Interface-Mediated Neurogenic Signaling: The Impact of Surface Geometry and Chemistry on Neural Cell Behavior for Regenerative and Brain-Machine Interfacing Applications.

Nanomaterial advancements have driven progress in central and peripheral nervous system applications such as tissue regeneration and brain-machine interfacing. Ideally, neural interfaces with native tissue shall seamlessly integrate, a process that is often mediated by the interfacial material properties. Surface topography and material chemistry are significant extracellular stimuli that can influence neural cell behavior to facilitate tissue integration and augment therapeutic outcomes. This review characterizes topographical modifications, including micropillars, microchannels, surface roughness, and porosity, implemented on regenerative scaffolding and brain-machine interfaces. Their impact on neural cell response is summarized through neurogenic outcome and mechanistic analysis. The effects of surface chemistry on neural cell signaling with common interfacing compounds like carbon-based nanomaterials, conductive polymers, and biologically inspired matrices are also reviewed. Finally, the impact of these extracellular mediated neural cues on intracellular signaling cascades is discussed to provide perspective on the manipulation of neuron and neuroglia cell microenvironments to drive therapeutic outcomes.

E41K Mutation Activates Bruton's Tyrosine Kinase by Stabilizing an Inositol Hexakisphosphate dependent Invisible Dimer.

Bruton's tyrosine kinase (BTK) regulates diverse cellular signaling of the innate and adaptive immune system in response to microbial pathogens. Downregulation or constitutive activation of BTK is reported in patients with autoimmune diseases or various B-cell leukemias. BTK is a multidomain protein tyrosine kinase that adopts an Src-like autoinhibited conformation maintained by the interaction between the kinase and PH-TH domains. The PH-TH domain plays a central role in regulating BTK function. BTK is activated by binding to PIP3 at the plasma membrane upon stimulation by the B-cell receptor (BCR). The PIP3 binding allows dimerization of the PH-TH domain and subsequent transphosphorylation of the activation loop. Alternatively, a recent study shows that the multivalent T-cell-independent (TI) antigen induces BCR response by activating BTK independently of PIP3 binding. It was proposed that a transiently stable IP6-dependent PH-TH dimer may activate BTK during BCR activation by the TI antigens. However, no IP6-dependent PH-TH dimer has been identified yet. Here, we investigated a constitutively active PH-TH mutant (E41K) to determine if the elusive IP6-dependent PH-TH dimer exists. We showed that the constitutively active E41K mutation activates BTK by stabilizing the IP6-dependent PH-TH dimer. We observed that a downregulating mutation in the PH-TH domain (R28H) linked to X-linked agammaglobulinemia impairs BTK activation at the membrane and in the cytosol by preventing PH-TH dimerization. We conclude that the IP6 dynamically remodels the BTK active fraction between the membrane and cytoplasm. Stimulating with IP6 increases the cytosolic fraction of the activated BTK.

Unveiling the Promising Role of Substance P/Neurokinin 1 Receptor in Cancer Cell Proliferation and Cell Cycle Regulation in Human Malignancies.

Neurokinin receptors are a family of G protein-coupled receptors that were first identified in the central and peripheral nervous systems. However these receptors were later found in other types of cells, therefore, new perspectives concerning their novel roles were described. Mammalian has three neurokinin receptors, among which neurokinin-1 receptors [NK1R] have been indicated to be involved in most, if not all, intracellular functions, primarily the regulation of cell proliferation. By interacting with its potent agonist, substance P [SP], NK1R can engage a variety of signaling pathways and serve as a platform for cells to proliferate by regulating the expression of the cell cycle-related genes. Furthermore, the activity of SP/NK1R is stimulated by various oncogenes, indicating the involvement of this pathway in human cancers. As a result, numerous NK1R antagonists have been investigated in oncology trials, and the promising anti-- cancer effect of these receptors has opened up new possibilities for incorporating these antagonists into cancer treatment. Considering these factors, gaining a deeper understanding of the SP/NK1R pathway could offer significant advantages for cancer patients. The more knowledge we acquire about this pathway, the greater the potential for exploiting it in the development of effective treatment strategies. Here, we present a comprehensive review of the current knowledge pertaining to the biological function of the SP/NK1R, with a specific emphasis on its recently discovered role in the regulation of cell proliferation. Moreover, we provide insights into the impact of this pathway in human cancers, along with an overview of the most significant NK1R antagonists currently utilized in cancer research studies.

