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.

Effects of Ca2+ and Mg2+ on ATP-dependent 45Ca2+ influx in A-431 human epidermoidal carcinoma cells

1991 ◽  
Vol 69 (1) ◽  
pp. 29-35 ◽  
Author(s):  
Kenji Sugita ◽  
Kazuo Hosoi ◽  
Yoshimi Shioda ◽  
Takao Ueha
Keyword(s):  
Calcium Ions ◽  
Calcium Influx ◽  
Inositol Trisphosphate ◽  
Cytosolic Calcium ◽  
Carcinoma Cells ◽  
Dependent Manner ◽  
Parallel Effect ◽  
Intracellular Calcium Ion ◽  
Preincubation Medium ◽  
In Cells

Upon stimulation with 10−6–10−3 M ATP, A-431 human epidermoidal carcinoma cells incorporated radioactive calcium from their medium in a temperature-dependent manner. The rate of incorporation of 45Ca2+ was rapid for the initial 5 min, but decreased immediately thereafter. The preincubation of cells for 2 h in medium depleted of both Ca2+ and Mg2+ abolished the ATP-dependent 45Ca2+ incorporation, irrespective of whether or not the subsequent incubation medium contained Mg2+ ions. ATP-dependent 45Ca2+ incorporation could be restored by a second preincubation (1 h) in medium containing 1 mM Mg2+, but no Ca2+. The Mg2+ ions in the second preincubation medium could be replaced by Ca2+, Co2+, or Cu2+ for restoration of such activity. Elevation of inositol trisphosphate (InsP3) was observed in cells depleted of either Ca2+ or Mg2+, but not in cells depleted of both ions. A parallel effect was observed in changes in [Ca2+]i. Since the concentration of cytosolic calcium ions does not change by incubation of cells in medium depleted of and (or) restored with calcium ions, we conclude that either calcium or magnesium ions associated with some cellular component(s) are responsible for production of InsP3, which then supposedly mobilizes Ca2+ and provokes 45Ca2+ influx.Key words: intracellular calcium ion, inositol trisphosphate, calcium influx.

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.

Protein tyrosine phosphatase-α complexes with the IGF-I receptor and undergoes IGF-I-stimulated tyrosine phosphorylation that mediates cell migration

2009 ◽  
Vol 297 (1) ◽  
pp. C133-C139 ◽  
Author(s):  
Shirley C. Chen ◽  
Ranvikram S. Khanna ◽  
Darrell C. Bessette ◽  
Lionel A. Samayawardhena ◽  
Catherine J. Pallen
Keyword(s):  
Cell Migration ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Kinases ◽  
Tyrosine Phosphatase ◽  
Phosphorylation Site ◽  
Protein Tyrosine ◽  
Fibroblast Migration ◽  
Igf I ◽  
In Cells

Protein tyrosine phosphatase-α (PTPα) is a widely expressed receptor-type phosphatase that functions in multiple signaling systems. The actions of PTPα can be regulated by its phosphorylation on serine and tyrosine residues, although little is known about the conditions that promote PTPα phosphorylation. In this study, we tested the ability of several extracellular factors to stimulate PTPα tyrosine phosphorylation. The growth factors IGF-I and acidic FGF induced the highest increase in PTPα phosphorylation at tyrosine 789, followed by PMA and lysophosphatidic acid, while EGF had little effect. Further investigation of IGF-I-induced PTPα tyrosine phosphorylation demonstrated that this occurs through a novel Src family kinase-independent mechanism that does not require focal adhesion kinase, phosphatidylinositol 3-kinase, or MEK. We also show that PTPα physically interacts with the IGF-I receptor. In contrast to IGF-I-induced PTPα phosphorylation, this association does not require IGF-I. The interaction of PTPα and the IGF-I receptor is independent of PTPα catalytic activity, and expression of exogenous PTPα does not promote IGF-I receptor tyrosine dephosphorylation, indicating that PTPα does not act as an IGF-I receptor phosphatase. However, PTPα mediates IGF-I signaling, because IGF-I-stimulated fibroblast migration was reduced by ∼50% in cells lacking PTPα or in cells with mutant PTPα lacking the tyrosine 789 phosphorylation site. Our results suggest that PTPα tyrosine phosphorylation can occur in response to diverse stimuli and can be mediated by various tyrosine kinases. In the case of IGF-I, we propose that IGF-I-induced tyrosine 789 phosphorylation of PTPα, possibly catalyzed by the PTPα-associated IGF-I receptor tyrosine kinase, is required for efficient cell migration in response to this growth factor.

scholarly journals Osmotic stress and the yeast cytoskeleton: phenotype-specific suppression of an actin mutation.

