Additionally, exchange kinetics of alternatively spliced isoforms of the intrinsically disordered region in SAP97/hDLG C-terminus shows differential sensitivities to Ca2+ bound Calmodulin, affirming that the molecular signatures of local phase transitions of SAP97/hDLG depends on their nanoscale heterogeneity and compositionality of isoforms.Synapse associated protein-97/Human Disk Large (SAP97/hDLG) is a conserved, alternatively spliced, modular, scaffolding protein critical in regulating the molecular organization of cell-cell junctions in vertebrates. We confirm that the molecular determinants of first order phase transition of SAP97/hDLG is controlled by morpho-functional changes in its nanoscale organization. Furthermore, the nanoscale molecular signatures of these signalling islands and phase transitions are altered in response to changes in cytosolic Ca 2+ bound Calmodulin, resulting in altered properties of nanocondensates and their real-time regulationSAP97/hDLG is a ubiquitous, alternatively spliced, and conserved modular scaffolding protein involved in the organization cell junctions and excitatory synapses. Here, authors confirm that SAP97/hDLG condenses in to nanosized molecular domains in both heterologous cells and hippocampal pyramidal neurons. Authors demonstrate that in vivo and in vitro condensation, molecular signatures of nanoscale condensates and exchange kinetics of SAP97/hDLG is modulated by the local availability of alternatively spliced isoforms. Additionally, SAP97/hDLG isoforms exhibits a differential sensitivity to Ca Discs large (DLG) subfamily of MAGUKs is considered pivotal in the molecular organization of cell adhesion complexes in neurons and epithelial cells. Members of the DLG family comprises of SAP97/hDLG, SAP90/PSD95, SAP102/NE-DLG, and PSD93/Chapsyn 1103. DLG family of molecules have a unique modular organization and is composed of multiple protein–protein interaction domains. All the DLG family of molecules, characterized to date, contain three PDZ (PSD95/DLG/ZO1) domains, an Src homology three domain (SH3) and a guanylate kinase domain3. Best characterized of these domains are the PDZ domains4, which binds with high affinity to the carboxyl terminal peptide motifs of several proteins, notably GluN2 subunits of NMDA receptors6, GluA1 subunit of AMPA receptors8 and the voltage-gated inwardly rectifying K+ channels10. The guanylate kinase like (GUK) domain of MAGUKs lacks vital amino acid residues required for ATP/GMP binding11. Previous reports confirm that instead of an enzymatic role, this domain is modified to be part of multiple protein–protein interactions13. Accordingly, interactions have been mapped to this region including that of multidomain proteins such as GKAP14 and SPAR15. It has been shown that the SH3 domain of MAGUKs has an atypical binding specificity to the GUK domain. Although this SH3 binding can occur intra/inter-molecularly, it is believed that the intramolecular mode is preferred17. Though not well understood, it is thought that the intramolecular interaction of SH3 domain is supported by other tertiary interactions when the SH3 and GUK domains are adjacent in the same polypeptide18.Membrane-associated guanylate kinases (MAGUKs) are multidomain scaffolding molecules that organize near membrane signaling complexes at the postsynaptic density of neurochemical synapses and other cell junctions19. Several isoforms of SAP97 and its human orthologue hDLG (human Disc large) have been identified, making it one of the most spliced members in the family of DLGs’. These isoforms contribute to the differential expression and targeting of this protein to different subcellular regions21. SAP97/hDLG exists either as a membrane-bound α- or β-isoform characterized by the presence of Lin2-Lin7 domains. An alternatively spliced proline-rich insertion called I1 is located between the N-terminal region of SAP97/hDLG and the first PDZ domain. A highly flexible intrinsically disordered “HOOK” region, separating SH3 and GUK domains has been characterized to contain two alternatively spliced insertions, namely I2 and I320. In the same region, a third alternatively spliced insertion has been described as I4, a brain specific isoform of SAP97/hDLG. The region separating insertion sites of I2/I3 and I4 is also alternatively spliced and given the name I520. I3 and PDZ 1-2 domains of SAP97/hDLG show similarly charged residues, forming binding sites for 4.1-like proteins14. These sites contribute to SAP97/hDLG localization at the sites of cell-cell contact. I3 is also known to be responsible for localizing the protein to the plasma membrane22. I2 is reported to be responsible for targeting SAP97/hDLG to the nucleus20, though contradictory results have been documented23. The intrinsically disordered HOOK region of SAP97/hDLG responds to elevated intracellular Ca2+ by interacting with the Ca2+-binding protein Calmodulin (CaM). In the absence of intermolecular interaction, the HOOK region masks protein–protein interaction sites in the SH3 region of SAP97/hDLG24. Interaction of the HOOK region of SAP97/hDLG to CaM alters its molecular conformation, thereby destabilizing the interaction between SH3 and GUK domains27. In vivo, and in vitro studies imply that binding of CaM or any other high-affinity ligands to the HOOK region can destabilize SAP97 as well as other MAGUKs27 Destabilizing intramolecular SH3-GUK interaction can facilitate the rapid formation of multi-protein complexes, further aiding PDZ interacting receptors to get immobilized on the membrane4. Among the different SAP97/hDLG isoforms, CaM has been confirmed to be associated only with the HOOK regions of I3 containing isoforms28. It is not well understood if the different C-terminal spliced variants of SAP97/hDLG respond differentially to Ca2+-bound CaM ([Ca2+.CaM])21.SAP97/hDLG is a crucial member of the DLG family of proteins involved in membrane scaffolding and activity-dependent cell morphology changes30. In parallel, many of these molecules involved in the formation of transient domains also participate in liquid–liquid phase separation (LLPS)33. In vitro and in vivo evaluation of LLPS also indicate the presence of nano to micron sized phase separated compartments with the potential to have altered kinetics of exchange within and outside such compartments35. In vivo, such differing kinetics arise as a response to cellular environment, modification that happens post translationally or because of multicomponent system within a cell. To get a better insight into this regulation in the context of SAP97/hDLG, we performed direct stochastic optical reconstruction microscopy (dSTORM), a super resolution technique, to understand the endogenous distribution of SAP97/hDLG in heterologous cells. We observed that nanoscale segregation of SAP97/hDLG was physiologically regulated by the availability of free Ca2+ and modulated through the Ca2+ sensing protein Calmodulin. Using single-molecule localization microscopy in combination with Ca2+ perturbation and analysis paradigms to evaluate the free energy change in protein aggregation, we show that the nanoscale condensation of SAP97/hDLG followed a first-order phase transition with spontaneous nucleation and growth. These nanocondensates displayed an altered phase transition in response to differential modulation of intracellular Ca2+ levels. Since SAP97/hDLG can associate with [Ca2+.CaM], we confirmed the presence of its C-terminal spliced isoforms in the hippocampal and cortical regions of rodent brains as well as in heterologous cell lines. These isoforms are known to alter the dynamics of several cell surface molecules including cell adhesion molecules, channels, and receptors such as AMPA receptors, which are known to form distinct subsynaptic nanodomains37. Next, using Fluorescence Recovery after Photobleaching (FRAP) in combination with Total Internal Reflection Fluorescence (TIRF) illumination in live neuroblastoma cells, we evaluated the exchange of SAP97/hDLG isoforms in a diffraction limited compartment. The rate of exchange of alternatively spliced C-terminal variants of SAP97/hDLG were distinct from each other. This local exchange of each isoform was influenced by cytoplasmic Ca2+ levels and was modulated by the interaction of the intrinsically disordered HOOK region of SAP97/hDLG to Ca2+-binding proteins. Furthermore, in vitro phase transition studies confirmed that the isoforms of SAP97/hDLG can transition into phase separated condensates and can co-condense Calmodulin in a Ca2+-dependent manner. We confirmed that these differential effects of isoform expression at micron scale is also conserved in vivo at nanoscale using dissociated hippocampal neurons and neuroblastoma cells.Over the last decade, our understanding of the formation, maintenance, and elimination of molecular clusters at nanoscale is getting re-evaluated vigorously in vitro and in vivo. Growing body of evidence confirms the existence of regulatory nanodomains, which are formed transiently in real-time at spatial scales of 10–200 nm in cellsHere, with the aid of a paradigm to evaluate the molecular fingerprints of first-order phase transition across spatial scales in vivo and in vitro, we confirm that the local compositionality of isoform expression has a direct consequence on the real-time local exchange and nanoscale condensation of SAP97/hDLG. Additionally, our results verify that cellular condensation of SAP97/hDLG is differentially regulated between its isoforms, and this variability is regulated by the spatial heterogeneity and exchange kinetics of isoforms present in near membrane SAP97/hDLG condensates.39. Neuro-2a cells were immunolabelled for SAP97/hDLG and imaged by dSTORM, revealing endogenous clusters of SAP97/hDLG. Widefield conventional epifluorescence illumination showed diffused distribution of SAP97/hDLG across different cellular compartments. dSTORM revealed a large population of sub-diffraction sized molecular aggregates of SAP97/hDLG  by ratiometric analysis between ensemble fluorescence intensity of clusters and isolated single molecules outside the clusters41. The evaluation paradigms that we used for quantifying the molecular content resulted in extracting similar copy numbers of selected molecules as reported previously, validating the robustness of the workflow40. Distribution of the number of molecules detected inside a cluster resulted in a histogram where the probability of occurrence decreases rapidly towards clusters with very high molecular content behaving non-uniformly as supramolecular aggregation patterns ) was determined from the distribution of cluster sizes, which was fit by the inverse of the probability distribution of molecules, allowing a direct estimation of the nucleation barrier (∆Gc) and critical cluster radius (Rc)  and saturation concentration (Csat) at equilibrium with the clustered phase. We then extracted the number of detected SAP97/hDLG molecules per nanoscale both from high-density molecular domains, which depicted zones of functional aggregation (nanodomains) and disperse molecules from extra-nanodomain regions. A positive (Camb < Csat) or negative (Camb > Csat) sign depicted the cluster’s saturation state, i.e., whether the system was in a sub- or a super-saturated state. Bulk energy of the system was extracted from the data by subtracting the surface energy from free energy, and the curve was observed to be linear with a negative slope, representing a super-saturated system  represents the modeled relative frequency distribution of cluster size (n), and nc is the critical cluster size attaining a maximum value of ΔG(nc), which is the nucleation barrier  from the polynomial fit, which was normalized (ΔG(nc) = 1.00 ± 0.08). The critical radius of cluster 3) of untreated control dataset was calculated and normalized (Rc = 0.98 ± 0.06). These results confirmed that the SAP97/hDLG cluster sizes were below the diffraction limit, and the formation of these nanoscale condensates were governed by single order phase transitions, emphasizing the requirement for super-resolution microscopy techniques to resolve and measure them.SAP97/hDLG is involved in several near membrane signaling complexes and is an integral scaffold for many transmembrane molecules. The local heterogeneity of SAP97/hDLG is critical for its association with different membrane-associated signaling complexes. To understand the fine variability of SAP97/hDLG nanoscale organization, we relied on direct stochastic optical reconstruction microscopy (dSTORM), which allows the reconstruction of a sub-diffraction limited image from several single-molecule localizationsDLG Fig. . Recent rns Fig. . The freRc) Fig. . Saturattem Fig. . P(n) reier Fig. . The dat2+ levels on the nanoscale organization and first-order phase transition of endogenous SAP97/hDLG, we relied on a pharmacological approach to modulate cytosolic Ca2+ levels. We incubated the cells with