The mechanisms and dynamics of αvβ3 integrin clustering in living cells

During cell migration, the physical link between the extracellular substrate and the actin cytoskeleton mediated by receptors of the integrin family is constantly modified. We analyzed the mechanisms that regulate the clustering and incorporation of activated αvβ3 integrins into focal adhesions. Manganese (Mn2+) or mutational activation of integrins induced the formation of de novo F-actin–independent integrin clusters. These clusters recruited talin, but not other focal adhesion adapters, and overexpression of the integrin-binding head domain of talin increased clustering. Integrin clustering required immobilized ligand and was prevented by the sequestration of phosphoinositole-4,5-bisphosphate (PI(4,5)P2). Fluorescence recovery after photobleaching analysis of Mn2+-induced integrin clusters revealed increased integrin turnover compared with mature focal contacts, whereas stabilization of the open conformation of the integrin ectodomain by mutagenesis reduced integrin turnover in focal contacts. Thus, integrin clustering requires the formation of the ternary complex consisting of activated integrins, immobilized ligands, talin, and PI(4,5)P2. The dynamic remodeling of this ternary complex controls cell motility.

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Synthesis and Characterization of ROSA Dye - A Rhodamine B-type Fluorophore, Suitable for Bioconjugation and Fluorescence Studies in Live Cells

Protein and Peptide Letters ◽

10.2174/0929866526666190619110430 ◽

2019 ◽

Vol 26 (10) ◽

pp. 758-767

Author(s):

Vicente Rubio ◽

Vijaya Iragavarapu ◽

Maciej J. Stawikowski

Keyword(s):

Quantum Yield ◽

Cancer Cells ◽

Fluorescent Probe ◽

Extinction Coefficient ◽

Rhodamine B ◽

Solid Phase ◽

Total Yield ◽

Molar Extinction Coefficient ◽

Αvβ3 Integrin ◽

Αvβ3 Integrins

Background: Herein we report the multigram-scale synthesis, characterization and application of a rhodamine B-based fluorophore (ROSA) suitable for fluorescent studies in biological applications. This fluorophore is devoid of rhodamine spirolactone formation and furthermore characterized by a high molar extinction coefficient (ϵ=87250 ± 1630 M-1cm-1) and quantum yield (φ) of 0.589 ± 0.070 in water. Reported here is also the application of ROSA towards synthesis of a ROSA-PEG-GRGDS-NH2 fluorescent probe suitable for live cell imaging of αvβ3 integrins for in vitro assays. Objective: The main objective of this study is to efficiently prepare rhodamine B derivative, devoid of spirolactone formation that would be suitable for bioconjugation and subsequent bioimaging. Methods: Rhodamine B was transformed into rhodamine B succinimide ester (RhoB-OSu) using N-hydroxysuccinimide. RhoB-OSu was further coupled to sarcosine to obtain rhodamine Bsarcosine dye (ROSA) in good yield. The ROSA dye was then coupled to a αvβ3 integrin binding sequence using standard solid-phase conditions. Resulting ROSA-PEG-GRGDS-NH2 probe was used to image integrins on cancer cells. Results: The rhodamine B-sarcosine dye (ROSA) was obtained in multigram scale in good total yield of 47%. Unlike rhodamine B, the ROSA dye does not undergo pH-dependent spirolactone/spirolactam formation as compared with rhodamine B-glycine. It is also characterized by excellent quantum yield (φ) of 0.589 ± 0.070 in water and high molar extinction coefficient of 87250 ± 1630 M-1cm-1. ROSA coupling to the RGD-like peptide was proved to be efficient and straightforward. Imaging using standard filters on multimode plate reader and confocal microscope was performed. The αvβ3 integrins present on the surface of live WM-266-4 (melanoma) and MCF- 7 (breast cancer) cells were successfully imaged. Conclusion: We successfully derivatized rhodamine B to create an inexpensive, stable and convenient to use fluorescent probe. The obtained derivative has excellent photochemical properties and it is suitable for bioconjugation and many imaging applications.

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

Communications Biology ◽

10.1038/s42003-021-01886-9 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Marta Ripamonti ◽

Nicolas Liaudet ◽

Latifeh Azizi ◽

Daniel Bouvard ◽

Vesa P. Hytönen ◽

...

