Tyrosine phosphorylation of Kv1.2 modulates its interaction with the actin-binding protein cortactin

Tyrosine phosphorylation evokes functional changes in a variety of ion channels. Modulation of the actin cytoskeleton also affects the function of some channels. Little is known about how these avenues of ion channel regulation may interact. We report that the potassium channel Kv1.2 associates with the actin-binding protein cortactin and that the binding is modulated by tyrosine phosphorylation. Immunocytochemical and biochemical analyses show that Kv1.2 and cortactin co-localize to the cortical actin cytoskeleton at the leading edges of the cell. Binding assays using purified recombinant proteins reveal a 19-amino acid span within the carboxyl terminus of Kv1.2 that is necessary for direct cortactin binding. Phosphorylation of specific tyrosines within the C terminus of Kv1.2 attenuates that binding. In HEK293 cells, activation of the M1 muscarinic acetylcholine receptor evokes tyrosine phosphorylation-dependent suppression of Kv1.2 ionic current. We show that M1 receptor activation also reduces the interaction of cortactin with Kv1.2 and that mutant Kv1.2 channels deficient for cortactin binding exhibit strongly attenuated ionic current. These results demonstrate a dynamic, phosphorylation-dependent interaction between Kv1.2 and the actin cytoskeleton-binding protein cortactin and suggest a role for that interaction in the regulation of Kv1.2 ionic current.     

Actin cytoskeleton remodelling in the anterior pituitary folliculostellate cell line TtT/GF: participation of the actin-binding protein cortactin

We have previously shown that the folliculostellate (FS) cells of the anterior pituitary change their shape from stellate (type I) to polygonal (type II) coincidently with variations in the secretory activity of the pituitary. To elucidate the mechanisms involved in this switch in phenotypes, here we studied the impact of serum factors on the morphology of the FS cell line TtT/GF. TtT/GF cells cultured in serum-containing medium displayed elongated shapes and membrane ruffles similarly to type I cells. Serum deprivation caused the loss of plasma membrane activity and the acquisition by the cells of a sedentary phenotype and of a polygonal shape typical of type II FS cells. Addition of serum to the starved cells induced the reappearance of membrane raffles and lamellipodia. The switch in phenotypes and the maintenance of a motile phenotype depended on tyrosine kinase but not on Erk activity. Because the transition between phenotypes involved the tyrosine kinase-dependent reorganization of cortical actin filaments, we studied the participation of the actin-binding protein, cortactin, a tyrosine kinase substrate. Cortactin and its tyrosine-phosphorylated form, pY421-cortactin, localized to membrane ruffles and lamellipodia in serum-cultured TtT/GF cells, while they were evenly distributed over the whole cell cortex in serum-starved cells. Serum treatment of starved cells induced a transient increase in pY421-cortactin levels and the clustering of pY421-cortactin in membrane regions where protrusions were developing. Both serum responses were blocked by a tyrosine kinase inhibitor. Together, the results indicate that the transition from a polygonal to an elongated shape entails the acquisition of a dynamic cortical actin cytoskeleton that involves the tyrosine kinase-dependent phosphorylation of cortactin and the translocation of cortical pY421-cortactin to sites of ruffle formation at the plasma membrane.     

Antagonistic interactions between the cAMP-dependent protein kinase and Tor signaling pathways modulate cell growth in Saccharomyces cerevisiae

In mammals, X-chromosome inactivation (XCI) equalizes X-linked gene expression between XY males and XX females and is controlled by a specialized region known as the X-inactivation center (Xic). The Xic harbors two chromatin interaction domains, one centered around the noncoding Xist gene and the other around the antisense Tsix counterpart. Previous work demonstrated the existence of a chromatin transitional zone between the two domains. Here, we investigate the region and discover a conserved element, RS14, that presents a strong binding site for Ctcf protein. RS14 possesses an insulatory function suggestive of a boundary element and is crucial for cell differentiation and growth. Knocking out RS14 results in compromised Xist induction and aberrant XCI in female cells. These data demonstrate that a junction element between Tsix and Xist contributes to the initiation of XCI.     