1992 ◽  
Vol 118 (3) ◽  
pp. 561-571 ◽  
Author(s):  
S Chowdhury ◽  
K W Smith ◽  
M C Gustin
Keyword(s):  
Osmotic Stress ◽  
Actin Filament ◽  
Physiological Process ◽  
Actin Binding ◽  
Temperature Sensitive ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Yeast Saccharomyces Cerevisiae ◽  
Rhodamine Phalloidin ◽  
Diploid Cells

In the yeast Saccharomyces cerevisiae, actin filaments function to direct cell growth to the emerging bud. Yeast has a single essential actin gene, ACT1. Diploid cells containing a single copy of ACT1 are osmosensitive (Osms), i.e., they fail to grow in high osmolarity media (D. Shortle, unpublished observations cited by Novick, P., and D. Botstein. 1985. Cell. 40:415-426). This phenotype suggests that an underlying physiological process involving actin is osmosensitive. Here, we demonstrate that this physiological process is a rapid and reversible change in actin filament organization in cells exposed to osmotic stress. Filamentous actin was stained using rhodamine phalloidin. Increasing external osmolarity caused a rapid loss of actin filament cables, followed by a slower redistribution of cortical actin filament patches. In the recovery phase, cables and patches were restored to their original levels and locations. Strains containing an act1-1 mutation are both Osms and temperature-sensitive (Ts) (Novick and Botstein, 1985). To identify genes whose products functionally interact with actin in cellular responses to osmotic stress, we have isolated extragenic suppressors which revert only the Osms but not the Ts phenotype of an act1-1 mutant. These suppressors identify three genes, RAH1-RAH3. Morphological and genetic properties of a dominant suppressor mutation suggest that the product of the wild-type allele, RAH3+, is an actin-binding protein that interacts with actin to allow reassembly of the cytoskeleton following osmotic stress.

Activation of c-Src in cells bearing v-Crk and its suppression by Csk

1992 ◽  
Vol 12 (10) ◽  
pp. 4706-4713
Author(s):  
H Sabe ◽  
M Okada ◽  
H Nakagawa ◽  
H Hanafusa
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Src Kinase ◽  
Cellular Protein ◽  
Morphological Transformation ◽  
Protein Tyrosine ◽  
In Cells

The protein product of the CT10 virus, p47gag-crk (v-Crk), which contains Src homology region 2 (SH2) and 3 (SH3) domains but lacks a kinase domain, is believed to cause an increase in cellular protein tyrosine phosphorylation. A candidate tyrosine kinase, Csk (C-terminal Src kinase), has been implicated in c-Src Tyr-527 phosphorylation, which negatively regulates the protein tyrosine kinase of pp60c-src (c-Src). To investigate how c-Src kinase activity is regulated in vivo, we first looked at whether v-Crk can activate c-Src kinase. We found that cooverexpression of v-Crk and c-Src caused elevation of c-Src kinase activity, resulting in an increase of tyrosine phosphorylation of cellular proteins and morphological transformation of rat 3Y1 fibroblasts. v-Crk and c-Src complexes were not detected, although v-Crk bound to a variety of tyrosine-phosphorylated proteins in cells overexpressing v-Crk and c-Src. Overexpression of Csk in these transformed cells caused reversion to normal phenotypes and also reduced the level of c-Src kinase activity. However, Csk did not cause reversion of cells transformed by v-Src or c-Src527F, in which Tyr-527 was changed to Phe. These results strongly suggest that Csk acts on Tyr-527 of c-Src and suppresses c-Src kinase activity in vivo. Because Csk can suppress transformation by cooverexpression of v-Crk and c-Src, we suggest that v-Crk causes activation of c-Src in vivo by altering the phosphorylation state of Tyr-527.

scholarly journals Signal transduction mechanisms involved in in vitro ram sperm capacitation

Reproduction ◽  
2006 ◽  
Vol 132 (5) ◽  
pp. 721-732 ◽  
Author(s):  
Patricia Grasa ◽  
José Álvaro Cebrián-Pérez ◽  
Teresa Muiño-Blanco
Keyword(s):  
Tyrosine Phosphorylation ◽  
Calcium Influx ◽  
Calcium Entry ◽  
Calcium Entry Blocker ◽  
Protein Tyrosine Phosphorylation ◽  
Sperm Capacitation ◽  
Protein Tyrosine ◽  
Entry Blocker ◽  
Ram Sperm

