Potassium channel gating in adhesion: from an oocyte–silicon to a neuron–astrocyte adhesion contact

In a neuron–astrocyte adhesion contact the ionic current due to the opening of voltage-dependent potassium channels has to flow along a narrow intercellular cleft, generating there an extracellular voltage. This voltage might be large enough to affect significantly the dependence of channel gating from the intracellular voltage. In order to test this hypothesis, we considered a Xenopus oocyte expressing voltage-dependent potassium channels adhering to a layer of silicon oxide as a simplified model of cell–cell adhesion; here the cell membrane and silicon oxide are separated by a narrow cleft and form a junction of circular shape. We measured directly the extracellular voltage along the diameter of the cleft and investigated its effect on channel gating using a linear array of field effect transistors integrated in the silicon substrate. On this experimental basis we demonstrated that the voltage dependence of potassium channels is strongly affected by adhesion, as can be predicted using a model of a two-dimensional cable and electrodiffusion theory. Computations based on the model showed that along a neuron–astrocyte adhesion contact the opening of voltage-dependent Kv2.1 potassium channels is significantly reduced with respect to identical channels facing an open extracellular space.     

On the role of synchrony for neuron–astrocyte interactions and perceptual conscious processing

Recent research on brain correlates of cognitive processes revealed the occurrence of global synchronization during conscious processing of sensory stimuli. In spite of technological progress in brain imaging, an explanation of the computational role of synchrony is still a highly controversial issue. In this study, we depart from an analysis of the usage of blood-oxygen-level-dependent functional magnetic resonance imaging for the study of cognitive processing, leading to the identification of evoked local field potentials as the vehicle for sensory patterns that compose conscious episodes. Assuming the “astrocentric hypothesis” formulated by James M. Robertson (astrocytes being the final stage of conscious processing), we propose that the role of global synchrony in perceptual conscious processing is to induce the transfer of information patterns embodied in local field potentials to astrocytic calcium waves, further suggesting that these waves are responsible for the “binding” of spatially distributed patterns into unitary conscious episodes.     

Subcellular location of astrocytic calcium stores favors extrasynaptic neuron–astrocyte communication

Neuron–astrocyte interactions are important for brain computations and synaptic plasticity. Perisynaptic astrocytic processes (PAPs) contain a high density of transporters that are responsible for neurotransmitter clearance. Metabotropic glutamate receptors are thought to trigger Ca²⁺ release from Ca²⁺ stores in PAPs in response to synaptic activity. Our ultrastructural study revealed that PAPs are actually devoid of Ca²⁺ stores and have a high surface-to-volume ratio favorable for uptake. Astrocytic processes containing Ca²⁺ stores were located further away from the synapses and could therefore respond to changes in ambient glutamate. Thus, the anatomic data do not support communication involving Ca²⁺ stores in tripartite synapses, but rather point to extrasynaptic communication.     

Interactions of neurons with topographic nano cues affect branching morphology mimicking neuron–neuron interactions

We study the effect of topographic nano-cues on neuronal growth-morphology using invertebrate neurons in culture. We use photolithography to fabricate substrates with repeatable line-pattern ridges of nano-scale heights of 10-150?nm. We plate leech neurons atop the patterned-substrates and compare their growth pattern to neurons plated atop non-patterned substrates. The model system allows us the analysis of single neurite-single ridge interactions. The use of high resolution electron microscopy reveals small filopodia processes that attach to the line-pattern ridges. These fine processes, that cannot be detected in light microscopy, add anchoring sites onto the side of the ridges, thus additional physical support. These interactions of the neuronal process dominantly affect the neuronal growth direction. We analyze the response of the entire neuronal branching tree to the patterned substrates and find significant effect on the growth patterns compared to non-patterned substrates. Moreover, interactions with the nano-cues trigger a growth strategy similarly to interactions with other neuronal cells, as reflected in their morphometric parameters. The number of branches and the number of neurites originating from the soma decrease following the interaction demonstrating a tendency to a more simplified neuronal branching tree. The effect of the nano-cues on the neuronal function deserves further investigation and will strengthen our understanding of the interplay between function and form.     

Split focal adhesion kinase for probing protein–protein interactions

Since protein–protein interactions (PPIs) regulate a variety of cellular processes, the detection of PPIs is crucial for elucidating the underlying molecular mechanisms as well as developing therapeutics. In this study, we propose a novel system to detect PPIs using the distinct domains of focal adhesion kinase (FAK). In this system named “split FAK”, the linker and kinase domains in native FAK are tethered separately to two target proteins of interest. The interaction between the target proteins brings the linker and kinase domains into proximity, which leads to phosphorylation at Y397 of the linker domain, recruitment of another tyrosine kinase Src, and phosphorylation at Y576 of the kinase domain. PPIs are readily detected by probing phosphorylation at Y397 and Y576 of these domains. To demonstrate this system, we designed a series of split FAK chimeras with different domain structures. Consequently, dimerizer-induced interaction between FK506-binding protein 12 (FKBP) and the T2098L mutant of FKBP12-rapamycin binding domain (FRB) was clearly detected by probing phosphorylation at the specific tyrosine residues of most of the split FAK chimeras. This is a novel PPI detection system based on a mechanism-inspired design of a trans-activated split kinase.     