Thapsigargin (Tg) (1 µM)44 for elevating intracellular Ca2+ and with the membrane-permeable BAPTA-AM (Bapta) (50 µM)45 to chelate the soluble Ca2+ present in the cell. To confirm differential sensitivity of SAP97/hDLG to [Ca2+.CaM], we inhibited the Ca2+ sensing ability of CaM using w7 (25 µM)47. The aforementioned treatments altered the intracellular Ca2+ levels and modulated the Ca2+-mediated interaction of Calmodulin to the intrinically disordered HOOK region of SAP97/hDLG within the cell. The consequential effect on SAP97/hDLG nucleation energetics was extracted from the analysis of single-molecule-based super resolution data as explained below.To study the effect of intracellular CaCamb) reduced on mobilizing SAP97/hDLG by application of Tg, resulting in an overall decrease in the bulk energy. The reduction in bulk energy upon Tg incubation without modulating the critical radius confirmed a reduction of the surface energy, an2/3. The normalized energy barrier and critical cluster radius were compared across the different conditions to that of control cells  = 1.08 ± 0.04  = 1.04 ± 0.08)  = 1.54 ± 0.12  = 1.49 ± 0.03  = 2.45 ± 0.23  = 2.38 ± 0.04  Fig. . However.12 Fig.  differed.03 Fig. . Applica.23 Fig.  and Rc .Fig. 2I2+ levels and SAP97/hDLG interaction with CaM. Increase in intracellular Ca2+ did not change the critical cluster radius, while there was a reduction in the slope of bulk energy, suggesting a decrease in the ambient monomer concentration. In this case, unaltered free energy of the system with an increase in entropy points to a local regulation with reduced monomer fraction and enhanced population of segregated/clustered molecules. The perturbation with the CaM inhibitor resulted in a significant increase in the critical radius of the cluster as well as the nucleation barrier. In this case, the slope of bulk energy was further reduced due to an increase in saturation concentration of monomer. Furthermore, when a chelator blocked the intracellular Ca2+, both the nucleation barrier and the critical radius increased, while the bulk energy reduced substantially. The results proposed an alteration in the clustering of SAP97/hDLG, forming numerous sub-saturated clusters. In parallel, we observed an increase in the dispersed phase by lowering the bulk energy. The model proposes a higher nucleation barrier and critical cluster radius in these conditions, implying that the cells are in a sub-saturated state, where clusters exist though they are spontaneously dispersed to form monomers.In summary, we evaluated the perturbation kinetics of first-order phase transitions under different pharmacological treatments in Neuro-2a cells that altered the intracellular Ca48 in these different conditions. The actual scaling factor of the average intensity of nanodomains in the treated vs. control cells were estimated. For the purpose, the datapoints from each treated condition (Tg/w7/Bapta) was divided by 100 values within the range of the arbitrary scaling factor, keeping the arbitrary value as the median. The resultant datasets were named as scaled-treated data. To determine the actual scaling factor, the control and the scaled-treated datasets were tested for significance using Kolmogorov–Smirnov test (K–S test)48. The actual scaling factor should give highest p-value, as the difference in the datasets would be non- significant. The method was repeated 100 times with each dataset. The results provided us with 100 p-values for each condition, and they were plotted against their corresponding scaling factors  (10 min), w7 (25 µM) (30 min) and Bapta (50 µM) (20 min) resulted in an enhancement of both average intensity Fig.  and areaors Fig. . The scaely Fig. . Thus, tely Fig. . When thely Fig. . In para2+ levels . The fold-change in expression of each SAP97/hDLG spliced variant was normalized to the expression of β-2-microglobulin (B2M) and double normalized to the cortical expression. The N-terminal and C-terminal spliced variants of SAP97/hDLG are I1A, I1B, and I2, I3, respectively . I1AB isoform was found to include both I1A and I1B isoforms of the N-terminal I1 splicing. I2 and I3 splicing were observed to be present in comparable levels in both hippocampus and cortex. We did not find a case were both I2 and I3 splicing coexisted, consistent with previous observations stating that a construct with both I2 and I3 resulted in a truncated product by induction of a stop codon after I320. It has been previously reported that all isoforms of SAP97/hDLG are known to express an additional C-terminal splicing in the HOOK region, referred to as I5 splicing, except for the prematurely terminated I4 isoform, which is expressed in very low amounts only in the mammalian brain50. Additionally, the non-membrane-bound β-isoform  or β-I1AB-I3-I5 (referred to as I1AB-I3-I5) to quantify the protein trafficking dynamics in live cells.Among the different isoforms expressed in neuronal cells, α- and β-isoforms of SAP97/hDLG have been well characterizedI1AB-I2-I5 or GFP:: I1AB-I3-I5 constructs  of hDLG isoforms. Confocal microscopy revealed an isoform-specific difference in the localization of the protein in cells  experiments in total internal reflection fluorescence (TIRF) mode were conducted on Neuro-2a cells after ectopically expressing GFP:: I2 & I3 isoforms displayed significant differences in protein mobility when Ca2+ levels were perturbed. The application of Tg did not affect the mobile fraction of I2 , while the halftime of recovery was increased     . The reduced recovery and enhanced halftime demonstrated a high-bound fraction of I2-hDLG upon reducing cytosolic Ca2+ levels in cells.The fluorescence recovery curves of both  s) Fig. . CaM bei02) Fig.  along wi s) Fig. . Decreas02) Fig. . In thisI3, Tg application resulted in a higher mobile fraction  with a shortened halftime of recovery     with an extended halftime of recovery , similar to the I2 isoform  Fig. . The res02) Fig. , but not s) Fig. . The resorm Fig. .I2 isoform by Ca2+-bound Calmodulin, the cells were treated with w7 followed by Tg. The results showed a compelling difference in the mobile fraction of I2 − Tg + w7 , while the halftime was significantly increased   on the dynamics of SAP97/hDLG isoforms, the cells were treated with Tg followed by Bapta. The cells expressing I2 showed no major shift in their mobile fraction      without a significant difference in its mobile fraction , compared to Tg application  Fig. , substan02) Fig. , while t s) Fig. . Cells e05) Fig. , while t01) Fig. . When thion Fig. . These rI2 was significantly higher with respect to I3 . I2 applied with Tg, w7, and Bapta showed identical levels of mobile fraction compared to I3  . When SOCE was induced followed by Ca2+ inhibition, I3 was found to be significantly confined with respect to I2  , while with Tg incubation, I3 showed faster kinetics with respect to I2 . CaM blockade with w7 shifted I2 towards faster kinetics compared to I3 . In the presence of Bapta, though there was a significant delay in the halftime for both isoforms, I2 showed faster kinetics than I3  . On the contrary, I2 isoform showed a faster recovery on depleting Ca2+ stores as well as on chelating the intracellular free Ca2+, compared to I3   Fig. . However02) Fig. . The hal s) Fig. . Elevati04) Fig. . Additio04) Fig. . These rI2 or I3 insert, we compared the electrostatic distribution of the SH3-HOOK-GUK region of SAP97/hDLG and Calmodulin  for endogenously expressed SAP97/hDLG were different between neurons and Neuro-2a cells  isoforms, while it was significantly different between DIV-14 neurons and untransfected Neuro-2a cells  , while it was significantly different between neurons and I2 isoforms , as well as from the untransfected control (ΔG Control:1.00 ± 0.02)  Fig. . The nuc02) Fig. . Our res52. The incorporation of these large scaffolding molecules into multi-protein complexes and their intrinsic ability to form multimers with the same or different family of proteins give them the flexibility to alter their compositionality and their signaling state instantaneously56. The last few years have highlighted the significance of fine tuning the molecular self-organization from nano to micron scale at real-time temporal resolution by confined phase transitions at high spatial and temporal precision58. Several multidisciplinary approaches have been used to evaluate these phase transitions as modulations in local biochemical interaction profiles, conformational changes, post-translational modifications and local exchange kinetics. However, majority of these studies are performed in a controlled environment or as ensemble observations to verify the existence of phase transitions in living cells60. These studies provide valuable insights into the nature of transient organization of molecules in subcellular compartments, but do not provide enough understanding on how it aids the processing and regulation of chemical information at the scale of molecular complexes64. Using SAP97/hDLG, we have evaluated how the local thermodynamic changes contribute to differential sensitivity of this scaffolding protein to [Ca2+.CaM]. Since SAP97/hDLG is one of the principal components of several cell adhesion machinery, we evaluated its nanoscale molecular distribution using a combination of direct stochastic optical reconstruction microscopy in total internal reflection mode on molecules close to the adherent membrane of the cell65. This method allowed us to quantify both the molecular heterogeneity and nanoscale topography of individual SAP97/hDLG molecules in sub-diffraction sized domains on the plasma membrane66. Our analysis on the clustering properties of SAP97/hDLG clubbed with the morphometric analysis of super resolution images showed first-order phase transitions at multiple scales. We observed that SAP97/hDLG monomers transition into large molecular domains and further into smaller tightly packed molecular domains within these large domains. Our results are consistent with previous observations for membrane-associated scaffolding complexes such as Shank-Homer platforms observed in excitatory synapses68.Molecular organization of multidomain scaffolding proteins such as SAP97/hDLG is important in subcellular compartments with heterogeneous molecular composition69. Currently there is no experimental evidence to suggest that SAP97 can directly bind Ca2+. Over the years, there has been a consensus on the response of SAP97/hDLG to modulation of intracellular Ca2+ through its association with [Ca2+.CaM]70. The binding of CaM is expected to open the conformation of SAP97/hDLG by exposing its multiple protein domains, thereby facilitating intermolecular interaction and oligomerization69. We evaluated the first-order phase transition of SAP97/hDLG upon altering the intracellular Ca2+. Multiple strategies were adopted including elevation of cytosolic Ca2+, blocking of CaM binding to SAP97/hDLG and sequestering of intracellular free Ca2+. Upon comparing these conditions to the untreated distribution of SAP97/hDLG, we found a relative increase in the entropy in all cases, resulting in an increased bulk energy component. These results showed that aggregates in untreated cells had the lowest entropy, which increased with any alteration to the system, consistent with the second law of thermodynamics. Though entropy was increased, the discrete alteration of the nucleation barrier and critical radius of clusters across the variable conditions suggested an altered self-organization of clusters. We evaluated how this self-organization could affect entropy by altering its molecular distribution, by comparing a rank-order analysis of both the molecular content and morphological parameters of SAP97/hDLG clusters in each condition with that of unperturbed cells. Elevation of intracellular Ca2+ did not alter the nucleation barrier or critical radius, instead increased the molecular content and size of SAP97/hDLG clusters. Such a molecular rearrangement is energetically favorable for large and stable multi-protein complexes and displays a condition with the least change in entropy, compared to the control conditions. While blocking Ca2+-binding protein CaM or on sequestering free Ca2+, we observed a significant increase in the nucleation barrier along with increased critical radius. The molecular fingerprints of the nanoscale organization of SAP97/hDLG confirmed that the local changes in chemical information processing could result in modifying entropy by altering the nanoscale aggregation properties in live cells. The smallest molecular domains that can be obtained by STORM is determined by the localization precision of the microscope72. This results in an inherent limitation to quantify the domains, which are below the resolution limit. Though our results indicate that most of the molecular domains are well above this pointing accuracy, there is a likelihood of under sampling a small fraction of domains, which