scholarly journals Disruption of F-actin stimulates hypertonic activation of the BGT1 transporter in MDCK cells

2003 ◽  
Vol 284 (5) ◽  
pp. F930-F937 ◽  
Author(s):  
Jeremy L. Bricker ◽  
Shaoyou Chu ◽  
Stephen A. Kempson
Keyword(s):  
Actin Cytoskeleton ◽  
Mdck Cells ◽  
Major Change ◽  
Cytochalasin D ◽  
Transport Systems ◽  
Microtubule Network ◽  
Cell Monolayers ◽  
Latrunculin A ◽  
Membrane Transport Systems

Many membrane transport systems are altered by changes in the state of the actin cytoskeleton. Although an intact microtubule network is required for hypertonic activation of the betaine transporter (BGT1), the possible role of the actin cytoskeleton is unknown. BGT1 function in Madin-Darby canine kidney cell monolayers was assessed as Na+-dependent uptake of GABA, following disassembly of F-actin by cytochalasin D (1.0 μM) or latrunculin A (0.6 μM). Both drugs significantly increased ( P < 0.001) the activation of BGT1 transport by 24-h hypertonicity (500 mosmol/kgH2O). In contrast, the hypertonic upregulation of Na+-dependent alanine uptake remained unaltered by cytochalasin D. Disruption of F-actin did not interfere with downregulation of BGT1 transport when cells were transferred from hypertonic to isotonic medium. Immunofluorescence staining revealed colocalization of BGT1 and F-actin at the plasma membrane of hypertonic cells. Surface biotinylation revealed no major change in BGT1 protein abundance after cytochalasin D action, suggesting that stimulation of hypertonic activation of BGT1 transport is due to increased activity of existing BGT1 transporters.

The tricornered Gene, Which Is Required for the Integrity of Epidermal Cell Extensions, Encodes the Drosophila Nuclear DBF2-Related Kinase

Genetics ◽  
2000 ◽  
Vol 156 (4) ◽  
pp. 1817-1828 ◽  
Author(s):  
Wei Geng ◽  
Biao He ◽  
Mina Wang ◽  
Paul N Adler
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Cell Structure ◽  
Cytochalasin D ◽  
Epidermal Cells ◽  
Sense Organs ◽  
Latrunculin A ◽  
Cellular Extensions ◽  
Cuticular Structures ◽  
Cuticular Surface

Abstract During their differentiation epidermal cells of Drosophila form a rich variety of polarized structures. These include the epidermal hairs that decorate much of the adult cuticular surface, the shafts of the bristle sense organs, the lateral extensions of the arista, and the larval denticles. These cuticular structures are produced by cytoskeletal-mediated outgrowths of epidermal cells. Mutations in the tricornered gene result in the splitting or branching of all of these structures. Thus, tricornered function appears to be important for maintaining the integrity of the outgrowths. tricornered mutations however do not have major effects on the growth or shape of these cellular extensions. Inhibiting actin polymerization in differentiating cells by cytochalasin D or latrunculin A treatment also induces the splitting of hairs and bristles, suggesting that the actin cytoskeleton might be a target of tricornered. However, the drugs also result in short, fat, and occasionally malformed hairs and bristles. The data suggest that the function of the actin cytoskeleton is important for maintaining the integrity of cellular extensions as well as their growth and shape. Thus, if tricornered causes the splitting of cellular extensions by interacting with the actin cytoskeleton it likely does so in a subtle way. Consistent with this possibility we found that a weak tricornered mutant is hypersensitive to cytochalasin D. We have cloned the tricornered gene and found that it encodes the Drosophila NDR kinase. This is a conserved ser/thr protein kinase found in Caenorhabditis elegans and humans that is related to a number of kinases that have been found to be important in controlling cell structure and proliferation.

scholarly journals Transfection of nonmuscle beta- and gamma-actin genes into myoblasts elicits different feedback regulatory responses from endogenous actin genes

1992 ◽  
Vol 117 (4) ◽  
pp. 787-797 ◽  
Author(s):  
C Lloyd ◽  
G Schevzov ◽  
P Gunning
Keyword(s):  
Actin Cytoskeleton ◽  
Feedback Regulation ◽  
Cytochalasin D ◽  
Actin Gene ◽  
Down Regulation ◽  
Actin Genes ◽  
Beta Actin ◽  
Actin Mrna ◽  
Actin Protein