Neuron–astrocyte interactions in the medial nucleus of the trapezoid body

The calyx of Held (CoH) synapse serves as a model system to analyze basic mechanisms of synaptic transmission. Astrocyte processes are part of the synaptic structure and contact both pre- and postsynaptic membranes. In the medial nucleus of the trapezoid body (MNTB), midline stimulation evoked a current response that was not mediated by glutamate receptors or glutamate uptake, despite the fact that astrocytes express functional receptors and transporters. However, astrocytes showed spontaneous Ca²⁺ responses and neuronal slow inward currents (nSICs) were recorded in the postsynaptic principal neurons (PPNs) of the MNTB. These currents were correlated with astrocytic Ca²⁺ activity because dialysis of astrocytes with BAPTA abolished nSICs. Moreover, the frequency of these currents was increased when Ca²⁺ responses in astrocytes were elicited. NMDA antagonists selectively blocked nSICs while D-serine degradation significantly reduced NMDA-mediated currents. In contrast to previous studies in the hippocampus, these NMDA-mediated currents were rarely synchronized.     

Alternative modulation of protein–protein interactions by small molecules

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Granulocyte adhesion molecules — structure/functionrelationships

Neutrophil responses are regulated by cellular adhesion events, including interaction with. extracellular matrix and other cell types. The diversity of molecular structures which are included in the repertoire of cell adhesion molecules expressed by neutrophils and their subtle regulation allow fine tuning of cell adhesion processes to suit environmental demands. This article reviews some of the recent findings using biochemical, immunochemical and molecular techniques that allow the relationship between adhesion molecule structure and function to be examined. Understanding the molecular basis of cell adhesion events will allow development of novel strategies that allow manipulation of adhesion processes in a clinical setting.     

Modeling of cell adhesion and deformation mediated by receptor–ligand interactions

The current work is devoted to studying adhesion and deformation of biological cells mediated by receptors and ligands in order to enhance the existing models. Due to the sufficient in-plane continuity and fluidity of the phospholipid molecules, an isotropic continuum fluid membrane is proposed for modeling the cell membrane. The developed constitutive model accounts for the influence of the presence of receptors on the deformation and adhesion of the cell membrane through the introduction of spontaneous area dilation. Motivated by physics, a nonlinear receptor–ligand binding force is introduced based on charge-induced dipole interaction. Diffusion of the receptors on the membrane is governed by the receptor–ligand interaction via Fick’s Law and receptor-ligand interaction. The developed model is then applied to study the deformation and adhesion of a biological cell. The proposed model is used to study the role of the material, binding, spontaneous area dilation and environmental properties on the deformation and adhesion of the cell.     

Bioactivity of nitrolinoleate: effects on adhesion molecules and CD40–CD40L system

The vascular effects of nitrolinoleate (LNO₂), an endogenous product of linoleic acid (LA) nitration by nitric oxide-derived species and a potential nitrosating agent, were investigated on rat endothelial-leukocyte interactions. Confocal microscopy analysis demonstrated that LNO₂ was capable to deliver free radical nitric oxide (^·NO) into cells, 5 min after its administration to cultured cells, with a peak of liberation at 30 min. THP-1 monocytes incubated with LNO₂ for 5 min presented nitrosation of CD40, leading to its inactivation. Other anti-inflammatory actions of LNO₂ were observed in vivo by intravital microscopy assays. LNO₂ decreased the number of adhered leukocytes in postcapillary venules of the mesentery network. In addition to this, LNO₂ reduced mRNA and protein expression of β2-integrin in circulating leukocytes, as well as VCAM-1 in endothelial cells isolated from postcapillary venules, confirming its antiadhesive effects on both cell types. Moreover, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a nitric oxide scavenger, partially abolished the inhibitory action of LNO₂ on leukocyte-endothelium interaction, suggesting that the antiadhesion effects of LNO₂ involve a dual role in leukocyte adhesion, acting as a nitric oxide donor as well as through nitric oxide-independent mechanisms. In conclusion, LNO₂ inhibited adhesion molecules expression and promoted ^·NO inactivation of the CD40–CD40L system, both important processes of the inflammatory response.