Interaction of nonreceptor tyrosine-kinase Fer and p120 catenin is involved in neuronal polarization

The neuronal cytoskeleton is essential for establishment of neuronal polarity, but mechanisms controlling generation of polarity in the cytoskeleton are poorly understood. The nonreceptor tyrosine kinase, Fer, has been shown to bind to microtubules and to interact with several actin-regulatory proteins. Furthermore, Fer binds p120 catenin and has been shown to regulate cadherin function by modulating cadherin-beta-catenin interaction. Here we show involvement of Fer in neuronal polarization and neurite development. Fer is concentrated in growth cones together with cadherin, beta-catenin, and cortactin in stage 2 hippocampal neurons. Inhibition of Fer-p120 catenin interaction with a cell-permeable inhibitory peptide (FerP) increases neurite branching. In addition, the peptide significantly delays conversion of one of several dendrites into an axon in early stage hippocampal neurons. FerP-treated growth cones also exhibit modified localization of the microtubule and actin cytoskeleton. Together, this indicates that the Fer-p120 interaction is required for normal neuronal polarization and neurite development.     

Differential Effects of G- and F-Actin on the Plasma Membrane Calcium Pump Activity

We have previously shown that plasma membrane calcium ATPase (PMCA) pump activity is affected by the membrane protein concentration (Vanagas et al., Biochim Biophys Acta 1768:1641–1644, 2007). The results of this study provided evidence for the involvement of the actin cytoskeleton. In this study, we explored the relationship between the polymerization state of actin and its effects on purified PMCA activity. Our results show that PMCA associates with the actin cytoskeleton and this interaction causes a modulation of the catalytic activity involving the phosphorylated intermediate of the pump. The state of actin polymerization determines whether it acts as an activator or an inhibitor of the pump: G-actin and/or short oligomers activate the pump, while F-actin inhibits it. The effects of actin on PMCA are the consequence of direct interaction as demonstrated by immunoblotting and cosedimentation experiments. Taken together, these findings suggest that interactions with actin play a dynamic role in the regulation of PMCA-mediated Ca²⁺ extrusion through the membrane. Our results provide further evidence of the activation–inhibition phenomenon as a property of many cytoskeleton-associated membrane proteins where the cytoskeleton is no longer restricted to a mechanical function but is dynamically involved in modulating the activity of integral proteins with which it interacts.     

Cytochemical detection of actin in the structure of the synaptic apparatus of hippocampal field CA3

Ultrastructural distribution of actin in dendrites, dendritic spines and presynaptic boutons of the hippocampal area CA3 of the guinea pig was investigated using decoration and immunocytochemical methods. The distribution of actin was non-homogeneous in all the parts of neurons. The highest concentration of this contractile protein was revealed in the spine cytoplasm. Here actin forms a dense cytoskeleton meshwork and is present also in postsynaptic densities. An intimate interaction between the spine actin cytoskeleton and the postsynaptic actin densities has been revealed. This feature may indicate the involvement of actin cytoskeleton in the organization and maintenance of dimensions, location and geometry of active zones.     

Store‐operated Ca2+ entry: Vesicle fusion or reversible trafficking and de novo conformational coupling?

Store-operated Ca2+ entry (SOCE), a mechanism regulated by the filling state of the intracellular Ca2+ stores, is a major pathway for Ca2+ influx. Hypotheses to explain the communication between the Ca2+ stores and plasma membrane (PM) have considered both the existence of small messenger molecules, such as a Ca2+-influx factor (CIF), and both stable and de novo conformational coupling between proteins in the Ca2+ store and PM. Alternatively, a secretion-like coupling model based on vesicle fusion and channel insertion in the PM has been proposed, which shares some properties with the de novo conformational coupling model, such as the role of the actin cytoskeleton and soluble N-ethylmaleimide (NEM)-sensitive-factor attachment proteins receptor (SNARE) proteins. Here we review recent progress made in the characterization of the de novo conformational coupling and the secretion-like coupling models for SOCE. We pay particular attention into the involvement of SNARE proteins and the actin cytoskeleton in both SOCE models. SNAREs are recognized as proteins involved in exocytosis, participating in vesicle transport, membrane docking, and fusion. As with secretion, a role for the cortical actin network in Ca2+ entry has been demonstrated in a number of cell types. In resting cells, the cytoskeleton may prevent the interaction between the Ca2+ stores and the PM, or preventing fusion of vesicles containing Ca2+ channels with the PM. These are processes in which SNARE proteins might play a crucial role upon cell activation by directing a precise interaction between the membrane of the transported organelle and the PM.     

