Z-disc-associated,Alternatively Spliced,PDZ Motif-containing Protein (ZASP) Mutations in the Actin-binding Domain Cause Disruption of Skeletal Muscle Actin Filaments in Myofibrillar Myopathy

The core of skeletal muscle Z-discs consists of actin filaments from adjacent sarcomeres that are cross-linked by α-actinin homodimers. Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP)/Cypher interacts with α-actinin, myotilin, and other Z-disc proteins via the PDZ domain. However, these interactions are not sufficient to maintain the Z-disc structure. We show that ZASP directly interacts with skeletal actin filaments. The actin-binding domain is between the modular PDZ and LIM domains. This ZASP region is alternatively spliced so that each isoform has unique actin-binding domains. All ZASP isoforms contain the exon 6-encoded ZASP-like motif that is mutated in zaspopathy, a myofibrillar myopathy (MFM), whereas the exon 8–11 junction-encoded peptide is exclusive to the postnatal long ZASP isoform (ZASP-LΔex10). MFM is characterized by disruption of skeletal muscle Z-discs and accumulation of myofibrillar degradation products. Wild-type and mutant ZASP interact with α-actin, α-actinin, and myotilin. Expression of mutant, but not wild-type, ZASP leads to Z-disc disruption and F-actin accumulation in mouse skeletal muscle, as in MFM. Mutations in the actin-binding domain of ZASP-LΔex10, but not other isoforms, cause disruption of the actin cytoskeleton in muscle cells. These isoform-specific mutation effects highlight the essential role of the ZASP-LΔex10 isoform in F-actin organization. Our results show that MFM-associated ZASP mutations in the actin-binding domain have deleterious effects on the core structure of the Z-discs in skeletal muscle.     

Direct Interaction of CaVβ with Actin Up-regulates L-type Calcium Currents in HL-1 Cardiomyocytes

Expression of the β-subunit (Ca_Vβ) is required for normal function of cardiac L-type calcium channels, and its up-regulation is associated with heart failure. Ca_Vβ binds to the α₁ pore-forming subunit of L-type channels and augments calcium current density by facilitating channel opening and increasing the number of channels in the plasma membrane, by a poorly understood mechanism. Actin, a key component of the intracellular trafficking machinery, interacts with Src homology 3 domains in different proteins. Although Ca_Vβ encompasses a highly conserved Src homology 3 domain, association with actin has not yet been explored. Here, using co-sedimentation assays and FRET experiments, we uncover a direct interaction between Ca_Vβ and actin filaments. Consistently, single-molecule localization analysis reveals streaklike structures composed by Ca_Vβ₂ that distribute over several micrometers along actin filaments in HL-1 cardiomyocytes. Overexpression of Ca_Vβ₂-N3 in HL-1 cells induces an increase in L-type current without altering voltage-dependent activation, thus reflecting an increased number of channels in the plasma membrane. Ca_Vβ mediated L-type up-regulation, and Ca_Vβ-actin association is prevented by disruption of the actin cytoskeleton with cytochalasin D. Our study reveals for the first time an interacting partner of Ca_Vβ that is directly involved in vesicular trafficking. We propose a model in which Ca_Vβ promotes anterograde trafficking of the L-type channels by anchoring them to actin filaments in their itinerary to the plasma membrane.     

Differential Actin-regulatory Activities of Tropomodulin1 and Tropomodulin3 with Diverse Tropomyosin and Actin Isoforms

