Peripheral F-actin Research Articles

Human T-cell receptor triggering requires inactivation of Lim kinase-1 by Slingshot-1 phosphatase

Actin dynamics control early T-cell receptor (TCR) signalling during T-cell activation. However, the precise regulation of initial actin rearrangements is not completely understood. Here, we have investigated the regulatory role of the phosphatase Slingshot-1 (SSH1) in this process. Our data show that SSH1 rapidly polarises to nascent cognate synaptic contacts and later relocalises to peripheral F-actin networks organised at the mature immunological synapse. Knockdown of SSH1 expression by CRISPR/Cas9-mediated genome editing or small interfering RNA reveal a regulatory role for SSH1 in CD3ε conformational change, allowing Nck binding and proper downstream signalling and immunological synapse organisation. TCR triggering induces SSH1-mediated activation of actin dynamics through a mechanism mediated by Limk-1 inactivation. These data suggest that during early TCR activation, SSH1 is required for rapid F-actin rearrangements that mediate initial conformational changes of the TCR, integrin organisation and proximal signalling events for proper synapse organisation. Therefore, the SSH1 and Limk-1 axis is a key regulatory element for full T cell activation.

WAVE2 Regulates Actin-Dependent Processes Induced by the B Cell Antigen Receptor and Integrins.

B cell antigen receptor (BCR) signaling induces actin cytoskeleton remodeling by stimulating actin severing, actin polymerization, and the nucleation of branched actin networks via the Arp2/3 complex. This enables B cells to spread on antigen-bearing surfaces in order to increase antigen encounters and to form an immune synapse (IS) when interacting with antigen-presenting cells (APCs). Although the WASp, N-WASp, and WAVE nucleation-promoting factors activate the Arp2/3 complex, the role of WAVE2 in B cells has not been directly assessed. We now show that both WAVE2 and the Arp2/3 complex localize to the peripheral ring of branched F-actin when B cells spread on immobilized anti-Ig antibodies. The siRNA-mediated depletion of WAVE2 reduced and delayed B cell spreading on immobilized anti-Ig, and this was associated with a thinner peripheral F-actin ring and reduced actin retrograde flow compared to control cells. Depleting WAVE2 also impaired integrin-mediated B cell spreading on fibronectin and the LFA-1-induced formation of actomyosin arcs. Actin retrograde flow amplifies BCR signaling at the IS, and we found that depleting WAVE2 reduced microcluster-based BCR signaling and signal amplification at the IS, as well as B cell activation in response to antigen-bearing cells. Hence, WAVE2 contributes to multiple actin-dependent processes in B lymphocytes.

The dipeptide prolyl-hydroxyproline promotes cellular homeostasis and lamellipodia-driven motility via active β1-integrin in adult tendon cells

Collagen-derived hydroxyproline (Hyp)-containing peptides have a variety of biological effects on cells. These bioactive collagen peptides are locally generated by the degradation of endogenous collagen in response to injury. However, no comprehensive study has yet explored the functional links between Hyp-containing peptides and cellular behavior. Here, we show that the dipeptide prolyl-4-hydroxyproline (Pro-Hyp) exhibits pronounced effects on mouse tendon cells. Pro-Hyp promotes differentiation/maturation of tendon cells with modulation of lineage-specific factors and induces significant chemotactic activity in vitro. In addition, Pro-Hyp has profound effects on cell proliferation, with significantly upregulated extracellular signal–regulated kinase phosphorylation and extracellular matrix production and increased type I collagen network organization. Using proteomics, we have predicted molecular transport, cellular assembly and organization, and cellular movement as potential linked-network pathways that could be altered in response to Pro-Hyp. Mechanistically, cells treated with Pro-Hyp demonstrate increased directional persistence and significantly increased directed motility and migration velocity. They are accompanied by elongated lamellipodial protrusions with increased levels of active β1-integrin–containing focal contacts, as well as reorganization of thicker peripheral F-actin fibrils. Pro-Hyp–mediated chemotactic activity is significantly reduced (p < 0.001) in cells treated with the mitogen-activated protein kinase kinase 1/2 inhibitor PD98059 or the α5β1-integrin antagonist ATN-161. Furthermore, ATN-161 significantly inhibits uptake of Pro-Hyp into adult tenocytes. Thus, our findings document the molecular basis of the functional benefits of the Pro-Hyp dipeptide in cellular behavior. These dynamic properties of collagen-derived Pro-Hyp dipeptide could lead the way to its application in translational medicine.