Actin Cytoskeleton and the Shape of the Plant Cell (A Review)

Recent advances in the study of the cytoskeleton, actin cytoskeleton mainly, its involvement in plant-cell growth of various types, the creation of their specific shape, and also the pathways of intra- and extracellular signal transduction to the actin cytoskeleton are briefly considered. More detail information and the review of earlier publications may be found in numerous comprehensive reviews [1–6] and many others.     

Investigation of the adaptor protein PLIC-2 in multiple pathways

PLIC, Protein Linking IAP (CD47) to Cytoskeleton, have long since been implicated in connecting the extracellular membrane to the intracellular cell cytoskeleton. This phenomenon is supposedly achieved by bridging a receptor protein CD47 to vimentin, an intermediate filament, which in turn regulates integrin dependent cell spreading. Since the discovery of these proteins, the molecular details of the above-mentioned interactions and the underlying complexes are yet to be characterized. Several independent studies have together emphasized PLIC/Ubiquilin’s role in the proteasomal degradation pathway. This seems to be in contrast to the purported initial discovery of PLIC as a cytoskeletal adaptor protein. In an effort to reconcile the different roles associated with the ubiquitous PLIC proteins, we tested the involvement of PLIC-2 both in the proteasomal degradation pathway and as a protein linking the cell cytoskeleton to the cytoplasmic tail of CD47. This was achieved thorough an in vitro investigation of their binding interface using a combination of biophysical techniques. Our results show that the two terminal domains of PLIC-2 interact weakly with each other, while the C-terminal UBA domain interacts strongly with ubiquitin. Interestingly, no perceptible interaction was observed for PLIC-2 with the cytoplasmic tail of CD47 questioning its role as a “PLIC” protein linking the cell membrane to the cytoskeleton.     

Entamoeba histolytica and Entamoeba dispar: Morphological and Behavioral Differences Induced by Fibronectin through GTPases Activation and Actin‐Binding Proteins

Early steps of tissue invasion by Entamoeba histolytica are mediated by adhesion and migration through matrix components such as fibronectin with the participation of the actin cytoskeleton. Striking differences in their produced structures, movement, and migration were found. These observations suggest differential changes in their ability to organize the actin cytoskeleton and, therefore, to modify its morphology after adhesion to fibronectin. To understand these observations, we explore deeper the cytoskeleton pathway of E. histolytica compared to Entamoeba dispar, analyzing the activation and involvement of actin cytoskeleton regulatory proteins such as small GTPases (Rho, Rac1 and Cdc42), myosin IB, paxillin, alpha‐actinin, and ARP2/3 during interaction with fibronectin. Results showed a higher activation of Rac1 in E. histolytica compared to E. dispar, while Cdc42 and RhoA were equally activated in both amebae; besides, variations in the amount of myosin IB, paxillin, and ARP2/3 were detected among these species, coinciding and reflected in formation of lamellipodia in E. histolytica and filopodia in E. dispar. These could partially explain the higher invasive capacity of E. histolytica compared to E. dispar, due to its pleomorphic ability, high motility, migration, activation, and abundance of proteins involved in the cytoskeleton arrangement.     

Identification of a novel sequence in PDZ-RhoGEF that mediates interaction with the actin cytoskeleton

Small GTPases of the Rho family are crucial regulators of actin cytoskeleton rearrangements. Rho is activated by members of the Rho guanine-nucleotide exchange factor (GEF) family; however, mechanisms that regulate RhoGEFs are not well understood. This report demonstrates that PDZ-RhoGEF, a member of a subfamily of RhoGEFs that contain regulator of G protein signaling domains, is partially localized at or near the plasma membranes in 293T, COS-7, and Neuro2a cells, and this localization is coincident with cortical actin. Disruption of the cortical actin cytoskeleton in cells by using latrunculin B prevents the peri-plasma membrane localization of PDZ-RhoGEF. Coimmunoprecipitation and F-actin cosedimentation assays demonstrate that PDZ-RhoGEF binds to actin. Extensive deletion mutagenesis revealed the presence of a novel 25-amino acid sequence in PDZ-RhoGEF, located at amino acids 561-585, that is necessary and sufficient for localization to the actin cytoskeleton and interaction with actin. Last, PDZ-RhoGEF mutants that fail to interact with the actin cytoskeleton display enhanced Rho-dependent signaling compared with wild-type PDZ-RhoGEF. These results identify interaction with the actin cytoskeleton as a novel function for PDZ-RhoGEF, thus implicating actin interaction in organizing PDZ-RhoGEF signaling.     

