Calcium Oscillations

Calcium signaling results from a complex interplay between activation and inactivation of intracellular and extracellular calcium permeable channels. This complexity is obvious from the pattern of calcium signals observed with modest, physiological concentrations of calcium-mobilizing agonists, which typically present as sequential regenerative discharges of stored calcium, a process referred to as calcium oscillations. In this review, we discuss recent advances in understanding the underlying mechanism of calcium oscillations through the power of mathematical modeling. We also summarize recent findings on the role of calcium entry through store-operated channels in sustaining calcium oscillations and in the mechanism by which calcium oscillations couple to downstream effectors.Calcium ions participate in a multiplicity of physiological and pathological functions. Among the most intensely studied, and the major focus of this article, is the role of Ca²⁺ as a cellular signal. Elevations in cytoplasmic Ca²⁺ mediate a plethora of cellular responses, ranging from extremely rapid events (muscle contraction, neurosecretion), to slower more subtle responses (cell division, differentiation, apoptosis). In contrast to most cellular signals, it is a relatively simple matter to observe changes in cytoplasmic Ca²⁺ in real time in living cells. As a result, the truly complex nature of Ca²⁺ signaling pathways has been revealed. The challenge is to understand what regulates these signals and what the biological significance of their complexity is.In the majority of laboratory experiments examining effects of various stimulants on Ca²⁺ signaling, supramaximal concentrations of activating agonists are employed resulting in rapid, robust, and often sustained increases in cytoplasmic Ca²⁺. It has long been appreciated that these signals result from a coordinated release of intracellular stores and increased Ca²⁺ influx across the plasma membrane (Bohr, 1973; Putney et al. 1981). The intracellular release of Ca²⁺ most commonly results from the Ca²⁺ releasing action of the phospholipase C-derived second messenger, inositol 1,4,5-trisphosphate (InsP₃) (Streb et al. 1983), whereas the entry of Ca²⁺ is because of the activation of store-operated channels in the plasma membrane (Putney 1986). However, it is becoming increasingly clear that these large sustained elevations seldom occur with physiological levels of stimulants. Rather the more common pattern of Ca²⁺ signaling, in both excitable and nonexcitable cells is a pattern of periodic discharges and/or entry of Ca²⁺. In excitable cells, such as the heart for example, these may be comprised of, or initiated by regenerative all-or-none plasma membrane channel activation, the Ca²⁺ action potential (Tsien et al. 1986) with amplification by intracellular Ca²⁺ release (Fabiato 1983). In nonexcitable cells, these spikes of cytoplasmic Ca²⁺ arise from regenerative discharge of stored Ca²⁺, a process generally termed Ca²⁺ oscillations (Prince and Berridge 1973; Woods et al. 1986). Like Ca²⁺ action potentials, these all-or-none discharges of Ca²⁺ represent a form of excitable behavior of the intracellular Ca²⁺ release signaling mechanism. However, because it is not possible to easily monitor and control the transmembrane chemical and biophysical parameters, as is the case for excitable plasma membrane behavior, it has been more difficult to fully understand the basic mechanisms by which these Ca²⁺ oscillations arise. Thus, although the question has been exhaustively studied for well over twenty years, there is still uncertainty and controversy over the underlying processes that give rise to Ca²⁺ oscillations. A number of reviews have discussed these issues at some length (Berridge and Galione 1988; Rink and Jacob 1989; Berridge 1990; Petersen and Wakui 1990; Berridge 1991; Cuthbertson and Cobbold 1991; Meyer and Stryer 1991; Hellman et al. 1992; Tepikin and Petersen 1992; Thomas et al. 1992; Dupont and Goldbeter 1993; Keizer 1993; Sneyd et al. 1994; Li et al. 1995; Thomas et al. 1996; Shuttleworth 1999; Lewis 2003; Dupont et al. 2007). In the current treatment, we have chosen to focus on two important aspects of Ca²⁺ oscillations. First, we review the available evidence for various computational models of Ca²⁺ oscillations that employ a quantitative approach to validate or repudiate specific mechanisms. Second, we consider the interrelationship between Ca²⁺ oscillations and plasma membrane Ca²⁺ influx mechanisms, with the view that we may learn more of the physiological function that these intracellular discharges of Ca²⁺ provide.     

