organization of β3 integrin-FAs.

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Structural and functional analysis of LIM domain-dependent recruitment of paxillin to focal adhesions

10.21203/rs.3.rs-42943/v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Marta Ripamonti ◽

Nicolas Liaudet ◽

Bernhard Wehrle-Haller

Keyword(s):

Plasma Membrane ◽

Focal Adhesions ◽

Bimolecular Fluorescence Complementation ◽

Molecular Organization ◽

Structural Function ◽

Lim Domain ◽

Mechanical Coupling ◽

Lim Domains ◽

Β3 Integrin ◽

Structural Insights

Abstract The LIM domain-dependent localization of the adapter protein paxillin to focal adhesions (FAs) is not mechanistically understood. Here, by combining molecular biology with photoactivation and FA-isolation experiments, we demonstrate a specific contribution of each LIM domain and reveal the existence of multiple paxillin docking sites in the FA-complex. Mutation of β3 integrin at a putative paxillin binding site leads to rapid inward sliding of FAs, correlating with enhanced paxillin dissociation rates. Mechanical coupling of paxillin to integrins or the plasma membrane arrests the FAs sliding, thereby disclosing an essential structural function of the LIM-array for the maturation of integrin/talin clusters. Moreover, via bimolecular fluorescence complementation, we determine a precise spatial orientation of the paxillin LIM domains, juxtaposing the positively charged LIM4 to the plasma membrane and the extremity of the β3 integrin tail, providing structural insights into the molecular organization of FAs.

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αvβ3 integrin spatially regulates VASP and RIAM to control adhesion dynamics and migration

The Journal of Cell Biology ◽

10.1083/jcb.200912014 ◽

2010 ◽

Vol 189 (2) ◽

pp. 369-383 ◽

Cited By ~ 63

Author(s):

Daniel C. Worth ◽

Kairbaan Hodivala-Dilke ◽

Stephen D. Robinson ◽

Samantha J. King ◽

Penny E. Morton ◽

...

Keyword(s):

Regulatory Protein ◽

Focal Adhesions ◽

Αvβ3 Integrin ◽

Β1 Integrin ◽

Downstream Signaling ◽

Β3 Integrin ◽

And Migration ◽

2D And 3D

Integrins are fundamental to the control of protrusion and motility in adherent cells. However, the mechanisms by which specific members of this receptor family cooperate in signaling to cytoskeletal and adhesion dynamics are poorly understood. Here, we show that the loss of β3 integrin in fibroblasts results in enhanced focal adhesion turnover and migration speed but impaired directional motility on both 2D and 3D matrices. These motility defects are coupled with an increased rate of actin-based protrusion. Analysis of downstream signaling events reveals that loss of β3 integrin results in a loss of protein kinase A–dependent phosphorylation of the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP). Dephosphorylated VASP in β3-null cells is preferentially associated with Rap1-GTP–interacting adaptor molecule (RIAM) both in vitro and in vivo, which leads to enhanced formation of a VASP–RIAM complex at focal adhesions and subsequent increased binding of talin to β1 integrin. These data demonstrate a novel mechanism by which αvβ3 integrin acts to locally suppress β1 integrin activation and regulate protrusion, adhesion dynamics, and persistent migration.

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Absence of the αvβ3 Integrin Dictates the Time-Course of Angiogenesis in the Hypoxic Central Nervous System: Accelerated Endothelial Proliferation Correlates with Compensatory Increases in α5β1 Integrin Expression

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2009.276 ◽

2010 ◽

Vol 30 (5) ◽

pp. 1031-1043 ◽

Cited By ~ 27

Author(s):

Longxuan Li ◽

Jennifer V Welser ◽

Richard Milner

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Time Course ◽

Αvβ3 Integrin ◽

Integrin Expression ◽

Brain Endothelial Cells ◽

Endothelial Proliferation ◽

Β3 Integrin ◽

Α5β1 Integrin

Cerebral angiogenesis is an important adaptive response to hypoxia. As the αvβ3 integrin is induced on angiogenic vessels in the ischemic central nervous system (CNS), and the suggested angiogenic role for this integrin in other systems, it is important to determine whether the αvβ3 integrin is an important mediator of cerebral angiogenesis. αvβ3 integrin expression was examined in a model of cerebral hypoxia, in which mice were subject to hypoxia (8% O2) for 0, 4, 7, or 14 days. Immunofluorescence and western blot analysis revealed that in the hypoxic CNS, αvβ3 integrin was strongly induced on angiogenic brain endothelial cells (BEC), along with its ligand vitronectin. In the hypoxia model, β3 integrin-null mice showed no obvious defect in cerebral angiogenesis. However, early in the angiogenic process, BEC in these mice showed an increased mitotic index that correlated closely with increased α5 integrin expression. In vitro experiments confirmed α5 integrin upregulation on β3 integrin-null BEC, which also correlated with increased BEC proliferation on fibronectin. These studies confirm hypoxic induction of αvβ3 integrin on angiogenic vessels, but suggest distinct roles for the BEC integrins αvβ3 and α5β1 in cerebral angiogenesis, with αvβ3 having a nonessential role, and α5β1 promoting BEC proliferation.

