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.

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.

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.

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.