KRAP regulates mitochondrial Ca2+ uptake by licensing IP3 receptor activity and stabilizing ER-mitochondrial junctions.

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are high-conductance channels that allow the regulated redistribution of Ca2+ from the endoplasmic reticulum (ER) to the cytosol and, at specialized membrane contact sites (MCSs), to other organelles. Only a subset of IP3Rs release Ca2+ to the cytosol in response to IP3. These 'licensed' IP3Rs are associated with Kras-induced actin-interacting protein (KRAP, also known as ITPRID2) beneath the plasma membrane. It is unclear whether KRAP regulates IP3Rs at MCSs. We show, using simultaneous measurements of Ca2+ concentration in the cytosol and mitochondrial matrix, that KRAP also licenses IP3Rs to release Ca2+ to mitochondria. Loss of KRAP abolishes cytosolic and mitochondrial Ca2+ signals evoked by stimulation of IP3Rs via endogenous receptors. KRAP is located at ER-mitochondrial membrane contact sites (ERMCSs) populated by IP3R clusters. Using a proximity ligation assay between IP3R and voltage-dependent anion channel 1 (VDAC1), we show that loss of KRAP reduces the number of ERMCSs. We conclude that KRAP regulates Ca2+ transfer from IP3Rs to mitochondria by both licensing IP3R activity and stabilizing ERMCSs.

IP3R at ER-Mitochondrial Contact Sites: Beyond the IP3R-GRP75-VDAC1 Ca2+ Funnel.

Membrane contact sites (MCS) circumvent the topological constraints of functional coupling between different membrane-bound organelles by providing a means of communication and exchange of materials. One of the most characterised contact sites in the cell is that between the endoplasmic reticulum and the mitochondrial (ERMCS) whose function is to couple cellular Ca2+ homeostasis and mitochondrial function. Inositol 1,4,5-trisphosphate receptors (IP3Rs) on the ER, glucose-regulated protein 75 (GRP 75) and voltage-dependent anion channel 1 (VDAC1) on the outer mitochondrial membrane are the canonical component of the Ca2+ transfer unit at ERMCS. These are often reported to form a Ca2+ funnel that fuels the mitochondrial low-affinity Ca2+ uptake system. We assess the available evidence on the IP3R subtype selectivity at the ERMCS and consider if IP3Rs have other roles at the ERMCS beyond providing Ca2+. Growing evidence suggests that all three IP3R subtypes can localise and regulate Ca2+ signalling at ERMCS. Furthermore, IP3Rs may be structurally important for assembly of the ERMCS in addition to their role in providing Ca2+ at these sites. Evidence that various binding partners regulate the assembly and Ca2+ transfer at ERMCS populated by IP3R-GRP75-VDAC1, suggesting that cells have evolved mechanisms that stabilise these junctions forming a Ca2+ microdomain that is required to fuel mitochondrial Ca2+ uptake.

Phosphatidylinositol 4,5-bisphosphate and calcium at ER-PM junctions - Complex interplay of simple messengers.

Endoplasmic reticulum-plasma membrane contact sites (ER-PM MCS) are a specialised domain involved in the control of Ca2+ dynamics and various Ca2+-dependent cellular processes. Intracellular Ca2+ signals are broadly supported by Ca2+ release from intracellular Ca2+ channels such as inositol 1,4,5-trisphosphate receptors (IP3Rs) and subsequent store-operated Ca2+ entry (SOCE) across the PM to replenish store content. IP3Rs sit in close proximity to the PM where they can easily access newly synthesised IP3, interact with binding partners such as actin, and localise adjacent to ER-PM MCS populated by the SOCE machinery, STIM1-2 and Orai1-3, to possibly form a locally regulated unit of Ca2+ influx. PtdIns(4,5)P2 is a multiplex regulator of Ca2+ signalling at the ER-PM MCS interacting with multiple proteins at these junctions such as actin and STIM1, whilst also being consumed as a substrate for phospholipase C to produce IP3 in response to extracellular stimuli. In this review, we consider the mechanisms regulating the synthesis and turnover of PtdIns(4,5)P2 via the phosphoinositide cycle and its significance for sustained signalling at the ER-PM MCS. Furthermore, we highlight recent insights into the role of PtdIns(4,5)P2 in the spatiotemporal organization of signalling at ER-PM junctions and raise outstanding questions on how this multi-faceted regulation occurs.

iRhom pseudoproteases regulate ER stress-induced cell death through IP3 receptors and BCL-2.

