scholarly journals Characterization of Integrin–Tetraspanin Adhesion Complexes

1999 ◽  
Vol 146 (2) ◽  
pp. 477-492 ◽  
Author(s):  
Fedor Berditchevski ◽  
Elena Odintsova
Keyword(s):  
Actin Cytoskeleton ◽  
Tyrosine Phosphorylation ◽  
Cellular Localization ◽  
Protein Complexes ◽  
Ectopic Expression ◽  
Cell Periphery ◽  
Integrin Receptors ◽  
Cortical Actin ◽  
In Cells

Tetraspanins (or proteins from the transmembrane 4 superfamily, TM4SF) form membrane complexes with integrin receptors and are implicated in integrin-mediated cell migration. Here we characterized cellular localization, structural composition, and signaling properties of α3β1–TM4SF adhesion complexes. Double-immunofluorescence staining showed that various TM4SF proteins, including CD9, CD63, CD81, CD82, and CD151 are colocalized within dot-like structures that are particularly abundant at the cell periphery. Differential extraction in conjunction with chemical cross-linking indicated that the cell surface fraction of α3β1–TM4SF protein complexes may not be directly linked to the cytoskeleton. However, in cells treated with cytochalasin B α3β1–TM4SF protein complexes are relocated into intracellular vesicles suggesting that actin cytoskeleton plays an important role in the distribution of tetraspanins into adhesion structures. Talin and MARCKS are partially codistributed with TM4SF proteins, whereas vinculin is not detected within the tetraspanin-containing adhesion structures. Attachment of serum-starved cells to the immobilized anti-TM4SF mAbs induced dephosphorylation of focal adhesion kinase (FAK). On the other hand, clustering of tetraspanins in cells attached to collagen enhanced tyrosine phosphorylation of FAK. Furthermore, ectopic expression of CD9 in fibrosarcoma cells affected adhesion-induced tyrosine phosphorylation of FAK, that correlated with the reorganization of the cortical actin cytoskeleton. These results show that tetraspanins can modulate integrin signaling, and point to a mechanism by which TM4SF proteins regulate cell motility.

scholarly journals Two Sides of the Coin: Ezrin/Radixin/Moesin and Merlin Control Membrane Structure and Contact Inhibition

2019 ◽  
Vol 20 (8) ◽  
pp. 1996 ◽  
Author(s):  
Katharine A. Michie ◽  
Adam Bermeister ◽  
Neil O. Robertson ◽  
Sophia C. Goodchild ◽  
Paul M. G. Curmi
Keyword(s):  
Actin Cytoskeleton ◽  
Protein Complexes ◽  
Membrane Vesicle ◽  
Contact Inhibition ◽  
Cell Junctions ◽  
Cellular Membranes ◽  
Membrane Structures ◽  
Cortical Actin ◽  
Erm Proteins ◽  
Structural Conservation

The merlin-ERM (ezrin, radixin, moesin) family of proteins plays a central role in linking the cellular membranes to the cortical actin cytoskeleton. Merlin regulates contact inhibition and is an integral part of cell–cell junctions, while ERM proteins, ezrin, radixin and moesin, assist in the formation and maintenance of specialized plasma membrane structures and membrane vesicle structures. These two protein families share a common evolutionary history, having arisen and separated via gene duplication near the origin of metazoa. During approximately 0.5 billion years of evolution, the merlin and ERM family proteins have maintained both sequence and structural conservation to an extraordinary level. Comparing crystal structures of merlin-ERM proteins and their complexes, a picture emerges of the merlin-ERM proteins acting as switchable interaction hubs, assembling protein complexes on cellular membranes and linking them to the actin cytoskeleton. Given the high level of structural conservation between the merlin and ERM family proteins we speculate that they may function together.