We have examined the role of feedback-regulation in the expression of the nonmuscle actin genes. C2 mouse myoblasts were transfected with the human beta- and gamma-actin genes. In gamma-actin transfectants we found that the total actin mRNA and protein pools remained unchanged. Increasing levels of human gamma-actin expression resulted in a progressive down-regulation of mouse beta- and gamma-actin mRNAs. Transfection of the beta-actin gene resulted in an increase in the total actin mRNA and protein pools and induced an increase in the levels of mouse beta-actin mRNA. In contrast, transfection of a beta-actin gene carrying a single-point mutation (beta sm) produced a feedback-regulatory response similar to that of the gamma-actin gene. Expression of a beta-actin gene encoding an unstable actin protein had no impact on the endogenous mouse actin genes. This suggests that the nature of the encoded actin protein determines the feedback-regulatory response of the mouse genes. The role of the actin cytoskeleton in mediating this feedback-regulation was evaluated by disruption of the actin network with Cytochalasin D. We found that treatment with Cytochalasin D abolished the down-regulation of mouse gamma-actin in both the gamma- and beta sm-actin transfectants. In contrast, a similar level of increase was observed for the mouse beta-actin mRNA in both control and transfected cells. These experiments suggest that the down-regulation of mouse gamma-actin mRNA is dependent on the organization of the actin cytoskeleton. In addition, the mechanism responsible for the down-regulation of beta-actin may be distinct from that governing gamma-actin. We conclude that actin feedback-regulation provides a biochemical assay for differences between the two nonmuscle actin genes.

Oxygen-sensitive membrane transporters in vertebrate red cells

2000 ◽  
Vol 203 (9) ◽  
pp. 1395-1407 ◽  
Author(s):  
J.S. Gibson ◽  
A.R. Cossins ◽  
J.C. Ellory
Keyword(s):  
Membrane Transporters ◽  
Red Cells ◽  
Transport Systems ◽  
Red Cell ◽  
Organic Systems ◽  
Wide Range ◽  
Oxygen Sensitive ◽  
Radical Generation ◽  
Membrane Transport Systems

Oxygen is essential for all higher forms of animal life. It is required for oxidative phosphorylation, which forms the bulk of the energy supply of most animals. In many vertebrates, transport of O(2) from respiratory to other tissues, and of CO(2) in the opposite direction, involves red cells. These are highly specialised, adapted for their respiratory function. Intracellular haemoglobin, carbonic anhydrase and the membrane anion exchanger (AE1) increase the effective O(2)- and CO(2)-carrying capacity of red cells by approximately 100-fold. O(2) also has a pathological role. It is a very reactive species chemically, and oxidation, free radical generation and peroxide formation can be major hazards. Cells that come into contact with potentially damaging levels of O(2) have a variety of systems to protect them against oxidative damage. Those in red cells include catalase, superoxide dismutase and glutathione. In this review, we focus on a third role of O(2), as a regulator of membrane transport systems, a role with important consequences for the homeostasis of the red cell and also the organism as a whole. We show that regulation of red cell transporters by O(2) is widespread throughout the vertebrate kingdom. The effect of O(2) is selective but involves a wide range of transporters, including inorganic and organic systems, and both electroneutral and conductive pathways. Finally, we discuss what is known about the mechanism of the O(2) effect and comment on its physiological and pathological roles.

scholarly journals The obligatory role of the actin cytoskeleton on inward remodeling induced by dithiothreitol activation of endogenous transglutaminase in isolated arterioles

2014 ◽  
Vol 306 (4) ◽  
pp. H485-H495 ◽  
Author(s):  
Jorge A. Castorena-Gonzalez ◽  
Marius C. Staiculescu ◽  
Christopher A. Foote ◽  
Luis Polo-Parada ◽  
Luis A. Martinez-Lemus
Keyword(s):  
Structural Change ◽  
Actin Cytoskeleton ◽  
Wall Thickness ◽  
Actin Polymerization ◽  
Cytochalasin D ◽  
Actin Dynamics ◽  
Resistance Arteries ◽  
Early Stages ◽  
Elastic Characteristics