fall below the resolution limit or do not achieve the desired molecular intensity.Many of the interactions of SAP97/hDLG with other molecules have been mapped to specific domains because of its modular architecture2+ as well as inhibition of interaction of [Ca2+.CaM] resulted in differential exchange kinetics for the SAP97/hDLG C-terminal isoforms from the unperturbed condition. In the presence of CaM inhibitor, the phase transition of SAP97/hDLG in Neuro-2a cells increased the molecular content of clusters along with more cytosolic monomer fraction. The reduction in the slope of bulk energy (b ∝ Log(Camb/Csat)) suggested an elevation of the ambient concentration of dispersed phase of SAP97/hDLG. This reduction in bulk energy component correlates to an increase in the entropy of the system. This is consistent with the observation that the mobile fraction of I2 SAP97/hDLG isoform recovered with a shortened halftime of recovery causing an augmented mobility, consistent with the observation of endogenous SAP97/hDLG organization by super-resolution microscopy. In Neuro-2a cells, the mRNA of I2 is almost six-fold more expressed than I3, consistent with the recovery of ectopically expressed I2 isoform of SAP97/hDLG. The [Ca2+.CaM] independent membrane recruitment of I2 would be facilitated better when CaM gets inhibited. While the monomer fraction of such a model would complement the nucleation barrier to be heightened and the super-saturation of molecules to be less favored, attaining it requires spending more energy. In the case of ectopically expressed I3, the mobile fraction increased without any change in the halftime when CaM was inhibited.Endogenous SAP97/hDLG modulation resulted in distinct nanoscale fingerprints in multiple conditions. Elevation and sequestering of intracellular Ca2+ did not affect the mobile fraction of the I2 isoform, while the halftime was significantly increased, this property being influenced by local interactions during the recovery of I2-SAP97/hDLG. Though for I2, this occurred without affecting the nucleation barrier and critical radius of clusters, bulk energy was reduced. This reduction of bulk energy defines an increase in the entropy of the system, but a corresponding change in its nanoscale organization resulted in unchanged nucleation barrier. I2 interacts with CASK, which senses [Ca2+.CaM] through its Ca2+/Calmodulin-dependent protein Kinase (CAMK) domain. Since the cytoskeletal interaction of CASK is independent of [Ca2+.CaM], the immobilization of I2 is modulated by the activity-dependent interactions of CASK by Mint1/X11α/Lin10 and Liprin α74. Additionally, CASK is also known to form a tripartite complex with Mint1 and Velis/Lin7 in the rodent brain75. This response of I2-SAP97/hDLG would explain a Ca2+-mediated decrease in its local recycling rates. Meanwhile, I3-SAP97/hDLG displayed a significant increase in its local recovery rate in the presence of high levels of Ca2+. Though the I3 isoform, I3-SAP97/hDLG, is known to associate with both Calmodulin and Protein 4.1, it is unclear whether they could form a tripartite complex potentially dependent on the cytosolic Ca2+ level. However, the faster recovery kinetics of I3-SAP97/hDLG at high Ca2+ levels indicated a closed conformation, decreasing intermolecular interactions. The mobile fractions of I2 and I3 in this condition were similar to each other. The affinity of [Ca2+.CaM] to CAMK domain is known to be 35-fold higher than intramolecular interactions of CAMK76, resulting in differential intramolecular alterations and recycling dynamics for SAP97/hDLG isoforms upon elevation of intracellular Ca2+.Elevation of intracellular Ca2+ was investigated by chelating intracellular free Ca2+. Depletion of free Ca2+ in the cells resulted in an elevated nucleation barrier and critical cluster radius due to a steep decline in the slope of bulk energy, b. In comparison to elevated Ca2+, the decreased slope of bulk energy indicated a decrease in the ability of SAP97/hDLG to nucleate a tight molecular organization, resulting in dispersed clusters. This reduction in energy could lead to a transient phase with cluster formation and dispersion of this protein. In this case, both the mobile fraction and halftime increased sharply, concurring with the dynamic state of the molecules. Further, to attain super-saturation in this case, the system would need to overcome a higher energy barrier beyond the limit of the experimental system, confirming the availability of Ca2+ as a prerequisite for first-order phase transitions that control nanoscale clustering of SAP97/hDLG. In the absence of free Ca2+, the system is in a sub-saturated state, complementing the dispersed fraction of SAP97/hDLG compared to a clustered one. FRAP experiments showed a decrease in the mobile fraction of I2: SAP97/hDLG upon chelating Ca2+. The rank-order analysis also showed an increase in the recruitment of molecules into an organized domain without similar modifications in the cluster area. In the absence of Ca2+, fluorescence recovery of I3 was rescued and reached the levels of I2-SAP97/hDLG, but the halftime of I3-SAP97/hDLG was observed to be significantly higher, confirming an enhanced intermolecular interaction. Association of SAP97/hDLG with [Ca2+.CaM], would increase its propensity to form multimeric protein complexes by unmasking its binding sites. Thus, the compositionality of isoforms would contribute to altering the local kinetics of first-order phase transition, clustering properties and global heterogeneity of this critical scaffolding molecule in response to the flow of chemical information and instantaneous signaling.The differential regulation of SAP97/hDLG isoforms by Ca2+-bound Calmodulin engages with the C-terminal isoforms of SAP97/hDLG differentially. We evaluated this differential dependence by independently inhibiting CaM using w7 and by a stimulation paradigm combining CaM inhibition with elevation of cytosolic Ca2+ after activation of SOCE. Upon inhibition of CaM, I2-SAP97/hDLG showed a decrease in its mobile fraction with faster recovery of free molecules. However, elevation of Ca2+ along with inhibition of CaM resulted in a slower recovery for I2-SAP97/hDLG, confirming an increase in intermolecular interactions independent of [Ca2+.CaM]. I3-SAP97/hDLG behaved similar to I2-SAP97/hDLG upon CaM inhibition as well as on co-stimulation by elevating the intracellular Ca2+. However, a significant difference in the rate of recovery was observed between the two isoforms. The increase in the recovery time of I3 can be attributed to the large presence of endogenous I2 isoforms in Neuro-2a cells interacting with ectopically expressed I3-SAP97/hDLG, resulting in intermolecular interactions of both isoforms. Deprivation of Ca2+ after inducing SOCE did not alter the mobile fractions of I2 and I3 isoforms from basal conditions. However, both isoforms recovered significantly slower compared to the control. These observations confirmed that I2 and I3 isoforms are significantly affected by the availability of free Ca2+, with I3 being preferred via its CaM interaction.CaI3 isoforms occurred at a lower concentration than that of I2, indicating distinct fingerprints for phase separation allowing differential recruitment of proteins to form co-condensates. In contrast, Calmodulin did not self-nucleate, or phase separate into condensates either exclusively in presence of Ca2+ or in the presence of SAP97/hDLG. However, Calmodulin was recruited into SAP97/hDLG condensates when co-incubated with Ca2+. The condensates of SAP97/hDLG nucleated Calmodulin by LLPS in an isoform-specific manner. Formation of co-condensates of the I3 isoform of SAP97/hDLG occurred at a lower concentration than that of I2, indicating a Ca2+ response window arising from properties of their intrinsically disordered HOOK region. These observations confirmed that SAP97/hDLG can not only form condensates regulated by the HOOK region, but also recruit other proteins into it in response to physiological stimuli such as intracellular elevation of Ca2+ or local Ca2+ transients. Using Neuro-2a cells and hippocampal pyramidal neurons, we verified that the combination of isoforms in a cell governs its nucleation and saturation kinetics of nanoscale condensates. Though the critical cluster radius was different for all conditions between Neuro-2a cells and neurons, the nucleation barrier remained similar when neurons and Neuro-2a cells had comparable ratio of I3 vs. I2 isoforms. This allowed us to summarize that the nucleation barrier might be a result of intrinsic properties derived from the local compositionality of SAP97/hDLG isoforms, but the critical cluster radius could be influenced by its heterogeneity in interactome, as many interacting proteins of SAP97/hDLG such as synaptic molecules are not present in Neuro-2a cells.Additionally, we showed that SAP97/hDLG spontaneously nucleate into micron scale condensates in vitro using LLPS at physiologically relevant concentrations. The nucleation of condensates of 2+, resulting in first-order phase transitions of this scaffolding protein. In vitro, LLPS of SAP97/hDLG into condensates occur by homotypic interactions, while for Calmodulin it is heterotypic and requires the presence of both SAP97/hDLG and Ca2+. In vivo, this phase transition is dependent on the nanoscale organization of endogenous SAP97/hDLG, where they are organized in a dispersed phase, a liquid phase, and a gel phase with increased packing density per unit area. These different phases have distinct nanoscale signatures. The phase transition of SAP97/hDLG contributing to its local heterogeneity on the membrane would decide its recycling rates, as verified by the kinetics of the two isoforms, which are distinct only in the intrinsically disordered region at the C-terminus. SAP97/hDLG is a ubiquitous member of the MAGUK family and is widely expressed in most of the cell types. The combination of its isoforms distributed across the different subcellular compartments decide not only the scaffolding of integral membrane proteins, but also serves as a locus for several signaling pathways determining cell fate and function. The differing spatio-temporal properties of SAP97/hDLG isoforms render different molecular fingerprints for each nanodomain, where the compositionality of these domains decides the nature of first-order phase transitions resulting in self-organization of the molecules. Our results show that in spite of similar phase transition dynamics, the organization and exchange kinetics leading to such changes are distinct. Similar studies correlating local thermodynamic variables with organizational deficits would be useful in modeling cell adhesion mechanisms, where flow of chemical signals to transmit information is critical. It remains to be seen whether the thermodynamic regulation of molecular organization would follow classical rules in cell junctions such as synapses. Here, in addition to the flow of chemical information, storage, retrieval and elimination of information translates to memory, potentially sustaining longer than an instantaneous alteration in the chemical behavior of independent molecules.Our observations highlight the intrinsic modulation of SAP97/hDLG in response to alterations in intracellular CaAll the procedures in this study were performed according to the rules and guidelines declared in the Compendium of CPCSEA 2018 by the Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA), Ministry of Fisheries, Animal Husbandry and Dairying, India. The research protocol (CAF/Ethics/659 and CAF/Ethics/790) was approved by the Institutional Animal Ethics Committee (IAEC) of the Indian Institute of Science.21.The hDLG constructs tagged with enhanced green fluorescent protein (eGFP) at the N-terminus of the protein sequence were ectopically expressed in Neuro-2a cells for the FRAP experiments. The sequences can be found in the public domain of GenBank data base (accession code: U13896 and U13897 [10.1073/pnas.91.21.9818])2 incubator on coverslips  of 18 mm diameter and 0.17 mm thickness for 48 h and transfected with Turbofect 77. Before transfection, the cells were incubated in 1 ml serum-free DMEM for 1 h. Plasmid (1 μg) was mixed with Turbofect in 100 µl of DMEM. The cells were then incubated for 5 h at 37 °C, followed by the addition of 2x-FBS supplemented growth media (1 ml) and imaged after 24 h.Neuroblastoma cells  were cultured in DMEM supplemented with Glutamax, 1% fetal bovine serum and 1% penicillin–streptomycin at 37 °C. Cells were grown in a 5% CO80. Hippocampi were dissected out using a stereomicroscope  in chilled Hibernate-A medium (Gibco-A12475-01) supplemented with B-27-(Gibco-17504044), glutaMAX (Gibco-35050061) and antibiotic-antimycotic (Gibco-240062). The tissue was minced and incubated for 10 min at 37 °C in 0.25% trypsin. The sample was then triturated and centrifuged at 800 x g for 5 min. The resultant pellet was