A novel mouse model of diffuse midline glioma initiated in neonatal oligodendrocyte progenitor cells highlights cell-of-origin dependent effects of H3K27M.

Diffuse midline glioma (DMG) is a type of lethal brain tumor that develops mainly in children. The majority of DMG harbor the K27M mutation in histone H3. Oligodendrocyte progenitor cells (OPCs) in the brainstem are candidate cells-of-origin for DMG, yet there is no genetically engineered mouse model of DMG initiated in OPCs. Here, we used the RCAS/Tv-a avian retroviral system to generate DMG in Olig2-expressing progenitors and Nestin-expressing progenitors in the neonatal mouse brainstem. PDGF-A or PDGF-B overexpression, along with p53 deletion, resulted in gliomas in both models. Exogenous overexpression of H3.3K27M had a significant effect on tumor latency and tumor cell proliferation when compared with H3.3WT in Nestin+ cells but not in Olig2+ cells. Further, the fraction of H3.3K27M-positive cells was significantly lower in DMGs initiated in Olig2+ cells relative to Nestin+ cells, both in PDGF-A and PDGF-B-driven models, suggesting that the requirement for H3.3K27M is reduced when tumorigenesis is initiated in Olig2+ cells. RNA-sequencing analysis revealed that the differentially expressed genes in H3.3K27M tumors were non-overlapping between Olig2;PDGF-B, Olig2;PDGF-A, and Nestin;PDGF-A models. GSEA analysis of PDGFA tumors confirmed that the transcriptomal effects of H3.3K27M are cell-of-origin dependent with H3.3K27M promoting epithelial-to-mesenchymal transition (EMT) and angiogenesis when Olig2 marks the cell-of-origin and inhibiting EMT and angiogenesis when Nestin marks the cell-of-origin. We did observe some overlap with H3.3K27M promoting negative enrichment of TNFA_Signaling_Via_NFKB in both models. Our study suggests that the tumorigenic effects of H3.3K27M are cell-of-origin dependent, with H3.3K27M being more oncogenic in Nestin+ cells than Olig2+ cells.

Clathrin-mediated endocytosis cooperates with bulk endocytosis to generate vesicles.

Clathrin-mediated endocytosis, the most prominent endocytic mode, is thought to be generated primarily from relatively flat patches of the plasma membrane. By employing conventional and platinum replica electron microscopy and super-resolution STED microscopy in neuroendocrine chromaffin cells, we found that large Ω-shaped or dome-shaped plasma membrane invaginations, previously thought of as the precursor of bulk endocytosis, are primary sites for clathrin-coated pit generation after depolarization. Clathrin-coated pits are more densely packed at invaginations rather than flat membranes, suggesting that invaginations are preferred sites for clathrin-coated pit formation, likely because their positive curvature facilitates coated-pit formation. Thus, clathrin-mediated endocytosis closely collaborates with bulk endocytosis to enhance endocytic capacity in active secretory cells. This direct collaboration between two classically independent endocytic pathways is of broad importance given the central role of both clathrin-mediated endocytosis and bulk endocytosis in neurons, endocrine cells, immune cells, and many other cell types throughout the body.

Prenatal overexpression of platelet-derived growth factor receptor A results in central nervous system hypomyelination.

BACKGROUND: Platelet-derived growth factor (PDGF) signaling, through the ligand PDGF-A and its receptor PDGFRA, is important for the growth and maintenance of oligodendrocyte progenitor cells (OPCs) in the central nervous system (CNS). PDGFRA signaling is downregulated prior to OPC differentiation into mature myelinating oligodendrocytes. By contrast, PDGFRA is often genetically amplified or mutated in many types of gliomas, including diffuse midline glioma (DMG) where OPCs are considered the most likely cell-of-origin. The cellular and molecular changes that occur in OPCs in response to unregulated PDGFRA expression, however, are not known. METHODS: Here, we created a conditional knock-in (KI) mouse that overexpresses wild type (WT) human PDGFRA (hPDGFRA) in prenatal Olig2-expressing progenitors, and examined in vivo cellular and molecular consequences. RESULTS: The KI mice exhibited stunted growth, ataxia, and a severe loss of myelination in the brain and spinal cord. When combined with the loss of p53, a tumor suppressor gene whose activity is decreased in DMG, the KI mice failed to develop tumors but still exhibited hypomyelination. RNA-sequencing analysis revealed decreased myelination gene signatures, indicating a defect in oligodendroglial development. Mice overexpressing PDGFRA in prenatal GFAP-expressing progenitors, which give rise to a broader lineage of cells than Olig2-progenitors, also developed myelination defects. CONCLUSION: Our results suggest that embryonic overexpression of hPDGFRA in Olig2- or GFAP-progenitors is deleterious to OPC development and leads to CNS hypomyelination.

