Differential Control of Small-conductance Calcium-activated Potassium Channel Diffusion by Actin in Different Neuronal Subcompartments.

Small-conductance calcium-activated potassium (SK) channels show a ubiquitous distribution on neurons, in both somatodendritic and axonal regions. SK channels are associated with neuronal activity regulating action potential frequency, dendritic excitability, and synaptic plasticity. Although the physiology of SK channels and the mechanisms that control their surface expression levels have been investigated extensively, little is known about what controls SK channel diffusion in the neuronal plasma membrane. This aspect is important, as the diffusion of SK channels at the surface may control their localization and proximity to calcium channels, hence increasing the likelihood of SK channel activation by calcium. In this study, we successfully investigated the diffusion of SK channels labeled with quantum dots on human embryonic kidney cells and dissociated hippocampal neurons by combining a single-particle tracking method with total internal reflection fluorescence microscopy. We observed that actin filaments interfere with SK mobility, decreasing their diffusion coefficient. We also found that during neuronal maturation, SK channel diffusion was gradually inhibited in somatodendritic compartments. Importantly, we observed that axon barriers formed at approximately days in vitro 6 and restricted the diffusion of SK channels on the axon initial segment (AIS). However, after neuron maturation, SK channels on the AIS were strongly immobilized, even after disruption of the actin network, suggesting that crowding may cause this effect. Altogether, our work provides insight into how SK channels diffuse on the neuronal plasma membrane and how actin and membrane crowding impacts SK channel diffusion.

Dynamics of the axon plasma membrane skeleton.

It was recently revealed via super-resolution microscopy experiments that the axon plasma membrane skeleton (APMS) comprises a series of periodically arranged azimuthal actin rings connected via longitudinal spectrin filaments forming an orthotropic network. The common perception is that APMS enhances structural stability of the axon but its impact on axon deformation is unknown. To investigate the response of the APMS to extension, we introduce a coarse-grain molecular dynamics model consisting of actin particles forming rings and chains of particles representing spectrin tetramers with repeats than can unfold. We observe that the shape of force-extension curve is initially linear and the force level depends on the extension rate. Even during the initial deformation stage, unfolding of spectrin repeats occurs, but the saw-tooth shape of the corresponding force-extension curve observed in the case of one spectrin tetramer does not appear in the case of the entire APMS. The reason is that spectrin unfolding is not synchronized across filaments during extension. If actin-spectrin associations remain intact, the force-extension response reaches a perfectly plastic region because of increased spectrin unfolding frequency. However, when actin-spectrin links dissociate, which can happen at moderate and high extension rates, APMS softens and the resistance force decreases linearly as the axon elongates until it reaches a point where the APMS is completely severed. Furthermore, when the ring-to-ring distance is maintained fixed under stretch, the resistance force relaxes exponentially as a function of time due to additional unfolding of spectrin tetramers following the Kelvin-Voigt representation of the Zener model.

The periodic axon membrane skeleton leads to Na nanodomains but does not impact action potentials.

Recent work has established that axons have a periodic skeleton structure comprising of azimuthal actin rings connected via longitudinal spectrin tetramer filaments. This structure endows the axon with structural integrity and mechanical stability. Additionally, voltage-gated sodium channels follow the periodicity of the active-spectrin arrangement, spaced ∼190 nm segments apart. The impact of this periodic arrangement of sodium channels on the generation and propagation of action potentials is unknown. To address this question, we simulated an action potential using the Hodgkin-Huxley formalism in a cylindrical compartment, but instead of using a homogeneous distribution of voltage-gated sodium channels in the membrane, we applied the experimentally determined periodic arrangement. We found that the periodic distribution of voltage-gated sodium channels does not significantly affect the generation or propagation of action potentials but instead leads to large, localized sodium action currents caused by high-density sodium nanodomains. Additionally, our simulations show that the distance between periodic sodium channel strips could control axonal excitability, suggesting a previously underappreciated mechanism to regulate neuronal firing properties. Together, this work provides a critical new insight into the role of the periodic arrangement of sodium channels in axons, providing a foundation for future experimental studies.

A Theoretical Iteration for Predicting the Feasibility for Immediate Functional Dental Implant Loading.