resuspended in Hibernate-A and the supernatant was discarded. This procedure was repeated twice and then the cells were resuspended in the complete Neurobasal-A (Gibco-10888022) media . The cells were counted and seeded at the density of 1 × 105 cells/ml on 18 mm  glass coverslips (coated with poly-d-lysine at a concentration of 100 μg/ml) in a 12-well cell culture plate. Cultures were maintained at 37 °C and 5% CO2 at optimum humidity conditions. Media was complemented at an interval of 7 days. Hippocampal pyramidal neurons were transfected with Lipofectamine 3000 (Invitrogen) I3-hDLg::mEos; where I3-hDLG was fused with mEOS2.3, at 13 days in vitro and imaged within 12–24 h after78.Primary hippocampal neurons were cultured from neonatal Sprague-Dawley rat pups aged postnatal day 0–1 (P0–P1)n = 9) and from Neuro-2a cells cultured in T75 flask (N = 4) using TrizolTM extraction method . The primers used for the experiments are mentioned in the Supplementary Table N = 2−∆∆Ct  of endogenous control and the sample, ∆∆Ct = Difference between ∆Ctsample1 – ∆Ctsample2. Here, sample1 was taken to be the cortex).Total ribonucleic acid (RNA) was extracted from the hippocampus and cortex of P0 mice (81. Imaging was done within 24 h of completion of labeling procedure.Neuro-2a cells were transfected with a reaction mixture of fusion constructs of SAP97/hDLG (1 μg) using Turbofect (2 μl) in 100 μl of DMEM. For dSTORM imaging, Neuro-2a cells and DIV-14 primary hippocampal neurons were fixed using 4% paraformaldehyde and 4% sucrose in PBS for 10 min at 4 °C, quenched with 0.1 M glycine in PBS at room temperature, permeabilized with 0.25% TritonX-100 for 5 min and blocked with 10% BSA in PBS solution for 30 min at room temperature. The cells were incubated with the primary antibodies  diluted in 3% BSA (1:500) for 1 h at room temperature in a humidified chamber. The cells were washed four times (5 min each) with 3% BSA and then incubated with suitable secondary antibody for 45 min followed by 4-time washes of 5 min each. The 12-well plates were wrapped in an aluminum foil and stored in PBS at 4 °C for dSTORM imaging. The antibody specific for SAP97/hDLG was validated using western blot of whole protein extract from rodent brain lysate . The results are illustrated in supplementary information (eGFP (GeneBank: U13896 [10.1073/pnas.91.21.9818]) was PCR amplified to introduce 5’-XhoI and 3’-NotI restriction sites and cloned as a XhoI-NotI fragment in the bacterial expression vector pET-28a vector  to generate pET28a-GFP. The primers used for the experiments are mentioned in the Supplementary Table E. coli strain BL21(DE3) was transformed with pET-28a-SH3-I2-GUK::GFP, pET-28a-SH3-I3-GUK::GFP and with pET-28a-CaM::mCherry plasmid DNA. The cells were grown at 37 °C to an optical density of 0.8 at 600 nm and protein expression was induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside ((IPTG) Thermo Fisher Scientific, USA) overnight. The next day, the cells were centrifuged at 4000 x g for 10 min and the pellets were stored at −80 °C. The bacterial cells were lysed by sonication  with Tris buffer ). As the proteins were predominantly present in the soluble fraction, the sonicated samples were centrifuged at 30,000 x g for 1 h to remove cellular debris. The proteins were purified by Ni2+-nitrilotriacetic acid (NTA)-agarose affinity chromatography. The supernatant was passed through Ni2+-NTA-agarose resin (Qiagen) and the protein-bound beads were washed with 4 column volumes (~25 ml) of wash buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 50 mM imidazole. 4 ml elution buffer (10 mM Tris-Cl (pH 7.4), 150 mM NaCl and 500 mM imidazole) was added to elute the target proteins. The eluted proteins were concentrated to 500 μl  and was ultracentrifuged at 35,000 x g at 4 °C for 30 min to pellet out the protein aggregates.®6 increase 10/300GL column was pre-equilibrated with 3 column volumes of saline buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl and 2% glycerol) and the protein samples were injected. The whole process was performed in Akta pure® HPLC system using Unicorn7.3 software. The purity of isolated protein fractions of 500 μl were confirmed using SDS-PAGE. The concentration of proteins was measured using NanoDrop (Thermo Scientific). The absorbance measurement at 280 (A280) and the extinction coefficients of the respective proteins were used to calculate their concentration.The SuperoseThe SDS-PAGE gel was prepared with 10% resolving gel , 0.1% SDS, 0.1%APS, TEMED) and 4% stacking gel (0.126 M tris (6.8 pH)), 0.1% SDS, 0.1% APS, TEMED). The casting was done with 1.5 mm thickness glass plates (Bio-Rad). 15 µg total protein in 1x Laemmli buffer was loaded per lane and the electrophoresis was carried out in the running buffer  using the mini-Protean tetra cell system (Bio-Rad). The gels were stained with Coomassie Blue, destained twice with the washing solution  for 1 h, and were imaged using a Gel Doc EZ imager (Bio-Rad).I2-GUK::GFP, SH3-I3-GUK::GFP and CaM::mCherry, respectively. The reconstruction of the models was performed using UCSF-ChimeraX (Version-1.3). The electrostatic potential map was generated from APBS, an open server from NIH, USA.The amino acid sequences of fusion constructs were uploaded to i-TASSER(Iterative Threading ASSEmbly Refinement) server to obtain the structure predictions with high confidence. The predicted models with the highest c-score of −2.20, −2.00, and −1.00 were chosen for SH3-All LLPS experiments were carried out using acid-treated coverslips. The coverslips (Blue star) of 18 mm diameter were kept in concentrated HCL for 12 h and thoroughly washed with deionized water  and sterilized. The LLPS solution was prepared from varying concentrations of SEC purified proteins  and a molecular crowding agent polyethylene glycol (PEG-4000) ) in saline buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl and 2% glycerol) to determine the phase regime. The reaction mixture was loaded in a Ludin chamber  for imaging. The images were acquired at an ambient temperature of 25 °C. Calcium chloride was added to the reaction mixture at 2 mM concentration.2+ levels of Neuro-2a cells. Thapsigargin , a drug capable of blocking all the four isoforms of the SERCA pump, was applied. Stock solution of 5 mM Thapsigargin was prepared in dimethyl sulphoxide (DMSO) and working concentration of 1 μM in HEPES buffer (extracellular solution/ECS), wherein the cells were incubated for 10 min. Calmodulin (CaM), one of the significant Ca2+ sensors in living cells, was blocked by a CaM antagonist w7 (N-(6-Aminohexyl)−5-chloro-1-naphthalene sulfonamide hydrochloride) . The stock concentration of w7 was prepared in DMSO (100 mM) and working concentration of 25 μM in ECS, wherein the cells were incubated for 30 min. Concentrations of w7 above 50 μM was found to be lethal to cells. 50 mM stock solution of a cell permeable Ca2+ chelator named 1, 2-Bis (2-aminophenoxy) ethane-N, N, N′, N′-tetra acetic acid tetrakis (acetoxymethyl ester)  was prepared in DMSO and its working concentration of 50 μM in ECS. The cells were incubated for 20 min to chelate the intracellular Ca2+. All the treatments of Neuro-2a cells were performed at 37 °C.The pharmacological treatments were performed to alter the intracellular CaThe Ludin chamber loaded with the sample was mounted on an Axio observer Z1 inverted microscope  equipped with an Apotome 2 module. The image acquisition was performed using a plan-apochromat 63x/1.4 NA oil immersion objective and excitation/emission filters of GFP and DsRed . Zen 2 blue edition was used for image acquisition  software. The images were obtained using an Axiocam 506 mono 6 Mp CCD camera with a frame size of 2752p x 2208p (as czi or tif files) at 8-bit depth. The image processing was done using Fiji  and MetaMorph .Z-stacks of total volume (≈2000 μm3) were acquired with single optical sections of thickness 100 nm. ZEN 2011 LSM imaging software was used for acquisition and the image analysis was performed using Metamorph.Neuro-2a cells over-expressing SAP97/hDLG isoforms were imaged on a Zeiss LSM 780  confocal inverted motorized microscope equipped with a 63x oil objective of refractive index1.5 and NA1.4. The cells were imaged at a sampling of ≈88 nm/pixel. 3D Confocal 2, 10 mM HEPES). The Ludin chamber was mounted on a motorized inverted Olympus microscope (IX83) and was imaged at 37 °C . GFP excitation was performed using a 488 nm excitation laser in total internal reflection fluorescence (TIRF) mode. Image acquisition was done using a 100x objective  and an EMCCD . A pulsed 488 nm laser of 100 mW average power was used for bleaching within a region of interest (ROI) of 3 μm diameter at an exposure of 20 ms. After acquiring ten pre-bleach images, the ROI was bleached, and 400 post-bleach images were acquired. The image acquisition and data collection were performed using Metamorph software.FRAP experiments were performed on Neuro-2a cells to study the ectopic expression of alternative splice variants of hDLG fused with eGFP. The cells were cultured on 18 mm coverslips and were loaded on a Ludin Chamber  supplemented with an extracellular solution suitable for live cell imaging  × 100]/34 . A positive (Camb < Csat) or negative (Camb > Csat) sign depicts the cluster’s saturation state, i.e., whether the system is in a sub-saturated or super-saturated state. The term “bn” was extracted from the data by subtracting “an2/3” from ΔG, and the curve was observed to be linear with a negative slope, representing a super-saturated system. The negative bulk energy minimizes a system’s free energy, while the positive surface energy tries to maximize it. The balance of these two terms defines the nucleation barrier, above which the cluster formation tends to be spontaneous. P(n) represents the relative frequency distribution of cluster size (n) and nc is the critical cluster size attaining a maximum value of ΔG(nc), which is the nucleation barrier. The analysis showed an offset in the curve fit with –LogP(n), which was contributed by Log A, the y-intercept (c) of the curve. From the curve fit, the inflection points of the curve gave the critical cluster size, nc = (2a/3b)3, above which the clusters spontaneously grow at the expense of monomer concentration. The data fit gave the parameter values of a, b, and c (best fit mean ± s.e.m). On substituting these parameters in the free energy equation, we were able to extract the nucleation barrier ΔG(nc) and the critical cluster size nc ∝ (Rc)3. ΔG(nc) and critical radius of cluster (Rc) were normalized with respect to untreated control dataset.Since the number of molecules  were used to correct for 2D drift occurring during SMLM acquisition. The clusters were identified based on the minimum average intensity threshold of 3–5 detection events. The total intensity of single molecules was acquired from a binary mask after thresholding the image using the integrated morphometry analysis (IMA) plugin in MetaMorph. Detection of events was thresholded to 42 nm of cluster diameter (21 nm of radius), and the median of total intensities from single-molecule events was taken as the total intensity of a single molecule. From this, the number of molecules in a cluster was obtained by dividing the total intensity of clusters by the median of the total intensity of a single molecule41. The numbers thus obtained were correlated with numbers of standard scaffolding molecules such as PSD95, Bassoon, AMPA Receptors, Amyloid Precursor Protein and beta secretase molecule BACE1 in primary hippocampal neuronal cultures and APP and beta secretase molecule BACE1 in Neuro-2a cells81.A typical single-cell SMLM  experiment acquired with the microscope setup as described previously produced a set of 20,000 images that were analyzed to extract single-molecule position and dynamics. Single-molecule fluorescence events were localized in each image frame and tracked over time by a wavelet segmentation algorithm and simulated annealing, respectively. The algorithm used to derive quantitative data on protein localization and dynamics was run using the PALM-TRACER48. The bin size of the probability distribution for different pharmacological conditions was chosen between ∆n = 40, without the function hitting a noise floor. After a trial-and-error approach with various bin sizes, the range of bins was decided for the different datasets. The cluster numbers in all conditions ranged from 1200 to 1500. The critical cluster size (Rc) and nucleation barrier (∆Gc) for all conditions were calculated and were normalized to the mean of untreated control data.The derivation of the polynomial curve fitting of the normalized probability distribution function of SAP97/hDLG cluster size was followed from a previous studyα error prob of 0.1 and power of 0.9 from 2 to 4 groups. For the FRAP experiments, sample sizes were calculated using effect size of 0.7, α error prob of 0.1 and power