The structure and spontaneous curvature of clathrin lattices at the plasma membrane.

Clathrin-mediated endocytosis is the primary pathway for receptor and cargo internalization in eukaryotic cells. It is characterized by a polyhedral clathrin lattice that coats budding membranes. The mechanism and control of lattice assembly, curvature, and vesicle formation at the plasma membrane has been a matter of long-standing debate. Here, we use platinum replica and cryoelectron microscopy and tomography to present a structural framework of the pathway. We determine the shape and size parameters common to clathrin-mediated endocytosis. We show that clathrin sites maintain a constant surface area during curvature across multiple cell lines. Flat clathrin is present in all cells and spontaneously curves into coated pits without additional energy sources or recruited factors. Finally, we attribute curvature generation to loosely connected and pentagon-containing flat lattices that can rapidly curve when a flattening force is released. Together, these data present a universal mechanistic model of clathrin-mediated endocytosis.

Local protein dynamics during microvesicle exocytosis in neuroendocrine cells.

Calcium-triggered exocytosis is key to many physiological processes, including neurotransmitter and hormone release by neurons and endocrine cells. Dozens of proteins regulate exocytosis, yet the temporal and spatial dynamics of these factors during vesicle fusion remain unclear. Here we use total internal reflection fluorescence microscopy to visualize local protein dynamics at single sites of exocytosis of small synaptic-like microvesicles in live cultured neuroendocrine PC12 cells. We employ two-color imaging to simultaneously observe membrane fusion (using vesicular acetylcholine ACh transporter tagged to pHluorin) and the dynamics of associated proteins at the moments surrounding exocytosis. Our experiments show that many proteins, including the SNAREs syntaxin1 and VAMP2, the SNARE modulator tomosyn, and Rab proteins, are preclustered at fusion sites and rapidly lost at fusion. The ATPase N-ethylmaleimide-sensitive factor is locally recruited at fusion. Interestingly, the endocytic Bin-Amphiphysin-Rvs domain-containing proteins amphiphysin1, syndapin2, and endophilins are dynamically recruited to fusion sites and slow the loss of vesicle membrane-bound cargo from fusion sites. A similar effect on vesicle membrane protein dynamics was seen with the overexpression of the GTPases dynamin1 and dynamin2. These results suggest that proteins involved in classical clathrin-mediated endocytosis can regulate exocytosis of synaptic-like microvesicles. Our findings provide insights into the dynamics, assembly, and mechanistic roles of many key factors of exocytosis and endocytosis at single sites of microvesicle fusion in live cells.

Semisynthetic fluorescent pH sensors for imaging exocytosis and endocytosis.

The GFP-based superecliptic pHluorin (SEP) enables detection of exocytosis and endocytosis, but its performance has not been duplicated in red fluorescent protein scaffolds. Here we describe "semisynthetic" pH-sensitive protein conjugates with organic fluorophores, carbofluorescein, and Virginia Orange that match the properties of SEP. Conjugation to genetically encoded self-labeling tags or antibodies allows visualization of both exocytosis and endocytosis, constituting new bright sensors for these key steps of synaptic transmission.

Store-operated CRAC channels regulate gene expression and proliferation in neural progenitor cells.

Calcium signals regulate many critical processes during vertebrate brain development including neurogenesis, neurotransmitter specification, and axonal outgrowth. However, the identity of the ion channels mediating Ca(2+) signaling in the developing nervous system is not well defined. Here, we report that embryonic and adult mouse neural stem/progenitor cells (NSCs/NPCs) exhibit store-operated Ca(2+) entry (SOCE) mediated by Ca(2+) release-activated Ca(2+) (CRAC) channels. SOCE in NPCs was blocked by the CRAC channel inhibitors La(3+), BTP2, and 2-APB and Western blots revealed the presence of the canonical CRAC channel proteins STIM1 and Orai1. Knock down of STIM1 or Orai1 significantly diminished SOCE in NPCs, and SOCE was lost in NPCs from transgenic mice lacking Orai1 or STIM1 and in knock-in mice expressing the loss-of-function Orai1 mutant, R93W. Therefore, STIM1 and Orai1 make essential contributions to SOCE in NPCs. SOCE in NPCs was activated by epidermal growth factor and acetylcholine, the latter occurring through muscarinic receptors. Activation of SOCE stimulated gene transcription through calcineurin/NFAT (nuclear factor of activated T cells) signaling through a mechanism consistent with local Ca(2+) signaling by Ca(2+) microdomains near CRAC channels. Importantly, suppression or deletion of STIM1 and Orai1 expression significantly attenuated proliferation of embryonic and adult NPCs cultured as neurospheres and, in vivo, in the subventricular zone of adult mice. These findings show that CRAC channels serve as a major route of Ca(2+) entry in NPCs and regulate key effector functions including gene expression and proliferation, indicating that CRAC channels are important regulators of mammalian neurogenesis.