Keyword(s):

Focal Adhesions ◽

Bimolecular Fluorescence Complementation ◽

Molecular Organization ◽

Structural Function ◽

Αvβ3 Integrin ◽

Lim Domain ◽

Dissociation Kinetics ◽

Mechanical Coupling ◽

Β3 Integrin

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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Structure and Function of a Vimentin-associated Matrix Adhesion in Endothelial Cells

Molecular Biology of the Cell ◽

10.1091/mbc.12.1.85 ◽

2001 ◽

Vol 12 (1) ◽

pp. 85-100 ◽

Cited By ~ 111

Author(s):

Meredith Gonzales ◽

Babette Weksler ◽

Daisuke Tsuruta ◽

Robert D. Goldman ◽

Kristine J. Yoon ◽

...

Keyword(s):

Endothelial Cells ◽

Growth Factors ◽

Endothelial Cell ◽

Branching Morphogenesis ◽

Basement Membranes ◽

Αvβ3 Integrin ◽

Dependent Manner ◽

Matrix Adhesion ◽

Focal Contacts

The α4 laminin subunit is a component of endothelial cell basement membranes. An antibody (2A3) against the α4 laminin G domain stains focal contact-like structures in transformed and primary microvascular endothelial cells (TrHBMECs and HMVECs, respectively), provided the latter cells are activated with growth factors. The 2A3 antibody staining colocalizes with that generated by αv and β3 integrin antibodies and, consistent with this localization, TrHBMECs and HMVECs adhere to the α4 laminin subunit G domain in an αvβ3-integrin–dependent manner. The αvβ3 integrin/2A3 antibody positively stained focal contacts are recognized by vinculin antibodies as well as by antibodies against plectin. Unusually, vimentin intermediate filaments, in addition to microfilament bundles, interact with many of the αvβ3 integrin-positive focal contacts. We have investigated the function of α4-laminin and αvβ3-integrin, which are at the core of these focal contacts, in cultured endothelial cells. Antibodies against these proteins inhibit branching morphogenesis of TrHBMECs and HMVECs in vitro, as well as their ability to repopulate in vitro wounds. Thus, we have characterized an endothelial cell matrix adhesion, which shows complex cytoskeletal interactions and whose assembly is regulated by growth factors. Our data indicate that this adhesion structure may play a role in angiogenesis.

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Significance of RGD Loop and C-Terminal Domain of Echistatin for Recognition of αIIbβ3 and αvβ3 Integrins and Expression of Ligand-Induced Binding Site

Blood ◽

10.1182/blood.v90.4.1565 ◽

1997 ◽

Vol 90 (4) ◽

pp. 1565-1575 ◽

Cited By ~ 82

Author(s):

Cezary Marcinkiewicz ◽

Senadhi Vijay-Kumar ◽

Mary Ann McLane ◽

Stefan Niewiarowski

Keyword(s):

Binding Site ◽

Conformational Changes ◽

Inhibitory Effect ◽

Selective Recognition ◽

Αvβ3 Integrin ◽

Inhibit Platelet Aggregation ◽

Disulfide Bridges ◽

Terminal Domain ◽

Αvβ3 Integrins ◽

Terminal Amino

Abstract Echistatin is a viper venom disintegrin containing RGD loop maintained by disulfide bridges. It binds with a high affinity to αvβ3 and αIIbβ3 and it induces extensive conformational changes in these integrins resulting in expression of ligand-induced binding site (LIBS) epitopes. We investigated the activities of echistatin and its three analogues (R24A, D27W, echistatin 1-41). R24A echistatin did not react with αIIbβ3 and αvβ3 integrins and did not cause LIBS effect. D27W echistatin showed increased binding to αIIbβ3 and decreased binding to αvβ3. This substitution impaired the ability of echistatin to induce LIBS in αvβ3 integrin. Deletion of nine C-terminal amino acids of echistatin decreased its ability to bind αIIbβ3 and inhibit platelet aggregation. Truncated echistatin failed to induce LIBS epitopes on cells transfected with αIIbβ3 and αvβ3 genes. The ability of echistatin 1-41 to compete with binding of vitronectin to immobilized αvβ3 and monoclonal antibody 7E3 to platelets and to VNRC3 cells was decreased, although this analogue, after immobilization, retained its ability to bind purified αvβ3. We propose a hypothesis in which echistatin's RGD loop determines selective recognition of αIIbβ3 and αvβ3 integrin, whereas the C-terminal domain supports its binding to resting integrin and significantly contributes to the expression of LIBS epitope and to conformational changes of the receptor, leading to a further increase of the binding affinity of echistatin and of the inhibitory effect.