of 0.9 from 2 to 4 groups84.All the sample sizes were calculated using G*Power (Version 3.1.9.7). For the morphometric data from SMLM, sample sizes were calculated using effect size of 0.1, I2 and I3 variants were statistically analyzed and tested with one-way Welch’s t-test for two sample comparison and Brown–Forsythe and Welch’s ANOVA with Dunn’s correction for multiple comparison. Quantification of FRAP is shown as normalized average fluorescence intensity and of q-PCR as the fold-change of expression normalized to endogenous control. Values are expressed as mean ± s.e.m.FRAP experiments of hDLG-Rc) and nucleation barrier (∆Gc) data from multiple conditions were compared and tested for significance using Brown–Forsythe and Welch’s ANOVA with Dunn’s correction, with p < 0.001 as indicated by ****. Linear regression of bn was performed for all pharmacological conditions to explore the value and slope of the bulk energy component.The critical cluster size  were included in the analysis  > minimum of control). The datasets were analyzed by K–S test to compare the scaled-treated vs. the control distribution. The scaling factor corresponding to the highest p-value was considered as the accurate one. This process was repeated 100 times to obtain the scaling factor ± standard error of the mean.The normality of distributions of dSTORM data was checked by D’Agostino & Pearson omnibus normality test and was found to be non-normal. The frequency distributions for constructing the probability density function histograms and the cumulative probability distribution function curves were normalized. K–S test was used to compare the distributions of control, Tg, w7 and BAPTA datasets. For K–S test, the confidence level was set to 0.001 due to the high sample size and Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Anti-HEV antibody titers were significantly higher in 50 and 10 μg VLP-vaccinated rabbits than in the negative control rabbits at weeks 8 and 11, respectively. Serum IFN-γ and IL-12 levels were significantly higher (p < 0.01) in rabbits vaccinated with 50 and 100 μg VLP than in the negative control rabbits at weeks 4 and 6. Liver tissues of 50 and 100 μg VLP-vaccinated rabbits displayed significantly less (p < 0.05) fibrosis than those of the positive control rabbits. The prepared VLP vaccine demonstrated dose-dependent immunogenicity sufficient for inducing anti-HEV antibody production, thus protecting rabbits against swine HEV-3.Here, rabbits were immunized with a virus-like particle (VLP) vaccine prepared by expressing 239 amino acids of the swine hepatitis E virus (HEV)-3 capsid protein using a baculovirus system. Thirty specific-pathogen-free rabbits were divided into five groups . Positive control group rabbits showed viremia and fecal viral shedding, whereas rabbits vaccinated with 10 μg VLP showed transient fecal viral shedding, and rabbits vaccinated with 50 and 100 μg VLP did not show viremia or fecal viral shedding. Serum anti-HEV antibody titers increased in a dose-dependent manner. Anti-HEV antibody titers were significantly higher ( Hepeviridae, Orthohepevirus, and Orthohepevirus A and is a non-enveloped virus with a 7.2-kb positive single-stranded RNA [Hepatitis E virus (HEV) belongs to the families nded RNA . The virnded RNA . To datended RNA . The zoonded RNA ,6.The HEV genome consists of three open reading frames (ORFs) . ORF1, lEscherichia coli expression system and the baculovirus insect cell expression system [E. coli expression system, a large amount of protein can be obtained quickly at a low production cost. However, as a prokaryotic expression system, the E. coli system cannot translate multiple proteins and post-translational modifications. The baculovirus expression system can produce particles that are morphologically and antigenically similar to the native virus particle through post-translational modification. However, this system takes a relatively long time to produce particles compared to the E. coli expression system [E. coli system, has already been verified for its antigenicity, immunogenicity, and protectivity against HEV genotypes 1 and 4 in primates [There have been many attempts to evaluate the efficacy of a vaccine by expressing HEV VLPs using the n system ,14,15,16n system ,18. The primates . In addiprimates ,16,19. Tprimates . ClinicaResearch on HEV VLP vaccines has mainly focused on antigenicity and humoral immune responses, such as immunogenicity and vaccine protection efficacy against HEV ,13,15. SThe VLP vaccine described herein was used in a previous study . Briefly6 genome copies/mL in 1% bovine serum albumin (Sigma-Aldrich). The HEV-3-239 VLP vaccine was administered into the biceps femoris, and swine HEV-3 was injected into the ear vein. All rabbits were euthanized with potassium chloride after zolazepam administration at week 12.Thirty 12-week-old female specific-pathogen-free rabbits were purchased from SAMTAKO . All animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Konkuk University, South Korea (KU19071). Prior to conducting the experiment, the presence of HEV and anti-HEV antibodies was verified in the feces and serum samples obtained from all rabbits. Rabbits were divided into five groups, namely the negative control; positive control ; and 10 μg, 50 μg, and 100 μg VLP-vaccinated groups, with six rabbits in each group. The experiment was conducted for 12 weeks. Rabbits in the vaccine groups received the 1st and 2nd vaccine dose at week 0 (W0) and week 2 (W2), respectively. The vaccination schedule was determined based on our and other previous studies ,23,24. Ag, 20 min). Feces collected from the rabbits were dissolved in phosphate-buffered saline (PBS) at a ratio of 1:10 and released using a vortex mixer. The supernatant containing HEV was separated from the fecal solution using centrifugation . Liver samples from the sacrificed rabbits were cut into small pieces, and the excised liver tissue was prepared as a suspension using a cell strainer and PBS containing RNase. All serum, fecal supernatant, and liver samples were stored at −70 °C. HEV RNA was extracted from the collected serum and fecal supernatant using the Patho Gene-spin DNA/RNA Extraction kit . Nested RT-PCR was performed with two pairs of primers [Blood collected from the rabbits was centrifuged to separate the serum . The test was repeated twice, and the average value was calculated. The levels of interleukin (IL)-10, IL-12, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) were measured in serum collected at weeks 0, 2, 4, 6, 8, 10, and 12 using commercially available ELISA kits—rabbit IL-10 ELISA kit , rabbit IL-12 ELISA kit , rabbit TNF-α DuoSet ELISA , and rabbit IFN-γ Do-It-Yourself ELISA —according to the manufacturer’s instructions.® .Livers obtained from rabbits euthanized at week 12 were fixed with 10% neutral buffered formalin and embedded in paraffin. Tissues were stained with hematoxylin and eosin and Masson’s trichrome to assess the overall lesion and fibrosis in the liver. All Masson’s trichrome stained livers were observed at 40× magnification, and the ratio of the fibrotic tissue area to the liver tissue area was calculated using MetaMorphp < 0.05.GraphPad Prism software version 8.0.2  was used for statistical analyses and generating graphs. All data are expressed as the means ± SDs of six replicates. The data were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test as the post hoc test. The statistical significance for each test was set at The detection of HEV-3 RNA was confirmed in fecal and serum samples collected weekly from rabbits for 12 weeks and in liver samples obtained from euthanized rabbits at week 12. As expected, HEV-3 RNA was not detected in any fecal, serum, or liver suspension samples obtained from rabbits in the negative control group during the experimental period . By contp < 0.05) than that of rabbits in the negative control group. The anti-HEV antibody titers of rabbits vaccinated with 100 μg VLP were only significantly higher (p < 0.05 or p < 0.001) than those of rabbits in the positive control group from weeks 4 to 10  from week 11 (p < 0.05) from week 8 (p < 0.05) from week 4  than those of rabbits in positive control groups at week 4. The mean serum IL-12 level of rabbits in the negative and positive control groups was 1126 pg/mL and 1122 pg/mL, respectively, whereas that of the 50 and 100 μg VLP-vaccinated groups was 1529 and 1471 pg/mL, respectively, at week 4 (p < 0.05) and 100 μg VLP (p < 0.01) increased significantly compared to those in the positive control at week 4. The mean serum IFN-γ levels of negative control rabbits were 557.8 pg/mL at week 4. The mean serum IFN-γ levels of positive control rabbits were 576.8 pg/mL at week 4. The mean serum IFN-γ levels of 50 and 100 μg VLP-vaccinated rabbits increased sharply to 1547 and 1727 pg/mL, respectively, at week 4  higher than that in the livers of negative control rabbits. The percentage of fibrosis in the livers of rabbits vaccinated with 50 and 100 μg VLP was significantly (p < 0.05) lower than that in the livers of positive control rabbits. Although the degree of liver fibrosis in rabbits vaccinated with 10 μg VLP was higher than that of negative control rabbits and rabbits vaccinated with the two other concentrations of VLP, the difference was not statistically significant , an extension of the 92 amino acids in the verified . In addiverified ,16,31. Sverified . Aligninp < 0.05) from week 4 compared to that of the negative and positive control rabbits. Compared to that in positive control rabbits, the titer of anti-HEV antibodies in rabbits vaccinated with 100 μg VLP significantly increased from week 4, suggesting that anti-HEV antibodies were generated following the two vaccine injections. The titer of anti-HEV antibodies in rabbits vaccinated with 50 and 10 μg VLP increased significantly (p < 0.05) at weeks 8 and 11, respectively. In this study, we only determined the O.D. values of serum HEV antibodies obtained from rabbits without measuring the end-point titers of antibodies. If the end-point antibody titers were determined, we could identify the exact amounts of antibodies in each serum. It would be a limitation of this study. Fecal shedding was observed for two weeks, from week 8 to 9, in one rabbit vaccinated with 10 μg of the VLP vaccine. However, rabbits vaccinated with 50 and 100 μg VLP did not show viremia or fecal shedding. According to the experimental results, the anti-HEV antibody titers in the serum of rhesus monkeys vaccinated with 10 and 20 μg HEV p239 vaccine, expressed by using the E. coli system, increased gradually from week 2, whereas the anti-HEV antibody titers in the serum of monkeys vaccinated with 5 μg VLP increased from week 4. In addition, virus fecal shedding was observed for three weeks and one week in monkeys vaccinated with 5 and 10 μg VLP, respectively [The anti-HEV antibody titer of rabbits vaccinated with the HEV-3-239 VLP vaccine increased in a dose-dependent manner. The titer of anti-HEV antibodies in rabbits vaccinated with 100 μg VLP increased significantly (ectively . Therefoectively . The vacectively . We expeThe serum ALT and AST levels of the positive control rabbits infected with swine HEV-3 did not increase significantly and were similar to those of the negative control and vaccinated rabbits. Serum ALT and AST levels of rabbits in all groups were maintained within the normal ranges of 45–80 U/L and 35–130 U/L, respectively, throughout the experimental period. Similarly, ALT levels increased in rabbits infected with swine HEV-3 and rabbit HEV but did not increase significantly and were within the normal range ,34. HoweIn this study, after two VLP vaccinations and one HEV-3 challenge, we confirmed the induction of cytokine production associated with Th1- and Th2-type immune response in the sera of rabbits from all groups. The levels of Th1-type cytokines, IL-12 and IFN-γ, were significantly increased in rabbits immunized with 50 and 100 μg VLP vaccine. By contrast, the serum concentration of IL-10 significantly decreased in all groups at the end of the experimental period. Similarly, IL-12 and IFN-γ levels were significantly increased in the serum of rabbits immunized with the nanogel vaccine based on rabbit HEV . PeripheIn this study, fibrosis of the liver tissue of rabbits infected with swine HEV-3 was determined using Masson’s trichrome staining. The area of fibrotic tissue was calculated as a percentage based on previous studies ,43,44. AThe HEV-3-239-VLP vaccine, prepared by expressing 239 amino acids of swine HEV-3 capsid protein using a baculovirus system, prevented HEV-3 infection in a dose-dependent manner. Anti-HEV antibodies and Th1-type cytokines may be associated with HEV-3 protection. Fibrosis of the liver tissues could be another indicator of HEV-3 infection. The HEV-3-239 VLP vaccine may be applicable to humans and other animal species in the future to prevent HEV infection.