Tropomodulins (Tmods) are F-actin pointed end capping proteins that interact with tropomyosins (TMs) and cap TM-coated filaments with higher affinity than TM-free filaments. Here, we tested whether differences in recognition of TM or actin isoforms by Tmod1 and Tmod3 contribute to the distinct cellular functions of these Tmods. We found that Tmod3 bound ∼5-fold more weakly than Tmod1 to α/βTM, TM5b, and TM5NM1. However, surprisingly, Tmod3 was as effective as Tmod1 at capping pointed ends of skeletal muscle α-actin (α_sk-actin) filaments coated with α/βTM, TM5b, or TM5NM1. Tmod3 only capped TM-coated α_sk-actin filaments more weakly than Tmod1 in the presence of recombinant αTM2, which is unacetylated at its NH₂ terminus, binds F-actin weakly, and has a disabled Tmod-binding site. Moreover, both Tmod1 and Tmod3 were similarly effective at capping pointed ends of platelet β/cytoplasmic γ (γ_cyto)-actin filaments coated with TM5NM1. In the absence of TMs, both Tmod1 and Tmod3 had similarly weak abilities to nucleate β/γ_cyto-actin filament assembly, but only Tmod3 could sequester cytoplasmic β- and γ_cyto-actin (but not α_sk-actin) monomers and prevent polymerization under physiological conditions. Thus, differences in TM binding by Tmod1 and Tmod3 do not appear to regulate the abilities of these Tmods to cap TM-α_sk-actin or TM-β/γ_cyto-actin pointed ends and, thus, are unlikely to determine selective co-assembly of Tmod, TM, and actin isoforms in different cell types and cytoskeletal structures. The ability of Tmod3 to sequester β- and γ_cyto-actin (but not α_sk-actin) monomers in the absence of TMs suggests a novel function for Tmod3 in regulating actin remodeling or turnover in cells.     

Serine 59 Phosphorylation of αB-Crystallin Down-regulates Its Anti-apoptotic Function by Binding and Sequestering Bcl-2 in Breast Cancer Cells

The small heat shock protein (sHSP) αB-crystallin is a new oncoprotein in breast carcinoma that predicts poor clinical outcome in breast cancer. However, although several reports have demonstrated that phosphorylation of sHSPs modify their structural and functional properties, the significance of αB-crystallin phosphorylation in cancer cells has not yet been investigated. In this study, we have characterized the phosphorylation status of αB-crystallin in breast epithelial carcinoma cells line MCF7 submitted to anti-cancer agents like vinblastine. We have showed that the main phosphorylation site of αB-crystallin in response to vinblastine is serine 59 and determined a correlation between this post-translational modification and higher apoptosis level. The overexpression of the serine 59 “pseudophosphorylated” mutant (S59E) induces a significant increase in the apoptosis level of vinblastine-treated MCF7 cells. In contrast, overexpression of wild-type αB-crystallin or “nonphosphorylatable” mutant (S59A) result in a resistance to this microtubule-depolymerizing agent, while inhibition of endogenous levels of αB-crystallin by expression of shRNA lowers it. Analyzing further the molecular mechanism of this phenomenon, we report for the first time that phosphorylated αB-crystallin preferentially interacts with Bcl-2, an anti-apoptotic protein, and this interaction prevents the translocation of Bcl-2 to mitochondria. Hence, this study identifies serine 59 phosphorylation as an important key in the down-regulation of αB-crystallin anti-apoptotic function in breast cancer and suggests new strategies to improve anti-cancer treatments.     

Human myocytes are protected from titin aggregation-induced stiffening by small heat shock proteins

In myocytes, small heat shock proteins (sHSPs) are preferentially translocated under stress to the sarcomeres. The functional implications of this translocation are poorly understood. We show here that HSP27 and αB-crystallin associated with immunoglobulin-like (Ig) domain-containing regions, but not the disordered PEVK domain (titin region rich in proline, glutamate, valine, and lysine), of the titin springs. In sarcomeres, sHSP binding to titin was actin filament independent and promoted by factors that increased titin Ig unfolding, including sarcomere stretch and the expression of stiff titin isoforms. Titin spring elements behaved predominantly as monomers in vitro. However, unfolded Ig segments aggregated, preferentially under acidic conditions, and αB-crystallin prevented this aggregation. Disordered regions did not aggregate. Promoting titin Ig unfolding in cardiomyocytes caused elevated stiffness under acidic stress, but HSP27 or αB-crystallin suppressed this stiffening. In diseased human muscle and heart, both sHSPs associated with the titin springs, in contrast to the cytosolic/Z-disk localization seen in healthy muscle/heart. We conclude that aggregation of unfolded titin Ig domains stiffens myocytes and that sHSPs translocate to these domains to prevent this aggregation.     