In vivo Roles of Rab27 and Its Effectors in Exocytosis.

The monomeric GTPase Rab27 regulates exocytosis of a broad range of vesicles in multicellular organisms. Several effectors bind GTP-bound Rab27a and/or Rab27b on secretory vesicles to execute a series of exocytic steps, such as vesicle maturation, movement along microtubules, anchoring within the peripheral F-actin network, and tethering to the plasma membrane, via interactions with specific proteins and membrane lipids in a local milieu. Although Rab27 effectors generally promote exocytosis, they can also temporarily restrict it when they are involved in the rate-limiting step. Genetic alterations in Rab27-related molecules cause discrete diseases manifesting pigment dilution and immunodeficiency, and can also affect common diseases such as diabetes and cancer in complex ways. Although the function and mechanism of action of these effectors have been explored, it is unclear how multiple effectors act in coordination within a cell to regulate the secretory process as a whole. It seems that Rab27 and various effectors constitutively reside on individual vesicles to perform consecutive exocytic steps. The present review describes the unique properties and in vivo roles of the Rab27 system, and the functional relationship among different effectors coexpressed in single cells, with pancreatic beta cells used as an example.Key words: membrane trafficking, regulated exocytosis, insulin granules, pancreatic beta cells.

Beneficial and detrimental effects of the phytochemical naringenin on rainbow trout intestinal epithelial cells

Abstract Monolayer cultures of the rainbow trout cell line, RTgutGC, were used to study actions on the intestinal epithelium of a potential feed additive, the flavanone naringenin. Although having little effect on viability or morphology, naringenin altered the F-actin staining pattern. Stress fibers disappeared and peripheral actin became more intense. Naringenin enhanced barrier function, as measured by transepithelial electrical resistance (TEER) and Lucifer yellow permeability. When cell-free gaps or wounds were created, cells in L15/FBS migrated into and covered the gap but more slowly with naringenin. By contrast in L15, cells only partially migrated into the gap and high naringenin concentrations blocked migration. These cells in naringenin had fewer protrusion and stress fibers but some had prominent peripheral F-actin staining on the side facing the gap. Therefore, naringenin might be enhancing barrier function and impeding cell migration by a common mechanism, modulating actin dynamics.

Control of nuclear organization by F-actin binding proteins

ABSTRACTThe regulation of nuclear shape and deformability is a key factor in controlling diverse events from embryonic development to cancer cell metastasis, but the mechanisms governing this process are still unclear. Our recent study demonstrated an unexpected role for the F-actin bundling protein fascin in controlling nuclear plasticity through a direct interaction with Nesprin-2. Nesprin-2 is a component of the LINC complex that is known to couple the F-actin cytoskeleton to the nuclear envelope. We demonstrated that fascin, which is predominantly associated with peripheral F-actin rich filopodia, binds directly to Nesprin-2 at the nuclear envelope in a range of cell types. Depleting fascin or specifically blocking the fascin-Nesprin-2 complex leads to defects in nuclear polarization, movement and cell invasion. These studies reveal a novel role for an F-actin bundling protein in control of nuclear plasticity and underline the importance of defining nuclear-associated roles for F-actin binding proteins in future.

Transient Intervals of Hyper-Gravity Enhance Endothelial Barrier Integrity: Impact of Mechanical and Gravitational Forces Measured Electrically.