We investigated the effect of hypoxia on [Ca2+]i in isolated and quiescent VSMCs from C57BL/10SnJ (WT) and C57BL/10ScSn-Dmd (mdx) male mice. [Ca2+]i was measured using Ca2+-selective microelectrodes under normoxic conditions  and after hypoxia (glucose-free solution aerated with 95% N2-5% CO2 for 30 min). [Ca2+]i in mdx VSMCs was significantly elevated compared to WT under normoxia. Hypoxia-induced [Ca2+]i overload, which was significantly greater in mdx than in WT VSMCs. A low Ca2+ solution caused a reduction in [Ca2+]i and prevented [Ca2+]i overload secondary to hypoxia. Nifedipine (10 µM), a Ca2+ channel blocker, did not modify resting [Ca2+]i in VSMCs but partially prevented the hypoxia-induced elevation of [Ca2+]i in both genotypes. SAR7334 (1 µM), an antagonist of TRPC3 and TRPC6, reduced the basal and [Ca2+]i overload caused by hypoxia. Cell viability, assessed by tetrazolium salt -2,5-diphenyltetrazolium bromide, was significantly reduced in mdx compared to WT VSMCs. Pretreatment with SAR7341 increases cell viability in normoxic mdx (p < 0.001) and during hypoxia in WT and mdx VSMCs. These results provide evidence that the lack of dystrophin makes VSMCs more susceptible to hypoxia-induced [Ca2+]i overload, which appears to be mediated by increased Ca2+ entry through L-type Ca2+ and TRPC channels.Duchenne muscular dystrophy (DMD) is an inherited muscular disorder caused by mutations in the dystrophin gene. DMD patients have hypoxemic events due to sleep-disordered breathing. We reported an anomalous regulation of resting intracellular Ca We have previously described how the absence of dystrophin caused a chronic increase in [Ca2+]i and sodium concentration ([Na+]i) in skeletal [2+]i appears to be mediated by increased leakage of ryanodine and inositol trisphosphate receptors in the sarcoplasmic-endoplasmic reticulum and an increased influx of Ca2+ through transient receptor potential canonical (TRPC) channels. Studies from our laboratory have shown that reduction in [Ca2+]i by pharmacological and non-pharmacological approaches reduced [Ca2+]i and provoked protection in dystrophic muscle cells and neurons i in WT and mdx VSMCs. We found that mdx VSMCs are more susceptible to hypoxic stress than WT. Susceptibility appears to depend on the anomalous increase in [Ca2+]i, and the subsequent reduction in cell viability of mdx VSMCs compared to WT VSMCs. Furthermore, Ca2+ influx can play an essential role as a contributor to intracellular Ca2+ overload observed during acute hypoxia.In this study, we tested the effects of acute hypoxic stress on i was measured under normoxic conditions  and after 45 min of hypoxia.Hypoxia was induced by perfusing WT and mdx VSMCs with a glucose-free solution saturated with 95% NThe viability of WT and mdx VSMCs was determined under normoxic and hypoxia conditions using the 3 -2,5-diphenyltetrazolium bromide (MTT) assay according to the manufacturer’s kit instructions . Furthermore, the viability of the VSMC was determined in both genotypes pretreated with nifedipine or SR7334 and then subjected to hypoxia. Data collected from treated and untreated WT and mdx VSMCs are represented as a reduction in the MTT signal relative to untreated WT VSMCs.2, 1 MgCl2, 5 glucose, and 3.6 NaHCO3 (pH 7.4). The Ringer solution was aerated with a mixture of 95% O2 and 5% CO2 under normoxic conditions. For the low Ca2+ solution, CaCl2 was omitted and instead 2 mM MgCl2 and 1 mM ethylene glycol tetraacetic acid (EGTA) were added, reducing [Ca2+]e to 35 ± 8 nM as determined with Ca2+ selective microelectrodes. Nifedipine (10 µM) and SAR7334 (1 µM) were prepared from stock solutions.The Ringer solution had the following composition (in mM): 135 NaCl, 5 KCl, 1.8 CaClp < 0.05 was considered significant. nmice: number of mice and ncell: number of measurements. A GraphPad Prism 9  was used for the statistical analysis. Data are presented as a mean ± standard deviation of the mean. One-way analysis of variance (ANOVA) on the ranks was used, followed by Tukey’s post hoc tests. 2+]i is the net result of influxes, sequestration, and calcium extrusion. We confirm that under normoxia conditions, there was a significant difference in resting membrane potential and [Ca2+]i in mdx VSMCs compared to WT [p < 0.001 compared to WT) (2+]i in WT VSMCs was (123 ± 3 nM) while in mdx it was (285 ± 31 nM) (p < 0.001 compared to WT)  A. [Ca2+]d to WT) B. 2+]i overload in WT and mdx VSMCs, however, resulted in the magnitude being significantly greater in mdx than in WT VSMC. In WT VSMCs, hypoxia caused a consistent depolarization of the RMP from −63 ± 2 mV (normoxic) to −54 ± 3 mV, while in mdx VSMCs, it went from −54 ± 3 mV to −41 ± 3 (2+]i overload in WT VMSCs increased from 123 ± 3 nM (normoxic) to 980 ± 119 nM and in mdx VSMCs from 285 ± 31 nM to 2962 ± 287 nM (2+] induced by hypoxia caused the development of hypercontracture and death in mdx VSMCs.Acute hypoxia-induced muscle membrane depolarization and [Ca −41 ± 3 A. Simila± 287 nM B. A pers2+ concentration ([Ca2+]e) on [Ca2+]i was evaluated under normoxia and acute hypoxia in VSMCs from WT and mdx mice. We confirmed that the reduction in [Ca2+]e caused a significant decrease in resting [Ca2+]i in WT and mdx VSMCs and that the magnitude was greater in mdx than in the WT VMSCs [2+]i was reduced by 55% , while in WT, it was reduced by 23%  in VSMCs  Ca2+ solution markedly reduced the increase in [Ca2+]i overload secondary to hypoxia in both genotypes (compared with 2+]e prevented the development of hypoxia-induced hypercontracture in mdx VSMCs.The effect of a low (35 ± 8 nM) extracellular CaWT VMSCs . In mdx in VSMCs . In addi2+]i during hypoxia, in another set of experiments, we studied the effects of nifedipine, an L-type Ca2+ channels blocker, on [Ca2+]i in WT and mdx VSMCs under normoxic and hypoxic conditions. Under normoxic conditions, incubation in nifedipine 10 µM for 10 min did not modify resting [Ca2+]i in WT or mdx VSMCs compared to untreated VSMCs (2+ current in VSMCs (Lopez unpublished results). However, nifedipine partially prevented the magnitude of elevation of [Ca2+]i associated with hypoxia in both genotypes, with effects on mdx VSMCs greater than WT  while mdx VSMCs to 2013 ± 366 nM  (2+]i during hypoxia were obtained when WT and mdx VSMCs were pretreated with amlodipine and other L-type Ca2+ channel blockers .To further elucidate the molecular mechanisms that result in elevation i in VSMCs [2+]i in WT VSMCs from 122 ± 3 nM to 100 ± 6 nM (18% reduction) and mdx VSMCs from 285 ± 31 nM to 154 ± 15 (45% reduction)  and in mdx VSMCs to 837 ± 95 nM (2013 ± 366 nM in untreated cells) (2+]i by 64% compared to untreated WT VSMCs (p < 0.001) and 72% in mdx VSMCs compared to untreated mdx VSMCs (p < 0.001)  . Furtherd cells) . SAR7334< 0.001)  insert. p < 0.001) without any effect on WT VSMCs (p = 0.91). Furthermore, SAR7334 increased cell viability in both groups during hypoxia compared to the untreated WT and mdx VSMCs .As shown in 2+] in smooth muscle cells associated with hypoxia have been suggested, this is the first study to examine the effect of acute hypoxia on [Ca2+]i in mdx VSMCs. The main findings of the present study are (i) hypoxia elicited depolarization of the muscle resting membrane potential and an elevation in [Ca2+]i in both WT and mdx, whose magnitude is much higher in mdx than in WT VSMCs. (ii) The removal of extracellular Ca2+ blocked intracellular Ca2+ elevation caused by hypoxia. (iii) Pretreatment with nifedipine reduced the amplitude of hypoxia-induced increase in [Ca2+]i in both genotypes. (iv) Blockade of the TRPC3 and -6 channels abolished the increase in [Ca2+]i elicited by hypoxia. (v) Cell viability was significantly reduced in mdx compared to WT VSMCs. Hypoxia decreased cell viability in both genotypes, and pretreatment with SAR7334 increased cell viability in normoxic mdx and WT and mdx during hypoxia.Although abnormalities in intracellular [CaDMD is a progressive muscle-wasting disease caused by the absence of the protein dystrophin . Dystrop2+]i in striated and non-striated muscle cells. Therefore, we characterize the acute effects of hypoxia on [Ca2+]i in VSMCs isolated from WT and mdx mice.Sleep disorder breathing with hypoxia is a dominant pathological component of the complex changes observed in patients with DMD; however, no studies have examined the role of hypoxia on [Ca2+]i is maintained at low concentrations (100–120 nM) against a large extracellular concentration gradient [2+]i is normally maintained by a complex equilibrium between the activities of Ca2+ release channels, sarco-endoplasmic reticulum-ATPase reuptake pumps, a Ca2+/Na+ exchanger, intracellular Ca2+ buffers, and surface membrane channels [2+]i is generally accepted to be a crucial event in dystrophic pathogenesis [2+] compared to non-dystrophic WT VSMCs. Failure to maintain a low [Ca2+]i in VSMCs may activate enzymes such as proteases, nucleases, and lipases that impair energy production, compromise muscle function and cause cell death [2+]i has been observed in striated muscle cells from patients with DMD and mdx mice [2+]i in normoxic VSMCs compared to WT is quantitatively similar to those observed in skeletal muscle cells from patients with DMD [2+]i in dystrophic muscle cells, including VSMCs reduced oxidative stress, calpain activity, muscle damage, and improved muscle cell viability and function [2+]i plays a crucial role in the pathogenesis of DMD, and preventing its overload reduces muscle injury and minimizes hypoxia-induced cell damage. In excitable cells i in both genotypes; however, the rise was more significant in mdx than in WT VSMCs. This result is consistent with an increase in sarcolemmal Ca2+ influx in both genotypes; however, it can also be associated with a decrease in Ca2+ uptake by the sarcoplasmic reticulum and/or a decrease in Ca2+ efflux due to inhibition of the Na+/Ca2+ exchanger. Due to these possibilities, we explored the contribution of the sarcolemmal Ca2+ influx to intracellular Ca2+ overload in WT and mdx VSMCs. Incubation in low Ca2+-solution prevented hypoxia-induced elevation of [Ca2+]i in WT (86% inhibition) and mdx VSMC (93% in inhibition). Although the other Ca2+ pathways were not studied, it is fair to conclude that most of the Ca2+ involved in the intracellular overload observed during hypoxia is related to nifedipine-SAR 7334 sensitive sarcolemmal Ca2+ influx.We found that hypoxia was associated with muscle membrane depolarization in WT and mdx; however, the magnitude was much greater in mdx than in WT VSMCs. Previous evidence implies that hypoxia blocks voltage-gated Krization . Hypoxia2+ sarcolemmal influx associated with hypoxia, WT and mdx VSMCs were treated with nifedipine. Nifedipine is an L-type Ca2+ channel blocker, which prevents the entry of Ca2+, resulting in a reduction in oxidative stress, improved muscle function, and an antiproliferative effect in VSMCs [2+]i in normoxic WT and mdx VSMCs, but partially prevented elevation in [Ca2+]i caused by hypoxia (22% in WT versus 32% mdx)  see . The eff hypoxia . Nifedip2+ influx, we studied the effects of SAR7334, a TRPC3 and TRPC6 blocker