Tropomodulin1 is required for membrane skeleton organization and hexagonal geometry of fiber cells in the mouse lens

Hexagonal packing geometry is a hallmark of close-packed epithelial cells in metazoans. Here, we used fiber cells of the vertebrate eye lens as a model system to determine how the membrane skeleton controls hexagonal packing of post-mitotic cells. The membrane skeleton consists of spectrin tetramers linked to actin filaments (F-actin), which are capped by tropomodulin1 (Tmod1) and stabilized by tropomyosin (TM). In mouse lenses lacking Tmod1, initial fiber cell morphogenesis is normal, but fiber cell hexagonal shapes and packing geometry are not maintained as fiber cells mature. Absence of Tmod1 leads to decreased γTM levels, loss of F-actin from membranes, and disrupted distribution of β2-spectrin along fiber cell membranes. Regular interlocking membrane protrusions on fiber cells are replaced by irregularly spaced and misshapen protrusions. We conclude that Tmod1 and γTM regulation of F-actin stability on fiber cell membranes is critical for the long-range connectivity of the spectrin–actin network, which functions to maintain regular fiber cell hexagonal morphology and packing geometry.     

α-Actinin and Filamin Cooperatively Enhance the Stiffness of Actin Filament Networks

Background

The close subcellular proximity of different actin filament crosslinking proteins suggests that these proteins may cooperate to organize F-actin structures to drive complex cellular functions during cell adhesion, motility and division. Here we hypothesize that α-actinin and filamin, two major F-actin crosslinking proteins that are both present in the lamella of adherent cells, display synergistic mechanical functions.

Methodology/Principal Findings

Using quantitative rheology, we find that combining α-actinin and filamin is much more effective at producing elastic, solid-like actin filament networks than α-actinin and filamin separately. Moreover, F-actin networks assembled in the presence of α-actinin and filamin strain-harden more readily than networks in the presence of either α-actinin or filamin.

Significance

These results suggest that cells combine auxiliary proteins with similar ability to crosslink filaments to generate stiff cytoskeletal structures, which are required for the production of internal propulsive forces for cell migration, and that these proteins do not have redundant mechanical functions.     

Dynamin2 Organizes Lamellipodial Actin Networks to Orchestrate Lamellar Actomyosin

Actin networks in migrating cells exist as several interdependent structures: sheet-like networks of branched actin filaments in lamellipodia; arrays of bundled actin filaments co-assembled with myosin II in lamellae; and actin filaments that engage focal adhesions. How these dynamic networks are integrated and coordinated to maintain a coherent actin cytoskeleton in migrating cells is not known. We show that the large GTPase dynamin2 is enriched in the distal lamellipod where it regulates lamellipodial actin networks as they form and flow in U2-OS cells. Within lamellipodia, dynamin2 regulated the spatiotemporal distributions of α-actinin and cortactin, two actin-binding proteins that specify actin network architecture. Dynamin2''s action on lamellipodial F-actin influenced the formation and retrograde flow of lamellar actomyosin via direct and indirect interactions with actin filaments and a finely tuned GTP hydrolysis activity. Expression in dynamin2-depleted cells of a mutant dynamin2 protein that restores endocytic activity, but not activities that remodel actin filaments, demonstrated that actin filament remodeling by dynamin2 did not depend of its functions in endocytosis. Thus, dynamin2 acts within lamellipodia to organize actin filaments and regulate assembly and flow of lamellar actomyosin. We hypothesize that through its actions on lamellipodial F-actin, dynamin2 generates F-actin structures that give rise to lamellar actomyosin and for efficient coupling of F-actin at focal adhesions. In this way, dynamin2 orchestrates the global actin cytoskeleton.     

Analysis of turnover dynamics of the submembranous actin cortex

The cell cortex is a thin network of actin, myosin motors, and associated proteins that underlies the plasma membrane in most eukaryotic cells. It enables cells to resist extracellular stresses, perform mechanical work, and change shape. Cortical structural and mechanical properties depend strongly on the relative turnover rates of its constituents, but quantitative data on these rates remain elusive. Using photobleaching experiments, we analyzed the dynamics of three classes of proteins within the cortex of living cells: a scaffold protein (actin), a cross-linker (α-actinin), and a motor (myosin). We found that two filament subpopulations with very different turnover rates composed the actin cortex: one with fast turnover dynamics and polymerization resulting from addition of monomers to free barbed ends, and one with slow turnover dynamics with polymerization resulting from formin-mediated filament growth. Our data suggest that filaments in the second subpopulation are on average longer than those in the first and that cofilin-mediated severing of formin-capped filaments contributes to replenishing the filament subpopulation with free barbed ends. Furthermore, α-actinin and myosin minifilaments turned over significantly faster than F-actin. Surprisingly, only one-fourth of α-actinin dimers were bound to two actin filaments. Taken together, our results provide a quantitative characterization of essential mechanisms under­lying actin cortex homeostasis.     