BackgroundEndothelial cells (EC) guard vascular functions by forming a dynamic barrier throughout the vascular system that sensitively adapts to ‘classical’ biomechanical forces, such as fluid shear stress and hydrostatic pressure. Alterations in gravitational forces might similarly affect EC integrity, but remain insufficiently studied.MethodsIn an unique approach, we utilized Electric Cell-substrate Impedance Sensing (ECIS) in the gravity-simulators at the European Space Agency (ESA) to study dynamic responses of human EC to simulated micro- and hyper-gravity as well as to classical forces.ResultsShort intervals of micro- or hyper-gravity evoked distinct endothelial responses. Stimulated micro-gravity led to decreased endothelial barrier integrity, whereas hyper-gravity caused sustained barrier enhancement by rapid improvement of cell-cell integrity, evidenced by a significant junctional accumulation of VE-cadherin (p = 0.011), significant enforcement of peripheral F-actin (p = 0.008) and accompanied by a slower enhancement of cell-matrix interactions. The hyper-gravity triggered EC responses were force dependent and nitric-oxide (NO) mediated showing a maximal resistance increase of 29.2±4.8 ohms at 2g and 60.9±6.2 ohms at 4g vs. baseline values that was significantly suppressed by NO blockage (p = 0.011).ConclusionIn conclusion, short-term application of hyper-gravity caused a sustained improvement of endothelial barrier integrity, whereas simulated micro-gravity weakened the endothelium. In clear contrast, classical forces of shear stress and hydrostatic pressure induced either short-lived or no changes to the EC barrier. Here, ECIS has proven a powerful tool to characterize subtle and distinct EC gravity-responses due to its high temporal resolution, wherefore ECIS has a great potential for the study of gravity-responses such as in real space flights providing quantitative assessment of a variety of cell biological characteristics of any adherent growing cell type in an automated and continuous fashion.

HSP27 phosphorylation protects against endothelial barrier dysfunction under burn serum challenge

F-actin rearrangement is an early event in burn-induced endothelial barrier dysfunction. HSP27, a target of p38 MAPK/MK2 pathway, plays an important role in actin dynamics through phosphorylation. The question of whether HSP27 participates in burn-related endothelial barrier dysfunction has not been identified yet. Here, we showed that burn serum induced a temporal appearance of central F-actin stress fibers followed by a formation of irregular dense peripheral F-actin in pulmonary endothelial monolayer, concomitant with a transient increase of HSP27 phosphorylation that conflicted with the persistent activation of p38 MAPK/MK2 unexpectedly. The appearance of F-actin stress fibers and transient increase of HSP27 phosphorylation occurred prior to the burn serum-induced endothelial hyperpermeability. Overexpressing phospho-mimicking HSP27 (HSP27(Asp)) reversed the burn serum-induced peripheral F-actin rearrangement with the augmentation of central F-actin stress fibers, and more importantly, attenuated the burn serum-induced endothelial hyperpermeability; such effects were not observed by HSP27(Ala), a non-phosphorylated mutant of HSP27. HSP27(Asp) overexpression also rendered the monolayer more resistant to barrier disruption caused by Cytochalasin D, a chemical reagent that depolymerizes F-actin specifically. Further study showed that phosphatases and sumoylation-inhibited MK2 activity contributed to the blunting of HSP27 phosphorylation during the burn serum-induced endothelial hyperpermeability. Our study identifies HSP27 phosphorylation as a protective response against burn serum-induced endothelial barrier dysfunction, and suggests that targeting HSP27 wound be a promising therapeutic strategy in ameliorating burn-induced lung edema and shock development.

Interferon-Gamma Increases Endothelial Permeability by Causing Activation of p38 MAP Kinase and Actin Cytoskeleton Alteration.

Interferon-gamma (IFN-γ) is known to potentiate the progression of inflammatory diseases, such as inflammatory bowel disease and atherosclerosis. IFN-γ has been found to disrupt the barrier integrity of epithelial and endothelial cell both in vivo and in vitro. However, the mechanisms of IFN-γ underlying increased endothelial cell permeability have not been extensively elucidated. We reported that IFN-γ exhibits a biphasic nature in increasing endothelial permeability. The changes observed in the first phase (4-8 h) involve cell retraction and rounding in addition to condensed peripheral F-actin without a significant change in the F-/G-actin ratio. However, cell elongation, stress fiber formation, and an increased F-/G-actin ratio were noticed in the second phase (16-24 h). Consistent with our finding from the permeability assay, IFN-γ induced the formation of intercellular gaps in both phases. A delayed phase of increased permeability was observed at 12 h, which paralleled the onset of cell elongation, stress fiber formation, and increased F-/G-actin ratio. In addition, IFN-γ stimulated p38 mitogen-activated protein (MAP) kinase phosphorylation over a 24 h period. Inhibition of p38 MAP kinase by SB203580 prevented increases in paracellular permeability, actin rearrangement, and increases in the F-/G-actin ratio caused by IFN-γ. Our results suggest that p38 MAP kinase is activated in response to IFN-γ and causes actin rearrangement and altered cell morphology, which in turn mediates endothelial cell hyperpermeability. The F-/G-actin ratio might be involved in the regulation of actin distribution and cell morphology rather than the increased permeability induced by IFN-γ.

