The Potential of PIP3 in Enhancing Wound Healing.

Given the role of phosphatidylinositol 3,4,5-trisphosphate (PIP3) in modulating cellular processes such as proliferation, survival, and migration, we hypothesized its potential as a novel therapeutic agent for wound closure enhancement. In this study, PIP3 was examined in its free form or as a complex with cationic starch (Q-starch) as a carrier. The intracellular bioactivity and localization of free PIP3 and the Q-starch/PIP3 complexes were examined. Our results present the capability of Q-starch to form complexes with PIP3, facilitate its cellular membrane internalization, and activate intracellular paths leading to enhanced wound healing. Both free PIP3 and Q-starch/PIP3 complexes enhanced monolayer gap closure in scratch assays and induced amplified collagen production within HaCAT and BJ fibroblast cells. Western blot presented enhanced AKT activation by free or complexed PIP3 in BJ fibroblasts in which endogenous PIP3 production was pharmacologically inhibited. Furthermore, both free PIP3 and Q-starch/PIP3 complexes expedited wound closure in mice, after single or daily dermal injections into the wound margins. Free PIP3 and the Q-starch/PIP3 complexes inherently activated the AKT signaling pathway, which is responsible for crucial wound healing processes such as migration; this was also observed in wound assays in mice. PIP3 was identified as a promising molecule for enhancing wound healing, and its ability to circumvent PI3K inhibition suggests possible implications for chronic wound healing.

In vitro inhibition of cancer angiogenesis and migration by a nanobody that targets the orphan receptor Tie1.

The human signaling molecules Tie1 and Tie2 receptor tyrosine kinases (RTKs) play important pathophysiological roles in many diseases, including different cancers. The activity of Tie1 is mediated mainly through the downstream angiopoietin-1 (Ang1)-dependent activation of Tie2, rendering both Tie 1 and the Tie1/Tie2/Ang1 axis attractive putative targets for therapeutic intervention. However, the development of inhibitors that target Tie1 and an understanding of their effect on Tie2 and on the Tie1/Tie2/Ang1 axis remain unfulfilled tasks, due, largely, to the facts that Tie1 is an orphan receptor and is difficult to produce and use in the quantities required for immune antibody library screens. In a search for a selective inhibitor of this orphan receptor, we sought to exploit the advantages (e.g., small size that allows binding to hidden epitopes) of non-immune nanobodies and to simultaneously overcome their limitations (i.e., low expression and stability). We thus performed expression, stability, and affinity screens of yeast-surface-displayed naïve and predesigned synthetic (non-immune) nanobody libraries against the Tie1 extracellular domain. The screens yielded a nanobody with high expression and good affinity and specificity for Tie1, thereby yielding preferential binding for Tie1 over Tie2. The stability, selectivity, potency, and therapeutic potential of this synthetic nanobody were profiled using in vitro and cell-based assays. The nanobody triggered Tie1-dependent inhibition of RTK (Tie2, Akt, and Fak) phosphorylation and angiogenesis in endothelial cells, as well as suppression of human glioblastoma cell viability and migration. This study opens the way to developing nanobodies as therapeutics for different cancers associated with Tie1 activation.

A Missense Variation in PHACTR2 Associates with Impaired Actin Dynamics, Dilated Cardiomyopathy, and Left Ventricular Non-Compaction in Humans.

Dilated cardiomyopathy (DCM) with left ventricular non-compaction (LVNC) is a primary myocardial disease leading to contractile dysfunction, progressive heart failure, and excessive risk of sudden cardiac death. Using whole-exome sequencing to investigate a possible genetic cause of DCM with LVNC in a consanguineous child, a homozygous nucleotide change c.1532G>A causing p.Arg511His in PHACTR2 was found. The missense change can affect the binding of PHACTR2 to actin by eliminating the hydrogen bonds between them. The amino acid change does not change PHACTR2 localization to the cytoplasm. The patient's fibroblasts showed a decreased globular to fibrillary actin ratio compared to the control fibroblasts. The re-polymerization of fibrillary actin after treatment with cytochalasin D, which disrupts the actin filaments, was slower in the patient's fibroblasts. Finally, the patient's fibroblasts bridged a scar gap slower than the control fibroblasts because of slower and indirect movement. This is the first report of a human variation in this PHACTR family member. The knock-out mouse model presented no significant phenotype. Our data underscore the importance of PHACTR2 in regulating the monomeric actin pool, the kinetics of actin polymerization, and cell movement, emphasizing the importance of actin regulation for the normal function of the human heart.