i and prevented the influx of Ca2+ induced by 1-oleoyl-2-acetyl-sn-glycerol in WT and mdx VSMCs; furthermore, mdx VSMCs showed a significant up-regulation of TRPC-3, -6 proteins compared to WT VSMCs [2+]i in WT and mdx VSMCs [2+]i in both genotypes. To gain insight into hypoxia-induced Caignaling ,36. Muscignaling ,34. Dysrignaling . We have2+]i in both genotypes appears to be independent of the effect on the resting membrane potential, because it did not reverse the depolarization associated with hypoxia in WT and mdx VSMCs . SAR7334 was used at a high concentration (1 µM) to block both TRPC3 and 6 channels since concentrations <1 µM block preferentially TRPC6 channels but not TRPC3 [2+], it appears to account for most of the response, as shown in those VSMCs pretreated with SAR7334. These findings suggest that a Ca2+ influx mediated through TRPC channels plays a critical role in the elevation of [Ca2+]i observed during acute hypoxia in WT and mdx VSMCs, a contribution that appears to be more important in mdx than in WT. Therefore, it is possible that blocking TRPC3 and 6 may have a therapeutic potential to prevent intracellular Ca2+ muscle overload in patients with DMD.The underlying mechanism of SAR7334 on [Ca2+] has been suggested as a vital regulator of cell survival and cell death in response to stress conditions. Chronic increases in intracellular [Ca2+] can induce the production of reactive oxygen species (ROS), activation of proteases as calpain, and the release of factors that promote apoptosis [2+ and ROS signaling are closely integrated [2+ can modify mitochondrial ROS generation and vice versa [2+]i and subsequent increases in intracellular ROS generation may explain this low cell viability. Using the DCFDA fluorescence probe for measurements of oxidant levels, we found that mdx VSMCs had a significantly increased DCF fluorescence signal rate compared to controls (Lopez unpublished results). Hypoxia caused a reduction in cell viability in WT VSMCs and a further reduction in mdx VSMCs compared to untreated cells. Pretreatment with SAR7334 significantly increased cell viability in WT and mdx VSMCs. It is reasonable to suggest that the effects of SAR7334 could be related to a reduction in [Ca2+]i, which prevents the activation of pro-apoptotic signaling pathways, which in turn will reduce cell viability. Intracellular i induced by hypoxia may be a common mediator contributing to cell injury in VSMCs and other tissues, such as cardiac cells and neurons. In mdx cardiac cells, acute hypoxia causes diastolic and systolic dysfunction i in WT and mdx VSMCs. The lack of dystrophin made mdx VSMCs more susceptible to hypoxia-induced Ca2+ overload than WT VSMCs. The [Ca2+]i overload associated with the hypoxic episode appears to be mediated by two independent Ca2+ entry pathways, the TRPC channel (SAR7334 experiments) and the L-type Ca2+ channels (nifedipine experiments). However, TRPC channels appear to be the main path responsible for intracellular Ca2+ elevation. Mechanistically, muscle membrane depolarization does not appear to be the primary mechanism for hypoxia-induced elevation of [Ca2+]i, because nifedipine and SAR7334 ameliorate or prevent elevation of [Ca2+]i without reversing muscle depolarization.These studies demonstrate that hypoxia caused muscle membrane depolarization and elevation of [Ca


Adenosine triphosphate (ATP) and its metabolites drive microglia migration and cytokine production by activating P2X- and P2Y- class purinergic receptors. Purinergic receptor activation gives rise to diverse intracellular calcium (Ca2+ signals, or waveforms, that differ in amplitude, duration, and frequency. Whether and how these characteristics of diverse waveforms influence microglia function is not well-established. We developed a computational model trained with data from published primary murine microglia studies. We simulate how purinoreceptors influence Ca2+ signaling and migration, as well as, how purinoreceptor expression modifies these processes. Our simulation confirmed that P2 receptors encode the amplitude and duration of the ATP-induced Ca2+ waveforms. Our simulations also implicate CD39, an ectonucleotidase that rapidly degrades ATP, as a regulator of purinergic receptor-induced Ca2+ responses. Namely, it was necessary to account for CD39 metabolism of ATP to align the model’s predicted purinoreceptor responses with published experimental data. In addition, our modeling results indicate that small Ca2+ transients accompany migration, while large and sustained transients are needed for cytokine responses. Lastly, as a proof-of-principal, we predict Ca2+ transients and cell membrane displacements in a BV2 microglia cell line using published P2 receptor mRNA data to illustrate how our computer model may be extrapolated to other microglia subtypes. These findings provide important insights into how differences in purinergic receptor expression influence microglial responses to ATP. Microglia are the macrophages of the central nervous system (CNS). They contribute to homeostatic and innate immune responses when subject to a spectrum of molecular stimuli, including those associated with infection and cellular damage . Microgl2+ fluctuations in many cells. These fluctuations are frequently and interchangeably referred to as ‘transients’ or ‘waveforms’ in the literature. In other cell types, the waveform of an induced Ca2+ signal, that is, its duration, amplitude, and frequency, has been shown to selectively control intracellular processes including phosphorylation, gene transcription and mechanical responses  and metabotropic (P2Y) receptors. Ionotropic P2X receptors are non-selective cation channels widely expressed in cells throughout the CNS including microglia. Of these, P2X7 and P2X4 tend to be the most highly-expressed P2X-class receptors in microglia  that can mediate pathways including ER Ca2+ release , and uridine triphosphate (UTP). Among these are P2Y2 (ATP/UTP), P2Y6 (UDP), P2Y12, and P2Y13 (ATP/ADP) (ATP-dependent responses in microglia are mediated by purinergic (P2) receptors. P2 receptors are broadly categorized into two classes: ionotropic  . In prin2+ transients that coincide with directed migration have been observed in microglia  activity. The activation of IP3 receptors by IP3 induces ERCa2+ release. We model this as a deterministic process, for simplicity, although the Ca2+ spiking that results resembles that which is observed experimentally  to activate P2X4 versus P2X7 receptors. For model validation, we converted the ratiometric data to concentrations by normalizing the cytosolic fluorescent intensity of resting microglia to 100 nM. We neglect SOCE given that this mechanism of Ca2+ entry occurs well after Ca2+ currents mediated directly by 5–10 min after ATP stimulation  and Gotran (ver. 2020.2.0.), see , see  for deta0.), see , the Geniff ODEs  was usedeveloped  to iterahttps://github.com/huskeypm/pkh-lab-analyses/tree/master/2021-P2Y-microglia. Generated data are available upon request.All code written and simulation input files in support of this publication are publicly available at 2+ imaging, 96 well plates were used for BV2 cell culture. Cells were plated in a density of 5,000 per well 2 days prior to imaging. On the day of Ca2+ imaging, the cells were incubated at 37°C with 5% CO2 with 1 μg/ml Fluo-4 AM (Thermo Fisher Scientific) in Leibovitz’s L-15 medium for 45 min. Fluo-4 was used to measure the intracellular Ca2+ concentration due to its lower binding affinity relative to indicators like fluo-8 (a.k.a. Fluo-2 medium affinity)  . This alsolution . Excess 2+ measurements were conducted at 0.5 Hz. For each ATP dose, approximately 48 cells were selected to calculate the Ca2+ transients (as indicated by pixel intensity). For each cell, the ATP-induced Ca2+ transient was normalized with respect to the control stage; namely, the average pixel intensity of the control stage was subtracted from the trace of the entire time course.For data in via the aforementioned Ca2+ imaging protocol were processed via custom python routines. The TIF file from the experiment was processed as a 3D matrix with a specific shape defined by  where T denotes the time-index, M × N was the image dimensionality (in the unit of pixels). The pixel intensity  was stored in 16-bit color depth. As the first step, the cell detection protocol identified cells from the image. Specifically, the intensity at each pixel of the image was summed up along the time-index to get a total image with shape (M × N). The total image was then subject to a log transformation and normalization. The histogram of pixel intensities of the normalized image was plotted to help identify a custom thresholding value. Parameters used for acquisition of these signals are embedded in the source code provided with this project. After determining the thresholding value, the normalized total image was converted into a binary image with intensities greater than the thresholding value identified as cell bodies with the rest as background. The detected cells in the binary image satisfying the aforementioned condition were segmented and labeled. Using this information, the original TIF gray-scale image (dark-field image) was utilized to record the change in the pixel intensity at each cell location, the average trace of which later represented the ATP-mediated Ca2+ transient of each cell.The recorded images 2+ transients before and after addition of different concentrations of ATP by taking time lapse images using widefield excitation. To normalize the data, we first calculated the average Ca2+ signal or fluorescence intensity in the control phase, the first 10 min without ATP. We then identified Ca2+ transients by dividing the entire time trace by the average fluorescence intensity. This was used to identify changes in the fluorescence signal.We subsequently analyzed the CaUsing the bright-field data collected for ATP treated BV2 cells, we selected cells that exhibited displacements within the first 20 frames directly after ATP treatment, which corresponds to 600 s in total. To measure displacements, we manually selected reference points within a given BV2 cell process at the initial time point and its approximate position at the final (10th) frame, from which a vector was defined. The resolution of the brightfield data was approximately 1.6 µm/pixel, therefore the length in pixels of the displacement vector was converted into micrometers.P2Y-class) and ionotropic (P2X-class) purinergic receptors to simulate Ca2+ signaling in microglia  to produce Inositol trisphosphate (IP3) and Diacylglycerol (DAG) from PIP2  to match the Ikeda et al data in MG5 microglial cells treated with 1 mM ATP  relative to the experimentally-measured transients from Ikeda et al (dashed blue). After this initial peak, our model predicts an oscillatory Ca2+ waveform that is complemented by decreases in ER Ca2+ owing to IP3 receptor activation  and removal of extracellular Ca2+via excess EGTA all reduce pAkt levels  P2X4 in particular generated prominent Ca2+ transients with micromolar ATP treatments  ttivation . Indeed,pAkt. Without additional