F-actin bundles direct the initiation and orientation of lamellipodia through adhesion-based signaling.

Mesenchymal cells such as fibroblasts are weakly polarized and reorient directionality by a lamellipodial branching mechanism that is stabilized by phosphoinositide 3-kinase (PI3K) signaling. However, the mechanisms by which new lamellipodia are initiated and directed are unknown. Using total internal reflection fluorescence microscopy to monitor cytoskeletal and signaling dynamics in migrating cells, we show that peripheral F-actin bundles/filopodia containing fascin-1 serve as templates for formation and orientation of lamellipodia. Accordingly, modulation of fascin-1 expression tunes cell shape, quantified as the number of morphological extensions. Ratiometric imaging reveals that F-actin bundles/filopodia play both structural and signaling roles, as they prime the activation of PI3K signaling mediated by integrins and focal adhesion kinase. Depletion of fascin-1 ablated fibroblast haptotaxis on fibronectin but not platelet-derived growth factor chemotaxis. Based on these findings, we conceptualize haptotactic sensing as an exploration, with F-actin bundles directing and lamellipodia propagating the process and with signaling mediated by adhesions playing the role of integrator.

An investigation into plasmolysis in the oomyceteAchlya bisexualisreveals that membrane–wall attachment points are sensitive to peptides containing the sequence RGD and that cell wall deposition can occur despite retraction of the protoplast

The structure and function of membrane-wall attachment sites in walled cells, and how these relate to animal focal adhesions, is an area that is poorly understood. In view of this, we investigated how membrane-wall attachments that form upon plasmolysis, respond to peptides that disrupt animal focal adhesions. The degree of cytoplasmic disruption during plasmolysis was also investigated. Upon hyperosmotic challenge, the protoplast in hyphae of the oomycete Achlya bisexualis typically retracted incompletely due to membrane-wall attachments. The inclusion, in the plasmolysing solution, of peptides containing the sequence RGD disrupted these attachments in a dose-dependent manner. In some hyphae, protoplast retraction stopped temporarily at attachment points - upon resumption of retraction, material was left that traced the outline of the static protoplast. Staining of this material with fluorescence brightener indicated the presence of cellulose, which suggests that wall deposition was able to occur despite plasmolysis. The F-actin cytoskeleton was disrupted during plasmolysis; peripheral F-actin staining was observed, but there was no distinct F-actin cap; staining was more diffuse; and there were fewer plaques compared with nonplasmolysed hyphae. Our data indicate that membrane-wall attachment points are sensitive to RGD-containing peptides and that wall deposition continues despite protoplast retraction and F-actin disruption.

Fussel-15, a New Player in Wound Healing, Is Deregulated in Keloid and Localized Scleroderma

Dermal wound healing depends on highly complex interplay among various cytokines and cell types. Disruption of this process can result in impaired healing in the form of excessive scarring, as is the case in fibrotic diseases such as keloid and scleroderma. In the present study, we found Fussel-15, a new member of the Ski/Sno family of TGF-β/BMP signaling repressors, to be expressed in early wound healing and constantly overexpressed in keloid-derived and scleroderma-derived fibroblasts. Comparing the results of three-dimensional free-floating and attached-released in vitro wound healing assays, we observed that Fussel-15 is expressed during the migratory phase in the free-floating assay, indicating that Fussel-15 might play a role during fibroblast migration. Fussel-15-transfected fibroblasts showed greater migration ability in a scratch wound healing assay, compared with control-transfected cells. This migratory phenotype due to Fussel-15 was confirmed by increased peripheral F-actin localization and modifications in size, amount, and distribution of focal adhesion complexes, which were observed using F-actin and focal adhesion kinase (FAK) immunofluorescence staining, respectively. The present results suggest that expression of Fussel-15 during wound healing might promote fibroblast migration. Permanent expression of Fussel-15 in keloid and skin sclerosis fibroblasts could be involved in the pathogenesis of these conditions, but the molecular mechanism underlying this up-regulation remains to be determined.