Betacap73-ARF6 interactions modulate cell shape and motility after injury in vitro

To understand the role that ARF6 plays in regulating isoactin dynamics and cell motility, we transfected endothelial cells (EC) with HA-tagged ARF6: the wild-type form (WT), a constitutively-active form unable to hydrolyze GTP (Q67L), and two dominant-negative forms, which are either unable to release GDP (T27N) or fail to bind nucleotide (N122I). Motility was assessed by digital imaging microscopy before Western blot analysis, coimmunoprecipitation, or colocalization studies using ARF6, beta-actin, or beta-actin-binding protein-specific antibodies. EC expressing ARF6-Q67L spread and close in vitro wounds at twice the control rates. EC expressing dominant-negative ARF6 fail to develop a leading edge, are unable to ruffle their membranes (N122I), and possess arborized processes. Colocalization studies reveal that the Q67L and WT ARF6-HA are enriched at the leading edge with beta-actin; but T27N and N122I ARF6-HA are localized on endosomes together with the beta-actin capping protein, betacap73. Coimmunoprecipitation and Western blot analyses reveal the direct association of ARF6-HA with betacap73, defining a role for ARF6 in signaling cytoskeletal remodeling during motility. Knowledge of the role that ARF6 plays in orchestrating membrane and beta-actin dynamics will help to reveal molecular mechanisms regulating actin-based motility during development and disease.     

Phosphatidylinositol phosphates modulate interactions between the StarD4 sterol trafficking protein and lipid membranes

There is substantial evidence for extensive nonvesicular sterol transport in cells. For example, lipid transfer by the steroidogenic acute regulator-related proteins (StarD) containing a StarT domain has been shown to involve several pathways of nonvesicular trafficking. Among the soluble StarT domain–containing proteins, StarD4 is expressed in most tissues and has been shown to be an effective sterol transfer protein. However, it was unclear whether the lipid composition of donor or acceptor membranes played a role in modulating StarD4-mediated transport. Here, we used fluorescence-based assays to demonstrate a phosphatidylinositol phosphate (PIP)-selective mechanism by which StarD4 can preferentially extract sterol from liposome membranes containing certain PIPs (especially, PI(4,5)P₂ and to a lesser degree PI(3,5)P2). Monophosphorylated PIPs and other anionic lipids had a smaller effect on sterol transport. This enhancement of transport was less effective when the same PIPs were present in the acceptor membranes. Furthermore, using molecular dynamics (MD) simulations, we mapped the key interaction sites of StarD4 with PIP-containing membranes and identified residues that are important for this interaction and for accelerated sterol transport activity. We show that StarD4 recognizes membrane-specific PIPs through specific interaction with the geometry of the PIP headgroup as well as the surrounding membrane environment. Finally, we also observed that StarD4 can deform membranes upon longer incubations. Taken together, these results suggest a mechanism by which PIPs modulate cholesterol transfer activity via StarD4.     

Intra-membrane ligand diffusion and cell shape modulate juxtacrine patterning

A key problem in developmental biology is how pattern and planar polarity are transmitted in epithelial structures. Examples include Drosophila neuronal differentiation, ommatidia formation in the compound eye, and wing hair polarization. A key component for the generation of such patterns is direct cell-cell signalling by transmembrane ligands, called juxtacrine signalling. Previous models for this mode of communication have considered homogeneous distributions in the cell membrane, and the role of polarity has been largely ignored. In this paper we determine the role of inhomogeneous protein and receptor distributions in juxtacrine signalling. We explicitly include individual membrane segments, diffusive transport of proteins and receptors between these segments, and production terms with a combination of local and global responses to ligand binding. Our analysis shows that intra-membrane ligand transport is vital for the generation of long wavelength patterns. Moreover, with no ligand transport, there is no pattern formation for lateral induction, a process in which receptor activation up-regulates ligand production. Biased production of ligand also modulates patterning bifurcations and predicted wavelengths. In addition, biased ligand and receptor trafficking can lead to regular polarity across a lattice, in which each cell has the same orientation-directly analogous to patterns of hairs in the Drosophila wing. We confirm the trends in pattern wavelengths previously observed for patterns with cellular homogeneity-lateral inhibition tends to give short-range patterns, while lateral induction can give patterns with much longer wavelengths. Moreover, the original model can be recovered if intra-membrane bound receptor diffusion is included and rapid equilibriation between the sides is considered. Finally, we consider the role of irregular cell shapes and waves in such networks, including wave propagation past clones of non-signalling cells.     

