Project description:Lysosome damage activates multiple pathways to prevent lysosome-dependent cell death, including a repair mechanism involving ER-lysosome membrane contact sites, phosphatidylinositol 4-kinase-2a (PI4K2A), phosphatidylinositol-4 phosphate (PI4P) and oxysterol-binding protein-related proteins (ORPs) lipid transfer proteins. PI4K2A localizes to trans-Golgi network and endosomes yet how it is delivered to damaged lysosomes remains unknown. During acute sterile damage, and damage caused by intracellular bacteria, we show that ATG9A-containing vesicles perform a critical role in delivering PI4K2A to damaged lysosomes. ADP ribosylation factor interacting protein 2 (ARFIP2), a component of ATG9A vesicles, binds and sequesters PI4P on lysosomes, balancing ORP-dependent lipid transfer and promoting retrieval of ATG9A vesicles through recruitment of the adaptor protein complex-3 (AP-3). Our results reveal a role for mobilized ATG9A vesicles and ARFIP2 in lysosome homeostasis after damage and bacterial infection.
Project description:Lysosome damage activates multiple pathways to prevent lysosome-dependent cell death, including a repair mechanism involving ER-lysosome membrane contact sites, phosphatidylinositol 4-kinase-2a (PI4K2A), phosphatidylinositol-4 phosphate (PI4P) and oxysterol-binding protein-related proteins (ORPs) lipid transfer proteins. PI4K2A localizes to trans-Golgi network and endosomes yet how it is delivered to damaged lysosomes remains unknown. During acute sterile damage, and damage caused by intracellular bacteria, we show that ATG9A-containing vesicles perform a critical role in delivering PI4K2A to damaged lysosomes. ADP ribosylation factor interacting protein 2 (ARFIP2), a component of ATG9A vesicles, binds and sequesters PI4P on lysosomes, balancing ORP-dependent lipid transfer and promoting retrieval of ATG9A vesicles through recruitment of the adaptor protein complex-3 (AP-3). Our results reveal a role for mobilized ATG9A vesicles and ARFIP2 in lysosome homeostasis after damage and bacterial infection.
Project description:The neural behavior of glioblastoma, including the formation of tumor microtubes and synaptic circuitry, is increasingly understood to be pivotal for disease manifestation (Osswald et al. 2015; Venkatesh et al. 2015; Weil et al. 2017; Venkataramani et al. 2019; Venkatesh et al. 2019; Alcantara Llaguno et al. 2019; Venkataramani et al. 2022). Nonetheless, the few approved treatments for glioblastoma target its oncological nature, while its neural vulnerabilities remain incompletely mapped and clinically unexploited. Here, we systematically survey the neural molecular dependencies and cellular heterogeneity across 27 glioblastoma patients and diverse model systems. In patient tumor samples taken directly after surgery, we identify a spectrum of neural stem cell morphologies indicative of poor prognosis, and discover a set of repurposable neuroactive drugs with unexpected and consistent anti-glioma efficacy. Glioblastoma cells exhibit functional dependencies on highly expressed drug targets including neurological ion channels and receptors, while interpretable molecular machine learning reveals downstream convergence on secondary drug targets (COSTAR) involving AP-1-driven tumor suppression. COSTAR enables in silico drug screening on >1 million compounds that are validated with high accuracy. Multi-omic profiling of drug-treated glioblastoma cells confirms rapid Ca2+-driven AP-1 pathway induction to represent a tumor-intrinsic vulnerability at the intersection of oncogenesis and neural activity-dependent signaling. Finally, the consistent anti-glioma activity across patients and model systems is epitomized by the antidepressant Vortioxetine, which synergizes in vivo with approved glioblastoma chemotherapies. In all, our global analysis reveals that the neural vulnerabilities of glioblastoma converge on an AP-1 mediated gene regulatory network with direct translatable potential.