Modelling condensation and the initiation of chondrogenesis in chick wing bud mesenchymal cells levitated in an ultrasound trap

Chick wing bud mesenchymal cell micromass culture allows the study of a variety of developmental mechanisms, ranging from cell adhesion to pattern formation. However, many cells remain in contact with an artificial substratum, which can influence cytoskeletal organisation and differentiation. An ultrasound standing wave trap facilitates the rapid formation of 2-D monolayer cell aggregates with a defined zero time-point, independent from contact with a surface. Aggregates formed rapidly (within 2 min) and intercellular membrane spreading occurred at points of contact. This was associated with an increase in peripheral F-actin within 10 min of cell-cell contact and aggregates had obtained physical integrity by 30 min. The chondrogenic transcription factor Sox9 could be detected in cells in the ultrasound trap within 3 h (ultrasound exposure alone was not responsible for this effect). This approach facilitates the study of the initial cell-cell interactions that occur during condensation formation and demonstrates that a combination of cell shape and cytoskeletal organisation is required for the initiation and maintenance of a differentiated phenotype, which is lost when these phenomena are influenced by contact with an artificial substrate.

Abi plays an opposing role to Abl inDrosophilaaxonogenesis and synaptogenesis

Abl tyrosine kinase (Abl) regulates axon guidance by modulating actin dynamics. Abelson interacting protein (Abi), originally identified as a kinase substrate of Abl, also plays a key role in actin dynamics, yet its role with respect to Abl in the developing nervous system remains unclear. Here we show that mutations in abi disrupt axonal patterning in the developing Drosophila central nervous system (CNS). However, reducing abi gene dosage by half substantially rescues Abl mutant phenotypes in pupal lethality, axonal guidance defects and locomotion deficits. Moreover, we show that mutations in Abl increase synaptic growth and spontaneous synaptic transmission frequency at the neuromuscular junction. Double heterozygosity for abi and enabled (ena) also suppresses the synaptic overgrowth phenotypes of Abl mutants, suggesting that Abi acts cooperatively with Ena to antagonize Abl function in synaptogenesis. Intriguingly, overexpressing Abi or Ena alone in cultured cells dramatically redistributed peripheral F-actin to the cytoplasm, with aggregates colocalizing with Abi and/or Ena, and resulted in a reduction in neurite extension. However, co-expressing Abl with Abi or Ena redistributed cytoplasmic F-actin back to the cell periphery and restored bipolar cell morphology. These data suggest that abi and Abl have an antagonistic interaction in Drosophila axonogenesis and synaptogenesis, which possibly occurs through the modulation of F-actin reorganization.

Myosin II regulates the shape of three-dimensional intestinal epithelial cysts.

The development of luminal organs begins with the formation of spherical cysts composed of a single layer of epithelial cells. Using a model three-dimensional cell culture, this study examines the role of a cytoskeletal motor, myosin II, in cyst formation. Caco-2 and SK-CO15 intestinal epithelial cells were embedded into Matrigel, and myosin II was inhibited by blebbistatin or siRNA-mediated knockdown. Whereas control cells formed spherical cysts with a smooth surface, inhibition of myosin II induced the outgrowth of F-actin-rich surface protrusions. The development of these protrusions was abrogated after inhibition of F-actin polymerization or of phospholipase C (PLC) activity, as well as after overexpression of a dominant-negative ADF/cofilin. Surface protrusions were enriched in microtubules and their formation was prevented by microtubule depolymerization. Myosin II inhibition caused a loss of peripheral F-actin bundles and a submembranous extension of cortical microtubules. Our findings suggest that inhibition of myosin II eliminates the cortical F-actin barrier, allowing microtubules to reach and activate PLC at the plasma membrane. PLC-dependent stimulation of ADF/cofilin creates actin-filament barbed ends and promotes the outgrowth of F-actin-rich protrusions. We conclude that myosin II regulates the spherical shape of epithelial cysts by controlling actin polymerization at the cyst surface.