αB-Crystallin Interacts with Cytoplasmic Intermediate Filament Bundles during Mitosis

The small heat-shock protein αB-crystallin interacts with intermediate filament proteins. Using cosedimentation assay, we showed previously that in vitro binding of αB-crystallin to peripherin and vimentin was temperature-dependent. Furthermore, when NIH 3T3 cells were submitted to different stress conditions a dynamic reorganization of the intermediate filament network was observed concomitantly with the recruitment of αB-crystallins on the intermediate filament proteins. Thus, the intracellular state of αB-crystallin correlated directly with the remodeling of the intermediate filament network in response to stress. Here, we show data suggesting that αB-crystallin is implicated in remodeling of intermediate filaments during cell division. We investigated the intracellular distribution of αB-crystallin in naturally occurring mitotic NIH 3T3 cells and in neuroblastoma N2a and N1E115 cells. In NIH 3T3 cells, αB-crystallin remained diffused throughout the cell cycle. Subcellular fractionation of αB-crystallin showed that αB-crystallin remained in the cytosolic compartment during mitosis. Furthermore, αB-crystallin accumulated in mitotically arrested NIH 3T3 cells. This increased level of αB-crystallin protein was due to an increased level of αB-crystallin mRNA in mitotic NIH 3T3 cells. In the neuroblastoma cells, the intermediate filaments were rearranged into thick cable-like structures and αB-crystallin was recruited onto them. In neuroblastoma N2a cells the level of expression did not change during the cell cycle. However, a small fraction of αB-crystallin switched onto the insoluble fraction in mitotically arrested N2a cells. Our results suggested that depending on the state of rearrangement of the intermediate filament network during mitosis αB-crystallin was either recruited onto the intermediate filaments or upregulated in the cytosolic compartment.     

Adducins Regulate Remodeling of Apical Junctions in Human Epithelial Cells

Epithelial adherens junctions (AJs) and tight junctions (TJs) are dynamic structures that readily undergo disintegration and reassembly. Remodeling of the AJs and TJs depends on the orchestrated dynamics of the plasma membrane with its underlying F-actin cytoskeleton, and the membrane–cytoskeleton interface may play a key role in junctional regulation. Spectrin–adducin–ankyrin complexes link membranes to the actin cytoskeleton where adducins mediate specrtrin–actin interactions. This study elucidates roles of adducins in the remodeling of epithelial junctions in human SK-CO15 colonic and HPAF-II pancreatic epithelial cell monolayers. These cells expressed the α and γ isoforms of adducin that positively regulated each others protein level and colocalized with E-cadherin and β-catenin at mature, internalized and newly assembled AJs. Small interfering RNA-mediated down-regulation of α- or γ-adducin expression significantly attenuated calcium-dependent AJ and TJ assembly and accelerated junctional disassembly triggered by activation of protein kinase C. Two mechanisms were found to mediate the impaired AJ and TJ assembly in adducin-depleted cells. One mechanism involved diminished expression and junctional recruitment of βII-spectrin, and the other mechanism involved the decrease in the amount of cellular F-actin and impaired assembly of perijunctional actin bundles. These findings suggest novel roles for adducins in stabilization of epithelial junctions and regulation of junctional remodeling.     

Mechanism of Cdc42-induced Actin Polymerization in Neutrophil Extracts

Cdc42, activated with GTPγS, induces actin polymerization in supernatants of lysed neutrophils. This polymerization, like that induced by agonists, requires elongation at filament barbed ends. To determine if creation of free barbed ends was sufficient to induce actin polymerization, free barbed ends in the form of spectrin-actin seeds or sheared F-actin filaments were added to cell supernatants. Neither induced polymerization. Furthermore, the presence of spectrin-actin seeds did not increase the rate of Cdc42-induced polymerization, suggesting that the presence of Cdc42 did not facilitate polymerization from spectrin-actin seeds such as might have been the case if Cdc42 inhibited capping or released G-actin from a sequestered pool.Electron microscopy revealed that Cdc42-induced filaments elongated rapidly, achieving a mean length greater than 1 μm in 15 s. The mean length of filaments formed from spectrin-actin seeds was <0.4 μm. Had spectrin-actin seeds elongated at comparable rates before they were capped, they would have induced longer filaments. There was little change in mean length of Cdc42-induced filaments between 15 s and 5 min, suggesting that the increase in F-actin over this time was due to an increase in filament number. These data suggest that Cdc42 induction of actin polymerization requires both creation of free barbed ends and facilitated elongation at these ends.     