Non-pigmented ciliary epithelium derived extracellular vesicles uptake mechanism by the trabecular meshwork.

Consistent with increasing findings, extracellular vesicles (EVs), consider as a major constituents of the aqueous humor, have a role as signaling mediators in glaucoma. Following secretion, EVs hold immense promise for utilization as bio-therapeutics and drug delivery vehicles due to their nature as biological nanoparticles that facilitate intercellular molecular transport. Yet, the specific pathway utilizing for transferring signals by EVs in the ocular drainage system is not fully understood. Hence, the objective of this study was to examine internalization mechanisms by which Non-Pigmented Ciliary Epithelium (NPCE)-derived EVs deliver their signals to the Trabecular Meshwork (TM) cells. EVs were isolated and size and concentration were determined. Internalization study of treated EVs with Proteinase-K to achieve removal of surface membrane proteins on EVs was conducted. Energy dependent uptake mechanism was examined under various temperatures. Using uptake inhibitors endocytosis, phagocytosis, and Wnt-TGFß2 signaling were investigated. TM cells exposed to NPCE EVs demonstrate a significant decrease in the levels of two proteins in two Wnt-TGFb2 signaling proteins levels: p-GSK3ß and ß-catenin. A significant decrease in the uptake by TM cells of Proteinase-K-treated EVs was found, followed by attenuation of the Wnt-TGFß2 proteins expression. Energy dependent uptake revealed a significant decrease in EVs internalization. The exposure of TM cells to endocytosis uptake inhibitors abolished the decrease of the Wnt-TGFß2 proteins levels. Exposure to phagocytosis uptake inhibitor resulted in a partial inhibition of NPCE EVs effect in TM cells. The attenuation of proteins expression levels following uptake inhibitors treatment or EVs membrane proteins removal indicates that Wnt-TGFß2 signaling in TM cells is mediated through NPCE EVs surface proteins in an active manner that involves endocytosis-dependent routes.

Imaging Flow Cytometry Illuminates New Dimensions of Amyloid Peptide-Membrane Interactions.

Membrane interactions of amyloidogenic proteins constitute central determinants both in protein aggregation as well as in amyloid cytotoxicity. Most reported studies of amyloid peptide-membrane interactions have employed model membrane systems combined with application of spectroscopy methods or microscopy analysis of individual binding events. Here, we applied for the first time, to our knowledge, imaging flow cytometry for investigating interactions of representative amyloidogenic peptides, namely, the 106-126 fragment of prion protein (PrP(106-126)) and the human islet amyloid polypeptide (hIAPP), with giant lipid vesicles. Imaging flow cytometry was also applied to examine the inhibition of PrP(106-126)-membrane interactions by epigallocatechin gallate, a known modulator of amyloid peptide aggregation. We show that imaging flow cytometry provided comprehensive population-based statistical information upon morphology changes of the vesicles induced by PrP(106-126) and hIAPP. Specifically, the experiments reveal that both PrP(106-126) and hIAPP induced dramatic transformations of the vesicles, specifically disruption of the spherical shapes, reduction of vesicle circularity, lobe formation, and modulation of vesicle compactness. Interesting differences, however, were apparent between the impact of the two peptides upon the model membranes. The morphology analysis also showed that epigallocatechin gallate ameliorated vesicle disruption by PrP(106-126). Overall, this study demonstrates that imaging flow cytometry provides powerful means for disclosing population-based morphological membrane transformations induced by amyloidogenic peptides and their inhibition by aggregation modulators.

Dimerization of NKp46 receptor is essential for NKp46-mediated lysis: characterization of the dimerization site by epitope mapping.

NKp46 is a primary activating receptor of NK cells that is involved in lysis of target cells by NK cells. Previous studies showed that the membrane-proximal domain of NKp46 (NKp46D2) retained the binding of NKp46 to its ligands and is involved in lysis. We studied NKp46D2 by using a peptide-based epitope mapping approach and identified an NKp46D2-derived linear epitope that inhibited NKp46-mediated lysis. The epitope, designated as pep4 (aa 136-155), interacted with NKp46, and lysis by NK cells was inhibited by the presence of pep4. Through modeling and mutagenesis, we showed that pep4 could be involved in NKp46 homodimerization. R145 and D147 contribute to the function of pep4, and R145Q mutation in recombinant NKp46 reduced its binding to target cells. At the cellular level, fluorescent resonance energy transfer analysis revealed that pep4 is indeed involved in dimerization of cell membrane-associated NKp46. We suggest that the NKp46-derived pep4 site is part of the dimerization surface of NKp46 and that NKp46 dimerization contributes to NKp46-mediated lysis by NK cells.