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eIF2α interactions with mRNA control accurate start codon selection by the translation preinitiation complex

Nucleic Acids Research ◽

10.1093/nar/gkaa761 ◽

2020 ◽

Vol 48 (18) ◽

pp. 10280-10296

Author(s):

Anil Thakur ◽

Swati Gaikwad ◽

Anil K Vijjamarri ◽

Alan G Hinnebusch

Keyword(s):

Translation Initiation ◽

Ternary Complex ◽

Start Codon ◽

Preinitiation Complex ◽

Closed Conformation ◽

Open Conformation ◽

Kozak Context ◽

Codon Selection

Abstract In translation initiation, AUG recognition triggers rearrangement of the 48S preinitiation complex (PIC) from an open conformation to a closed state with more tightly-bound Met-tRNAi. Cryo-EM structures have revealed interactions unique to the closed complex between arginines R55/R57 of eIF2α with mRNA, including the −3 nucleotide of the ‘Kozak’ context. We found that R55/R57 substitutions reduced recognition of a UUG start codon at HIS4 in Sui− cells (Ssu− phenotype); and in vitro, R55G-R57E accelerated dissociation of the eIF2·GTP·Met-tRNAi ternary complex (TC) from reconstituted PICs with a UUG start codon, indicating destabilization of the closed complex. R55/R57 substitutions also decreased usage of poor-context AUGs in SUI1 and GCN4 mRNAs in vivo. In contrast, eIF2α-R53 interacts with the rRNA backbone only in the open complex, and the R53E substitution enhanced initiation at a UUG codon (Sui− phenotype) and poor-context AUGs, while reducing the rate of TC loading (Gcd− phenotype) in vivo. Consistently, R53E slowed TC binding to the PIC while decreasing TC dissociation at UUG codons in vitro, indicating destabilization of the open complex. Thus, distinct interactions of eIF2α with rRNA or mRNA stabilize first the open, and then closed, conformation of the PIC to influence the accuracy of initiation in vivo.

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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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A ternary complex comprising FAK, PTPα and IP3 receptor 1 functionally engages focal adhesions and the endoplasmic reticulum to mediate IL-1-induced Ca2+ signalling in fibroblasts

Biochemical Journal ◽

10.1042/bj20150907 ◽

2016 ◽

Vol 473 (4) ◽

pp. 397-410 ◽

Cited By ~ 9

Author(s):

Qin Wang ◽

Yongqiang Wang ◽

Gregory P. Downey ◽

Sergey Plotnikov ◽

Christopher A. McCulloch

Keyword(s):

Endoplasmic Reticulum ◽

Ternary Complex ◽

Focal Adhesions ◽

Eukaryotic Cells ◽

Temporal Control ◽

Ip3 Receptor ◽

Cell Adhesions ◽

Spatio Temporal ◽

Mechanistic Basis

Our studies provide a mechanistic basis by which Ca2+ release is linked to the formation of cell adhesions, thereby providing a tightly sequestered system in eukaryotic cells that enables fine spatio-temporal control of signalling.

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Immunological characterization of eristostatin and echistatin binding sites on αIIb β3 and αVβ3 integrins

Biochemical Journal ◽

10.1042/bj3170817 ◽

1996 ◽

Vol 317 (3) ◽

pp. 817-825 ◽

Cited By ~ 43

Author(s):

Cezary MARCINKIEWICZ ◽

Louis A. ROSENTHAL ◽

David M. MOSSER ◽

Thomas J. KUNICKI ◽

Stefan NIEWIAROWSKI

Keyword(s):