We validate the chlortetracycline (CTC) technique for the evaluation of capacitation and acrosome reaction-like changes in ram sperm, carrying out a double estimation of the acrosome status after treatment with lysophosphatidylcholine, using fluorescein isocyanate (FITC)-RCA/ethidium homodimer 1 (EthD-1) and CTC/EthD-1. Highly consistent results and a positive correlation between the results of acrosome-reacted sperm evaluated with both techniques were obtained. In this study, we evaluate the effects of ram sperm capacitation of BSA, Ca2+, NaHCO3and cAMP agonists and their influence on the associated protein tyrosine phosphorylation. We found a time-dependent increase in capacitation related to protein tyrosine phosphorylation, either in the absence or the presence of BSA. The addition of an increasing concentration of cholesterol to samples containing BSA did not influence results. The effect of bicarbonate was concentration-dependent, with a significantly lowered value of non-capacitated sperm in the presence 18 and 25 mM. The addition of extracellular calcium did not significantly increase either the proportion of capacitated sperm or the protein tyrosine phosphorylation signalling, although a significantly higher value of acrosome-reacted sperm was found in samples containing 4 mM Ca2+. cAMP agonists increased capacitated sperm and protein tyrosine phosphorylation signalling. The inhibition of protein kinase A by H-89 caused a decrease in sperm capacitation. Addition of a calcium-entry blocker (Verapamil; Sigma) did not influence results, which suggests that the calcium entry blocker was unable to inhibit the calcium influx associated with capacitation in ram sperm. Our findings might benefit our understanding of the biochemical mechanisms involved in mammalian sperm capacitation and ultimately, fertility.

scholarly journals Small GTP-binding Protein TC10 Differentially Regulates Two Distinct Populations of Filamentous Actin in 3T3L1 Adipocytes

2002 ◽  
Vol 13 (7) ◽  
pp. 2334-2346 ◽  
Author(s):  
Makoto Kanzaki ◽  
Robert T. Watson ◽  
June Chunqiu Hou ◽  
Mark Stamnes ◽  
Alan R. Saltiel ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Actin Polymerization ◽  
Coat Proteins ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Amino Terminal ◽  
Gtp Binding ◽  
Subcellular Compartmentalization ◽  
Constitutively Active

TC10 is a member of the Rho family of small GTP-binding proteins that has previously been implicated in the regulation of insulin-stimulated GLUT4 translocation in adipocytes. In a manner similar to Cdc42-stimulated actin-based motility, we have observed that constitutively active TC10 (TC10/Q75L) can induce actin comet tails in Xenopus oocyte extracts in vitro and extensive actin polymerization in the perinuclear region when expressed in 3T3L1 adipocytes. In contrast, expression of TC10/Q75L completely disrupted adipocyte cortical actin, which was specific for TC10, because expression of constitutively active Cdc42 was without effect. The effect of TC10/Q75L to disrupt cortical actin was abrogated after deletion of the amino terminal extension (ΔN-TC10/Q75L), whereas this deletion retained the ability to induce perinuclear actin polymerization. In addition, alteration of perinuclear actin by expression of TC10/Q75L, a dominant-interfering TC10/T31N mutant or a mutant N-WASP protein (N-WASP/ΔVCA) reduced the rate of VSV G protein trafficking to the plasma membrane. Furthermore, TC10 directly bound to Golgi COPI coat proteins through a dilysine motif in the carboxyl terminal domain consistent with a role for TC10 regulating actin polymerization on membrane transport vesicles. Together, these data demonstrate that TC10 can differentially regulate two types of filamentous actin in adipocytes dependent on distinct functional domains and its subcellular compartmentalization.

scholarly journals Mechanosensitive calcium signaling promotes epithelial tight junction remodeling by activating RhoA

2021 ◽  
Author(s):  
Saranyaraajan Varadarajan ◽  
Rachel E. Stephenson ◽  
Eileen R. Misterovich ◽  
Jessica L. Wu ◽  
Ivan S. Erofeev ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intracellular Calcium ◽  
Tight Junctions ◽  
Calcium Influx ◽  
Cell Junctions ◽  
Mechanical Stimuli ◽  
Local Repair ◽  
Transient Activation ◽  
Epithelial Tight Junction ◽  
Sense And Respond