Monitoring the Interaction between β2-Microglobulin and the Molecular Chaperone αB-crystallin by NMR and Mass Spectrometry: αB-CRYSTALLIN DISSOCIATES β2-MICROGLOBULIN OLIGOMERS*

The interaction at neutral pH between wild-type and a variant form (R3A) of the amyloid fibril-forming protein β₂-microglobulin (β2m) and the molecular chaperone αB-crystallin was investigated by thioflavin T fluorescence, NMR spectroscopy, and mass spectrometry. Fibril formation of R3Aβ2m was potently prevented by αB-crystallin. αB-crystallin also prevented the unfolding and nonfibrillar aggregation of R3Aβ2m. From analysis of the NMR spectra collected at various R3Aβ2m to αB-crystallin molar subunit ratios, it is concluded that the structured β-sheet core and the apical loops of R3Aβ2m interact in a nonspecific manner with the αB-crystallin. Complementary information was derived from NMR diffusion coefficient measurements of wild-type β2m at a 100-fold concentration excess with respect to αB-crystallin. Mass spectrometry acquired in the native state showed that the onset of wild-type β2m oligomerization was effectively reduced by αB-crystallin. Furthermore, and most importantly, αB-crystallin reversibly dissociated β2m oligomers formed spontaneously in aged samples. These results, coupled with our previous studies, highlight the potent effectiveness of αB-crystallin in preventing β2m aggregation at the various stages of its aggregation pathway. Our findings are highly relevant to the emerging view that molecular chaperone action is intimately involved in the prevention of in vivo amyloid fibril formation.     

Danio rerio αE-catenin Is a Monomeric F-actin Binding Protein with Distinct Properties from Mus musculus αE-catenin

It is unknown whether homologs of the cadherin·catenin complex have conserved structures and functions across the Metazoa. Mammalian αE-catenin is an allosterically regulated actin-binding protein that binds the cadherin·β-catenin complex as a monomer and whose dimerization potentiates F-actin association. We tested whether these functional properties are conserved in another vertebrate, the zebrafish Danio rerio. Here we show, despite 90% sequence identity, that Danio rerio and Mus musculus αE-catenin have striking functional differences. We demonstrate that D. rerio αE-catenin is monomeric by size exclusion chromatography, native PAGE, and small angle x-ray scattering. D. rerio αE-catenin binds F-actin in cosedimentation assays as a monomer and as an α/β-catenin heterodimer complex. D. rerio αE-catenin also bundles F-actin, as shown by negative stained transmission electron microscopy, and does not inhibit Arp2/3 complex-mediated actin nucleation in bulk polymerization assays. Thus, core properties of α-catenin function, F-actin and β-catenin binding, are conserved between mouse and zebrafish. We speculate that unique regulatory properties have evolved to match specific developmental requirements.     

Sro9, a Multicopy Suppressor of the Bud Growth Defect in the Saccharomyces Cerevisiae Rho3-Deficient Cells, Shows Strong Genetic Interactions with Tropomyosin Genes, Suggesting Its Role in Organization of the Actin Cytoskeleton