Arabidopsis phosphatidylinositol phosphate kinase 1 binds F-actin and recruits phosphatidylinositol 4-kinase beta1 to the actin cytoskeleton

The actin cytoskeleton can be influenced by phospholipids and lipid-modifying enzymes. In animals the phosphatidylinositol phosphate kinases (PIPKs) are associated with the cytoskeleton through a scaffold of proteins; however, in plants such an interaction was not clear. Our approach was to determine which of the plant PIPKs interact with actin and determine whether the PIPK-actin interaction is direct. Our results indicate that AtPIPK1 interacts directly with actin and that the binding is mediated through a predicted linker region in the lipid kinase. AtPIPK1 also recruits AtPI4Kbeta1 to the cytoskeleton. Recruitment of AtPI4Kbeta1 to F-actin was dependent on the C-terminal catalytic domain of phosphatidylinositol-4-phosphate 5-kinase but did not require the presence of the N-terminal 251 amino acids, which includes 7 putative membrane occupation and recognition nexus motifs. In vivo studies confirm the interaction of plant lipid kinases with the cytoskeleton and suggest a role for actin in targeting PIPKs to the membrane.     

The KSR2-calcineurin complex regulates STIM1-ORAI1 dynamics and store-operated calcium entry (SOCE)

Store-operated calcium entry (SOCE) is the predominant Ca²⁺ entry mechanism in nonexcitable cells and controls a variety of physiological and pathological processes. Although significant progress has been made in identifying the components required for SOCE, the molecular mechanisms underlying it are elusive. The present study provides evidence for a direct involvement of kinase suppressor of Ras 2 (KSR2) in SOCE. Using lymphocytes and fibroblasts from ksr2^−/− mice and shKSR2-depleted cells, we find that KSR2 is critical for the elevation of cytosolic Ca²⁺ concentration. Specifically, our results show that although it is dispensable for Ca²⁺-store depletion, KSR2 is required for optimal calcium entry. We observe that KSR2 deficiency affects stromal interaction molecule 1 (STIM1)/ORAI1 puncta formation, which is correlated with cytoskeleton disorganization. Of interest, we find that KSR2-associated calcineurin is crucial for SOCE. Blocking calcineurin activity impairs STIM1/ORAI1 puncta-like formation and cytoskeleton organization. In addition, we observe that calcineurin activity and its role in SOCE are both KSR2 dependent.     

Cleavage of SNAP-25 and VAMP-2 impairs store-operated Ca2+ entry in mouse pancreatic acinar cells

We recently reported that store-operated Ca²⁺ entry (SOCE) in nonexcitable cells is likely to be mediated by a reversible interaction between Ca²⁺ channels in the plasma membrane and the endoplasmic reticulum, a mechanism known as "secretion-like coupling." As for secretion, in this model the actin cytoskeleton plays a key regulatory role. In the present study we have explored the involvement of the secretory proteins synaptosome-associated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) in SOCE in pancreatic acinar cells. Cleavage of SNAP-25 and VAMPs by treatment with botulinum toxin A (BoNT A) and tetanus toxin (TeTx), respectively, effectively inhibited amylase secretion stimulated by the physiological agonist CCK-8. BoNT A significantly reduced Ca²⁺ entry induced by store depletion using thapsigargin or CCK-8. In addition, treatment with BoNT A once SOCE had been activated reduced Ca²⁺ influx, indicating that SNAP-25 is needed for both the activation and maintenance of SOCE in pancreatic acinar cells. VAMP-2 and VAMP-3 are expressed in mouse pancreatic acinar cells. Both proteins associate with the cytoskeleton upon Ca²⁺ store depletion, although only VAMP-2 seems to be sensitive to TeTx. Treatment of pancreatic acinar cells with TeTx reduced the activation of SOCE without affecting its maintenance. These findings support a role for SNAP-25 and VAMP-2 in the activation of SOCE in pancreatic acinar cells and show parallels between this process and secretion in a specialized secretory cell type. synaptosome-associated protein; vesicle-associated membrane protein; pancreatic acinar cells; cytoskeleton; calcium entry     

Interaction of epidermal growth factor receptors with the cytoskeleton is related to receptor clustering

Recently it has been established that cytoskeleton-associated epidermal growth factor (EGF) receptors are predominantly of the high-affinity class and that EGF induces a recruitment of low-affinity receptors to the cytoskeleton. The nature of this EGF-induced receptor-cytoskeleton interaction, however, is still unknown. Therefore, we have studied the association of mutated EGF receptors with the cytoskeleton. Receptor deletion mutants lacking almost all intracellular amino acid residues displayed no interaction with the cytoskeleton, demonstrating that the cytoplasmic receptor domain is involved in this interaction. Further analysis revealed that receptor-cytoskeleton interaction is independent of receptor kinase activity and the C-terminal 126 amino acid residues, which include the auto-phosphorylation sites. Furthermore, it is shown that the high-affinity receptor subclass is not essential for association of low-affinity receptors to the cytoskeleton. EGF receptor-cytoskeleton interaction was increased, however, by treatment with sphingomyelinase, an enzyme known to induce membrane protein clustering, indicating that EGF receptor clustering may cause the association to the cytoskeleton.     