Epithelia maintain an effective barrier by remodeling cell-cell junctions in response to mechanical stimuli. Cells often respond to mechanical stress through activating RhoA and remodeling actomyosin. Previously, we found that local leaks in the barrier are rapidly repaired by localized, transient activation of RhoA – ″Rho flares″ – but how Rho flares are initiated remains unknown. Here, we discovered that intracellular calcium flashes occur in Xenopus laevis epithelial cells undergoing Rho flare-mediated remodeling of tight junctions. Calcium flashes originate at the site of barrier leaks and propagate into the cell. Depletion of intracellular calcium or inhibition of mechanosensitive calcium channels (MSC) reduced the amplitude of calcium flashes and diminished the activation of Rho flares. Furthermore, MSC-dependent calcium influx was necessary to maintain global barrier function by regulating local repair of tight junctions through efficient junction contraction. We propose that MSC-dependent calcium flashes are an important mechanism allowing epithelial cells to sense and respond to local leaks induced by mechanical stimuli.

scholarly journals Dual pools of actin at presynaptic terminals

2012 ◽  
Vol 107 (12) ◽  
pp. 3479-3492 ◽  
Author(s):  
Adam Bleckert ◽  
Huzefa Photowala ◽  
Simon Alford
Keyword(s):  
Synaptic Transmission ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Repetitive Stimulation ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Vesicle Recycling ◽  
Latrunculin A ◽  
Kinetics Of

We investigated actin's function in vesicle recycling and exocytosis at lamprey synapses and show that FM1-43 puncta and phalloidin-labeled filamentous actin (F-actin) structures are colocalized, yet recycling vesicles are not contained within F-actin clusters. Additionally, phalloidin also labels a plasma membrane-associated cortical actin. Injection of fluorescent G-actin revealed activity-independent dynamic actin incorporation into presynaptic synaptic vesicle clusters but not into cortical actin. Latrunculin-A, which sequesters G-actin, dispersed vesicle-associated actin structures and prevented subsequent labeled G-actin and phalloidin accumulation at presynaptic puncta, yet cortical phalloidin labeling persisted. Dispersal of presynaptic F-actin structures by latrunculin-A did not disrupt vesicle clustering or recycling or alter the amplitude or kinetics of excitatory postsynaptic currents (EPSCs). However, it slightly enhanced release during repetitive stimulation. While dispersal of presynaptic actin puncta with latrunculin-A failed to disperse synaptic vesicles or inhibit synaptic transmission, presynaptic phalloidin injection blocked exocytosis and reduced endocytosis measured by action potential-evoked FM1-43 staining. Furthermore, phalloidin stabilization of only cortical actin following pretreatment with latrunculin-A was sufficient to inhibit synaptic transmission. Conversely, treatment of axons with jasplakinolide, which induces F-actin accumulation but disrupts F-actin structures in vivo, resulted in increased synaptic transmission accompanied by a loss of phalloidin labeling of cortical actin but no loss of actin labeling within vesicle clusters. Marked synaptic deficits seen with phalloidin stabilization of cortical F-actin, in contrast to the minimal effects of disruption of a synaptic vesicle-associated F-actin, led us to conclude that two structurally and functionally distinct pools of actin exist at presynaptic sites.

Anti-calmodulin agents affect osmotic and angiotensin II-induced vasopressin release

1989 ◽  
Vol 256 (4) ◽  
pp. E516-E523 ◽  
Author(s):  
N. F. Rossi ◽  
R. W. Schrier
Keyword(s):  
Angiotensin Ii ◽  
Calcium Ions ◽  
Calcium Influx ◽  
Alternative Mechanism ◽  
Hormone Release ◽  
Ang Ii ◽  
Vasopressin Release ◽  
Blocking Effects ◽  
Osmotic Stimulation ◽  
Crucial Part

Calcium ions and particularly calcium influx play a crucial part in initiating the intracellular events that result in arginine vasopressin (AVP) release to both osmotic and nonosmotic stimuli. Calmodulin appears to modulate the effects of calcium on synaptic transmission and hormone release in other systems. This study tested the effects of three distinct classes of anti-calmodulin agents on the release of AVP to either a rise in osmolality of 20 mosmol/kg water or to 1 X 10(-5) angiotensin II (ANG II) in cultured hypothalamo-neurohypophysical complexes. Micromolar concentrations of R 24571, the active naphthalenesulfonamides, W 7 and W 13, and trifluoperazine (TFP) inhibited AVP release to osmotic stimulation. In contrast, W 5, a severalfold less active anti-calmodulin agent, had no effect on osmotically stimulated AVP release. The active naphthalenesulfonamides, but not R 24571 or TFP, blocked release of AVP to ANG II. In contrast, neither R 24571 nor TFP inhibited AVP release to ANG II stimulation. Collectively, the data demonstrated a dissociation between inhibition of AVP release and the anti-calmodulin properties of the drugs, thereby suggesting that nonspecific actions masked the calmodulin-blocking effects of the drugs or that the inhibition occurred by some alternative mechanism(s).

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