When planning an implant-supported restoration, the dentist is faced with surgical and prosthetic technical issues as well as the patient's expectations. Many patients wish an immediate solution to an edentulous condition. This may be especially true in the esthetic zone, and that zone is determined by the patient. The dentist may consider when it is feasible to load the supporting implants with definitive or provisional prosthetics. In this work, many parameters were theoretically assessed for inclusion: bone density, cortical thickness, insertion torque, parafunction, bite load capacity, number of implants under load, implant/crown ratio, implant diameter, and length. After assessment, the most influential parameters were selected. An iteration, using patient age, implant diameter, bite load capacity, and cortical thickness, is now presented to aid the implant dentist in determining the feasibility for immediate functional loading of a just-placed dental implant in a healed site. Extensive testing is required to develop this concept. According to this iteration, most immediate functional loaded implants would fail. A future refined and definitive formula may enable the clinician to safely and immediately functionally load an implant with a definitive prosthesis. For access to the applet, please go to https://implantloading.shinyapps.io/shiny_app/.

Atomic force microscopy imaging for nanoscale and microscale assessments of extracellular matrix in intervertebral disc and degeneration.

Degeneration of the intervertebral disc (IVD) is a condition that is often associated with debilitating back pain. There are no disease-modifying treatments available to halt the progression of this ubiquitous disorder. This is partly due to a lack of understanding of extracellular matrix (ECM) changes that occur at the micro- and nanometer size scales as the disease progresses. Over the past decade, atomic force microscopy (AFM) has been utilized as a tool to investigate the impact of disease on nanoscale structure of ECM in bone, skin, tendon, and dentin. We have expanded this methodology to include the IVD and report the first quantitative analysis of ECM structure at submicron size scales in a murine model for progressive IVD degeneration. Collagen D-spacing, a metric of nanoscale structure at the fibril level, was observed as a distribution of values with an overall average value of 62.5 ± 2.5 nm. In degenerative discs, the fibril D-spacing distribution shifted towards higher values in both the annulus fibrosus and nucleus pulposus (NP) (P < .05). A novel microstructural feature, collagen toroids, defined by a topographical pit enclosed by fibril-forming matrix was observed in the NP. With degeneration, these microstructures became more numerous and the morphology was altered from circular (aspect ratio 1.0 ± 0.1) to oval (aspect ratio 1.5 ± 0.4), P < .005. These analyses provide ECM structural details of the IVD at size scales that have historically been missing in studies of disc degeneration. Knowledge gained from these insights may aid the development of novel disease-modifying therapeutics.

Modeling of the axon plasma membrane structure and its effects on protein diffusion.

The axon plasma membrane consists of the membrane skeleton, which comprises ring-like actin filaments connected to each other by spectrin tetramers, and the lipid bilayer, which is tethered to the skeleton via, at least, ankyrin. Currently it is unknown whether this unique axon plasma membrane skeleton (APMS) sets the diffusion rules of lipids and proteins in the axon. To answer this question, we developed a coarse-grain molecular dynamics model for the axon that includes the APMS, the phospholipid bilayer, transmembrane proteins (TMPs), and integral monotopic proteins (IMPs) in both the inner and outer lipid layers. We first showed that actin rings limit the longitudinal diffusion of TMPs and the IMPs of the inner leaflet but not of the IMPs of the outer leaflet. To reconcile the experimental observations, which show restricted diffusion of IMPs of the outer leaflet, with our simulations, we conjectured the existence of actin-anchored proteins that form a fence which restricts the longitudinal diffusion of IMPs of the outer leaflet. We also showed that spectrin filaments could modify transverse diffusion of TMPs and IMPs of the inner leaflet, depending on the strength of the association between lipids and spectrin. For instance, in areas where spectrin binds to the lipid bilayer, spectrin filaments would restrict diffusion of proteins within the skeleton corrals. In contrast, in areas where spectrin and lipids are not associated, spectrin modifies the diffusion of TMPs and IMPs of the inner leaflet from normal to confined-hop diffusion. Overall, we showed that diffusion of axon plasma membrane proteins is deeply anisotropic, as longitudinal diffusion is of different type than transverse diffusion. Finally, we investigated how accumulation of TMPs affects diffusion of TMPs and IMPs of both the inner and outer leaflets by changing the density of TMPs. We showed that the APMS structure acts as a fence that restricts the diffusion of TMPs and IMPs of the inner leaflet within the membrane skeleton corrals. Our findings provide insight into how the axon skeleton acts as diffusion barrier and maintains neuronal polarity.