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Marching at the front and dragging behind

The Journal of Cell Biology ◽

10.1083/jcb.200107107 ◽

2001 ◽

Vol 155 (7) ◽

pp. 1319-1332 ◽

Cited By ~ 245

Author(s):

Christoph Ballestrem ◽

Boris Hinz ◽

Beat A. Imhof ◽

Bernhard Wehrle-Haller

Keyword(s):

Fluorescent Protein ◽

Focal Adhesions ◽

High Density ◽

Αvβ3 Integrin ◽

Low Density ◽

Adhesion Sites ◽

Cell Front ◽

Β3 Integrin ◽

Green Fluorescent ◽

Migrating Cells

Integrins are cell–substrate adhesion molecules that provide the essential link between the actin cytoskeleton and the extracellular matrix during cell migration. We have analyzed αVβ3-integrin dynamics in migrating cells using a green fluorescent protein–tagged β3-integrin chain. At the cell front, adhesion sites containing αVβ3-integrin remain stationary, whereas at the rear of the cell they slide inward. The integrin fluorescence intensity within these different focal adhesions, and hence the relative integrin density, is directly related to their mobility. Integrin density is as much as threefold higher in sliding compared with stationary focal adhesions. High intracellular tension under the control of RhoA induced the formation of high-density contacts. Low-density adhesion sites were induced by Rac1 and low intracellular tension. Photobleaching experiments demonstrated a slow turnover of β3-integrins in low-density contacts, which may account for their stationary nature. In contrast, the fast β3-integrin turnover observed in high-density contacts suggests that their apparent sliding may be caused by a polarized renewal of focal contacts. Therefore, differential acto-myosin–dependent integrin turnover and focal adhesion densities may explain the mechanical and behavioral differences between cell adhesion sites formed at the front, and those that move in the retracting rear of migrating cells.

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Intermediate filaments control collective migration by restricting traction forces and sustaining cell-cell contacts

10.1101/328609 ◽

2018 ◽

Author(s):

Chiara De Pascalis ◽

Carlos Pérez-González ◽

Shailaja Seetharaman ◽

Batiste Boëda ◽

Benoit Vianay ◽

...

Keyword(s):

Adherens Junctions ◽

Cell Monolayer ◽

Focal Adhesions ◽

Collective Migration ◽

Traction Forces ◽

Mechanical Coupling ◽

Directed Migration ◽

Cell Protrusion ◽

Cell Cell

AbstractMesenchymal cell migration relies on the coordinated regulation of the actin and microtubule networks which participate in polarised cell protrusion, adhesion and contraction. During collective migration, most of the traction forces are generated by the acto-myosin network linked to focal adhesions at the front of leader cells, which transmit these pulling forces to the followers. Here, using an in vitro wound healing assay to induce polarisation and collective directed migration of primary astrocytes, we show that the intermediate filament (IF) network composed of vimentin, GFAP and nestin contributes to directed collective movement by controlling the distribution of forces in the migrating cell monolayer. Together with the cytoskeletal linker plectin, these IFs control the organisation and dynamics of the acto-myosin network, promoting the actin-driven treadmilling of adherens junctions, thereby facilitating the polarisation of leader cells. Independently of their effect on adherens junctions, IFs influence the dynamics and localisation of focal adhesions and limit their mechanical coupling to the acto-myosin network. We thus conclude that IFs promote collective directed migration by restricting the generation of traction forces to the front of leader cells, preventing aberrant tractions in the followers and by contributing to the maintenance of lateral cell-cell interactions.

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Integrin αvβ3 mediates K1735 murine melanoma cell motility in vivo and in vitro

Journal of Cell Science ◽

10.1242/jcs.114.14.2665 ◽

2001 ◽

Vol 114 (14) ◽

pp. 2665-2672 ◽

Cited By ~ 1

Author(s):

Xiaowu Li ◽

Joseph Regezi ◽

F. Patrick Ross ◽

Scott Blystone ◽

Duško Ilić ◽

...