Calcium ions and tyrosine phosphorylation interact coordinately with actin to regulate cytoprotective responses to stretching

1997 ◽  
Vol 110 (1) ◽  
pp. 11-21 ◽  
Author(s):  
M. Glogauer ◽  
P. Arora ◽  
G. Yao ◽  
I. Sokholov ◽  
J. Ferrier ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Calcium Ions ◽  
Calcium Influx ◽  
Magnetic Bead ◽  
Mechanical Stimuli ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Force Application ◽  
Membrane Rigidity ◽  
In Cells

The actin-dependent sensory and response elements of stromal cells that are involved in mechanical signal transduction are poorly understood. To study mechanotransduction we have described previously a collagen-magnetic bead model in which application of well-defined forces to integrins induces an immediate (< 1 second) calcium influx. In this report we used the model to determine the role of calcium ions and tyrosine-phosphorylation in the regulation of force-mediated actin assembly and the resulting change in membrane rigidity. Collagen-beads were bound to cells through the focal adhesion-associated proteins talin, vinculin, alpha 2-integrin and beta-actin, indicating that force application was mediated through cytoskeletal elements. When force (2 N/m2) was applied to collagen beads, confocal microscopy showed a marked vertical extension of the cell which was counteracted by an actin-mediated retraction. Immunoblotting showed that force application induced F-actin accumulation at the bead-membrane complex but vinculin, talin and alpha 2-integrin remained unchanged. Atomic force microscopy showed that membrane rigidity increased 6-fold in the vicinity of beads which had been exposed to force. Force also induced tyrosine phosphorylation of several cytoplasmic proteins including paxillin. The force-induced actin accumulation was blocked in cells loaded with BAPTA/AM or in cells preincubated with genistein, an inhibitor of tyrosine phosphorylation. Repeated force application progressively inhibited the amplitude of force-induced calcium ion flux. As force-induced actin reorganization was dependent on calcium and tyrosine phosphorylation, and as progressive increases of filamentous actin in the submembrane cortex were correlated with increased membrane rigidity and dampened calcium influx, we suggest that cortical actin regulates stretch-activated cation permeable channel activity and provides a desensitization mechanism for cells exposed to repeated long-term mechanical stimuli. The actin response may be cytoprotective since it counteracts the initial force-mediated membrane extension and potentially strengthens cytoskeletal integrity at force-transfer points.

scholarly journals Conformational states during vinculin unlocking differentially regulate focal adhesion properties

2017 ◽  
Author(s):  
Dror S. Chorev ◽  
Tova Volberg ◽  
Ariel Livne ◽  
Miriam Eisenstein ◽  
Bruno Martins ◽  
...  
Keyword(s):  
Conformational Transition ◽  
Protein Complexes ◽  
Focal Adhesions ◽  
Active Conformation ◽  
Integrin Receptors ◽  
Adhesion Properties ◽  
Structure And Dynamics ◽  
Direct Demonstration ◽  
Growth Stability

AbstractFocal adhesions (FAs) are multi-protein complexes that connect the actin cytoskeleton to the extracellular matrix, via integrin receptors. The growth, stability and adhesive functionality of these structures are tightly regulated by mechanical stress, yet, despite the extensive characterization of the integrin adhesome, the mechanisms underlying FA mechanosensitivity are still poorly understood. One of the key candidates for regulating FA-associated mechanosensing is vinculin, a prominent FA component, which was proposed to possess either closed (“auto-inhibited”) or open (active) conformations. However, a direct demonstration of the nature of conformational transition between the two states is still absent. In this study we combined multiple structural and biological approaches to probe the transition from auto-inhibited to active conformation, and determine its effects on FA structure and dynamics. We further show here that the closed to open transition requires two sequential steps that can differentially regulate FA growth and stability.

scholarly journals Differential Phosphatidylinositol-3-Kinase-Akt-mTOR Activation by Semliki Forest and Chikungunya Viruses Is Dependent on nsP3 and Connected to Replication Complex Internalization

2015 ◽  
Vol 89 (22) ◽  
pp. 11420-11437 ◽  
Author(s):  
Bastian Thaa ◽  
Roberta Biasiotto ◽  
Kai Eng ◽  
Maarit Neuvonen ◽  
Benjamin Götte ◽  
...  
Keyword(s):  
Cell Biology ◽  
Nonstructural Protein ◽  
Ectopic Expression ◽  
Mtor Pathway ◽  
Cell Periphery ◽  
Phosphatidylinositol 3 Kinase ◽  
Replication Complex ◽  
Acidic Domain ◽  
In Cells ◽  
Phosphatidylinositol 3