Regulation of Hyperoxia-induced NADPH Oxidase Activation in Human Lung Endothelial Cells by the Actin Cytoskeleton and Cortactin

Although the actin cytoskeleton has been implicated in the control of NADPH oxidase in phagocytosis, very little is known about the cytoskeletal regulation of endothelial NADPH oxidase assembly and activation. Here, we report a role for cortactin and the tyrosine phosphorylation of cortactin in hyperoxia-induced NADPH oxidase activation and ROS production in human pulmonary artery ECs (HPAECs). Exposure of HPAECs to hyperoxia for 3 h induced NADPH oxidase activation, as demonstrated by enhanced superoxide production. Hyperoxia also caused a thickening of the subcortical dense peripheral F-actin band and increased the localization of cortactin in the cortical regions and lamellipodia at cell-cell borders that protruded under neighboring cells. Pretreatment of HPAECs with the actin-stabilizing agent phallacidin attenuated hyperoxia-induced cortical actin thickening and ROS production, whereas cytochalasin D and latrunculin A enhanced basal and hyperoxia-induced ROS formation. In HPAECs, a 3-h hyperoxic exposure enhanced the tyrosine phosphorylation of cortactin and interaction between cortactin and p47(phox), a subcomponent of the EC NADPH oxidase, when compared with normoxic cells. Furthermore, transfection of HPAECs with cortactin small interfering RNA or myristoylated cortactin Src homology domain 3 blocking peptide attenuated ROS production and the hyperoxia-induced translocation of p47(phox) to the cell periphery. Similarly, down-regulation of Src with Src small interfering RNA attenuated the hyperoxia-mediated phosphorylation of cortactin tyrosines and blocked the association of cortactin with actin and p47(phox). In addition, the hyperoxia-induced generation of ROS was significantly lower in ECs expressing a tyrosine-deficient mutant of cortactin than in vector control or wild-type cells. These data demonstrate a novel function for cortactin and actin in hyperoxia-induced activation of NADPH oxidase and ROS generation in human lung endothelial cells.

Prostaglandins PGE 2 and PGI 2 promote endothelial barrier enhancement via PKA- and Epac1/Rap1-dependent Rac activation

Prostaglandin E 2 (PGE 2) and prostacyclin are lipid mediators produced by cyclooxygenase and implicated in the regulation of vascular function, wound repair, inflammatory processes, and acute lung injury. Although protective effects of these prostaglandins (PGs) are associated with stimulation of intracellular cAMP production, the crosstalk between cAMP-activated signal pathways in the regulation of endothelial cell (EC) permeability is not well understood. We studied involvement of cAMP-dependent kinase (PKA), cAMP–Epac–Rap1 pathway, and small GTPase Rac in the PGs-induced EC barrier protective effects and cytoskeletal remodeling. PGE 2 and PGI 2 synthetic analog beraprost increased transendothelial electrical resistance and decreased dextran permeability, enhanced peripheral F-actin rim and increased intercellular adherens junction areas reflecting EC barrier-protective response. Furthermore, beraprost dramatically attenuated thrombin-induced Rho activation, MLC phosphorylation and EC barrier dysfunction. In vivo, beraprost attenuated lung barrier dysfunction induced by high tidal volume mechanical ventilation. Both PGs caused cAMP-mediated activation of PKA-, Epac/Rap1- and Tiam1/Vav2-dependent pathways of Rac1 activation and EC barrier regulation. Knockdown of Epac, Rap1, Rac-specific exchange factors Tiam1 and Vav2 using siRNA approach, or inhibition of PKA activity decreased Rac1 activation and PG-induced EC barrier enhancement. Thus, our results show that barrier-protective effects of PGE 2 and prostacyclin on pulmonary EC are mediated by PKA and Epac/Rap pathways, which converge on Rac activation and lead to enhancement of peripheral actin cytoskeleton and adherens junctions. These mechanisms may mediate protective effects of PGs against agonist-induced lung vascular barrier dysfunction in vitro and against mechanical stress-induced lung injury in vivo.