Diverse Fgfr1 signaling pathways and endocytic trafficking regulate mesoderm development.

The fibroblast growth factor (FGF) pathway is a conserved signaling pathway required for embryonic development. Activated FGF receptor 1 (FGFR1) drives multiple intracellular signaling cascade pathways, including ERK/MAPK and PI3K/AKT, collectively termed canonical signaling. However, unlike Fgfr1-null embryos, embryos containing hypomorphic mutations in Fgfr1 lacking the ability to activate canonical downstream signals are still able to develop to birth but exhibit severe defects in all mesodermal-derived tissues. The introduction of an additional signaling mutation further reduces the activity of Fgfr1, leading to earlier lethality, reduced somitogenesis, and more severe changes in transcriptional outputs. Genes involved in migration, ECM interaction, and phosphoinositol signaling were significantly downregulated, proteomic analysis identified changes in interactions with endocytic pathway components, and cells expressing mutant receptors show changes in endocytic trafficking. Together, we identified processes regulating early mesoderm development by mechanisms involving both canonical and noncanonical Fgfr1 pathways, including direct interaction with cell adhesion components and endocytic regulation.

Chemical Proteomic Profiling of Protein Dopaminylation in Colorectal Cancer Cells.

Histone dopaminylation is a newly identified epigenetic mark that plays a role in the regulation of gene transcription, where an isopeptide bond is formed between the fifth amino acid of H3 (i.e., glutamine) and dopamine. Recently, we developed a chemical probe to specifically label and enrich histone dopaminylation via bioorthogonal chemistry. Given this powerful tool, we found that histone H3 glutamine 5 dopaminylation (H3Q5dop) was highly enriched in colorectal tumors, which could be attributed to the high expression level of its regulator, transglutaminase 2 (TGM2), in colon cancer cells. Due to the enzyme promiscuity of TGM2, nonhistone proteins have also been identified as dopaminylation targets; however, the dopaminylated proteome in cancer cells still remains elusive. Here, we utilized our chemical probe to enrich dopaminylated proteins from colorectal cancer cells in a bioorthogonal manner and performed the chemical proteomics analysis. Therefore, 425 dopaminylated proteins were identified, many of which are involved in nucleic acid metabolism and transcription pathways. More importantly, a number of dopaminylation sites were identified and attributed to the successful application of our chemical probe. Overall, these findings shed light on the significant association between cellular protein dopaminylation and cancer development, further suggesting that targeting these pathways may become a promising anticancer strategy.

Microfluidics single-cell encapsulation reveals that poly-l-lysine-mediated stem cell adhesion to alginate microgels is crucial for cell-cell crosstalk and its self-renewal.