Proliferating cell nuclear antigen is a novel inhibitory ligand for the natural cytotoxicity receptor NKp44.

NK cells play an important role in the early immune response to cancer. The NKp44 activating receptor is the only natural cytotoxicity receptor that is expressed exclusively by primate NK cells, yet its cellular ligands remain largely unknown. Proliferating cell nuclear Ag (PCNA) is overexpressed in cancer cells. In this study, we show that the NKp44 receptor recognizes PCNA. Their interaction inhibits NK cell function through NKp44/ITIM. The physical interaction of NKp44 and PCNA is enabled by recruitment of target cell PCNA to the NK immunological synapse. We demonstrate that PCNA promotes cancer survival by immune evasion through inhibition of NKp44-mediated NK cell attack.

Imaging Flow Cytometry to Study Microbial Autoaggregation.

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The Lactobacillaceae family consists of Gram-positive bacteria with confirmed probiotic properties. This study utilizes Lactobacillaceae species as a model to demonstrate the effectiveness of single-cell high throughput analysis in studying cellular aggregation. The focus is on analyzing the response of these beneficial species to simple carbohydrates from the diet. The study showcases how Imaging Flow Cytometry (IFC) can overcome the fundamental differences in the assembly of probiotic bacteria in the presence and absence of carbohydrates. IFC combines the power and speed of conventional flow cytometry with the spatial resolution of microscopy, enabling high-rate complex morphometric measurements in a phenotypically defined manner across a library of beneficial bacterial strains and conditions. This protocol provides insights into the autoaggregation of Lactobacillaceae species and sheds light on their response to dietary carbohydrates, contributing to understanding the mechanisms behind the beneficial effects of these probiotic bacteria.

Dual fluorescent labelling of the human malaria parasite Plasmodium falciparum for the analysis of the ABC type transporter pfmdr2.

BACKGROUND: The study of the Plasmodium falciparum heavy metal transporter gene pfmdr2 employed radioactive labelled heavy metal. As the use of radioactive isotopes shrank considerably during the last few years, resulting in the cessation of the production of some isotopes, amongst them Cadmium109 which was used for that purpose, a different approach had to be developed. Herein, a dual fluorescent labelling of heavy metals accumulation in the P. falciparum parasite is proposed as an alternative to the use of radioactive labelled heavy metals. METHODS: Plasmodium falciparum Cd resistant and sensitive strains at the trophozoite stage were used in this study. The cells were cultured at different CdCl2 concentrations and for different time periods followed by staining of the infected red blood cells with Fluo-3/AM for Cd detection and Hoechst 33342 for parasite DNA labelling. The fluorescent analysis was done by flow cytometry and confocal microscopy. RESULTS: The results show that the sensitive strain has a higher Fluo-3/AM fluorescence in a Cd concentration and time dependent manner, whereas in the resistant strain Fluo-3/AM fluorescence levels were negligible and increased only at high concentrations of Cd and at long incubation periods, but to a much lesser extent than the sensitive strain. No Cd uptake is observed in uninfected red blood cells populations originating from cultures infected with either sensitive or resistant strain. In addition, confocal microscopy overlay of Fluo-3/AM and Hoechst staining shows that the Cd metal accumulates in the parasite itself. CONCLUSIONS: The dual fluorescent labelling is a valid method for detecting heavy metal accumulation in P. falciparum. Furthermore, in contrast to the use of radioactive labelled heavy metal, the fluorescent labelling enables us to differentiate between the different populations existing in a P. falciparum infected red blood cells cultures and thus actually study a phenomenon at the level of a single cell.

Defining the timeline of periostin upregulation in cardiac fibrosis following acute myocardial infarction in mice.