RHO3 encodes a Rho-type small GTPase in the yeast Saccharomyces cerevisiae and is involved in the proper organization of the actin cytoskeleton required for bud growth. SRO9 (YCL37c) was isolated as a multicopy suppressor of a rho3δ mutation. An Sro9p domain required for function is similar to a domain in the La protein (an RNA-binding protein). Disruption of SRO9 did not affect vegetative growth, even with the simultaneous disruption of an SRO9 homologue, SRO99. However, sro9δ was synthetically lethal with a disruption of TPM1, which encodes tropomyosin; sro9δ tpm1δ cells did not distribute cortical actin patches properly and lysed. We isolated TPM2, the other gene for tropomyosin, as a multicopy suppressor of a tpm1δ sro9δ double mutant. Genetic analysis suggests that TPM2 is functionally related to TPM1 and that tropomyosin is important but not essential for cell growth. Overexpression of SRO9 suppressed the growth defect in tpm1δ tpm2δ cells, disappearance of cables of actin filaments in both rho3δ cells and tpm1δ cells, and temperature sensitivity of actin mutant cells (act1-1 cells), suggesting that Sro9p has a function that overlaps or is related to tropomyosin function. Unlike tropomyosin, Sro9p does not colocalize with actin cables but is diffusely cytoplasmic. These results suggest that Sro9p is a new cytoplasmic factor involved in the organization of actin filaments.     

A Highlights from MBoC Selection: αE-catenin actin-binding domain alters actin filament conformation and regulates binding of nucleation and disassembly factors

The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell–cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments and that binding is accompanied by a conformational change in the actin protomer that affects filament structure. αE-catenin ABD binding limits barbed-end growth, especially in actin filament bundles. αE-catenin ABD inhibits actin filament branching by the Arp2/3 complex and severing by cofilin, both of which contact regions of the actin protomer that are structurally altered by αE-catenin ABD binding. In epithelial cells, there is little correlation between the distribution of αE-catenin and the Arp2/3 complex at developing cell–cell contacts. Our results indicate that αE-catenin binding to filamentous actin favors assembly of unbranched filament bundles that are protected from severing over more dynamic, branched filament arrays.     

Molecular basis of F-actin regulation and sarcomere assembly via myotilin

Sarcomeres, the basic contractile units of striated muscle cells, contain arrays of thin (actin) and thick (myosin) filaments that slide past each other during contraction. The Ig-like domain-containing protein myotilin provides structural integrity to Z-discs—the boundaries between adjacent sarcomeres. Myotilin binds to Z-disc components, including F-actin and α-actinin-2, but the molecular mechanism of binding and implications of these interactions on Z-disc integrity are still elusive. To illuminate them, we used a combination of small-angle X-ray scattering, cross-linking mass spectrometry, and biochemical and molecular biophysics approaches. We discovered that myotilin displays conformational ensembles in solution. We generated a structural model of the F-actin:myotilin complex that revealed how myotilin interacts with and stabilizes F-actin via its Ig-like domains and flanking regions. Mutant myotilin designed with impaired F-actin binding showed increased dynamics in cells. Structural analyses and competition assays uncovered that myotilin displaces tropomyosin from F-actin. Our findings suggest a novel role of myotilin as a co-organizer of Z-disc assembly and advance our mechanistic understanding of myotilin’s structural role in Z-discs.

Myotilin is a scaffold protein in the Z-disc, the boundary between adjacent sarcomeres, aiding structural integrity via multiple interactions, including F-actin and α-actinin-2. An integrative structural model of the complex between myotilin and F-actin reveals that myotilin displaces tropomyosin from F-actin, implying a novel role of myotilin in sarcomere biogenesis beyond a mere interaction hub.     

An Autoinhibited Structure of α-Catenin and Its Implications for Vinculin Recruitment to Adherens Junctions

α-Catenin is an actin- and vinculin-binding protein that regulates cell-cell adhesion by interacting with cadherin adhesion receptors through β-catenin, but the mechanisms by which it anchors the cadherin-catenin complex to the actin cytoskeleton at adherens junctions remain unclear. Here we determined crystal structures of αE-catenin in the autoinhibited state and the actin-binding domain of αN-catenin. Together with the small-angle x-ray scattering analysis of full-length αN-catenin, we deduced an elongated multidomain assembly of monomeric α-catenin that structurally and functionally couples the vinculin- and actin-binding mechanisms. Cellular and biochemical studies of αE- and αN-catenins show that αE-catenin recruits vinculin to adherens junctions more effectively than αN-catenin, partly because of its higher affinity for actin filaments. We propose a molecular switch mechanism involving multistate conformational changes of α-catenin. This would be driven by actomyosin-generated tension to dynamically regulate the vinculin-assisted linkage between adherens junctions and the actin cytoskeleton.     