ABSTRACTMany viruses affect or exploit the phosphatidylinositol-3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway, a crucial prosurvival signaling cascade. We report that this pathway was strongly activated in cells upon infection with the Old World alphavirus Semliki Forest virus (SFV), even under conditions of complete nutrient starvation. We mapped this activation to the hyperphosphorylated/acidic domain in the C-terminal tail of SFV nonstructural protein nsP3. Viruses with a deletion of this domain (SFV-Δ50) but not of other regions in nsP3 displayed a clearly delayed and reduced capacity of Akt stimulation. Ectopic expression of the nsP3 of SFV wild type (nsP3-wt), but not nsP3-Δ50, equipped with a membrane anchor was sufficient to activate Akt. We linked PI3K-Akt-mTOR stimulation to the intracellular dynamics of viral replication complexes, which are formed at the plasma membrane and subsequently internalized in a process blocked by the PI3K inhibitor wortmannin. Replication complex internalization was observed upon infection of cells with SFV-wt and SFV mutants with deletions in nsP3 but not with SFV-Δ50, where replication complexes were typically accumulated at the cell periphery. In cells infected with the closely related chikungunya virus (CHIKV), the PI3K-Akt-mTOR pathway was only moderately activated. Replication complexes of CHIKV were predominantly located at the cell periphery. Exchanging the hypervariable C-terminal tail of nsP3 between SFV and CHIKV induced the phenotype of strong PI3K-Akt-mTOR activation and replication complex internalization in CHIKV. In conclusion, infection with SFV but not CHIKV boosts PI3K-Akt-mTOR through the hyperphosphorylated/acidic domain of nsP3 to drive replication complex internalization.IMPORTANCESFV and CHIKV are very similar in terms of molecular and cell biology, e.g., regarding replication and molecular interactions, but are strikingly different regarding pathology: CHIKV is a relevant human pathogen, causing high fever and joint pain, while SFV is a low-pathogenic model virus, albeit neuropathogenic in mice. We show that both SFV and CHIKV activate the prosurvival PI3K-Akt-mTOR pathway in cells but greatly differ in their capacities to do so: Akt is strongly and persistently activated by SFV infection but only moderately activated by CHIKV. We mapped this activation capacity to a region in nonstructural protein 3 (nsP3) of SFV and could functionally transfer this region to CHIKV. Akt activation is linked to the subcellular dynamics of replication complexes, which are efficiently internalized from the cell periphery for SFV but not CHIKV. This difference in signal pathway stimulation and replication complex localization may have implications for pathology.

scholarly journals Cortactin Tyrosine Phosphorylation Requires Rac1 Activity and Association with the Cortical Actin Cytoskeleton

2003 ◽  
Vol 14 (8) ◽  
pp. 3216-3229 ◽  
Author(s):  
Julie A. Head ◽  
Dongyan Jiang ◽  
Min Li ◽  
Lynda J. Zorn ◽  
Erik M. Schaefer ◽  
...  
Keyword(s):  
Cell Motility ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Growth Factor Receptor ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Cell Periphery ◽  
Carboxy Terminus ◽  
Cortical Actin ◽  
Cortactin Tyrosine Phosphorylation

Cortactin is an F-actin binding protein that activates actin-related protein 2/3 complex and is localized within lamellipodia. Cortactin is a substrate for Src and other protein tyrosine kinases involved in cell motility, where its phosphorylation on tyrosines 421, 466, and 482 in the carboxy terminus is required for cell movement and metastasis. In spite of the importance of cortactin tyrosine phosphorylation in cell motility, little is known regarding the structural, spatial, or signaling requirements regulating cortactin tyrosine phosphorylation. Herein, we report that phosphorylation of cortactin tyrosine residues in the carboxy terminus requires the aminoterminal domain and Rac1-mediated localization to the cell periphery. Phosphorylation-specific antibodies directed against tyrosine 421 and 466 were produced to study the regulation and localization of tyrosine phosphorylated cortactin. Phosphorylation of cortactin tyrosine 421 and 466 was elevated in response to Src, epidermal growth factor receptor and Rac1 activation, and tyrosine 421 phosphorylated cortactin localized with F-actin in lamellipodia and podosomes. Cortactin tyrosine phosphorylation is progressive, with tyrosine 421 phosphorylation required for phosphorylation of tyrosine 466. These results indicate that cortactin tyrosine phosphorylation requires Rac1-induced cortactin targeting to cortical actin networks, where it is tyrosine phosphorylated in hierarchical manner that is closely coordinated with its ability to regulate actin dynamics.

scholarly journals Cytoskeleton assembly at endothelial cell–cell contacts is regulated by αII-spectrin–VASP complexes