After myocardial infarction (MI), the heart's reparative response to the ischemic insult and the related loss of cardiomyocytes involves cardiac fibrosis, in which the damaged tissue is replaced with a fibrous scar. Although the scar is essential to prevent ventricular wall rupture in the infarction zone, it expands over time to remote, non-infarct areas, significantly increasing the extent of fibrosis and markedly altering cardiac structure. Cardiac function in this scenario deteriorates, thereby increasing the probability of heart failure and the risk of death. Recent works have suggested that the matricellular protein periostin, known to be involved in fibrosis, is a candidate therapeutic target for the regulation of MI-induced fibrosis and remodeling. Different strategies for the genetic manipulation of periostin have been proposed previously, yet those works did not properly address the time dependency between periostin activity and cardiac fibrosis. Our study aimed to fill that gap in knowledge and fully elucidate the explicit timing of cellular periostin upregulation in the infarcted heart to enable the safer and more effective post-MI targeting of periostin-producing cells. Surgical MI was performed in C57BL/6J and BALB/c mice by ligation of the left anterior descending coronary artery. Flow cytometry analyses of cells derived from the infarcted hearts and quantitative real-time PCR of the total cellular RNA revealed that periostin expression increased during days 2-7 and peaked on day 7 post-infarct, regardless of mouse strain. The established timeline for cellular periostin expression in the post-MI heart is a significant milestone toward the development of optimal periostin-targeted gene therapy.

Context-dependent differences in the functional responses of Lactobacillaceae strains to fermentable sugars.

Lactobacillaceae are Gram-positive rods, facultative anaerobes, and belong to the lactic acid bacteria (LAB) that frequently serve as probiotics. We systematically compared five LAB strains for the effects of different carbohydrates on their free-living and biofilm lifestyles. We found that fermentable sugars triggered an altered carrying capacity with strain specificity during planktonic growth. In addition, heterogeneous response to fermentable sugar was manifested in microbial aggregation (measured by imaging flow cytometry), colony development, and attachment to mucin. The acid production capacities of the strains were compatible and could not account for heterogeneity in their differential carrying capacity in liquid and on a solid medium. Among tested LAB strains, L. paracasei, and L. rhamnosus GG survived self-imposed acid stress while L. acidophilus was extremely sensitive to its own glucose utilization acidic products. The addition of a buffering system during growth on a solid medium significantly improved the survival of most tested probiotic strains during fermentation, but the formation of biofilms and aggregation capacity were responsive to the carbohydrate provided rather than to the acidity. We suggest that the optimal performance of the beneficial microbiota members belonging to Lactobacillaceae varies as a function of the growth model and the dependency on a buffering system.

Metaphase Cells Enrichment for Efficient Use in the Dicentric Chromosome Assay.

The dicentric chromosome assay (DCA), is considered the 'gold standard' for radiation biodosimetry. Yet, DCA, as currently implemented, may be impractical for emergency response applications, especially when time is of the essence, owing to its labor-intensive and time-consuming nature. The growth of a primary lymphocyte culture for 48 h in vitro is required for DCA, and manual scoring of dicentric chromosomes (DCs) requires an additional 24-48 h, resulting in an overall processing time of 72-96 h for dose estimation. In order to improve this timing. we introduce a protocol that will detect the metaphase cells in a population of cells, and then will harvest only those metaphase cells. Our metaphase enrichment approach is based on fixed human lymphocytes incubated with monoclonal, anti-phosphorylated H3 histone (ser 10). Antibodies against this histone have been shown to be specific for mitotic cells. Colcemid is used to arrest the mitotic cells in metaphase. Following that, a flow-cytometric sorting apparatus isolates the mitotic fraction from a large population of cells, in a few minutes. These mitotic cells are then spread onto a slide and treated with our C-Banding procedure [Gonen et al. 2022], to visualize the centromeres with DAPI. This reduces the chemical processing time to ~2 h. This reduces the time required for the DCA and makes it practical for a much wider set of applications, such as emergency response following exposure of a large population to ionizing radiation.

Heat stress increases immune cell function in Hexacorallia.

