Visualization and Manipulation of Plasma Membrane-Endoplasmic Reticulum Contact Sites Indicates the Presence of Additional Molecular Components within the STIM1-Orai1 Complex
ABSTRACT STIM1, a recently identified endoplasmic reticulum (ER) protein, rapidly translocates to a plasma membrane-adjacent ER compartment upon depletion of the ER Ca(2+) stores. Here we use a novel means, namely a chemically inducible bridge formation between the plasma and ER membranes, to highlight the plasma membrane-adjacent ER compartment and show that this is the site where STIM1 and its Ca(2+) channel partner, Orai1, form a productive interaction upon store depletion. By changing the length of the linkers connecting the plasma and ER membranes, we show that Orai1 requires a larger space than STIM1 between the two membranes. This finding suggests that Orai1 is part of a larger macromolecular cluster with an estimated 11-14-nm protrusion to the cytoplasm, whereas the cytoplasmic domain of STIM1 fits in a space calculated to be less than 6 nm. We finally show that agonist-induced translocation of STIM1 is rapidly reversible and only partially affects STIM1 in the juxtanuclear ER compartment. These studies are the first to detect juxtaposed areas between the ER and the plasma membrane in live cells, revealing novel details of STIM1-Orai1 interactions.
- SourceAvailable from: Yong Miao
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- "Research article (Liou et al., 2005; Zhang et al., 2005; Stathopulos et al., 2006; Wu et al., 2006; Liou et al., 2007; Varnai et al., 2007; Luik et al., 2008; Stathopulos et al., 2008), we hypothesized that this increase in cluster size could result from an increase in the density of Stim1 in the junctional ER or could reflect a specific defect in the ability of Stim1 to efficiently co-cluster Orai1. To distinguish between these possibilities, we first quantified the average intensity of CFP-Stim1 in individual Stim1–Orai1 clusters. "
ABSTRACT: eLife digest Calcium is an essential element for many biological functions. In particular, the movement of calcium ions through the cell membrane has a central role in many of the signalling pathways that cells use to communicate with other cells. Signals are produced by calcium ions both entering and leaving the cell, with information being contained in the rate, location, and duration of the flow of ions. Calcium is stored inside cells in a structure called the endoplasmic reticulum, and when stores of calcium are low, special channels in the cell membrane called CRAC (calcium release activated calcium) channels are used to ferry more calcium ions into the cell. This process, known as store-operated calcium entry, relies on two important groups of proteins: the Stim proteins that sense when calcium stores are low; and, the Orai structural proteins that form the actual channel. Previous work has shown that when the calcium stores are low, the Stim proteins—which reside in the endoplasmic reticulum—form clusters and these clusters then move to a part of the endoplasmic reticulum that is next to the cell membrane, where they join the Orai1 proteins to form larger clusters. However, to date it has been unclear whether Stim-Orai clustering at the cell membrane is sufficient for CRAC channels to open, or if additional steps are involved. Miao et al. now show that another protein is involved in the formation of functional CRAC channels. Working with fruit fly cells, Miao et al. used genetic techniques to prevent the expression of various proteins that were thought to have a role in the movement of calcium ions through the cell membrane. One of these candidates, a protein called α-SNAP that is found in the internal fluid of the cell, was identified as having a central role in the import of calcium ions into the cell. Further work showed that α-SNAP re-organizes the Stim and Orai proteins to produce working CRAC channels. DOI: http://dx.doi.org/10.7554/eLife.00802.002eLife Sciences 07/2013; 2:e00802. DOI:10.7554/eLife.00802 · 8.52 Impact Factor
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- "At first glance it seems counterintuitive that PI4P synthesis at distal sites can supply the plasma membrane during ligand-activated signalling. However, the recent discovery of stable inter-organelle membrane contacts which facilitate lipid transfer between different organelles [78,79] is worth considering in this context. Most strikingly, a precedent for such inter-organelle regulation of phosphoinositide concentrations has been demonstrated in yeast where the ER-associated PI4P phosphatase Sac1, can act in trans at inter-membrane contact sites to dephosphorylate PI4P at the plasma membrane . "
ABSTRACT: The four mammalian phosphatidylinositol 4-kinases modulate inter-organelle lipid trafficking, phosphoinositide signalling and intracellular vesicle trafficking. In addition to catalytic domains required for the synthesis of PI4P, the phosphatidylinositol 4-kinases also contain isoform-specific structural motifs that mediate interactions with proteins such as AP-3 and the E3 ubiquitin ligase Itch, and these determine isoform-specific roles in membrane trafficking. Moreover, different permutations of phosphatidylinositol 4-kinase isozymes may be required for a single cellular function such as occurs during GPCR signalling and in Golgi to lysosome trafficking. Phosphatidylinositol 4-kinases have recently been implicated in human disease. Emerging paradigms include increased phosphatidylinositol 4-kinase expression in some cancers, impaired functioning associated with neurological pathologies, the subversion of PI4P trafficking functions in bacterial infection and the activation of lipid kinase activity in viral disease. We discuss how the diverse and sometimes overlapping functions of the phosphatidylinositol 4-kinases present challenges for the design of isoform-specific inhibitors in a therapeutic context.Progress in lipid research 04/2013; 52(3). DOI:10.1016/j.plipres.2013.04.002 · 12.96 Impact Factor
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- "The fact that overexpression of the two proteins results in reconstituted CRAC currents has enabled us to address questions about their overall communication [54, 68, 107]. In principle, two pathways are possible as to how STIM transmits the signal of store depletion to the activation of Orai channels in the plasma membrane: either by a simple, direct interaction or via a further molecule [11, 22, 95, 105]. Meanwhile, several independent studies, however, have proven direct binding of soluble STIM1 fragments to Orai, thereby inducing constitutive CRAC currents [32, 34, 57, 58, 66, 106]. "
ABSTRACT: Store-operated Ca(2+) entry describes the phenomenon that connects a depletion of internal Ca(2+) stores to an activation of plasma membrane-located Ca(2+) selective ion channels. Tremendous progress towards the underlying molecular mechanism came with the discovery of the two respective limiting components, STIM and Orai. STIM1 represents the ER-located Ca(2+) sensor and transmits the signal of store depletion to the plasma membrane. Here it couples to and activates Orai, the highly Ca(2+)-selective pore-forming subunit of Ca(2+) release-activated Ca(2+) channels. In this review, we focus on the molecular steps that these two proteins undergo from store-depletion to their coupling, the activation, and regulation of Ca(2+) currents.Cellular and Molecular Life Sciences CMLS 07/2012; 69(24). DOI:10.1007/s00018-012-1072-8 · 5.86 Impact Factor