2008 ◽  
Vol 180 (1) ◽  
pp. 205-219 ◽  
Author(s):  
Peter M. Benz ◽  
Constanze Blume ◽  
Jan Moebius ◽  
Chris Oschatz ◽  
Kai Schuh ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Ectopic Expression ◽  
Sh3 Domain ◽  
Fiber Formation ◽  
Cell Junctions ◽  
Cortical Actin ◽  
Differential Proteomics ◽  
Cell Contacts ◽  
Actin Fiber ◽  
Cell Cell

Directed cortical actin assembly is the driving force for intercellular adhesion. Regulated by phosphorylation, vasodilator-stimulated phosphoprotein (VASP) participates in actin fiber formation. We screened for endothelial proteins, which bind to VASP, dependent on its phosphorylation status. Differential proteomics identified αII-spectrin as such a VASP-interacting protein. αII-Spectrin binds to the VASP triple GP5-motif via its SH3 domain. cAMP-dependent protein kinase–mediated VASP phosphorylation at Ser157 inhibits αII-spectrin–VASP binding. VASP is dephosphorylated upon formation of cell–cell contacts and in confluent, but not in sparse cells, αII-spectrin colocalizes with nonphosphorylated VASP at cell–cell junctions. Ectopic expression of the αII-spectrin SH3 domain at cell–cell contacts translocates VASP, initiates cortical actin cytoskeleton formation, stabilizes cell–cell contacts, and decreases endothelial permeability. Conversely, the permeability of VASP-deficient endothelial cells (ECs) and microvessels of VASP-null mice increases. Reconstitution of VASP-deficient ECs rescues barrier function, whereas αII-spectrin binding-deficient VASP mutants fail to restore elevated permeability. We propose that αII-spectrin–VASP complexes regulate cortical actin cytoskeleton assembly with implications for vascular permeability.

scholarly journals The Role of Rab27a in the Regulation of Melanosome Distribution within Retinal Pigment Epithelial Cells

2004 ◽  
Vol 15 (5) ◽  
pp. 2264-2275 ◽  
Author(s):  
Clare E. Futter ◽  
José S. Ramalho ◽  
Gesine B. Jaissle ◽  
Mathias W. Seeliger ◽  
Miguel C. Seabra
Keyword(s):  
Actin Cytoskeleton ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Adherens Junction ◽  
Retinal Pigment Epithelial Cells ◽  
Cell Periphery ◽  
Light Cycle ◽  
Cortical Actin ◽  
Myosin Va ◽  
Myosin Viia

Melanosomes within the retinal pigment epithelium (RPE) of mammals have long been thought to exhibit no movement in response to light, unlike fish and amphibian RPE. Here we show that the distribution of melanosomes within the mouse RPE undergoes modest but significant changes with the light cycle. Two hours after light onset, there is a threefold increase in the number of melanosomes in the apical processes that surround adjacent photoreceptors. In skin melanocytes, melanosomes are motile and evenly distributed throughout the cell periphery. This distribution is due to the interaction with the cortical actin cytoskeleton mediated by a tripartite complex of Rab27a, melanophilin, and myosin Va. In ashen (Rab27a null) mice RPE, melanosomes are unable to move beyond the adherens junction axis and do not enter apical processes, suggesting that Rab27a regulates melanosome distribution in the RPE. Unlike skin melanocytes, the effects of Rab27a are mediated through myosin VIIa in the RPE, as evidenced by the similar melanosome distribution phenotype observed in shaker-1 mice, defective in myosin VIIa. Rab27a and myosin VIIa are likely to be required for association with and movement through the apical actin cytoskeleton, which is a prerequisite for entry into the apical processes.

scholarly journals Adenovirus Endocytosis Requires Actin Cytoskeleton Reorganization Mediated by Rho Family GTPases

1998 ◽  
Vol 72 (11) ◽  
pp. 8806-8812 ◽  
Author(s):  
Erguang Li ◽  
Dwayne Stupack ◽  
Gary M. Bokoch ◽  
Glen R. Nemerow
Keyword(s):  
Actin Cytoskeleton ◽  
Dominant Negative ◽  
Cortical Actin ◽  
Lipid Kinase ◽  
Ras Protein ◽  
Cytoskeleton Reorganization ◽  
Pathway Activation ◽  
Rho Family Gtpases ◽  
Rho Family ◽  
In Cells