Climate change induced heat stress has increased coral bleaching events worldwide. Differentially regulated immune genes are one of the primary responses to heat stress suggesting that immune activation is critical. However, the cellular immune mechanisms of coral bleaching is currently unknown, and it is still not known if the immune response documented during heat stress is a consequence of bleaching or is directly caused by the heat stress itself. To address this question, we have used two model system sea anemones (Order: Actiniaria): Exaiptasia diaphana and Nematostella vectensis. E. diaphana is an established sea anemone model for algal symbiont interaction, while N. vectensis is an established sea anemone model that lacks the algal symbiont. Here, we examined the effect of increased temperature on phagocytic activity, as an indication of immune function. Our data shows that immune cell activity increases during heat stress, while small molecule pinocytosis remains unaffected. We observed an increase in cellular production of reactive oxygen species with increasing temperatures. We also found that the cellular immune activity was not affected by the presence of the Symbiodiniaceae. Our results suggest that the immune activity observed in heat-stress induced bleaching in corals is a fundamental and basic response independent of the bleaching effect. These results establish a foundation for improving our understanding of hexacorallian immune cell biology, and its potential role in coral bleaching.

Mechanical Regulation of the Cytotoxic Activity of Natural Killer Cells.

Mechanosensing has been recently explored for T cells and B cells and is believed to be a part of their activation mechanism. Here, we investigated the mechanosensing of the third type of lymphocyte - natural killer (NK) cells, by showing that they modulate their immune activity in response to changes in the stiffness of a stimulating surface. Interestingly, we found that this immune response is bell-shaped and peaks for a stiffness of a few hundreds of kPa. This bell-shaped behavior was observed only for surfaces functionalized with the activating ligand major histocompatibility complex class I polypeptide-related sequence A but not for control surfaces, lacking immunoactive functionalities. We found that stiffness does not affect uniformly all the cells but increases the size of a little group of extra-active cells, which in turn contributes to the overall activation effect of the entire cell population. We further imaged the clustering of costimulatory adapter protein DAP10 on the NK cell membrane and found the same bell-shaped dependence to surface stiffness. Our findings reveal what seems to be ″the tip of the iceberg″ of mechanosensation of NK cells and provide an important insight into the mechanism of their immune signaling.

Distinct features of the Leishmania cap-binding protein LeishIF4E2 revealed by CRISPR-Cas9 mediated hemizygous deletion.

Leishmania parasites cycle between sand-fly vectors and mammalian hosts adapting to alternating environments by stage-differentiation accompanied by changes in the proteome profiles. Translation regulation plays a central role in driving the differential program of gene expression since control of gene regulation in Leishmania is mostly post-transcriptional. The Leishmania genome encodes six eIF4E paralogs, some of which bind a dedicated eIF4G candidate, and each eIF4E is assumed to have specific functions with perhaps some overlaps. However, LeishIF4E2 does not bind any known eIF4G ortholog and was previously shown to comigrate with the polysomal fractions of sucrose gradients in contrast to the other initiation factors that usually comigrate with pre-initiation and initiation complexes. Here we deleted one of the two LeishIF4E2 gene copies using the CRISPR-Cas9 methodology. The deletion caused severe alterations in the morphology of the mutant cells that became round, small, and equipped with a very short flagellum that did not protrude from its pocket. Reduced expression of LeishIF4E2 had no global effect on translation and growth, unlike other LeishIF4Es; however, there was a change in the proteome profile of the LeishIF4E2(+/-) cells. Upregulated proteins were related mainly to general metabolic processes including enzymes involved in fatty acid metabolism, DNA repair and replication, signaling, and cellular motor activity. The downregulated proteins included flagellar rod and cytoskeletal proteins, as well as surface antigens involved in virulence. Moreover, the LeishIF4E2(+/-) cells were impaired in their ability to infect cultured macrophages. Overall, LeishIF4E2 does not behave like a general translation factor and its function remains elusive. Our results also suggest that the individual LeishIF4Es perform unique functions.

A transepithelial pathway delivers succinate to macrophages, thus perpetuating their pro-inflammatory metabolic state.

The gut metabolite composition determined by the microbiota has paramount impact on gastrointestinal physiology. However, the role that bacterial metabolites play in communicating with host cells during inflammatory diseases is poorly understood. Here, we aim to identify the microbiota-determined output of the pro-inflammatory metabolite, succinate, and to elucidate the pathways that control transepithelial succinate absorption and subsequent succinate delivery to macrophages. We show a significant increase of succinate uptake into pro-inflammatory macrophages, which is controlled by Na+-dependent succinate transporters in macrophages and epithelial cells. Furthermore, we find that fecal and serum succinate concentrations were markedly augmented in inflammatory bowel diseases (IBDs) and corresponded to changes in succinate-metabolizing gut bacteria. Together, our results describe a succinate production and transport pathway that controls the absorption of succinate generated by distinct gut bacteria and its delivery into macrophages. In IBD, this mechanism fails to protect against the succinate surge, which may result in chronic inflammation.