Collagen can modulate cell interactions with fibronectin

We have examined the effects of soluble collagen on the function of fibronectin in baby hamster kidney (BHK) cells. Collagen and its purified alpha1(l) chain noncompetitively inhibited cell spreading on substrates precoated with fibronectin or a 75,000-D cell-binding fragment of fibronectin. Neither preincubation of cells with collagen followed by washing nor the addition of collagen to previously spread cells had any inhibitory effect on cell spreading, which indicates a requirement for the concurrent presence of collagen during the process of spreading. Treatment of collagen or alpha1(l) chain with collagenase abolished the inhibitory effect on fibronectin-mediated cell spreading. However, direct attachment of BHK cells to fibronectin-coated or 75,000-D fragment-coated substrates was not inhibited by collagen or by the alpha1(l) chain. Moreover, the binding of [3H]fibronectin or the 3'-75,000-D fragment to cell surfaces was not inhibited by the presence of soluble collagen, whereas soluble fibronectin inhibited binding. Although the binding of [3H]fibronectin-coated beads to BHK cell surfaces was also not inhibited by collagen, the phagocytosis of such beads was inhibited by the presence of collagen. On the other hand, soluble fibronectin partially inhibited the binding of fibronectin-coated beads but did not inhibit phagocytosis of the beads that did bind. The mechanism of the inhibition of fibronectin function by collagen and the possible interactions of two different kinds of receptors on the cell surface are discussed.     

Green fluorescent protein impairs actin-myosin interactions by binding to the actin-binding site of myosin

Green fluorescent proteins (GFP) are widely used in biology for tracking purposes. Although expression of GFP is considered to be innocuous for the cells, deleterious effects have been reported. We recently demonstrated that expression of eGFP in muscle impairs its contractile properties (Agbulut, O., Coirault, C., Niederlander, N., Huet, A., Vicart, P., Hagege, A., Puceat, M., and Menasche, P. (2006) Nat. Meth. 3, 331). This prompted us to identify the molecular mechanisms linking eGFP expression to contractile dysfunction and, particularly, to test the hypothesis that eGFP could inhibit actin-myosin interactions. Therefore, we assessed the cellular, mechanical, enzymatic, biochemical, and structural properties of myosin in the presence of eGFP and F-actin. In vitro motility assays, the maximum actin-activated ATPase rate (V(max)) and the associated constant of myosin for actin (K(m)) were determined at 1:0.5, 1:1, and 1:3 myosin:eGFP molar ratios. At a myosin:eGFP ratio of 1:0.5, there was a nearly 10-fold elevation of K(m). As eGFP concentration increased relative to myosin, the percentage of moving filaments, the myosin-based velocity, and V(max) significantly decreased compared with controls. Moreover, myosin co-precipitated with eGFP. Crystal structures of myosin, actin, and GFP indicated that GFP and actin exhibited similar electrostatic surface patterns and the ClusPro docking model showed that GFP bound preferentially to the myosin head and especially to the actin-binding site. In conclusion, our data demonstrate that expression of eGFP in muscle resulted in the binding of eGFP to myosin, thereby disturbing the actin-myosin interaction and in turn the contractile function of the transduced cells. This potential adverse effect of eGFP should be kept in mind when using this marker to track cells following transplantation.     

Hydrophobic and electrostatic interactions modulate protein escape at the ribosomal exit tunnel

After translation, nascent proteins must escape the ribosomal exit tunnel to attain complete folding to their native states. This escape process also frees up the ribosome tunnel for a new translation job. In this study, we investigate the impacts of energetic interactions between the ribosomal exit tunnel and nascent proteins on the protein escape process by molecular dynamics simulations using partially coarse-grained models that incorporate hydrophobic and electrostatic interactions of the ribosome tunnel of Haloarcula marismortui with nascent proteins. We find that, in general, attractive interactions slow down the protein escape process, whereas repulsive interactions speed it up. For the small globular proteins considered, the median escape time correlates with both the number of hydrophobic residues, N_h, and the net charge, Q, of a nascent protein. A correlation coefficient exceeding 0.96 is found for the relation between the median escape time and a combined quantity of N_h + 5.9Q, suggesting that it is ∼6 times more efficient to modulate the escape time by changing the total charge than the number of hydrophobic residues. The estimated median escape times are found in the submillisecond-to-millisecond range, indicating that the escape does not delay the ribosome recycling. For various types of the tunnel model, with and without hydrophobic and electrostatic interactions, the escape time distribution always follows a simple diffusion model that describes the escape process as a downhill drift of a Brownian particle, suggesting that nascent proteins escape along barrier-less pathways at the ribosome tunnel.     