Valsartan impedes epinephrine-induced ICAM-4 activation on normal, sickle cell trait and sickle cell disease red blood cells.

Abnormal red blood cell (RBC) adhesion to endothelial αvß3 plays a crucial role in triggering vaso-occlusive episodes in sickle cell disease (SCD). It is known that epinephrine, a ß-adrenergic receptor (ß-AR) stimulator, increases the RBC surface density of active intercellular adhesion molecule-4 (ICAM-4) which binds to the endothelial αvß3. It has also been demonstrated that in human embryonic kidney 293 cells, mouse cardiomyocytes, and COS-7 cell lines, the ß-adrenergic and renin-angiotensin systems are interrelated and that there is a direct interaction and cross-regulation between ß-AR and angiotensin II type 1 receptor (AT1R). Selective blockade of AT1R reciprocally inhibits the downstream signaling of ß-ARs, similar to the inhibition observed in the presence of a ß-AR-blocker. However, it is not known if this mechanism is active in human RBCs. Here, we studied the effect of valsartan, an AT1R blocker, on the surface density of active ICAM-4 receptors in normal, sickle cell trait, and homozygous sickle RBCs. We applied single molecule force spectroscopy to detect active ICAM-4 receptors on the RBC plasma membrane with and without the presence of valsartan and epinephrine. We found that epinephrine significantly increased whereas valsartan decreased their surface density. Importantly, we found that pretreatment of RBCs with valsartan significantly impeded the activation of ICAM-4 receptors induced by epinephrine. The observed reduced expression of active ICAM-4 receptors on the RBC plasma membrane leads us to conjecture that valsartan may be used as a supporting remedy for the prevention and treatment of vaso-occlusive crisis in SCD.

KCa2 channel localization and regulation in the axon initial segment.

Small conductance calcium-activated potassium (KCa2) channels are expressed throughout the CNS and play a critical role in synaptic and neuronal excitability. KCa2 channels have a somatodendritic distribution with their highest expression in distal dendrites. It is unclear whether KCa2 channels are specifically present on the axon initial segment (AIS), the site at which action potentials are initiated in neurons. Through a powerful combination of toxin pharmacology, single-molecule atomic force microscopy, and dual-color fluorescence microscopy, we report here that KCa2 channels-predominantly the KCa2.3 subtype-are indeed present on the AIS. We also report that cAMP-PKA controls the axonal KCa2 channel surface expression. Surprisingly, and in contrast to KCa2 channels that were observed in the soma and dendrites, the inhibition of cAMP-PKA increased the surface expression of KCa2 channels without promoting nanoclustering. Lastly, we found that axonal KCa2 channels seem to undergo endocytosis in a dynamin-independent manner, unlike KCa2 channels in the soma and dendrites. Together, these novel results demonstrate that the distribution and membrane recycling of KCa2 channels differs among various neuronal subcompartments.-Abiraman, K., Tzingounis, A. V., Lykotrafitis, G. KCa2 channel localization and regulation in the axon initial segment.

Microfluidic experimental setup for adhesion and recovery measurements of red blood cells in sickle cell disease.

Current microfluidic assays, which aim at quantifying mechanical properties of sickle cell red blood cells (SS-RBCs), suffer from a number of drawbacks in functionalization and flow control. Specifically, physical adsorption functionalization techniques produce inconsistent functional surfaces, and common volumetric flow pumps cannot be used to adjust the flow inside microchannels with minimal delay. We have designed an experimental setup that alleviates these complications by implementing aspiration for microchannel assembly that enables the use of most functionalization techniques and a pressure controller that allows instant and precise changes in the microchannel flow. Utilizing this setup, we have quantified SS-RBC adhesion to the integrin αvß3, a specific adhesion protein expressed on the endothelium, as well as measured the shear modulus and viscosity of the SS-RBC plasma membrane.

Modeling of the axon membrane skeleton structure and implications for its mechanical properties.