Molecular-scale spatio-chemical control of the activating-inhibitory signal integration in NK cells.

The role of juxtaposition of activating and inhibitory receptors in signal inhibition of cytotoxic lymphocytes remains strongly debated. The challenge lies in the lack of tools that allow simultaneous spatial manipulation of signaling molecules. To circumvent this, we produced a nanoengineered multifunctional platform with molecular-scale spatial control of ligands, which was applied to elucidate KIR2DL1-mediated inhibition of NKG2D signaling-receptors of natural killer cells. This platform was conceived by bimetallic nanodot patterning with molecular-scale registry, followed by a ternary functionalization with distinct moieties. We found that a 40-nm gap between activating and inhibitory ligands provided optimal inhibitory conditions. Supported by theoretical modeling, we interpret these findings as a consequence of the size mismatch and conformational flexibility of ligands in their spatial interaction. This highly versatile approach provides an important insight into the spatial mechanism of inhibitory immune checkpoints, which is essential for the rational design of future immunotherapies.

Functional Characterization of Hexacorallia Phagocytic Cells.

Phagocytosis is the cellular defense mechanism used to eliminate antigens derived from dysregulated or damaged cells, and microbial pathogens. Phagocytosis is therefore a pillar of innate immunity, whereby foreign particles are engulfed and degraded in lysolitic vesicles. In hexacorallians, phagocytic mechanisms are poorly understood, though putative anthozoan phagocytic cells (amoebocytes) have been identified histologically. We identify and characterize phagocytes from the coral Pocillopora damicornis and the sea anemone Nematostella vectensis. Using fluorescence-activated cell sorting and microscopy, we show that distinct populations of phagocytic cells engulf bacteria, fungal antigens, and beads. In addition to pathogenic antigens, we show that phagocytic cells engulf self, damaged cells. We show that target antigens localize to low pH phagolysosomes, and that degradation is occurring within them. Inhibiting actin filament rearrangement interferes with efficient particle phagocytosis but does not affect small molecule pinocytosis. We also demonstrate that cellular markers for lysolitic vesicles and reactive oxygen species (ROS) correlate with hexacorallian phagocytes. These results establish a foundation for improving our understanding of hexacorallian immune cell biology.

Capillary bacterial migration on non-nutritive solid surfaces.

Here we describe an additional type of bacterial migration in which bacterial cells migrate vertically across a non-nutritive solid surface carried by capillary forces. Unlike standard motility experiments, these were run on a glass slide inserted into a Falcon tube, partly immersed in a nutrient medium and partly exposed to air. Observations revealed that capillary forces initiated upward cell migration when biofilm was formed at the border between liquid and air. The movement was facilitated by the production of extracellular polymeric substances (EPS). This motility differs from earlier described swarming, twitching, gliding, sliding, or surfing, although these types of movements are not excluded. We therefore propose to call it "capillary movement of biofilm". This phenomenon may be an ecologically important mode of bacterial motility on solid surfaces.

Molecular and cellular correlates in Kv channel clustering: entropy-based regulation of cluster ion channel density.

Scaffold protein-mediated ion channel clustering at unique membrane sites is important for electrical signaling. Yet, the mechanism(s) by which scaffold protein-ion channel interactions lead to channel clustering or how cluster ion channel density is regulated is mostly not known. The voltage-activated potassium channel (Kv) represents an excellent model to address these questions as the mechanism underlying its interaction with the post-synaptic density 95 (PSD-95) scaffold protein is known to be controlled by the length of the extended 'ball and chain' sequence comprising the C-terminal channel region. Here, using sub-diffraction high-resolution imaging microscopy, we show that Kv channel 'chain' length regulates Kv channel density with a 'bell'-shaped dependence, reflecting a balance between thermodynamic considerations controlling 'chain' recruitment by PSD-95 and steric hindrance due to the spatial proximity of multiple channel molecules. Our results thus reveal an entropy-based mode of channel cluster density regulation that mirrors the entropy-based regulation of the Kv channel-PSD-95 interaction. The implications of these findings for electrical signaling are discussed.