Inositol triphosphate–triggered calcium release from the endoplasmic reticulum induces lysosome biogenesis via TFEB/TFE3

Lysosomes serve as dynamic regulators of cell and organismal physiology by integrating the degradation of macromolecules with receptor and nutrient signaling. Previous studies have established that activation of the transcription factor EB (TFEB) and transcription factor E3 (TFE3) induces the expression of lysosomal genes and proteins in signaling-inactive starved cells, that is, under conditions when activity of the master regulator of nutrient-sensing signaling mechanistic target of rapamycin complex 1 is repressed. How lysosome biogenesis is triggered in signaling-active cells is incompletely understood. Here, we identify a role for calcium release from the lumen of the endoplasmic reticulum in the control of lysosome biogenesis that is independent of mechanistic target of rapamycin complex 1. We show using functional imaging that calcium efflux from endoplasmic reticulum stores induced by inositol triphosphate accumulation upon depletion of inositol polyphosphate-5-phosphatase A, an inositol 5-phosphatase downregulated in cancer and defective in spinocerebellar ataxia, or receptor-mediated phospholipase C activation leads to the induction of lysosome biogenesis. This mechanism involves calcineurin and the nuclear translocation and elevated transcriptional activity of TFEB/TFE3. Our findings reveal a crucial function for inositol polyphosphate-5-phosphatase A–mediated triphosphate hydrolysis in the control of lysosome biogenesis via TFEB/TFE3, thereby contributing to our understanding how cells are able to maintain their lysosome content under conditions of active receptor and nutrient signaling

Inositol triphosphate-triggered calcium release blocks lipid exchange at endoplasmic reticulum-Golgi contact sites

Vesicular traffic and membrane contact sites between organelles enable the exchange of proteins, lipids, and metabolites. Recruitment of tethers to contact sites between the endoplasmic reticulum (ER) and the plasma membrane is often triggered by calcium. Here we reveal a function for calcium in the repression of cholesterol export at membrane contact sites between the ER and the Golgi complex. We show that calcium efflux from ER stores induced by inositol-triphosphate [IP3] accumulation upon loss of the inositol 5-phosphatase INPP5A or receptor signaling triggers depletion of cholesterol and associated Gb3 from the cell surface, resulting in a blockade of clathrin-independent endocytosis (CIE) of Shiga toxin. This phenotype is caused by the calcium-induced dissociation of oxysterol binding protein (OSBP) from the Golgi complex and from VAP-containing membrane contact sites. Our findings reveal a crucial function for INPP5A-mediated IP3 hydrolysis in the control of lipid exchange at membrane contact sites

Pdro, a protein associated with late endosomes and lysosomes and implicated in cellular cholesterol homeostasis.

BACKGROUND: Cellular cholesterol is a vital component of the cell membrane. Its concentration is tightly controlled by mechanisms that remain only partially characterized. In this study, we describe a late endosome/lysosomes-associated protein whose expression level affects cellular free cholesterol content. METHODOLOGY/PRINCIPAL FINDINGS: Using a restricted proteomic analysis of detergent-resistant membranes (DRMs), we have identified a protein encoded by gene C11orf59. It is mainly localized to late endosome/lysosome (LE/LY) compartment through N-terminal myristoylation and palmitoylation. We named it Pdro for protein associated with DRMs and endosomes. Very recently, three studies have reported on the same protein under two other names: the human p27RF-Rho that regulates RhoA activation and actin dynamics, and its rodent orthologue p18 that controls both LE/LY dynamics through the MERK-ERK pathway and the lysosomal activation of mammalian target of rapamycin complex 1 by amino acids. We found that, consistent with the presence of sterol-responsive element consensus sequences in the promoter region of C11orf59, Pdro mRNA and protein expression levels are regulated positively by cellular cholesterol depletion and negatively by cellular cholesterol loading. Conversely, Pdro is involved in the regulation of cholesterol homeostasis, since its depletion by siRNA increases cellular free cholesterol content that is accompanied by an increased cholesterol efflux from cells. On the other hand, cells stably overexpressing Pdro display reduced cellular free cholesterol content. Pdro depletion-mediated excess cholesterol results, at least in part, from a stimulated low-density lipoprotein (LDL) uptake and an increased cholesterol egress from LE/LY. CONCLUSIONS/SIGNIFICANCE: LDL-derived cholesterol release involves LE/LY motility that is linked to actin dynamics. Because Pdro regulates these two processes, we propose that modulation of Pdro expression in response to sterol levels regulates LDL-derived cholesterol through both LDL uptake and LE/LY dynamics, to ultimately control free cholesterol homeostasis

Role of class IA PI3Ks in EGF-stimulated chemokinesis in MDA-MB 231 cells. Panel A.

<p>Individual MDA-MB 231 cells were tracked moving on Matrigel in stable gradients of EGF (the concentration of EGF in the reservoir was 0 ng/ml (a), 15 ng/ml (b), 30 ng/ml (c) and 60 ng/ml (d)) or expressing sh-CT (e), sh-p110 (f) or treated with LY294002 (g) in Dunn chambers. The data are presented centre-zeroed with the source of EGF at the top. Directionality was analysed using Mathmatica and significant directionality is denoted by a grey vector and arrow. The number of individual tracks analysed is shown (n) and were collected from at least 3 independent experiments. <b>Panel B.</b> The total accumulated distances moved by individual cells in the experiments shown in panel A are shown, the data presented are means. Parental MDA-MB 231 cells (some parental cells were pretreated with 10 µM LY294002) or derivatives expressing either control or p110α-directed shRNAi constructs (3 sh-CT (N2, N3 and N4, see Methods, separate cell lines expressing each construct were used and the data derived were pooled for presentation as they were indistinguishable) and 2 sh-p110α -expressing (A1 and A2, 2 cell lines expressing the individual constructs were used and the data from were pooled for presentation) independent, selected populations which were in the range 80–90% eGFP +ve) were exposed to EGF gradients (30 ng/ml EGF in the reservoir) in Dunn chambers. The cell tracks and their directionality are shown as in panel A. The number of cells tracked (n) is indicated and were collected from at least 4 independent experiments. The total accumulated distances moved by individual cells in the experiments shown in panel A are shown, the data presented are means. The data for all control shRNAi constructs and all p110α-directed constructs were pooled separately to enable an overall comparison of their effects. Statistical comparisons were conducted as in Fig. 4C.</p