Super-resolution microscopy recently revealed that, unlike the soma and dendrites, the axon membrane skeleton is structured as a series of actin rings connected by spectrin filaments that are held under tension. Currently, the structure-function relationship of the axonal structure is unclear. Here, we used atomic force microscopy (AFM) to show that the stiffness of the axon plasma membrane is significantly higher than the stiffnesses of dendrites and somata. To examine whether the structure of the axon plasma membrane determines its overall stiffness, we introduced a coarse-grain molecular dynamics model of the axon membrane skeleton that reproduces the structure identified by super-resolution microscopy. Our proposed computational model accurately simulates the median value of the Young's modulus of the axon plasma membrane determined by atomic force microscopy. It also predicts that because the spectrin filaments are under entropic tension, the thermal random motion of the voltage-gated sodium channels (Nav), which are bound to ankyrin particles, a critical axonal protein, is reduced compared to the thermal motion when spectrin filaments are held at equilibrium. Lastly, our model predicts that because spectrin filaments are under tension, any axonal injuries that lacerate spectrin filaments will likely lead to a permanent disruption of the membrane skeleton due to the inability of spectrin filaments to spontaneously form their initial under-tension configuration.

Regulation of Active ICAM-4 on Normal and Sickle Cell Disease RBCs via AKAPs Is Revealed by AFM.

Human healthy (wild-type (WT)) and homozygous sickle (SS) red blood cells (RBCs) express a large number of surface receptors that mediate cell adhesion between RBCs, and between RBCs and white blood cells, platelets, and the endothelium. In sickle cell disease (SCD), abnormal adhesion of RBCs to endothelial cells is mediated by the intercellular adhesion molecule-4 (ICAM-4), which appears on the RBC membrane and binds to the endothelial αvß3 integrin. This is a key factor in the initiation of vaso-occlusive episodes, the hallmark of SCD. A better understanding of the mechanisms that control RBC adhesion to endothelium may lead to novel approaches to both prevention and treatment of vaso-occlusive episodes in SCD. One important mechanism of ICAM-4 activation occurs via the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA)-dependent signaling pathway. Here, we employed an in vitro technique called single-molecule force spectroscopy to study the effect of modulation of the cAMP-PKA-dependent pathway on ICAM-4 receptor activation. We quantified the frequency of active ICAM-4 receptors on WT-RBC and SS-RBC membranes, as well as the median unbinding force between ICAM-4 and αvß3. We showed that the collective frequency of unbinding events in WT-RBCs is not significantly different from that of SS-RBCs. This result was confirmed by confocal microscopy experiments. In addition, we showed that incubation of normal RBCs and SS-RBCs with epinephrine, a catecholamine that binds to the ß-adrenergic receptor and activates the cAMP-PKA-dependent pathway, caused a significant increase in the frequency of active ICAM-4 receptors in both normal RBCs and SS-RBCs. However, the unbinding force between ICAM-4 and the corresponding ligand αvß3 remained the same. Furthermore, we demonstrated that forskolin, an adenylyl cyclase activator, significantly increased the frequency of ICAM-4 receptors in WT-RBCs and SS-RBCs, confirming that the activation of ICAM-4 is regulated by the cAMP-PKA pathway. Finally, we showed that A-kinase anchoring proteins play an essential role in ICAM-4 activation.

Biomechanics and biorheology of red blood cells in sickle cell anemia.

Sickle cell anemia (SCA) is an inherited blood disorder that causes painful crises due to vaso-occlusion of small blood vessels. The primary cause of the clinical phenotype of SCA is the intracellular polymerization of sickle hemoglobin resulting in sickling of red blood cells (RBCs) in deoxygenated conditions. In this review, we discuss the biomechanical and biorheological characteristics of sickle RBCs and sickle blood as well as their implications toward a better understanding of the pathophysiology and pathogenesis of SCA. Additionally, we highlight the adhesive heterogeneity of RBCs in SCA and their specific contribution to vaso-occlusive crisis.

Computational Biomechanics of Human Red Blood Cells in Hematological Disorders.

We review recent advances in multiscale modeling of the biomechanical characteristics of red blood cells (RBCs) in hematological diseases, and their relevance to the structure and dynamics of defective RBCs. We highlight examples of successful simulations of blood disorders including malaria and other hereditary disorders, such as sickle-cell anemia, spherocytosis, and elliptocytosis.