Microfluidic cell encapsulation has provided a platform for studying the behavior of individual cells and has become a turning point in single-cell analysis during the last decade. The engineered microenvironment, along with protecting the immune response, has led to increasingly presenting the results of practical and pre-clinical studies with the goals of disease treatment, tissue engineering, intelligent control of stem cell differentiation, and regenerative medicine. However, the significance of cell-substrate interaction versus cell-cell communications in the microgel is still unclear. In this study, monodisperse alginate microgels were generated using a flow-focusing microfluidic device to determine how the cell microenvironment can control human bone marrow-derived mesenchymal stem cells (hBMSCs) viability, proliferation, and biomechanical features in single-cell droplets versus multi-cell droplets. Collected results show insufficient cell proliferation (234 % and 329 %) in both single- and multi-cell alginate microgels. Alginate hydrogels supplemented with poly-l-lysine (PLL) showed a better proliferation rate (514 % and 780 %) in a comparison of free alginate hydrogels. Cell stiffness data illustrate that hBMSCs cultured in alginate hydrogels have higher membrane flexibility and migration potency (Young's modulus equal to 1.06 kPa), whereas PLL introduces more binding sites for cell attachment and causes lower flexibility and migration potency (Young's modulus equal to 1.83 kPa). Considering that cell adhesion is the most important parameter in tissue engineering, in which cells do not run away from a 3D substrate, PLL enhances cell stiffness and guarantees cell attachments. In conclusion, cell attachment to PLL-mediated alginate hydrogels is crucial for cell viability and proliferation. It suggests that cell-cell signaling is good enough for stem cell viability, but cell-PLL attachment alongside cell-cell signaling is crucial for stem cell proliferation and self-renewal.

Optical Control of Cell-Surface and Endomembrane-Exclusive ß-Adrenergic Receptor Signaling.

Beta-adrenergic receptors (ßARs) are G protein-coupled receptors (GPCRs) that mediate catecholamine hormone-induced stress responses, such as elevation of heart rate. Besides those that are plasma membrane-bound, endomembrane ßARs are also signaling competent. Dysregulation of ßAR pathways underlies severe pathological conditions. Emerging evidence indicates pathological molecular signatures in deeper endomembrane ßARs signaling, likely contributing to conditions such as cardiomyocyte hypertrophy and apoptosis. However, the lack of approaches to control endomembrane ß1ARs has impeded linking signaling with pathology. Informed by the ß1AR-catecholamine interactions, we engineered an efficient photo-labile pro-ligand (OptoIso) to trigger ßAR signaling exclusively in endomembrane regions using blue light stimulation. Not only does OptoIso undergo blue light deprotection in seconds, but also efficiently enters cells and allows examination of G protein heterotrimer activation exclusively at endomembranes. OptoIso also allows optical activation of plasma membrane ßAR signaling in selected single cells with native fidelity, which can be reversed by terminating blue light. Thus, OptoIso will be a valuable experimental tool to elicit spatial and temporal control of ßAR signaling in user-defined endomembrane or plasma membrane regions in unmodified cells with native fidelity.

TGF-ß signaling: critical nexus of fibrogenesis and cancer.

The transforming growth factor-beta (TGF-ß) signaling pathway is a vital regulator of cell proliferation, differentiation, apoptosis, and extracellular matrix production. It functions through canonical SMAD-mediated processes and noncanonical pathways involving MAPK cascades, PI3K/AKT, Rho-like GTPases, and NF-κB signaling. This intricate signaling system is finely tuned by interactions between canonical and noncanonical pathways and plays key roles in both physiologic and pathologic conditions including tissue homeostasis, fibrosis, and cancer progression. TGF-ß signaling is known to have paradoxical actions. Under normal physiologic conditions, TGF-ß signaling promotes cell quiescence and apoptosis, acting as a tumor suppressor. In contrast, in pathological states such as inflammation and cancer, it triggers processes that facilitate cancer progression and tissue remodeling, thus promoting tumor development and fibrosis. Here, we detail the role that TGF-ß plays in cancer and fibrosis and highlight the potential for future theranostics targeting this pathway.

Development of an miRFP680-Based Fluorescent Calcium Ion Biosensor Using End-Optimized Transposons.