Cho Cells ◽

Binding Sites ◽

Sequence Similarity ◽

Chinese Hamster ◽

Amino Acid Sequence Similarity ◽

Scatchard Analysis ◽

Αvβ3 Integrin ◽

Αvβ3 Integrins ◽

20 Nm ◽

High Degree

Two disintegrins with a high degree of amino acid sequence similarity, echistatin and eristostatin, showed a low level of interaction with Chinese hamster ovary (CHO) cells, but they bound to CHO cells transfected with αIIbβ3 genes (A5 cells) and to CHO cells transfected with αvβ3 genes (VNRC3 cells) in a reversible and saturable manner. Scatchard analysis revealed that eristostatin bound to 816000 sites per A5 cell (Kd 28 nM) and to 200000 sites (Kd 14 nM) per VNRC3 cell respectively. However, VNRC3 cells did not bind to immobilized eristostatin. Echistatin bound to 495000 sites (Kd 53 nM) per A5 cell and to 443000 sites (Kd 20 nM) per VNRC3 cell. As determined by flow cytometry, radiobinding assay and adhesion studies, binding of both disintegrins to A5 cells and resting platelets and binding of echistatin to VNRC3 cells resulted in the expression of ligand-induced binding sites (LIBS) on the β3 subunit. Eristostatin inhibited, more strongly than echistatin, the binding of three monoclonal antibodies: OPG2 (RGD motif dependent), A2A9 (αIIbβ3 complex dependent) and 7E3 (αIIbβ3 and αvβ3 complex dependent) to A5 cells, to resting and to activated platelets and to purified αIIbβ3. Experiments in which echistatin and eristostatin were used alone or in combination to inhibit the binding of 7E3 and OPG2 antibodies to resting platelets suggested that these two disintegrins bind to different but overlapping sites on αIIbβ3 integrin. Monoclonal antibody LM 609 and echistatin seemed to bind to different sites on αvβ3 integrin. However, echistatin inhibited binding of 7E3 antibody to VNRC3 cells and to purified αvβ3, suggesting that αvβ3 and αIIbβ3 might share the same epitope to which both echistatin and 7E3 bind. Eristostatin had no effect in these systems, providing further evidence that it binds to a different epitope on αvβ3.

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Gelsolin Deficiency Blocks Podosome Assembly and Produces Increased Bone Mass and Strength

The Journal of Cell Biology ◽

10.1083/jcb.148.4.665 ◽

2000 ◽

Vol 148 (4) ◽

pp. 665-678 ◽

Cited By ~ 173

Author(s):

Meenakshi Chellaiah ◽

Neil Kizer ◽

Matthew Silva ◽

Ulises Alvarez ◽

David Kwiatkowski ◽

...

Keyword(s):

Bone Resorption ◽

Bone Growth ◽

Skeletal Development ◽

Critical Role ◽

Focal Adhesions ◽

Αvβ3 Integrin ◽

Endochondral Bone ◽

Trabecular Thickness ◽

Signaling Complex ◽

Cartilage Proteoglycans

Osteoclasts are unique cells that utilize podosomes instead of focal adhesions for matrix attachment and cytoskeletal remodeling during motility. We have shown that osteopontin (OP) binding to the αvβ3 integrin of osteoclast podosomes stimulated cytoskeletal reorganization and bone resorption by activating a heteromultimeric signaling complex that includes gelsolin, pp60c-src, and phosphatidylinositol 3′-kinase. Here we demonstrate that gelsolin deficiency blocks podosome assembly and αvβ3-stimulated signaling related to motility in gelsolin-null mice. Gelsolin-deficient osteoclasts were hypomotile due to retarded remodeling of the actin cytoskeleton. They failed to respond to the autocrine factor, OP, with stimulation of motility and bone resorption. Gelsolin deficiency was associated with normal skeletal development and endochondral bone growth. However, gelsolin-null mice had mildly abnormal epiphyseal structure, retained cartilage proteoglycans in metaphyseal trabeculae, and increased trabecular thickness. With age, the gelsolin-deficient mice expressed increased trabecular and cortical bone thickness producing mechanically stronger bones. These observations demonstrate the critical role of gelsolin in podosome assembly, rapid cell movements, and signal transduction through the αvβ3 integrin.

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Focal adhesion kinase regulates early steps of myofibrillogenesis in cardiomyocytes

10.1101/261248 ◽

2018 ◽

Author(s):

Nilay Taneja ◽

Abigail C. Neininger ◽

Matthew R. Bersi ◽

W. David Merryman ◽

Dylan T. Burnette

Keyword(s):

Focal Adhesion Kinase ◽

Focal Adhesion ◽

De Novo ◽

Heart Function ◽

Focal Adhesions ◽

Induced Pluripotent Stem Cell ◽

Force Balance ◽

Human Cardiomyocytes ◽

Induced Pluripotent ◽

The Stability

AbstractForces generated by myofibrils within cardiomyocytes must be balanced by adhesion to the substrate and to other cardiomyocytes for proper heart function. Loss of this force balance results in cardiomyopathies that ultimately cause heart failure. How this force balance is first established during the assembly of myofibrils is poorly understood. Using human induced pluripotent stem cell derived cardiomyocytes, we show coupling of focal adhesions to myofibrils during early steps of de novo myofibrillogenesis is essential for myofibril maturation. We also establish a key role for Focal adhesion kinase (FAK), a known regulator of adhesion dynamics in non-muscle cells, in regulating focal adhesion dynamics in cardiomyocytes. Specifically, FAK inhibition increased the stability of vinculin in focal adhesions, allowing greater substrate coupling of assembling myofibrils. Furthermore, this coupling is critical for regulating myofibril tension and viscosity. Taken together, our findings uncover a fundamental mechanism regulating the maturation of myofibrils in human cardiomyocytes.

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