IMPETUS - Interactive MultiPhysics Environment for Unified Simulations.

We introduce IMPETUS - Interactive MultiPhysics Environment for Unified Simulations, an object oriented, easy-to-use, high performance, C++ program for three-dimensional simulations of complex physical systems that can benefit a large variety of research areas, especially in cell mechanics. The program implements cross-communication between locally interacting particles and continuum models residing in the same physical space while a network facilitates long-range particle interactions. Message Passing Interface is used for inter-processor communication for all simulations.

Tonic PKA Activity Regulates SK Channel Nanoclustering and Somatodendritic Distribution.

Small-conductance calcium-activated potassium (SK) channels mediate a potassium conductance in the brain and are involved in synaptic plasticity, learning, and memory. SK channels show a distinct subcellular localization that is crucial for their neuronal functions. However, the mechanisms that control this spatial distribution are unknown. We imaged SK channels labeled with fluorophore-tagged apamin and monitored SK channel nanoclustering at the single molecule level by combining atomic force microscopy and toxin (i.e., apamin) pharmacology. Using these two complementary approaches, we found that native SK channel distribution in pyramidal neurons, across the somatodendritic domain, depends on ongoing cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) levels, strongly limiting SK channel expression at the pyramidal neuron soma. Furthermore, tonic cAMP-PKA levels also controlled whether SK channels were expressed in nanodomains as single entities or as a group of multiple channels. Our study reveals a new level of regulation of SK channels by cAMP-PKA and suggests that ion channel topography and nanoclustering might be under the control of second messenger cascades.

FRAME (Force Review Automation Environment): MATLAB-based AFM data processor.

Data processing of force-displacement curves generated by atomic force microscopes (AFMs) for elastic moduli and unbinding event measurements is very time consuming and susceptible to user error or bias. There is an evident need for consistent, dependable, and easy-to-use AFM data processing software. We have developed an open-source software application, the force review automation environment (or FRAME), that provides users with an intuitive graphical user interface, automating data processing, and tools for expediting manual processing. We did not observe a significant difference between manually processed and automatically processed results from the same data sets.

Modeling of band-3 protein diffusion in the normal and defective red blood cell membrane.

We employ a two-component red blood cell (RBC) membrane model to simulate lateral diffusion of band-3 proteins in the normal RBC and in the RBC with defective membrane proteins. The defects reduce the connectivity between the lipid bilayer and the membrane skeleton (vertical connectivity), or the connectivity of the membrane skeleton itself (horizontal connectivity), and are associated with the blood disorders of hereditary spherocytosis (HS) and hereditary elliptocytosis (HE) respectively. Initially, we demonstrate that the cytoskeleton limits band-3 lateral mobility by measuring the band-3 macroscopic diffusion coefficients in the normal RBC membrane and in a lipid bilayer without the cytoskeleton. Then, we study band-3 diffusion in the defective RBC membrane and quantify the relation between band-3 diffusion coefficients and percentage of protein defects in HE RBCs. In addition, we illustrate that at low spectrin network connectivity (horizontal connectivity) band-3 subdiffusion can be approximated as anomalous diffusion, while at high horizontal connectivity band-3 diffusion is characterized as confined diffusion. Our simulations show that the band-3 anomalous diffusion exponent depends on the percentage of protein defects in the membrane cytoskeleton. We also confirm that the introduction of attraction between the lipid bilayer and the spectrin network reduces band-3 diffusion, but we show that this reduction is lower than predicted by the percolation theory. Furthermore, we predict that the attractive force between the spectrin filament and the lipid bilayer is at least 20 times smaller than the binding forces at band-3 and glycophorin C, the two major membrane binding sites. Finally, we explore diffusion of band-3 particles in the RBC membrane with defects related to vertical connectivity. We demonstrate that in this case band-3 diffusion can be approximated as confined diffusion for all attraction levels between the spectrin network and the lipid bilayer. By comparing the diffusion coefficients measured in horizontal vs. vertical defects, we conclude that band-3 mobility is primarily controlled by the horizontal connectivity.

Vesiculation of healthy and defective red blood cells.