Inward remodeling is the most prevalent structural change found in the resistance arteries and arterioles of hypertensive individuals. Separate studies have shown that the inward remodeling process requires transglutaminase activation and the polymerization of actin. Therefore, we hypothesize that inward remodeling induced via endogenous transglutaminase activation requires and depends on actin cytoskeletal structures. To test this hypothesis, isolated and cannulated rat cremaster arterioles were exposed to dithiothreitol (DTT) to activate endogenous transglutaminases. DTT induced concentration-dependent vasoconstriction that was suppressed by coincubation with cystamine or cytochalasin-D to inhibit tranglutaminase activity or actin polymerization, respectively. Prolonged (4 h) exposure to DTT caused arteriolar inward remodeling that was also blocked by the presence of cystamine or cytochalasin-D. DTT inwardly remodeled arterioles had reduced passive diameters, augmented wall thickness-to-lumen ratios and altered elastic characteristics that were reverted upon disruption of the actin cytoskeleton with mycalolide-B. In freshly isolated arterioles, exposure to mycalolide-B caused no changes in their passive diameters or their elastic characteristics. These results suggest that, in arterioles, the early stages of the inward remodeling process induced by prolonged endogenous transglutaminase activation require actin dynamics and depend on changes in actin cytoskeletal structures.

Cytoskeletal modulation of sodium current in human jejunal circular smooth muscle cells

2003 ◽  
Vol 284 (1) ◽  
pp. C60-C66 ◽  
Author(s):  
Peter R. Strege ◽  
Adrian N. Holm ◽  
Adam Rich ◽  
Steven M. Miller ◽  
Yijun Ou ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Smooth Muscle Cells ◽  
Actin Cytoskeleton ◽  
Intermediate Filaments ◽  
Sodium Current ◽  
Muscle Cells ◽  
Cytochalasin D ◽  
Circular Smooth Muscle

A Na+ current is present in human jejunal circular smooth muscle cells. The aim of the present study was to determine the role of the cytoskeleton in the regulation of the Na+ current. Whole cell currents were recorded by using standard patch-clamp techniques with Cs+ in the pipette to block K+currents. Cytochalasin D and gelsolin were used to disrupt the actin cytoskeleton and phalloidin to stabilize it. Colchicine was used to disassemble the microtubule cytoskeleton (and intermediate filaments) and paclitaxel to stabilize it. Acrylamide was used to disrupt the intermediate filament cytoskeleton. Perfusion of the recording chamber at 10 ml/min increased peak Na+ current recorded from jejunal smooth muscle cells by 27 ± 3%. Cytochalasin D and gelsolin abolished the perfusion-induced increase in Na+current, whereas incubation with phalloidin, colchicine, paclitaxel, or acrylamide had no effect. In conclusion, the Na+ current expressed in human jejunal circular smooth muscle cells appears to be regulated by the cytoskeleton. An intact actin cytoskeleton is required for perfusion-induced activation of the Na+ current.

scholarly journals c-Jun NH2-terminal kinase-2 mediates osmotic stress-induced tight junction disruption in the intestinal epithelium

2010 ◽  
Vol 299 (3) ◽  
pp. G572-G584 ◽  
Author(s):  
G. Samak ◽  
T. Suzuki ◽  
A. Bhargava ◽  
R. K. Rao
Keyword(s):  
Osmotic Stress ◽  
Actin Cytoskeleton ◽  
Restraint Stress ◽  
Paracellular Permeability ◽  
Cell Monolayers ◽  
Cold Restraint Stress ◽  
Jnk Activation ◽  
Mouse Ileum ◽  
Cold Restraint