The folding capacity of membrane and secretory proteins in the endoplasmic reticulum (ER) can be challenged by physiological and pathological perturbations, causing ER stress. If unresolved, this leads to cell death. We report a role for iRhom pseudoproteases in controlling apoptosis due to persistent ER stress. Loss of iRhoms causes cells to be resistant to ER stress-induced apoptosis. iRhom1 and iRhom2 interact with IP3 receptors, critical mediators of intracellular Ca2+ signalling, and regulate ER stress-induced transport of Ca2+ into mitochondria, a primary trigger of mitochondrial membrane depolarisation and cell death. iRhoms also bind to the anti-apoptotic regulator BCL-2, attenuating the inhibitory interaction between BCL-2 and IP3 receptors, which promotes ER Ca2+ release. The discovery of the participation of iRhoms in the control of ER stress-induced cell death further extends their potential pathological significance to include diseases dependent on protein misfolding and aggregation.

Current methods to analyze lysosome morphology, positioning, motility and function.

Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively degradative organelles, but more recent research has shown that they play essential roles in several other cellular functions, such as nutrient sensing, intracellular signalling and metabolism. Methodological advances played a key part in generating our current knowledge about the biology of this multifaceted organelle. In this review, we cover current methods used to analyze lysosome morphology, positioning, motility and function. We highlight the principles behind these methods, the methodological strategies and their advantages and limitations. To extract accurate information and avoid misinterpretations, we discuss the best strategies to identify lysosomes and assess their characteristics and functions. With this review, we aim to stimulate an increase in the quantity and quality of research on lysosomes and further ground-breaking discoveries on an organelle that continues to surprise and excite cell biologists.

P2X4 Receptors Mediate Ca2+ Release from Lysosomes in Response to Stimulation of P2X7 and H1 Histamine Receptors.

The P2X4 purinergic receptor is targeted to endolysosomes, where it mediates an inward current dependent on luminal ATP and pH. Activation of P2X4 receptors was previously shown to trigger lysosome fusion, but the regulation of P2X4 receptors and their role in lysosomal Ca2+ signaling are poorly understood. We show that lysosomal P2X4 receptors are activated downstream of plasma membrane P2X7 and H1 histamine receptor stimulation. When P2X4 receptors are expressed, the increase in near-lysosome cytosolic [Ca2+] is exaggerated, as detected with a low-affinity targeted Ca2+ sensor. P2X4-dependent changes in lysosome properties were triggered downstream of P2X7 receptor activation, including an enlargement of lysosomes indicative of homotypic fusion and a redistribution of lysosomes towards the periphery of the cell. Lysosomal P2X4 receptors, therefore, have a role in regulating lysosomal Ca2+ release and the regulation of lysosomal membrane trafficking.

KRAP tethers IP3 receptors to actin and licenses them to evoke cytosolic Ca2+ signals.

Regulation of IP3 receptors (IP3Rs) by IP3 and Ca2+ allows regenerative Ca2+ signals, the smallest being Ca2+ puffs, which arise from coordinated openings of a few clustered IP3Rs. Cells express thousands of mostly mobile IP3Rs, yet Ca2+ puffs occur at a few immobile IP3R clusters. By imaging cells with endogenous IP3Rs tagged with EGFP, we show that KRas-induced actin-interacting protein (KRAP) tethers IP3Rs to actin beneath the plasma membrane. Loss of KRAP abolishes Ca2+ puffs and the global increases in cytosolic Ca2+ concentration evoked by more intense stimulation. Over-expressing KRAP immobilizes additional IP3R clusters and results in more Ca2+ puffs and larger global Ca2+ signals. Endogenous KRAP determines which IP3Rs will respond: it tethers IP3R clusters to actin alongside sites where store-operated Ca2+ entry occurs, licenses IP3Rs to evoke Ca2+ puffs and global cytosolic Ca2+ signals, implicates the actin cytoskeleton in IP3R regulation and may allow local activation of Ca2+ entry.