The development of new or improved single fluorescent protein (FP)-based biosensors (SFPBs), particularly those with excitation and emission at near-infrared wavelengths, is important for the continued advancement of biological imaging applications. In an effort to accelerate the development of new SFPBs, we report modified transposons for the transposase-based creation of libraries of FPs randomly inserted into analyte binding domains, or vice versa. These modified transposons feature ends that are optimized to minimize the length of the linkers that connect the FP to the analyte binding domain. We rationalized that shorter linkers between the domains should result in more effective allosteric coupling between the analyte binding-dependent conformational change in the binding domain and the fluorescence modulation of the chromophore of the FP domain. As a proof of concept, we employed end-modified Mu transposons for the discovery of SFPB prototypes based on the insertion of two circularly permuted red FPs (mApple and FusionRed) into binding proteins for l-lactate and spermidine. Using an analogous approach, we discovered calcium ion (Ca2+)-specific SFPBs by random insertion of calmodulin (CaM)-RS20 into miRFP680, a particularly bright near-infrared (NIR) FP based on a biliverdin (BV)-binding fluorescent protein. Starting from an miRFP680-based Ca2+ biosensor prototype, we performed extensive directed evolution, including under BV-deficient conditions, to create highly optimized biosensors designated the NIR-GECO3 series. We have extensively characterized the NIR-GECO3 series and explored their utility for biological Ca2+ imaging. The methods described in this work will serve to accelerate SFPB development and open avenues for further exploration and optimization of SFPBs across a spectrum of biological applications.

A Simple and Rapid Microscale Method for Isolating Bacterial Lipopolysaccharides.

Bacterial endotoxins (lipopolysaccharides (LPSs)) are important mediators of inflammatory processes induced by Gram-negative microorganisms. LPSs are the key inducers of septic shock due to a Gram-negative bacterial infection; thus, the structure and functions of LPSs are of specific interest. Often, highly purified bacterial endotoxins must be isolated from small amounts of biological material. Each of the currently available methods for LPS extraction has certain limitations. Herein, we describe a rapid and simple microscale method for extracting LPSs. The method consists of the following steps: ultrasonic destruction of the bacterial material, LPS extraction via heating, LPS purification with organic solvents, and treatment with proteinase K. LPSs that were extracted by using this method contained less than 2-3% protein and 1% total nucleic acid. We also demonstrated the structural integrity of the O-antigen and lipid A via the sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) methods, respectively. We demonstrated the ability of the extracted LPSs to induce typical secretion of cytokines and chemokines by primary macrophages. Overall, this method may be used to isolate purified LPSs with preserved structures of both the O-antigen and lipid A and unchanged functional activity from small amounts of bacterial biomass.

Membrane Receptor-Mediated Disruption of Cellular Homeostasis: Changes in Intracellular Signaling Pathways Increase the Toxicity of Ochratoxin A.

Organisms maintain their cellular homeostatic balance by interacting with their environment through the use of their cell surface receptors. Membrane based receptors such as the transforming growth factor ß receptor (TGFR), the prolactin receptor (PRLR), and hepatocyte growth factor receptor (HGFR), along with their associated signaling cascade, play significant roles in retaining cellular homeostasis. While these receptors and related signaling pathways are essential for health of cell and organism, their dysregulation can lead to imbalance in cell function with severe pathological conditions such as cell death or cancer. Ochratoxin A (OTA) can disrupt cellular homeostasis by altering expression levels of these receptors and/or receptor-associated intracellular downstream signaling modulators and/or pattern and levels of their phosphorylation/dephosphorylation. Recent studies have shown that the activity of the TGFR, the PRLR, and HGFR and their associated signaling cascades change upon OTA exposure. A critical evaluation of these findings suggests that while increased activity of the HGFR and TGFR signaling pathways leads to an increase in cell survival and fibrosis, decreased activity of the PRLR signaling pathway leads to tissue damage. This review explores the roles of these receptors in OTA-related pathologies and effects on cellular homeostasis.

Mechanical force-activated CD109 on periodontal ligament stem cells governs osteogenesis and osteoclast to promote alveolar bone remodeling.