Project description:Abstract: RAS-like (RAL) GTPases function in Wnt signalling-dependent intestinal stem cell proliferation and regeneration. Whether RAL proteins work as canonical RAS effectors in the intestine, and the mechanisms of how they contribute to tumorigenesis remain unclear. Here, we show that RAL GTPases are necessary and sufficient to activate EGFR/MAPK signalling in the intestine. We identify non-canonical roles of RAL GTPases not as RAS effectors, but rather by acting upstream of RAS activation via induction of EGFR internalisation . Knocking down Drosophila RalA from intestinal stem and progenitor cells leads to increased levels of plasma membrane-associated EGFR and decreased MAPK pathway activation. Importantly, in addition to impacting stem cell proliferation and damage-induced intestinal regeneration, this function of RAL GTPases drives EGFR-dependent tumorigenic growth in the intestine and in human mammary epithelium. Altogether, our results reveal previously unrecognised cellular and molecular contexts where RAL GTPases become essential mediators of EGFR-driven tissue homeostasis and malignant transformation. Results: RalA is required within ISCs to induce midgut adult midgut regeneration following damage by oral infection with Erwinia carotovora carotovora 15 (Ecc15) (Johansson et al., 2019). To achieve a global view of intestinal pathways affected by RalA, we performed a transcriptomic analysis by RNAseq of whole midguts from vehicle treated (Mock) or damaged (Ecc15 fed) control animals or following RalA knockdown in intestinal stem and progenitor cells using the escargot-gal4 driver (ISC/EB>) (Micchelli and Perrimon, 2006). Consistent with its effect on ISC proliferation (Johansson et al., 2019), RalA knockdown significantly impaired damage-induced upregulation of cell cycle genes in the midgut. Additionally, levels of multiple transcriptional targets of the EGFR/MAPK pathway (Golembo et al., 1996; Hsu et al., 2001; Jin et al., 2015; Meng and Biteau, 2015), such as argos (aos), rhomboid (rho), Sox21a and string (stg) were increased following Ecc15 infection in control midguts. The upregulation of these target genes was significantly impaired upon RalA knockdown.
Project description:Το investigate the role of Rac1 and Rac3 GTPases in the development of MGE-derived cortical interneurons we generated a transgenic mice where both Rac1 and 3 were depleted from the medial ganglionic eminence. First we generated a conditional knockout for Rac1 (Vidaki et al. 2012) by crossing animals carrying a floxed allele of Rac1 (Rac1fl/fl) (the fourth and fifth exon of the Rac1 gene are flanked with loxP sites, Walmsley et al. 2003) to the Nkx2.1Tg(Cre) mice (Nkx2.1 transgenic Cre, Fogarty et al. 2007). The ROSA26fl-STOP-fl-YFP allele was also inserted as an independent marker (Srinivas et al. 2001). Next, we crossed the Rac1 conditional knockout with the Rac3 KO line (Corbetta et al. 2005) and obtained a mouse line where Rac1 and Rac3 were both depleted from the MGE-derived interneurons.
Project description:Cholesterol and phosphoinositides (PI) are two critically important lipids that are found in cellular membranes and dysregulated in many disorders. Therefore, uncovering molecular pathways connecting these essential lipids may offer new therapeutic insights. We report that loss of function of lysosomal Niemann-Pick Type C1 (NPC1) cholesterol transporter, which leads to neurodegenerative NPC disease, initiates a signaling cascade that alters the cholesterol/phosphatidylinositol 4-phosphate (PtdIns4P) countertransport cycle between Golgi-endoplasmic reticulum (ER), as well as lysosome-ER membrane contact sites (MCS). Central to these disruptions is increased recruitment of phosphatidylinositol 4-kinases-PI4KIIα and PI4KIIIβ-which boosts PtdIns4P metabolism at Golgi and lysosomal membranes. Aberrantly increased PtdIns4P levels elevate constitutive anterograde secretion from the Golgi complex, and mTORC1 recruitment to lysosomes. NPC1 disease mutations phenocopy the transporter loss of function and can be rescued by inhibition or knockdown of either key phosphoinositide enzymes or their recruiting partners. In summary, we show that the lysosomal NPC1 cholesterol transporter tunes the molecular content of Golgi and lysosome MCS to regulate intracellular trafficking and growth signaling in health and disease.
Project description:The small GTPase Rab5 has an essential role in sorting vesicles arriving to and leaving the early endosome. One key function of Rab5 in this process is the activation of the primordial phosphatidylinositol 3-kinase VPS34, a lipid kinase that phosphorylates phosphatidylinositol (PI) to generate PI3P.
Human VPS34 forms two heterotetrameric core complexes known as complexes I and II. Rab5a preferentially activates endocytic complex II in a membrane dependent manner.
To map Rab5a-GTP interaction sites in complex II, unnatural amino acid mediated crosslinking was employed by genetic code expansion. A Rab5a mutant, in which the S84 in was replaced by BrCO6K was used for crosslinking mass spectrometry analysis. BrCO6K is a lysine derivative that can crosslink to several amino acids such as cysteines, histidines, lysines and glutamates over distances up to 15 Å (Cigler et al., 2017; Nguyen et al., 2018). Both complex II and VPS34 alone incubated with Rab5a-GTP-BrCO6K gave rise to a cross-linked product that ran slightly above the 120 kDa molecular weight marker, consistent with 102 kDa VPS34 crosslinked to the 24 kDa Rab5a (126 kDa). The gel bands of this crosslinking product in both samples were cut and analysed by LC-MS/MS for identification of crosslinks.