Cell-wall lectins during winter wheat cold hardening

The polypeptide composition and functional activity of cell-wall lectins from roots of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) seedlings during cold hardening were studied. Several phases of lectin activity changes were observed, which indicates their involvement in the development of general adaptation syndrome of the cell. After 0.5-h low-temperature treatment, marked alterations occurred in the profile of protein elution: lectins with mol wts of 78 and 42.5 kD disappeared and new ones with mol wts of 72, 69, 37, and 34.5 kD appeared. It was established that 17.5-and 69-kD lectins and most lectins eluted with glucose were arabinogalactan proteins (AGP), which permitted a supposition that these lectins were involved in the interaction between the cell wall and cytoskeleton. After 7-day-long hardening, total protein content reduced and lectins with mol wts of 69 and 37 kD disappeared, which corresponded to reduced lectin activity by the end of hardening. A transient appearance of 37-and 69-kD lectins, which are AGP, might indicate their involvement in the triggering the development of plant-cell defense responses.     

Low levels of the pesticide, delta-hexachlorocyclohexane, lyses human erythrocytes and alters the organization of membrane lipids and proteins as revealed by Raman spectroscopy.

We studied the nature of the interaction of delta-hexachlorocyclohexane (delta-HCCH), a pesticide having a stereoisomeric structure similar to inositol, with red blood cells. Cell survival data, measured as percent of hemoglobin released by delta-HCCH, show that the cell lysis increases with post exposure time. delta-HCCH at 55-60 micrograms/ml causes about 70% cell lysis after 24 h of exposure. The nature of interaction of delta-HCCH with membrane components was evaluated by studying the thermotropic transitions and protein structure of ghosts using Raman spectroscopy. Control ghosts show transitions with onset/completion temperatures 30 degrees C/38 degrees C (high temperature transition) and 3 degrees C/10 degrees C (middle temperature transition) when monitored by the I2935/I2850 ratio. The interaction of delta-HCCH drastically broadens the high temperature transition and shifts it to the temperature range of 10-29 degrees C. The plots of (I2880-90/I2850) vs. temperature show two transitions for control ghosts, one extending from -10 degrees C to 3 degrees C (lower temperature transition) and the other from about 7 degrees C to about 15 degrees C (middle temperature transition). Ghosts lysed with delta-HCCH shows only a single and a very broad transition in the range of about -3 degrees C to about 15 degrees C. These changes in the thermal transition properties suggest that delta-HCCH alters lipid and lipid-protein phases of erythrocyte membranes. The comparison of Raman spectra in the amide I and III regions of erythrocyte ghosts and purified band 3 with several amidated compounds reveals that cytoskeleton proteins contain highly amidated residues (probably glutamine and asparagine). The interaction of delta-HCCH with erythrocytes drastically alters the environment of these amidated residues indicating the involvement of cytoskeleton proteins. We conclude that the interaction of delta-HCCH with red blood cells disrupt membrane structure and change the environment of cytoskeleton proteins that could cause cell lysis.     

Cross-linking of immunoglobulin E-receptor complexes induces their interaction with the cytoskeleton of rat basophilic leukemia cells

Bridging of immunoglobulin E (IgE)-receptor complexes on rat basophilic leukemia cells by polyclonal anti-IgE antibodies induces a detergent-resistant association of these complexes with the cellular cytoskeleton. In dose-response curves the extent of the cytoskeletal association appears to follow the extent of bridging, continuing to increase beyond where stimulated degranulation is maximal. This stable association is maintained after the aggregated IgE-receptor complexes have been internalized by the cell. Multivalent antigen and trimeric IgE cause less extensive receptor cross-linking than anti-IgE and stimulate degranulation; they also induce receptor association with the cytoskeleton that is revealed only after stabilization by addition of a chemical cross-linking reagent. The ability of a membrane impermeant chemical cross-linker to stabilize this association suggests that the receptor-cytoskeletal interaction may be mediated by a transmembrane protein that is exposed at the cell surface. Monomeric and dimeric IgE bound to receptors fail to induce a stable interaction with the cytoskeleton even in the presence of chemical cross-linkers and are poor (dimers) or insignificant (monomers) stimulators of cellular degranulation. These findings are consistent with a possible relationship between receptor attachment to the cytoskeleton, receptor immobilization as measured by fluorescence photobleaching recovery, and the triggering of cellular degranulation.     