Keyword(s):

Integrin Αvβ3 ◽

Melanoma Cells ◽

Αvβ3 Integrin ◽

Integrin Subunit ◽

Metastatic Colonization ◽

Murine Melanoma ◽

Cardiac Metastases ◽

Β3 Integrin

The integrin αvβ3 has been shown to be tightly linked to progression of human melanoma. In this study, using two clones from the K1735 murine melanoma system, we investigated the role of αvβ3 in metastasis. The highly metastatic K1735M2 cells express the αvβ3 integrin, whereas the poorly metastatic K1735C23 cells do not. When transduced with the β3 integrin subunit cDNA, the K1735C23 cells produced lung lesions and, in two animals, cardiac metastases, whereas the parental C23 cells did not. By contrast, transduction of the full-length β3 integrin antisense DNA into the K1735M2 cells suppressed metastatic colonization. To specifically investigate the activation of β3 integrin-mediated pathways, the β3-positive and the β3-negative K1735 cells were plated onto vitronectin, a major matrix molecule of both primary and metastatic melanomas. Tyr397 of FAK was phosphorylated several times higher in β3-expressing K1735 melanoma cells than in β3-negative cells. To determine whether phosphorylation of FAK was associated with K1735 melanoma motility, we expressed the FAK-related non-kinase (FRNK) in the highly metastatic K1735M2 cells. Exogenous expression of FRNK suppressed phosphorylation of FAK at Tyr397 and decreased the invasive ability of these cells. In addition, expression of a constitutively active mutant Src in poorly metastatic K1735C23 cells increased invasion in vitro; whereas expression of a kinase-inactive Src mutant suppressed invasion. Our results suggest that signals initiated by αvβ3 promote metastasis in K1735 melanoma cells through the phosphorylation of FAK and activation of Src.

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Intermediate filaments control collective migration by restricting traction forces and sustaining cell–cell contacts

The Journal of Cell Biology ◽

10.1083/jcb.201801162 ◽

2018 ◽

Vol 217 (9) ◽

pp. 3031-3044 ◽

Cited By ~ 47

Author(s):

Chiara De Pascalis ◽

Carlos Pérez-González ◽

Shailaja Seetharaman ◽

Batiste Boëda ◽

Benoit Vianay ◽

...

Keyword(s):

Adherens Junctions ◽

Cell Monolayer ◽

Focal Adhesions ◽

Collective Migration ◽

Traction Forces ◽

Mechanical Coupling ◽

Directed Migration ◽

Cell Protrusion ◽

Cell Cell

Mesenchymal cell migration relies on the coordinated regulation of the actin and microtubule networks that participate in polarized cell protrusion, adhesion, and contraction. During collective migration, most of the traction forces are generated by the acto-myosin network linked to focal adhesions at the front of leader cells, which transmit these pulling forces to the followers. Here, using an in vitro wound healing assay to induce polarization and collective directed migration of primary astrocytes, we show that the intermediate filament (IF) network composed of vimentin, glial fibrillary acidic protein, and nestin contributes to directed collective movement by controlling the distribution of forces in the migrating cell monolayer. Together with the cytoskeletal linker plectin, these IFs control the organization and dynamics of the acto-myosin network, promoting the actin-driven treadmilling of adherens junctions, thereby facilitating the polarization of leader cells. Independently of their effect on adherens junctions, IFs influence the dynamics and localization of focal adhesions and limit their mechanical coupling to the acto-myosin network. We thus conclude that IFs promote collective directed migration in astrocytes by restricting the generation of traction forces to the front of leader cells, preventing aberrant tractions in the followers, and by contributing to the maintenance of lateral cell–cell interactions.

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RNF141 interacts with KRAS to promote colorectal cancer progression

Oncogene ◽

10.1038/s41388-021-01877-4 ◽

2021 ◽

Author(s):

Jiuna Zhang ◽

Xiaoyu Jiang ◽

Jie Yin ◽

Shiying Dou ◽

Xiaoli Xie ◽

...

Keyword(s):