The C- and N-terminal STIM1 binding sites on Orai1 are required for both trapping and gating CRAC channels.

Ca(2+) release-activated Ca(2+) (CRAC) channels are activated through a mechanism wherein depletion of intracellular calcium stores results in the aggregation of stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca(2+) sensor, and Orai1, the CRAC channel protein, at overlapping sites in the ER and plasma membranes (PMs). The redistribution of CRAC channels is driven through direct STIM1-Orai1 binding, an important event that not only controls gating, but also regulates Orai1 ion selectivity. Orai1 harbours two STIM1 binding sites, one each on the intracellular C- and N-termini. Previous studies have proposed modular functions for these sites, with the C-terminal site thought to regulate STIM1-Orai1 binding and trapping of Orai1 at the ER-PM junctions, and the N-terminal site mediating gating. However, here we find that a variety of mutations in the N-terminal site impair the binding of Orai1 to STIM1 and to the soluble CRAC activation domain (CAD). Gating could be restored in several N- and C-terminal point mutants by directly tethering the minimal STIM1 activation domain (S) to Orai1 (Orai1-SS channels), indicating that loss of gating in these mutants by full-length STIM1 results from insufficient ligand binding. By contrast, gating could not be restored in mutant Orai1-SS channels carrying more drastic deletions that removed the STIM1 binding sites (1-85, 73-85, or 272-279 Orai1), suggesting that STIM1 binding to both sites is essential for channel activation. Moreover, analysis of ion selectivity indicated that the molecular requirements for gating and modulation of ion selectivity are similar, yet substantively different from those for Orai1 puncta formation, suggesting that ion selectivity and gating are mechanistically coupled in CRAC channels. Our results indicate that the C- and N-terminal STIM1 binding sites are both essential for multiple aspects of Orai1 function including STIM1-Orai1 association, Orai1 trapping, and channel activation.

Gated regulation of CRAC channel ion selectivity by STIM1.

Two defining functional features of ion channels are ion selectivity and channel gating. Ion selectivity is generally considered an immutable property of the open channel structure, whereas gating involves transitions between open and closed channel states, typically without changes in ion selectivity. In store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels, the molecular mechanism of channel gating by the CRAC channel activator, stromal interaction molecule 1 (STIM1), remains unknown. CRAC channels are distinguished by a very high Ca(2+) selectivity and are instrumental in generating sustained intracellular calcium concentration elevations that are necessary for gene expression and effector function in many eukaryotic cells. Here we probe the central features of the STIM1 gating mechanism in the human CRAC channel protein, ORAI1, and identify V102, a residue located in the extracellular region of the pore, as a candidate for the channel gate. Mutations at V102 produce constitutively active CRAC channels that are open even in the absence of STIM1. Unexpectedly, although STIM1-free V102 mutant channels are not Ca(2+)-selective, their Ca(2+) selectivity is dose-dependently boosted by interactions with STIM1. Similar enhancement of Ca(2+) selectivity is also seen in wild-type ORAI1 channels by increasing the number of STIM1 activation domains that are directly tethered to ORAI1 channels, or by increasing the relative expression of full-length STIM1. Thus, exquisite Ca(2+) selectivity is not an intrinsic property of CRAC channels but rather a tuneable feature that is bestowed on otherwise non-selective ORAI1 channels by STIM1. Our results demonstrate that STIM1-mediated gating of CRAC channels occurs through an unusual mechanism in which permeation and gating are closely coupled.

Permeation and gating mechanisms in store-operated CRAC channels.

Ca2+ is a ubiquitous signaling messenger mediating many essential cellular functions such as excitability, exocytosis and transcription. Among the different pathways by which cellular Ca2+ signals are generated, the entry of Ca2+ through store-operated Ca2+ release-activated Ca2+ (CRAC) channels has emerged as a widespread mechanism for regulating Ca2+ signaling in many eukaryotic cells. CRAC channels are implicated in the physiology and pathophysiology of numerous cell types, underlie several disease processes including a severe combined immunodeficiency syndrome, and have emerged as major targets for drug development. Although little was known of the molecular mechanisms of CRAC channels for several decades, the discovery of Orai1 as a prototypic CRAC channel pore-subunit, and the identification of STIM1 as the ER Ca2+ sensor, have led to rapid progress in our understanding of many aspects of CRAC channel behavior. This review examines the molecular features of the STIM and Orai proteins that regulate the activation and conduction mechanisms of CRAC channels.