refitting, our model predicted 40% and 15% reductions in pAkt levels solely from P2X4 and P2X7 inhibition, respectively. Our result for P2X4 was in close agreement with Ohsawa . These substantial reductions upon nullifying P2X4 contributions therefore implicate this ionotropic receptor in phosphorylating Akt. We additionally verified that P2Y12 −/− all but eliminates Akt phosphorylation. Lastly, we predict that blunted ectonucleotidase hydrolase function enhances pAkt phosphorylation, which suggests that prolonged Ca2+ waveforms further promote Akt activation  for 3 mM ATP after 6 h. Our model predictions indicate that both migration and TNFα increase with increasing Ca2+ waveform frequency and amplitude  was measured at 1 h, even with ATP treatments exceeding 1 mM (3 mM). Importantly, our model demonstrates that 1) both migration and TNFα production were positively correlated with the frequency and amplitude of the Ca2+ waveform and 2) migration could be triggered without significant TNFα responses when the Ca2+ waveforms were of short duration, low amplitude and low frequency.Our data thus far indicate that microglia have robust Cahowed in  that cytdel from . For refATP. Per , secretemplitude . Althoug2+ waveforms and Ca2+-dependent functions. This cell line was chosen, given that these cells are easier to culture and characterize compared to primary cells. Although mRNA expression levels do not necessarily directly correlate with protein expression  for the BV cells, while the cell-to-cell variance is represented by gray shaded regions. We found that the initial phase . Given that sustained increases in basal intracellular Ca2+ levels are associated with pathological states  and cytokine production. The ectonucleotidases CD39 and CD73 are the most highly expressed nucleotidases in microglia  P2Y12 silenced microglia migrate and 2) extracellular Ca2+ significantly enhances migration by promoting Akt phosphorylation  and war2+ signaling in promoting migration as well as TNFα synthesis. This raises the question as to whether ATP can stimulate microglia displacement associated with homeostatic functions, such as supporting neuron development and transmission  migration. We investigated this hypothesis by adapting P2 expression levels in the model based on published BV2 cell mRNA data. Model predictions of ATP-stimulated Ca2+ waveforms and migration were compared against experiments with BV2 cells.Our model suggests that the expression levels of P2 receptors enable microglia to regulate migration and pro-inflammatory responses to ATP . This ocaveforms , we hypoP2Y2 transcripts, but BV2 cells had fewer P2X4 mRNA transcripts. For simplicity, we assumed that the purinergic receptor activity in our model correlated with mRNA expression. Based on our assumptions the model predicted Ca2+ waveforms in BV2 cells that were qualitatively similar to those simulated for primary cells. In contrast, P2Y12 mRNA transcripts were reduced 30-fold in BV2 cells relative to primary cells . We did not investigate dependencies of migration or TNFα production on cAMP production in principle P2Y11 activation promotes Gs signaling, which would result in cAMP production  target  is also an established pathway contributing to cell migration and cytokine production  target . Similarent MLCK . P2Y2 -moduction . Altoget2+ waveforms in microglia have diverse properties, such as amplitude, duration and oscillatory behavior. These properties depend on which P2 receptor types are activated, in addition to ectonucleotidase activity. In this study, we developed a computational model to predict how P2 receptors and ectonucleotidase hydrolases control Ca2+ waveforms in microglia that in turn influence microglia migration and cytokine production. With this model, we examine the propensity for these diverse Ca2+ waveforms to drive these canonical microglial responses to ATP.ATP-induced CaP2X contributions to TNFα mRNA synthesis as a model for pro-inflammatory cytokine responses to ATP (2+ waveforms generated by P2X activation are typically high-amplitude and of finite duration. With the addition of P2Y, we predict a wider diversity of Ca2+ waveforms that can include stable and damped oscillations of low amplitude and frequency. Interestingly, our modeling results highlight a complementary role of ectonucleotidase activity, namely CD39, in controlling the ATP pool available to trigger such responses, by hydrolyzing ATP and ADP into AMP. This finding is particularly important since CD39 and its complementary CD73 ectonucleotidase, which hydrolyzes AMP, undergo significant expression changes when microglia are activated (We interpret these results in light of our previously published microglia model for probing s to ATP . In thatctivated .2+ waveforms shaped by P2 receptors and CD39 selectivity control downfield signaling pathways. We investigated this selective control by simulating migration versus cytokine production responses as a function of the Ca2+ waveforms generated by P2 receptors. Our model indicates that short-duration, oscillatory Ca2+ transients induced by P2Y receptors and P2X4 with micromolar ATP are sufficient to promote migration responses without significantly inducing TNFα production. On the other hand, millimolar ATP concentrations that activate P2X7 supported sustained cytosolic Ca2+ levels that could trigger TNFα release. In a similar vein, we can expect responses would vary with the types or combinations of nucleotides used to stimulate purinoreceptors. We demonstrate this as a proof-of-principle for UTP in P2Y2 receptors (P2Y6 receptors (2+ transients with spikes from IP3R activation, but lacked the high-amplitude fluctuations associated with P2X receptors; we anticipate that such transients would not be expected to induce significant TNFα production. Overall, these findings illustrate how microglia orchestrate complex cytokine and migration functions in response to ATP as well as other damage associated with molecular patterns like UTP (Our simulations indicate that the distinct Caeceptors  and P2Y6eceptors , while ieceptors . This relike UTP .P2X, P2Y, and ectonucleotidase proteins in mediating cellular responses to ATP further suggest how changes in gene expression shape microglia responses to stimuli. For instance, higher P2Y12 expression in resting relative to pro-inflammatory microglia likely favor migration responses to ATP in the former. Similarly, elevated P2X4 and P2X7 expression in pro-inflammatory microglia sensitize cytokine responses to ATP. These differences in microglia responses following changes in gene expression underscore the need for models to account for changes in protein activity.The concerted roles of in situ. For this reason, we tested if our model could leverage mRNA transcript data from the BV2 microglial cell line to approximate changes in P2 receptor activity. Using those mRNA data, we found that the model predicted Ca2+ waveforms and migration responses to ATP that were reasonably consistent with experiments we conducted using the BV2 microglia cell line. This raises the possibility that coupling models trained from cultured primary or immortalized cells in vitro with transcriptomic and proteomic data could enable predictions of cellular responses from in vivo or disease-associated microglia, for which extensive functional testing is not feasible. Related to this, since dysfunctional microglial responses are associated with neurological disorders including chronic pain, Alzheimer’s Disease and Parkinson’s Diseases (2+ could be helpful, such as cytoskeletal rearrangement (Robust characterization of detailed signaling networks in diverse microglia phenotypes remains a significant challenge. This is especially challenging for tissue resident microglia that are difficult to experimentally manipulate Diseases , our comangement , phagocyangement , and autangement .


However, GLRs in Brassica species have not yet been reported. In this study, 16 glutamate receptor-like channels (GLR) belonged to two groups were identified in the Brassica rapa (B. rapa) genome by bioinformatic analysis. Most members contain domains of ANF_receptor, Peripla_BP_6, Lig_chan, SBP_bac_3, and Lig_chan_Glu_bd that are closely related to glutamate receptor channels. This gene family contains many elements associated with drought stress, low temperature stress, methyl jasmonate (MeJA), salicylic acid (SA), and other stress resistance. Gene expression profiles showed that BraGLR genes were expressed in roots, stems, leaves, flowers, and siliques. BraGLR5 expression was elevated after drought stress in drought-sensitive plants. BraGLR1, BraGLR8, and BraGLR11 expression were significantly upregulated after salt stress. BraGLR3 expression is higher in the female sterile-line mutants than in the wild type. The expression levels of BraGLR6, BraGLR9, BraGLR12, and BraGLR13 were significantly higher in the male sterile-line mutants than in the wild type. The expression of most BraGLRs increased after self-pollination, with BraGLR9 exhibiting the greatest increase. These results suggest that BraGLRs play an important role in abiotic stress tolerance and sexual reproduction.Glutamate receptors (GLRs) are involved in multiple functions during the plant life cycle through affecting the Ca GLR1 and GLR2 from Arabidopsis thaliana . Subsequently, 20 members of the glutamate receptor-like gene family were identified in A. thaliana cyt can be decoded by calmodulins and further enhance plant heat tolerance cyt) gradient, thereby influencing Ca2+ inward flow-dependent pollen tube growth and function [2+]cyt in stigma papilla cells increases sharply after self-pollination in self-incompatible A. thaliana and can be effectively blocked by inhibitors of GLR. Additionally, in glutamate receptor-like mutants, the increase in Ca2+ caused by self-incompatibility is reduced, and GLRs can mediate Ca2+ influx, suggesting that GLR-mediated Ca2+ influx in stigma papilla cells is a key factor leading to plant rejection of its own pollen. Ca2+ was also reported to be involved in the self-incompatibility and self-compatibility response of Brassica napus cyt of papilla cells before CP decreases self-incompatibility; there is an increased self-incompatibility response in an AtGLR mutant with reduced Ca2+. Thus, GLRs can mediate Ca2+ inward flow, increase [Ca2+]cyt, and reject self-pollen cyt can be increased due to the direct inward flow of extracellular Ca2+ into the cytoplasm via the AtGLR3.3 channel [BraGLRs help mediate the Ca2+-related self-incompatibility signaling pathway, which could provide directions for exploring plant self-incompatibility signaling.Based on the transcriptome data analysis and qRT-PCR results, f-pollen . Given t channel . This anBraGLRs in the B. rapa genome. Based on the comprehensive analysis of sequence features, cis elements, expression profiles of different tissues, abiotic stress tolerance and sexual reproductive processes, and the published data, we further identified BraGLR1 and BraGLR9 as playing significant roles in regulating salt stress tolerance and sexual reproduction, respectively.In summary, we identified 16