Mechanical force-mediated bone remodeling is crucial for various physiological and pathological processes involving multiple factors, including stem cells and the immune response. However, it remains unclear how stem cells respond to mechanical stimuli to modulate the immune microenvironment and subsequent bone remodeling. Here, we found that mechanical force induced increased expression of CD109 on periodontal ligament stem cells (PDLSCs) in vitro and in periodontal tissues from the force-induced tooth movement rat model in vivo, accompanied by activated alveolar bone remodeling. Under mechanical force stimulation, CD109 suppressed the osteogenesis capacity of PDLSCs through the JAK/STAT3 signaling pathway, whereas it promoted PDLSC-induced osteoclast formation and M1 macrophage polarization through paracrine. Moreover, inhibition of CD109 in vivo by lentivirus-shRNA injection increased the osteogenic activity and bone density in periodontal tissues. On the contrary, it led to decreased osteoclast numbers and pro-inflammatory factor secretion in periodontal tissues and reduced tooth movement. Mechanistically, mechanical force-enhanced CD109 expression via the repression of miR-340-5p. Our findings uncover a CD109-mediated mechanical force response machinery on PDLSCs, which contributes to regulating the immune microenvironment and alveolar bone remodeling during tooth movement.

EGFRvIII Confers Sensitivity to Saracatinib in a STAT5-Dependent Manner in Glioblastoma.

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults, with few effective treatments. EGFR alterations, including expression of the truncated variant EGFRvIII, are among the most frequent genomic changes in these tumors. EGFRvIII is known to preferentially signal through STAT5 for oncogenic activation in GBM, yet targeting EGFRvIII has yielded limited clinical success to date. In this study, we employed patient-derived xenograft (PDX) models expressing EGFRvIII to determine the key points of therapeutic vulnerability within the EGFRvIII-STAT5 signaling axis in GBM. Our findings reveal that exogenous expression of paralogs STAT5A and STAT5B augments cell proliferation and that inhibition of STAT5 phosphorylation in vivo improves overall survival in combination with temozolomide (TMZ). STAT5 phosphorylation is independent of JAK1 and JAK2 signaling, instead requiring Src family kinase (SFK) activity. Saracatinib, an SFK inhibitor, attenuates phosphorylation of STAT5 and preferentially sensitizes EGFRvIII+ GBM cells to undergo apoptotic cell death relative to wild-type EGFR. Constitutively active STAT5A or STAT5B mitigates saracatinib sensitivity in EGFRvIII+ cells. In vivo, saracatinib treatment decreased survival in mice bearing EGFR WT tumors compared to the control, yet in EGFRvIII+ tumors, treatment with saracatinib in combination with TMZ preferentially improves survival.

Interplay between structure and signaling.

Modification of pectin, a component of the plant cell wall, is required to facilitate signaling by a RALF peptide, which is essential for many physiological and developmental processes.

Protective effects of Panax ginseng as a medical food against chemical toxic agents: molecular and cellular mechanisms.

Humans are exposed to different types of toxic agents, which may directly induce organ malfunction or indirectly alter gene expression, leading to carcinogenic and teratogenic effects, and eventually death. Ginseng (Panax ginseng) is the most valuable of all medicinal herbs. Nevertheless, specific data on the antidotal mechanisms of this golden herb are currently unavailable. Based on the findings of in vitro, in vivo, and clinical studies, this review focused on the probable protective mechanisms of ginseng and its major components, such as protopanaxadiols, protopanaxatriols, and pentacyclic ginsenosides against various chemical toxic agents. Relevant articles from 2000 to 2023 were gathered from PubMed/Medline, Scopus, and Google Scholar. This literature review shows that P. ginseng and its main components have protective and antidotal effects against the deteriorative effects of pesticides, pharmaceutical agents, including acetaminophen, doxorubicin, isoproterenol, cyclosporine A, tacrolimus, and gentamicin, ethanol, and some chemical agents. These improvements occur through multi-functional mechanisms. They exhibit antioxidant activity, induce anti-inflammatory action, and block intrinsic and extrinsic apoptotic pathways. However, relevant clinical trials are necessary to validate the mentioned effects and translate the knowledge from basic science to human benefit, fulfilling the fundamental goal of all toxicologists.