Mechanically tuning actin filaments to modulate the action of actin-binding proteins

In cells, the actin cytoskeleton is regulated by an interplay between mechanics and biochemistry. A key mechanism, which has emerged based on converging indications from structural, cellular, and biophysical data, depicts the actin filament as a mechanically tunable substrate: mechanical stress applied to an actin filament induces conformational changes, which modify the binding and the regulatory action of actin-binding proteins. For a long time, however, direct evidence of this mechanotransductive mechanism was very scarce. This situation is changing rapidly, and recent in vitro single-filament studies using different techniques have revealed that several actin-binding proteins are able to sense tension, curvature, and/or torsion, applied to actin filaments. Here, we discuss these recent advances and their possible implications.     

Direct interaction between caldesmon and cortactin

Actin polymerization and depolymerization plays a central role in controlling a wide spectrum of cellular processes. There are many actin-binding proteins in eukaryotic cells. Their roles in the remodeling of the actin architecture and whether they work cooperatively await further study. Caldesmon (CaD) is an actin-binding protein present in nearly all mammalian cells. Cortactin is another actin-binding protein found mainly in the cell cortex. There have been no reports suggesting that CaD and cortactin interact with each other or work as partners. Here, we present evidence that CaD binds cortactin directly by overlay, pull-down assays, ELISA, and by column chromatography. The interaction involves the N-terminal region of cortactin and the C-terminal region of CaD, and appears to be enhanced by divalent metal ions. Cortactin competes with both full-length CaD and its C-terminal fragment for actin binding. Binding of cortactin partially alleviates the inhibitory effect of CaD on the actomyosin ATPase activity. Not only can binding be demonstrated in vitro, the two proteins also co-localize in activated cells at the cortex. Whether such interactions bear any functional significance awaits further investigation.     

Identification of dynamin, a novel mechanochemical enzyme that mediates interactions between microtubules

We report that calf brain microtubules prepared without nucleotide contain, in addition to kinesin and dynein, a polypeptide of 100 kd that could be dissociated by nucleotide. The protein was selectively extracted from microtubules using a combination of GTP and AMP-PNP. The extract contained microtubule-stimulated (6-fold) MgATPase activity that partitioned into two components upon further purification: the 100 kd polypeptide and a soluble activating fraction. The 100 kd protein induced microtubules to form hexagonally packed bundles containing periodic cross bridges spaced 13 nm apart. In the presence of ATP and the activating fraction, bundles fragmented, elongated, and exhibited other behavior indicative of sliding between microtubules. These findings indicate that the 100 kd protein is part of a novel mechanochemical enzyme, which we term "dynamin", that may mediate microtubule sliding in vivo.     

Characterization of interactions of Nck with Sos and dynamin

One of the adaptor proteins, Nck, comprises a single SH2 domain and three SH3 domains that are important in protein-protein interactions. The in vivo association of Nck with the guanine nucleotide exchange factor Sos has been well documented; however, the precise nature of the interaction is unclear. To determine which SH3 domains are involved in the Nck-Sos interaction, individual SH3 domains of Nck were generated as glutathione S-transferase fusion proteins. We found that exclusively the third (C-terminal) SH3 domain of Nck has the ability to bind to Sos. In addition, in [35S]methionine labelled K562 cells, a 100,000 Mr protein was found to be associated with the third SH3 domain of Nck. This protein was identified as dynamin, a GTP-binding protein that has been implicated in clathrin-coated vesicle formation. Dynamin and Nck co-precipitated when cell lysates were immunoprecipitated with anti-Nck antibody. These data suggest that Nck may contribute to Ras activation and the function of dynamin in membrane trafficking through its third SH3 domain.     

The actin-binding ERM protein Moesin binds to and stabilizes microtubules at the cell cortex

Ezrin, Radixin, and Moesin (ERM) proteins play important roles in many cellular processes including cell division. Recent studies have highlighted the implications of their metastatic potential in cancers. ERM’s role in these processes is largely attributed to their ability to link actin filaments to the plasma membrane. In this paper, we show that the ERM protein Moesin directly binds to microtubules in vitro and stabilizes microtubules at the cell cortex in vivo. We identified two evolutionarily conserved residues in the FERM (4.1 protein and ERM) domains of ERMs that mediated the association with microtubules. This ERM–microtubule interaction was required for regulating spindle organization in metaphase and cell shape transformation after anaphase onset but was dispensable for bridging actin filaments to the metaphase cortex. These findings provide a molecular framework for understanding the complex functional interplay between the microtubule and actin cytoskeletons mediated by ERM proteins in mitosis and have broad implications in both physiological and pathological processes that require ERMs.     