ABSTRACT Adenovirus (Ad) endocytosis via αv integrins requires activation of the lipid kinase phosphatidylinositol-3-OH kinase (PI3K). Previous studies have linked PI3K activity to both the Ras and Rho signaling cascades, each of which has the capacity to alter the host cell actin cytoskeleton. Ad interaction with cells also stimulates reorganization of cortical actin filaments and the formation of membrane ruffles (lamellipodia). We demonstrate here that members of the Rho family of small GTP binding proteins, Rac and CDC42, act downstream of PI3K to promote Ad endocytosis. Ad internalization was significantly reduced in cells treated with Clostridium difficile toxin B and in cells expressing a dominant-negative Rac or CDC42 but not a H-Ras protein. Viral endocytosis was also inhibited by cytochalasin D as well as by expression of effector domain mutants of Rac or CDC42 that impair cytoskeletal function but not JNK/MAP kinase pathway activation. Thus, Ad endocytosis requires assembly of the actin cytoskeleton, an event initiated by activation of PI3K and, subsequently, Rac and CDC42.

scholarly journals Identification and Characterization of Schizosaccharomyces pombe asp1+, a Gene That Interacts with Mutations in the Arp2/3 Complex and Actin

Genetics ◽  
1999 ◽  
Vol 152 (3) ◽  
pp. 895-908
Author(s):  
Anna Feoktistova ◽  
Dannel McCollum ◽  
Ryoma Ohi ◽  
Kathleen L Gould
Keyword(s):  
Actin Cytoskeleton ◽  
Schizosaccharomyces Pombe ◽  
Elevated Temperatures ◽  
Genetic Interaction ◽  
Cortical Actin ◽  
Reading Frame ◽  
Growth Properties ◽  
Ca Ions ◽  
Synthetically Lethal

Abstract The Arp2/3 complex is an essential component of the actin cytoskeleton in yeast and is required for the movement of actin patches. In an attempt to identify proteins that interact with this complex in the fission yeast Schizosaccharomyces pombe, we sought high-copy suppressors of the S. pombe arp3-c1 mutant, and have identified one, which we have termed asp1+. The asp1+ open reading frame (ORF) predicts a highly conserved protein of 921 amino acids with a molecular mass of 106 kD that does not contain motifs of known function. Neither asp1+ nor its apparent Saccharomyces cerevisiae ortholog, VIP1, are essential genes. However, disruption of asp1+ leads to altered morphology and growth properties at elevated temperatures and defects in polarized growth. The asp1 disruption strain also is hypersensitive to Ca+ ions and to low pH conditions. Although Asp1p is not stably associated with the Arp2/3 complex nor localized in any discrete structure within the cytoplasm, the asp1 disruption mutant was synthetically lethal with mutations in components of the Arp2/3 complex, arp3-c1 and sop2-1, as well as with a mutation in actin, act1-48. Moreover, the vip1 disruption strain showed a negative genetic interaction with a las17Δ strain. We conclude that Asp1p/Vip1p is important for the function of the cortical actin cytoskeleton.

The Immuno-Electron Microscopic Localization of the Mo-Fe Protein Component of Nitrogenase in Cells of Azotobacter Vinelandii

1973 ◽  
Vol 31 ◽  
pp. 574-575
Author(s):  
J. T. Stasny ◽  
R. C. Burns ◽  
R. W. F. Hardy
Keyword(s):  
Cellular Localization ◽  
Direct Evidence ◽  
Azotobacter Vinelandii ◽  
Intracellular Localization ◽  
Protein Component ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Fe Protein ◽  
Intracytoplasmic Membranes

Structure-functlon studies of biological N2-fixation have correlated the presence of the enzyme nitrogenase with increased numbers of intracytoplasmic membranes in Azotobacter. However no direct evidence has been provided for the internal cellular localization of any nitrogenase. Recent advances concerned with the crystallizatiorTand the electron microscopic characterization of the Mo-Fe protein component of Azotobacter nitrogenase, prompted the use of this purified protein to obtain antibodies (Ab) to be conjugated to electron dense markers for the intracellular localization of the protein by electron microscopy. The present study describes the use of ferritin conjugated to goat antitMo-Fe protein immunoglobulin (IgG) and the observations following its topical application to thin sections of N2-grown Azotobacter.

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