Vesiculation of mature red blood cells (RBCs) contributes to removal of defective patches of the erythrocyte membrane. In blood disorders, which are related to defects in proteins of the RBC membrane, vesiculation of the plasma membrane is intensified. Several hypotheses have been proposed to explain RBC vesiculation but the exact underlying mechanisms and what determines the sizes of the vesicles are still not completely understood. In this work, we apply a two-component coarse-grained molecular dynamics RBC membrane model to study how RBC vesiculation is controlled by the membrane spontaneous curvature and by lateral compression of the membrane. Our simulation results show that the formation of small homogeneous vesicles with a diameter less than 40 nm can be attributed to a large spontaneous curvature of membrane domains. On the other hand, compression on the membrane can cause the formation of vesicles with heterogeneous composition and with sizes comparable with the size of the cytoskeleton corral. When spontaneous curvature and lateral compression are simultaneously considered, the compression on the membrane tends to facilitate formation of vesicles originating from curved membrane domains. We also simulate vesiculation of RBCs with membrane defects connected to hereditary elliptocytosis (HE) and to hereditary spherocytosis (HS). When the vertical connectivity between the lipid bilayer and the membrane skeleton is elevated, as in normal RBCs, multiple vesicles are shed from the compressed membrane with diameters similar to the cytoskeleton corral size. In HS RBCs, where the connectivity between the lipid bilayer and the cytoskeleton is reduced, larger-size vesicles are released under the same compression ratio as in normal RBCs. Lastly, we find that vesicles released from HE RBCs can contain cytoskeletal filaments due to fragmentation of the membrane skeleton while vesicles released from the HS RBCs are depleted of cytoskeletal filaments.

Dietary supplementation with docosahexanoic acid (DHA) increases red blood cell membrane flexibility in mice with sickle cell disease.

Humans and mice with sickle cell disease (SCD) have rigid red blood cells (RBCs). Omega-3 fatty acids, such as docosahexanoic acid (DHA), may influence RBC deformability via incorporation into the RBC membrane. In this study, sickle cell (SS) mice were fed natural ingredient rodent diets supplemented with 3% DHA (DHA diet) or a control diet matched in total fat (CTRL diet). After 8weeks of feeding, we examined the RBCs for: 1) stiffness, as measured by atomic force microscopy; 2) deformability, as measured by ektacytometry; and 3) percent irreversibly sickled RBCs on peripheral blood smears. Using atomic force microscopy, it is found that stiffness is increased and deformability decreased in RBCs from SS mice fed CTRL diet compared to wild-type mice. In contrast, RBCs from SS mice fed DHA diet had markedly decreased stiffness and increased deformability compared to RBCs from SS mice fed CTRL diet. Furthermore, examination of peripheral blood smears revealed less irreversibly sickled RBCs in SS mice fed DHA diet as compared to CTRL diet. In summary, our findings indicate that DHA supplementation improves RBC flexibility and reduces irreversibly sickled cells by 40% in SS mice. These results point to potential therapeutic benefits of dietary omega-3 fatty acids in SCD.

Single-cell force spectroscopy as a technique to quantify human red blood cell adhesion to subendothelial laminin.

Single-cell force spectroscopy (SCFS), an atomic force microscopy (AFM)-based assay, enables quantitative study of cell adhesion while maintaining the native state of surface receptors in physiological conditions. Human healthy and pathological red blood cells (RBCs) express a large number of surface proteins which mediate cell-cell interactions, or cell adhesion to the extracellular matrix. In particular, RBCs adhere with high affinity to subendothelial matrix laminin via the basal cell adhesion molecule and Lutheran protein (BCAM/Lu). Here, we established SCFS as an in vitro technique to study human RBC adhesion at baseline and following biochemical treatment. Using blood obtained from healthy human subjects, we recorded adhesion forces from single RBCs attached to AFM cantilevers as the cell was pulled-off of substrates coated with laminin protein. We found that an increase in the overall cell adhesion measured via SCFS is correlated with an increase in the resultant total force measured on 1 µm(2) areas of the RBC membrane. Further, we showed that SCFS can detect significant changes in the adhesive response of RBCs to modulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway. Lastly, we identified variability in the RBC adhesion force to laminin amongst the human subjects, suggesting that RBCs maintain diverse levels of active BCAM/Lu adhesion receptors. By using single-cell measurements, we established a powerful new method for the quantitative measurement of single RBC adhesion with specific receptor-mediated binding.