Progesterone resistance in endometriosis: A pathophysiological perspective and potential treatment alternatives.

Background: Endometriosis is a common gynecological disease affecting women of reproductive age. Patients with endometriosis frequently experience severe chronic pain and have higher chances to experience infertility. Progesterone resistance is a major problem that develops during the medical treatment of endometriosis, which often leads to treatment failure of hormonal therapies. Previous studies indicated that the dysregulation of progesterone receptors (PR) is the primary factor leading to progesterone resistance in endometriosis. Methods: This review article systematically reviewed and summarized findings extracted from previously published papers available on PubMed, encompassing both experimental studies and clinical trials. Main findings: Various determinants influencing PR expression in endometriosis have been identified, including the environmental toxins, microRNAs, cell signaling pathways, genetic mutations, and the pro-inflammatory cytokines. The selective estrogen/progesterone receptor modulators have emerged as novel therapeutic approaches for treating endometriosis, offering potential improvements in overcoming progesterone resistance. Conclusion: Concerns and limitations persist despite the newly developed drugs. Therefore, studies on unraveling new therapeutic targets based on the molecular mechanisms of progesterone resistance is warranted for the development potential alternatives to overcome hormonal treatment failure in endometriosis.

Modeling the reactive oxygen species (ROS) wave in Chlamydomonas reinhardtii colonies.

Reactive oxygen species (ROS) play a crucial role as signaling molecules in both plant and animal cells, enabling rapid responses to various stimuli. Among the many cellular mechanisms used to generate and transduce ROS signals, ROS-induced-ROS release (RIRR) is emerging as an important pathway involved in the responses of various multicellular and unicellular organisms to environmental stresses. In RIRR, one cellular compartment, organelle, or cell generates or releases ROS, triggering an increased ROS production and release by another compartment, organelle, or cell, thereby giving rise to a fast propagating ROS wave. This RIRR-based signal relay has been demonstrated to facilitate mitochondria-to-mitochondria communication in animal cells and long-distance systemic signaling in plants in response to biotic and abiotic stresses. More recently, it has been discovered that different unicellular microorganism communities also exhibit a RIRR cell-to-cell signaling process triggered by a localized stress treatment. However, the precise mechanism underlying the propagation of the ROS signal among cells within these unicellular communities remained elusive. In this study, we employed a reaction-diffusion model incorporating the RIRR mechanism to analyze the propagation of ROS-mediated signals. By effectively balancing production and scavenging processes, our model successfully reproduces the experimental ROS signal velocities observed in unicellular green algae (Chlamydomonas reinhardtii) colonies grown on agar plates, furthering our understanding of intercellular ROS communication.

The Role of Pericyte Migration and Osteogenesis in Periodontitis.

A ligature-induced periodontitis model was established in wild-type and CD146CreERT2; RosatdTomato mice to explore the function of pericytes in alveolar bone formation. We found that during periodontitis progression and periodontal wound healing, CD146+/NG2+ pericytes were enriched in the periodontal tissue areas, which could migrate to the alveolar bone surface and colocalize with ALP+/OCN+ osteoblasts. Chemokine C-X-C motif receptor 4 (CXCR4) inhibition using AMD3100 blocked CD146-Cre+ pericyte migration and osteogenesis, as well as further exacerbated periodontitis-associated bone loss. Next, primary pericytes were sorted out by magnetic-activated cell sorting and demonstrated that C-X-C motif chemokine ligand 12 (CXCL12) promotes pericyte migration and osteogenesis via CXCL12-CXCR4-Rac1 signaling. Finally, the local administration of an adeno-associated virus for Rac1 overexpression in NG2+ pericytes promotes osteoblast differentiation of pericytes and increases alveolar bone volume in periodontitis. Thus, our results provided the evidence that pericytes may migrate and osteogenesis via the CXCL12-CXCR4-Rac1 axis during the pathological process of periodontitis.