Differential Regulation of Pulmonary Endothelial Monolayer Integrity by Varying Degrees of Cyclic Stretch

Ventilator-induced lung injury is a life-threatening complication of mechanical ventilation at high-tidal volumes. Besides activation of proinflammatory cytokine production, excessive lung distension directly affects blood-gas barrier and lung vascular permeability. To investigate whether restoration of pulmonary endothelial cell (EC) monolayer integrity after agonist challenge is dependent on the magnitude of applied cyclic stretch (CS) and how these effects are linked to differential activation of small GTPases Rac and Rho, pulmonary ECs were subjected to physiologically (5% elongation) or pathologically (18% elongation) relevant levels of CS. Pathological CS enhanced thrombin-induced gap formation and delayed monolayer recovery, whereas physiological CS induced nearly complete EC recovery accompanied by peripheral redistribution of focal adhesions and cortactin after 50 minutes of thrombin. Consistent with differential effects on monolayer integrity, 18% CS enhanced thrombin-induced Rho activation, whereas 5% CS promoted Rac activation during the EC recovery phase. Rac inhibition dramatically attenuated restoration of monolayer integrity after thrombin challenge. Physiological CS preconditioning (5% CS, 24 hours) enhanced EC paracellular gap resolution after step-wise increase to 18% CS (30 minutes) and thrombin challenge. These results suggest a critical role for the CS amplitude and the balance between Rac and Rho in mechanochemical regulation of lung EC barrier.

Permselective Dysfunction of Podocyte-Podocyte Contact upon Angiotensin II Unravels the Molecular Target for Renoprotective Intervention

Ameliorating the function of the glomerular barrier to circulating proteins by blocking angiotensin II (Ang II) translates into less risk of progression toward end-stage renal failure in diabetic and nondiabetic nephropathies. However, the mechanisms underlying this barrier protection are not clear. Specialized contacts between adjacent podocytes are major candidate targets, and the actin cytoskeleton is emerging as a regulatory element. Here, we present data demonstrating that Ang II induced reorganization of F-actin fibers and redistribution of zonula occludens-1 (ZO-1) that is physically associated with actin in murine podocytes. These effects were paralleled by increased albumin permeability across podocyte monolayers. The F-actin stabilizer jasplakinolide prevented both ZO-1 redistribution and albumin leakage, suggesting that actin cytoskeleton rearrangement is instrumental to podocyte permselective dysfunction induced by Ang II. Changes in both F-actin and ZO-1 patterns were confirmed in glomeruli of rat isolated perfused kidneys on short infusion of Ang II, leading to increased protein excretion. Podocyte dysfunction was mediated by Ang II type 1 receptor and was partly dependent on Src kinase-phospholipase C activation. These data demonstrate that strategies aimed at stabilizing podocyte-podocyte contacts and targeting the relevant intracellular signal transduction are crucial to renoprotection.

Actin structure in the outflow tract of normal and glaucomatous eyes

Purpose. To characterize the in situ distribution of actin in Schlemm's canal endothelium (SCE) and juxtacanalicular tissue (JCT) cells in glaucomatous human eyes, and compare to the distribution in normal eyes. Methods. Fresh human eye bank eyes were perfused and fixed at pressure ( n=27 normal eyes and 22 confirmed glaucomatous eyes). Schlemm's canal was opened by microdissection and outflow tissues were labelled for confocal microscopy to visualize F-actin, nuclei, laminin and/or CD31. Images were acquired in Z-series from the inner wall of Schlemm's canal, juxtacanalicular tissue and outer corneoscleral meshwork. Results. In normal eyes, inner wall Schlemm's canal endothelial (SCE) cells showed a dense peripheral F-actin band, as previously described. JCT cells showed a more random and amorphous F-actin distribution. In glaucoma eyes, peripheral F-actin bands were less common in inner wall SCE cells; instead, F-actin was more centrally located within the cell and appeared ‘tangled’. These actin tangles were also prominent in JCT cells of glaucoma eyes. Glaucoma eyes also demonstrated structures with features of cross-linked actin networks (CLANs), and more frequent occurrence of punctuate actin concentrations. There was a significant degree of heterogeneity, with some regions from glaucomatous eyes appearing normal and vice versa. Conclusion. F-actin architecture in human outflow pathway cells in situ differs between normal and glaucoma eyes, with glaucomatous tissue showing a more ‘disordered’ actin architecture overall. Some of these changes are likely due to effects secondary to administration of anti-glaucoma medications. Most of the changes that we observed could potentially affect the biomechanical properties of the outflow pathway tissues in glaucoma, but their role in the pathogenesis of ocular hypertension remains unclear.