Mechanobiology of antigen-induced T cell arrest

To mount an immune response, T cells must first find rare antigens present at the surface of antigen-presenting cells (APCs). They achieve this by migrating rapidly through the crowded space of tissues and constantly sampling the surface of APCs. Upon antigen recognition, T cells decelerate and polarise towards the APC, ultimately forming a specialised interface known as the immunological synapse. These conjugates form as the result of the interaction between pairs of receptors/ligands that are under mechanical stress due to the continuously reorganising cell cytoskeleton. In this review, we discuss the involvement of mechanical forces during antigen recognition by migrating T cells. We will explore this question from a conceptual and technical perspective, with the aim of providing new insights into the emerging field of mechanobiology.     

Actin cytoskeleton and small heat shock proteins: how do they interact?

Actin and small heat shock proteins (sHsps) are ubiquitous and multifaceted proteins that exist in 2 reversible forms, monomers and multimers, ie, the microfilament of the cytoskeleton and oligomers of the sHsps, generally, supposed to be in a spherical and hollow form. Two situations are described in the literature, where the properties of actin are modulated by sHsps; the actin polymerization is inhibited in vitro by some sHsps acting as capping proteins, and the actin cytoskeleton is protected by some sHsps against the disruption induced by various stressful conditions. We propose that a direct actin-sHsp interaction occurs to inhibit actin polymerization and to participate in the in vivo regulation of actin filament dynamics. Protection of the actin cytoskeleton would result from an F-actin-sHsp interaction in which microfilaments would be coated by small oligomers of phosphorylated sHsps. Both proteins share common structural motives suggesting direct binding sites, but they remain to be demonstrated. Some sHsps would behave with the actin cytoskeleton as actin-binding proteins capable of either capping a microfilament when present as a nonphosphorylated monomer or stabilizing and protecting the microfilament when organized in small, phosphorylated oligomers.     

Role of actin cytoskeleton in dendritic spine morphogenesis

Dendritic spines are the postsynaptic receptive regions of most excitatory synapses, and their morphological plasticity play a pivotal role in higher brain functions, such as learning and memory. The dynamics of spine morphology is due to the actin cytoskeleton concentrated highly in spines. Filopodia, which are thin and headless protrusions, are thought to be precursors of dendritic spines. Drebrin, a spine-resident side-binding protein of filamentous actin (F-actin), is responsible for recruiting F-actin and PSD-95 into filopodia, and is suggested to govern spine morphogenesis. Interestingly, some recent studies on neurological disorders accompanied by cognitive deficits suggested that the loss of drebrin from dendritic spines is a common pathognomonic feature of synaptic dysfunction. In this review, to understand the importance of actin-binding proteins in spine morphogenesis, we first outline the well-established knowledge pertaining to the actin cytoskeleton in non-neuronal cells, such as the mechanism of regulation by small GTPases, the equilibrium between globular actin (G-actin) and F-actin, and the distinct roles of various actin-binding proteins. Then, we review the dynamic changes in the localization of drebrin during synaptogenesis and in response to glutamate receptor activation. Because side-binding proteins are located upstream of the regulatory pathway for actin organization via other actin-binding proteins, we discuss the significance of drebrin in the regulatory mechanism of spine morphology through the reorganization of the actin cytoskeleton. In addition, we discuss the possible involvement of an actin-myosin interaction in the morphological plasticity of spines.     

Interaction of alpha-subunit of the GTP-binding protein Go with cytoskeleton

It was found that about 30% of the alpha-subunit of the bovine brain GTP-binding protein, Go, is bound to the cytoskeleton. The efficiency of Go alpha binding to the cytoskeleton components depends on the nature of the guanyl nucleotides [GDP-beta-S, Gpp (NH) p] present in the incubation medium. It was shown that the alpha-subunit interaction with cytoskeleton components is controlled by ATP-dependent reactions. ATP diminishes the degree Go alpha adsorption. The non-hydrolysable ATP analog, App (NH) p, has no effect on the Go alpha interaction with the cytoskeleton. It was found that one of the cytoskeleton components capably of binding to Go alpha is tubulin. Similar interactions were detected in human neuroblastoma N2A cells.