Gastrointestinal epithelium faces osmotic stress, both at physiological and pathophysiological conditions. JNK activation is an immediate cellular response to osmotic stress. We investigated the effect of osmotic stress on intestinal epithelial barrier function and delineated the role of JNK2 in osmotic stress-induced tight junction (TJ) regulation in Caco-2 cell monolayers and ileum of Jnk −/− and Jnk2 −/− mice. The role of JNK activation in osmotic stress-induced TJ disruption was evaluated using JNK-specific inhibitor and antisense oligonucleotides. Furthermore, the effect of cold restraint stress in vivo on TJ integrity was determined in rats. Osmotic stress disrupted TJs and barrier function in Caco-2 cell monolayers without affecting cell viability. Osmotic stress activated JNK1 and JNK2 and the inhibition of JNK by SP600125 attenuated osmotic stress-induced TJ disruption. TJ disruption and barrier dysfunction by osmotic stress was associated with JNK-dependent remodeling of actin cytoskeleton. Knockdown of JNK2 accelerated TJ assembly and attenuated osmotic stress-induced TJ disruption in Caco-2 cell monolayers. In mouse ileum in vitro, osmotic stress increased paracellular permeability, which was attenuated by SP600125. Osmotic stress disrupted actin cytoskeleton and TJs and increased paracellular permeability in the ileum of wild-type and JNK1 −/− mice, but not in JNK2 −/− mouse ileum. Cold restraint stress activated JNK in rat ileum and caused JNK-dependent remodeling of actin cytoskeleton and redistribution of occludin and zona occluden-1 from the intercellular junctions. These results reveal the role of JNK2 in the mechanism of osmotic stress-induced TJ disruption in the intestinal epithelium.

scholarly journals Chlamydia trachomatis Induces Remodeling of the Actin Cytoskeleton during Attachment and Entry into HeLa Cells

2002 ◽  
Vol 70 (7) ◽  
pp. 3793-3803 ◽  
Author(s):  
Reynaldo A. Carabeo ◽  
Scott S. Grieshaber ◽  
Elizabeth Fischer ◽  
Ted Hackstadt
Keyword(s):  
Chlamydia Trachomatis ◽  
Cell Surface ◽  
Actin Cytoskeleton ◽  
Hela Cells ◽  
Actin Polymerization ◽  
Cytochalasin D ◽  
Actin Remodeling ◽  
Ultrastructural Studies ◽  
Infected Cells

ABSTRACT To elucidate the host cell machinery utilized by Chlamydia trachomatis to invade epithelial cells, we examined the role of the actin cytoskeleton in the internalization of chlamydial elementary bodies (EBs). Treatment of HeLa cells with cytochalasin D markedly inhibited the internalization of C. trachomatis serovar L2 and D EBs. Association of EBs with HeLa cells induced localized actin polymerization at the site of attachment, as visualized by either phalloidin staining of fixed cells or the active recruitment of GFP-actin in viable infected cells. The recruitment of actin to the specific site of attachment was accompanied by dramatic changes in the morphology of cell surface microvilli. Ultrastructural studies revealed a transient microvillar hypertrophy that was dependent upon C. trachomatis attachment, mediated by structural components on the EBs, and cytochalasin D sensitive. In addition, a mutant CHO cell line that does not support entry of C. trachomatis serovar L2 did not display such microvillar hypertrophy following exposure to L2 EBs, which is in contrast to infection with serovar D, to which it is susceptible. We propose that C. trachomatis entry is facilitated by an active actin remodeling process that is induced by the attachment of this pathogen, resulting in distinct microvillar reorganization throughout the cell surface and the formation of a pedestal-like structure at the immediate site of attachment and entry.

scholarly journals Evidence for secretion-like coupling involving pp60src in the activation and maintenance of store-mediated Ca2+ entry in mouse pancreatic acinar cells

2003 ◽  
Vol 370 (1) ◽  
pp. 255-263 ◽  
Author(s):  
Pedro C. REDONDO ◽  
Ana I. LAJAS ◽  
Ginés M. SALIDO ◽  
Antonio GONZALEZ ◽  
Juan A. ROSADO ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Acinar Cells ◽  
Coupling Model ◽  
Pancreatic Acinar Cells ◽  
Coupling Mechanism ◽  
Cell Type ◽  
Cortical Actin ◽  
Excitable Cells ◽  
Latrunculin A