Eph/ephrin interactions modulate muscle satellite cell motility and patterning

During development and regeneration, directed migration of cells, including neural crest cells, endothelial cells, axonal growth cones and many types of adult stem cells, to specific areas distant from their origin is necessary for their function. We have recently shown that adult skeletal muscle stem cells (satellite cells), once activated by isolation or injury, are a highly motile population with the potential to respond to multiple guidance cues, based on their expression of classical guidance receptors. We show here that, in vivo, differentiated and regenerating myofibers dynamically express a subset of ephrin guidance ligands, as well as Eph receptors. This expression has previously only been examined in the context of muscle-nerve interactions; however, we propose that it might also play a role in satellite cell-mediated muscle repair. Therefore, we investigated whether Eph-ephrin signaling would produce changes in satellite cell directional motility. Using a classical ephrin 'stripe' assay, we found that satellite cells respond to a subset of ephrins with repulsive behavior in vitro; patterning of differentiating myotubes is also parallel to ephrin stripes. This behavior can be replicated in a heterologous in vivo system, the hindbrain of the developing quail, in which neural crest cells are directed in streams to the branchial arches and to the forelimb of the developing quail, where presumptive limb myoblasts emigrate from the somite. We hypothesize that guidance signaling might impact multiple steps in muscle regeneration, including escape from the niche, directed migration to sites of injury, cell-cell interactions among satellite cell progeny, and differentiation and patterning of regenerated muscle.     

Ligands differentially modulate the protein interactions of the human estrogen receptors alpha and beta

The interactions of human estrogen receptor subtypes ERalpha and ERbeta with DNA and a 210 amino acid residue fragment of the coactivator protein SRC-1 bearing three nuclear receptor interaction motifs were investigated quantitatively using fluorescence anisotropy in the presence of agonist and antagonist ligands. ERalpha and ERbeta were found to bind in a similar manner to DNA, and both salt and temperature affected the affinity and/or stoichiometry of these interactions. The agonist ligands estradiol, estrone and estriol did not modify the binding of ERalpha to the fluorescein-labeled target estrogen response element. However, in the case of ERbeta, these ligands led to the formation of some higher-order protein-DNA complexes and a small decrease in affinity. The partial agonist 4-hydroxytamoxifen had little effect on either ER subtype, whereas the pure antagonist ICI 182,780 led to the cooperative formation of protein-DNA complexes of higher order than dimer, as further demonstrated by competition experiments and gel mobility-shift assays. In addition to DNA binding, the interaction of both ER subtypes with the Alexa488-labeled SRC-1 coactivator fragment was investigated by fluorescence anisotropy. The agonist ligands estrone, estradiol, estriol, genistein and ethynyl estradiol exhibited distinct capacities for inducing the recruitment of SRC-1 that were not correlated with their affinity for the receptor. Moreover, estrone and genistein exhibited subtype specificity in that they induced SRC-1 recruitment to ERbeta with much higher efficiency than in the case of ERalpha. The differential coactivator recruitment capacities of the ER agonists and their receptor subtype coactivator recruitment specificity may be linked to the molecular structure of the agonists with respect to their interactions with a specific histidine residue located at the back of the ligand-binding pocket. Altogether, these quantitative in vitro studies of ER interactions reveal the complex energetic and stoichiometric consequences of changes in the chemical structures of these proteins and their ligands.     

Changes in soil microbial communities modulate interactions between maize and weeds

Plant and Soil - Despite the importance of the soil microbiota to plant growth, the role of soil microorganisms in crop-weed competition remains largely unexplored. Here, we investigated the...     

The role of tropomyosin in the interactions of F-actin with caldesmon and actin-binding protein (or filamin)

The interactions of actin filaments with actin-binding protein (filamin) and caldesmon under the influence of tropomyosin were studied in detail using falling-ball viscometry, binding assay and electron microscopy. Caldesmon decreased the binding constant of filamin with F-actin. In contrast, the maximum binding ability of filamin to F-actin was decreased by tropomyosin. The filamin-induced gelation of actin filaments was inhibited by caldesmon. Tropomyosin also inhibited this gelation. The effect of caldesmon became stronger under the influence of tropomyosin. Furthermore, both caldesmon and tropomyosin additionally decreased the filamin binding to F-actin. From these results, caldesmon and tropomyosin appeared to influence filamin binding to F-actin with different modes of actin. In addition, there was no sign of direct interactions between filamin, caldesmon and tropomyosin as judged from gel filtration. Under the influence of caldesmon and tropomyosin, calmodulin conferred Ca2+ sensitivity on the filamin-induced gelation of actin filaments.