Colorectal Cancer ◽

Plasma Membrane ◽

Tumor Growth ◽

Cancer Progression ◽

Critical Role ◽

Ring Finger ◽

Immunohistochemical Analysis ◽

Bimolecular Fluorescence Complementation

AbstractRING finger proteins (RNFs) play a critical role in cancer initiation and progression. RNF141 is a member of RNFs family; however, its clinical significance, roles, and mechanism in colorectal cancer (CRC) remain poorly understood. Here, we examined the expression of RNF141 in 64 pairs of CRC and adjacent normal tissues by real-time PCR, Western blot, and immunohistochemical analysis. We found that there was more expression of RNF141 in CRC tissue compared with its adjacent normal tissue and high RNF141 expression associated with T stage. In vivo and in vitro functional experiments were conducted and revealed the oncogenic role of RNF141 in CRC. RNF141 knockdown suppressed proliferation, arrested the cell cycle in the G1 phase, inhibited migration, invasion and HUVEC tube formation but promoted apoptosis, whereas RNF141 overexpression exerted the opposite effects in CRC cells. The subcutaneous xenograft models showed that RNF141 knockdown reduced tumor growth, but its overexpression promoted tumor growth. Mechanistically, liquid chromatography-tandem mass spectrometry indicated RNF141 interacted with KRAS, which was confirmed by Co-immunoprecipitation, Immunofluorescence assay. Further analysis with bimolecular fluorescence complementation (BiFC) and Glutathione-S-transferase (GST) pull-down assays showed that RNF141 could directly bind to KRAS. Importantly, the upregulation of RNF141 increased GTP-bound KRAS, but its knockdown resulted in a reduction accordingly. Next, we demonstrated that RNF141 induced KRAS activation via increasing its enrichment on the plasma membrane not altering total KRAS expression, which was facilitated by the interaction with LYPLA1. Moreover, KRAS silencing partially abolished the effect of RNF141 on cell proliferation and apoptosis. In addition, our findings presented that RNF141 functioned as an oncogene by upregulating KRAS activity in a manner of promoting KRAS enrichment on the plasma membrane in CRC.

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Cyr61 promotes Schwann cell proliferation and migration via αvβ3 integrin

BMC Molecular and Cell Biology ◽

10.1186/s12860-021-00360-y ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Zhenghui Cheng ◽

Yawen Zhang ◽

Yinchao Tian ◽

Yuhan Chen ◽

Fei Ding ◽

...

Keyword(s):

Nerve Injury ◽

Peripheral Nerves ◽

Molecular Mechanisms ◽

Αvβ3 Integrin ◽

Promoting Effect ◽

Ccn Family ◽

And Migration ◽

Proliferation And Migration

Abstract Background Schwann cells (SCs) play a crucial role in the repair of peripheral nerves. This is due to their ability to proliferate, migrate, and provide trophic support to axon regrowth. During peripheral nerve injury, SCs de-differentiate and reprogram to gain the ability to repair nerves. Cysteine-rich 61 (Cyr61/CCN1) is a member of the CCN family of matrix cell proteins and have been reported to be abundant in the secretome of repair mediating SCs. In this study we investigate the function of Cyr61 in SCs. Results We observed Cyr61 was expressed both in vivo and in vitro. The promoting effect of Cyr61 on SC proliferation and migration was through autocrine and paracrine mechanisms. SCs expressed αvβ3 integrin and the effect of Cyr61 on SC proliferation and migration could be blocked via αvβ3 integrin. Cyr61 could influence c-Jun protein expression in cultured SCs. Conclusions In this study, we found that Cyr61 promotes SC proliferation and migration via αvβ3 integrin and regulates c-Jun expression. Our study contributes to the understanding of cellular and molecular mechanisms underlying SC’s function during nerve injury, and thus, may facilitate the regeneration of peripheral nerves after injury.

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Two LIM Domain Proteins and UNC-96 Link UNC-97/PINCH to Myosin Thick Filaments in Caenorhabditis elegans Muscle

Molecular Biology of the Cell ◽

10.1091/mbc.e07-03-0278 ◽

2007 ◽

Vol 18 (11) ◽

pp. 4317-4326 ◽

Cited By ~ 39

Author(s):

Hiroshi Qadota ◽

Kristina B. Mercer ◽

Rachel K. Miller ◽

Kozo Kaibuchi ◽

Guy M. Benian

Keyword(s):

Caenorhabditis Elegans ◽

Binding Assays ◽

Lim Domain ◽

Yeast Two Hybrid ◽

Lim Domains ◽

Thick Filaments ◽

Vitro Binding ◽

Two Hybrid ◽

Hybrid Screening

By yeast two-hybrid screening, we found three novel interactors (UNC-95, LIM-8, and LIM-9) for UNC-97/PINCH in Caenorhabditis elegans. All three proteins contain LIM domains that are required for binding. Among the three interactors, LIM-8 and LIM-9 also bind to UNC-96, a component of sarcomeric M-lines. UNC-96 and LIM-8 also bind to the C-terminal portion of a myosin heavy chain (MHC), MHC A, which resides in the middle of thick filaments in the proximity of M-lines. All interactions identified by yeast two-hybrid assays were confirmed by in vitro binding assays using purified proteins. All three novel UNC-97 interactors are expressed in body wall muscle and by antibodies localize to M-lines. Either a decreased or an increased dosage of UNC-96 results in disorganization of thick filaments. Our previous studies showed that UNC-98, a C2H2 Zn finger protein, acts as a linkage between UNC-97, an integrin-associated protein, and MHC A in myosin thick filaments. In this study, we demonstrate another mechanism by which this linkage occurs: from UNC-97 through LIM-8 or LIM-9/UNC-96 to myosin.

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