Store-mediated Ca2+ entry (SMCE) is one of the main pathways for Ca2+ influx in non-excitable cells. Recent studies favour a secretion-like coupling mechanism to explain SMCE, where Ca2+ entry is mediated by an interaction of the endoplasmic reticulum (ER) with the plasma membrane (PM) and is modulated by the actin cytoskeleton. To explore this possibility further we have now investigated the role of the actin cytoskeleton in the activation and maintenance of SMCE in pancreatic acinar cells, a more specialized secretory cell type which might be an ideal cellular model to investigate further the properties of the secretion-like coupling model. In these cells, the cytoskeletal disrupters cytochalasin D and latrunculin A inhibited both the activation and maintenance of SMCE. In addition, stabilization of a cortical actin barrier by jasplakinolide prevented the activation, but not the maintenance, of SMCE, suggesting that, as for secretion, the actin cytoskeleton plays a double role in SMCE as a negative modulator of the interaction between the ER and PM, but is also required for this mechanism, since the cytoskeleton disrupters impaired Ca2+ entry. Finally, depletion of the intracellular Ca2+ stores induces cytoskeletal association and activation of pp60src, which is independent on Ca2+ entry. pp60src activation requires the integrity of the actin cytoskeleton and participates in the initial phase of the activation of SMCE in pancreatic acinar cells.

scholarly journals Junctional ER Organization Affects Mechanotransduction at Cadherin-Mediated Adhesions

2021 ◽  
Vol 9 ◽  
Author(s):  
Michelle Joy-Immediato ◽  
Manuel J. Ramirez ◽  
Mauricio Cerda ◽  
Yusuke Toyama ◽  
Andrea Ravasio ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Actin Cytoskeleton ◽  
Structural Organization ◽  
Adherens Junctions ◽  
Conformational State ◽  
Microtubule Network ◽  
Close Proximity ◽  
Actomyosin Cytoskeleton

Cadherin-mediated adhesions (also known as adherens junctions) are adhesive complexes that connect neighboring cells in a tissue. While the role of the actin cytoskeleton in withstanding tension at these sites of contact is well documented, little is known about the involvement of microtubules and the associated endoplasmic reticulum (ER) network in cadherin mechanotransduction. Therefore, we investigated how the organization of ER extensions in close proximity of cadherin-mediated adhesions can affect such complexes, and vice versa. Here, we show that the extension of the ER to cadherin-mediated adhesions is tension dependent and appears to be cadherin-type specific. Furthermore, the different structural organization of the ER/microtubule network seems to affect the localization of ER-bound PTP1B at cadherin-mediated adhesions. This phosphatase is involved in the modulation of vinculin, a molecular clutch which enables differential engagement of the cadherin-catenin layer with the actomyosin cytoskeleton in response to tension. This suggests a link between structural organization of the ER/microtubule network around cadherin-specific adhesions, to control the mechanotransduction of adherens junctions by modulation of vinculin conformational state.

Actin cytoskeletal modulation of pressure-induced depolarization and Ca2+ influx in cerebral arteries

2002 ◽  
Vol 282 (4) ◽  
pp. H1410-H1420 ◽  
Author(s):  
Natalia I. Gokina ◽  
George Osol
Keyword(s):  
Actin Cytoskeleton ◽  
Smooth Muscle Actin ◽  
Cerebral Arteries ◽  
Membrane Depolarization ◽  
Cytochalasin D ◽  
Intraluminal Pressure ◽  
Induced Membrane ◽  
Latrunculin A ◽  
Cytoskeletal Dynamics ◽  
Actin Cytoskeletal Dynamics

The objective of this study was to examine the role of the actin cytoskeleton in the development of pressure-induced membrane depolarization and Ca2+ influx underlying myogenic constriction in cerebral arteries. Elevating intraluminal pressure from 10 to 60 mmHg induced membrane depolarization, increased intracellular cytosolic Ca2+ concentration ([Ca2+]i) and elicited myogenic constriction in both intact and denuded rat posterior cerebral arteries. Pretreatment with cytochalasin D (5 μM) or latrunculin A (3 μM) abolished constriction but enhanced the [Ca2+]i response; similarly, acute application of cytochalasin D to vessels with tone, or in the presence of 60 mM K+, elicited relaxation accompanied by an increase in [Ca2+]i. The effects of cytochalasin D were inhibited by nifedipine (3 μM), demonstrating that actin cytoskeletal disruption augments Ca2+ influx through voltage-sensitive L-type Ca2+ channels. Finally, pressure-induced depolarization was enhanced in the presence of cytochalasin D, further substantiating a role for the actin cytoskeleton in the modulation of ion channel function. Together, these results implicate vascular smooth muscle actin cytoskeletal dynamics in the control of cerebral artery diameter through their influence on membrane potential as well as via a direct effect on L-type Ca2+ channels.

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