Single Cell Profiling Distinguishes Leukemia-Selective Chemotypes.

A central challenge in chemical biology is to distinguish molecular families in which small structural changes trigger large changes in cell biology. Such families might be ideal scaffolds for developing cell-selective chemical effectors - for example, molecules that activate DNA damage responses in malignant cells while sparing healthy cells. Across closely related structural variants, subtle structural changes have the potential to result in contrasting bioactivity patterns across different cell types. Here, we tested a 600-compound Diversity Set of screening molecules from the Boston University Center for Molecular Discovery (BU-CMD) in a novel phospho-flow assay that tracked fundamental cell biological processes, including DNA damage response, apoptosis, M-phase cell cycle, and protein synthesis in MV411 leukemia cells. Among the chemotypes screened, synthetic congeners of the rocaglate family were especially bioactive. In follow-up studies, 37 rocaglates were selected and deeply characterized using 12 million additional cellular measurements across MV411 leukemia cells and healthy peripheral blood mononuclear cells. Of the selected rocaglates, 92% displayed significant bioactivity in human cells, and 65% selectively induced DNA damage responses in leukemia and not healthy human blood cells. Furthermore, the signaling and cell-type selectivity were connected to structural features of rocaglate subfamilies. In particular, three rocaglates from the rocaglate pyrimidinone (RP) structural subclass were the only molecules that activated exceptional DNA damage responses in leukemia cells without activating a detectable DNA damage response in healthy cells. These results indicate that the RP subset should be extensively characterized for anticancer therapeutic potential as it relates to the DNA damage response. This single cell profiling approach advances a chemical biology platform to dissect how systematic variations in chemical structure can profoundly and differentially impact basic functions of healthy and diseased cells.

A C-terminal motif containing a PKC phosphorylation site regulates γ-Protocadherin-mediated dendrite arborization in the cerebral cortex in vivo.

The Pcdhg gene cluster encodes 22 γ-Protocadherin (γ-Pcdh) cell adhesion molecules that critically regulate multiple aspects of neural development, including neuronal survival, dendritic and axonal arborization, and synapse formation and maturation. Each γ-Pcdh isoform has unique protein domains-a homophilically interacting extracellular domain and a juxtamembrane cytoplasmic domain-as well as a C-terminal cytoplasmic domain shared by all isoforms. The extent to which isoform-specific versus shared domains regulate distinct γ-Pcdh functions remains incompletely understood. Our previous in vitro studies identified protein kinase C (PKC) phosphorylation of a serine residue within a shared C-terminal motif as a mechanism through which γ-Pcdh promotion of dendrite arborization via myristoylated alanine-rich C-kinase substrate (MARCKS) is abrogated. Here, we used CRISPR/Cas9 genome editing to generate two new mouse lines expressing only non-phosphorylatable γ-Pcdhs, due either to a serine-to-alanine mutation (PcdhgS/A) or to a 15-amino acid C-terminal deletion resulting from insertion of an early stop codon (PcdhgCTD). Both lines are viable and fertile, and the density and maturation of dendritic spines remain unchanged in both PcdhgS/A and PcdhgCTD cortex. Dendrite arborization of cortical pyramidal neurons, however, is significantly increased in both lines, as are levels of active MARCKS. Intriguingly, despite having significantly reduced levels of γ-Pcdh proteins, the PcdhgCTD mutation yields the strongest phenotype, with even heterozygous mutants exhibiting increased arborization. The present study confirms that phosphorylation of a shared C-terminal motif is a key γ-Pcdh negative regulation point and contributes to a converging understanding of γ-Pcdh family function in which distinct roles are played by both individual isoforms and discrete protein domains.