TREK-1 Regulates Cytokine Secretion from Cultured Human Alveolar Epithelial Cells Independently of Cytoskeletal Rearrangements

BackgroundTREK-1 deficient alveolar epithelial cells (AECs) secrete less IL-6, more MCP-1, and contain less F-actin. Whether these alterations in cytokine secretion and F-actin content are related remains unknown. We now hypothesized that cytokine secretion from TREK-1-deficient AECs was regulated by cytoskeletal rearrangements.MethodsWe determined F-actin and α-tubulin contents of control, TREK-1-deficient and TREK-1-overexpressing human A549 cells by confocal microscopy and western blotting, and measured IL-6 and MCP-1 levels using real-time PCR and ELISA.ResultsCytochalasin D decreased the F-actin content of control cells. Jasplakinolide increased the F-actin content of TREK-1 deficient cells, similar to the effect of TREK-1 overexpression in control cells. Treatment of control and TREK-1 deficient cells with TNF-α, a strong stimulus for IL-6 and MCP-1 secretion, had no effect on F-actin structures. The combination of TNF-α+cytochalasin D or TNF-α+jasplakinolide had no additional effect on the F-actin content or architecture when compared to cytochalasin D or jasplakinolide alone. Although TREK-1 deficient AECs contained less F-actin at baseline, quantified biochemically, they contained more α-tubulin. Exposure to nocodazole disrupted α-tubulin filaments in control and TREK-1 deficient cells, but left the overall amount of α-tubulin unchanged. Although TNF-α had no effect on the F-actin or α-tubulin contents, it increased IL-6 and MCP-1 production and secretion from control and TREK-1 deficient cells. IL-6 and MCP-1 secretions from control and TREK-1 deficient cells after TNF-α+jasplakinolide or TNF-α+nocodazole treatment was similar to the effect of TNF-α alone. Interestingly, cytochalasin D decreased TNF-α-induced IL-6 but not MCP-1 secretion from control but not TREK-1 deficient cells.ConclusionAlthough cytochalasin D, jasplakinolide and nocodazole altered the F-actin and α-tubulin structures of control and TREK-1 deficient AEC, the changes in cytokine secretion from TREK-1 deficient cells cannot be explained by cytoskeletal rearrangements in these cells.     

Cytoplasmic and Nuclear Anti-Apoptotic Roles of αB-Crystallin in Retinal Pigment Epithelial Cells

In addition to its well-characterized role in the lens, αB-crystallin performs other functions. Methylglyoxal (MGO) can alter the function of the basement membrane of retinal pigment epithelial (RPE) cells. Thus, if MGO is not efficiently detoxified, it can induce adverse reactions in RPE cells. In this study, we examined the mechanisms underlying the anti-apoptotic activity of αB-crystallin in the human retinal pigment epithelial cell line ARPE-19 following MGO treatment using various assays, including nuclear staining, flow cytometry, DNA electrophoresis, pulse field gel electrophoresis, western blot analysis, confocal microscopy and co-immunoprecipitation assays. To directly assess the role of phosphorylation of αB-crystallin, we used site-directed mutagenesis to convert relevant serine residues to alanine residues. Using these techniques, we demonstrated that MGO induces apoptosis in ARPE-19 cells. Silencing αB-crystallin sensitized ARPE-19 cells to MGO-induced apoptosis, indicating that αB-crystallin protects ARPE-19 cells from MGO-induced apoptosis. Furthermore, we found that αB-crystallin interacts with the caspase subtypes, caspase-2L, -2S, -3, -4, -7, -8, -9 and -12 in untreated control ARPE-19 cells and that MGO treatment caused the dissociation of these caspase subtypes from αB-crystallin; transfection of S19A, S45A or S59A mutants caused the depletion of αB-crystallin from the nuclei of untreated control RPE cells leading to the release of caspase subtypes. Additionally, transfection of these mutants enhanced MGO-induced apoptosis in ARPE-19 cells, indicating that phosphorylation of nuclear αB-crystallin on serine residues 19, 45 and 59 plays a pivotal role in preventing apoptosis in ARPE-19 cells. Taken together, these results suggest that αB-crystallin prevents caspase activation by physically interacting with caspase subtypes in the cytoplasm and nucleus, thereby protecting RPE cells from MGO-induced apoptosis.