Competitive modulation of Ca2+ release-activated Ca2+ channel gating by STIM1 and 2-aminoethyldiphenyl borate.

Activation of Ca(2+) release-activated Ca(2+) channels by depletion of intracellular Ca(2+) stores involves physical interactions between the endoplasmic reticulum Ca(2+) sensor, STIM1, and the channels composed of Orai subunits. Recent studies indicate that the Orai3 subtype, in addition to being store-operated, is also activated in a store-independent manner by 2-aminoethyldiphenyl borate (2-APB), a small molecule with complex pharmacology. However, it is unknown whether the store-dependent and -independent activation modes of Orai3 channels operate independently or whether there is cross-talk between these activation states. Here we report that in addition to causing direct activation, 2-APB also regulates store-operated gating of Orai3 channels, causing potentiation at low doses and inhibition at high doses. Inhibition of store-operated gating by 2-APB was accompanied by the suppression of several modes of Orai3 channel regulation that depend on STIM1, suggesting that high doses of 2-APB interrupt STIM1-Orai3 coupling. Conversely, STIM1-bound Orai3 (and Orai1) channels resisted direct gating by high doses of 2-APB. The rate of direct 2-APB activation of Orai3 channels increased linearly with the degree of STIM1-Orai3 uncoupling, suggesting that 2-APB has to first disengage STIM1 before it can directly gate Orai3 channels. Collectively, our results indicate that the store-dependent and -independent modes of Ca(2+) release-activated Ca(2+) channel activation are mutually exclusive: channels bound to STIM1 resist 2-APB gating, whereas 2-APB antagonizes STIM1 gating.

The calcium ATPase SERCA2 regulates desmoplakin dynamics and intercellular adhesive strength through modulation of PKCα signaling.

Darier's disease (DD) is an inherited autosomal-dominant skin disorder characterized histologically by loss of adhesion between keratinocytes. DD is typically caused by mutations in sarcoendoplasmic reticulum Ca(2+)-ATPase isoform 2 (SERCA2), a major regulator of intracellular Ca(2+) homeostasis in the skin. However, a defined role for SERCA2 in regulating intercellular adhesion remains poorly understood. We found that diminution of SERCA2 function by pharmacological inhibition or siRNA silencing in multiple human epidermal-derived cell lines was sufficient to disrupt desmosome assembly and weaken intercellular adhesive strength. Specifically, SERCA2-deficient cells exhibited up to a 60% reduction in border translocation of desmoplakin (DP), the desmosomal cytolinker protein necessary for intermediate filament (IF) anchorage to sites of robust cell-cell adhesion. In addition, loss of SERCA2 impaired the membrane translocation of protein kinase C α (PKCα), a known regulator of DP-IF association and desmosome assembly, to the plasma membrane by up to 70%. Exogenous activation of PKCα in SERCA2-deficient cells was sufficient to rescue the defective DP localization, desmosome assembly, and intercellular adhesive strength to levels comparable to controls. Our findings indicate that SERCA2-deficiency is sufficient to impede desmosome assembly and weaken intercellular adhesive strength via a PKCα-dependent mechanism, implicating SERCA2 as a novel regulator of PKCα signaling.

STIM1-Orai1 interactions and Orai1 conformational changes revealed by live-cell FRET microscopy.

Ca(2+) entry through store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels initiates key functions such as gene expression and exocytosis of inflammatory mediators. Activation of CRAC channels by store depletion involves the redistribution of the ER Ca(2+) sensor, stromal interaction molecule 1 (STIM1), to peripheral sites where it co-clusters with the CRAC channel subunit, Orai1. However, how STIM1 communicates with the CRAC channel and initiates the subsequent events culminating in channel opening is unclear. Here, we show that redistribution of STIM1 and Orai1 occurs in parallel with a pronounced increase in fluorescence resonance energy transfer (FRET) between STIM1 and Orai1, supporting the idea that activation of CRAC channels occurs through physical interactions with STIM1. Co-expression of Orai1-CFP and Orai1-YFP results in a high degree of FRET in resting cells, indicating that Orai1 exists as a multimer. However, store depletion triggers molecular rearrangements in Orai1 resulting in a decline in Orai1-Orai1 FRET. The decline in Orai1-Orai1 FRET is not seen in the absence of STIM1 co-expression and is abolished in Orai1 mutants with impaired STIM1 interaction. Both the STIM1-Orai1 interaction as well as the molecular rearrangements in Orai1 are altered by two powerful modulators of CRAC channel activity: extracellular Ca(2+) and 2-APB. These studies identify a STIM1-dependent conformational change in Orai1 during the activation of CRAC channels and reveal that STIM1-Orai1 interaction and the downstream Orai1 conformational change can be independently modulated to fine-tune CRAC channel activity.