Store-Operated Calcium Channels: New Perspectives on Mechanism and Function

Store-operated calcium channels (SOCs) are a nearly ubiquitous Ca²⁺ entry pathway stimulated by numerous cell surface receptors via the reduction of Ca²⁺ concentration in the ER. The discovery of STIM proteins as ER Ca²⁺ sensors and Orai proteins as structural components of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel, a prototypic SOC, opened the floodgates for exploring the molecular mechanism of this pathway and its functions. This review focuses on recent advances made possible by the use of STIM and Orai as molecular tools. I will describe our current understanding of the store-operated Ca²⁺ entry mechanism and its emerging roles in physiology and disease, areas of uncertainty in which further progress is needed, and recent findings that are opening new directions for research in this rapidly growing field.Calcium is a remarkably multifunctional signaling ion, at the heart of diverse biological processes that direct the birth, development, function, and eventual death of cells, tissues, and organisms. Cells use a diverse array of transporters and channels to regulate intracellular Ca²⁺ concentration ([Ca²⁺]_i). A major pathway present in nearly all metazoan cells is the store-operated Ca²⁺ channel (SOC). The defining feature of SOCs, the one that distinguishes them from all other classes of Ca²⁺ channels discussed in this volume, is their activation by the reduction of Ca²⁺ concentration in the lumen of the ER ([Ca²⁺]_ER). Though they were originally described in nonexcitable cells (cells lacking the ability to fire action potentials), they are now known to be present in virtually all cells, including excitable cells like skeletal muscle and neurons (Parekh and Putney 2005).Physiologically, SOCs are most commonly activated by stimuli that release Ca²⁺ from the ER. This generally involves receptors that activate phospholipase C to produce inositol 1,4,5-trisphosphate (IP₃) and activate IP₃ receptors in the ER, but can also result from Ca²⁺-induced Ca²⁺ release through ER/SR ryanodine receptors. The notion that ER Ca²⁺ depletion can control Ca²⁺ entry was first formulated by Jim Putney 25 years ago as the “capacitative calcium entry” hypothesis based on observations that Ca²⁺ entry triggered by muscarinic agonists was more closely linked to the emptiness of the ER store than to IP₃ elevation or occupation of the muscarinic receptor (Putney 1986). The introduction of thapsigargin (TG) (Thastrup et al. 1989), a sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor that depletes ER Ca²⁺ independently of receptors and IP₃, and methods for measuring [Ca²⁺]_i in single cells (fura-2, indo-1, etc.) (Grynkiewicz et al. 1985) provided powerful tools that greatly accelerated progress in establishing store-operated Ca²⁺ entry (SOCE, as it was later renamed) as a ubiquitous Ca²⁺ entry pathway. TG-induced Ca²⁺ entry was soon shown to occur in dozens of cell types (Putney and Bird 1993; Parekh and Penner 1997), though nothing was known about the diversity of pathways that might be involved, let alone their molecular basis.A major step forward was the identification of store-operated Ca²⁺ currents in mast cells and T cells. This achievement arose initially from attempts to identify Ca²⁺ conductances triggered by secretory agonists in mast cells and antigen receptors in T cells (Penner et al. 1988; Lewis and Cahalan 1989). In both cases, extremely small currents were detected and linked to large [Ca²⁺]_i rises, suggesting a high Ca²⁺ selectivity. In T cells, the current was shown to activate spontaneously during whole-cell recordings and in response to the T-cell mitogen phytohemagglutinin in perforated-patch recordings (Lewis and Cahalan 1989). Soon after, fluorescence-based studies showed that Ca²⁺ influx triggered through T cell mitogens shared several features with TG-induced influx, suggesting that T cell receptor agonists activated the store-dependent pathway (Mason et al. 1991; Sarkadi et al. 1991). These two paths of research converged when Hoth and Penner described a highly Ca²⁺-selective current in mast cells that was activated in whole-cell recordings spontaneously (by Ca²⁺ chelators), by IP₃, and by ionomycin, and called it the Ca²⁺ release-activated Ca²⁺ (CRAC) current (Hoth and Penner 1992). In Jurkat T cells, Zweifach and Lewis showed that TG activated a similar current, which appeared to be identical to the mitogen-stimulated current described earlier, and made the first estimate of its characteristically tiny conductance (∼20 femtosiemens, far too small to resolve single-channel currents) (Zweifach and Lewis 1993). These initial studies defined a membrane conductance that over the next decade would be described biophysically and pharmacologically in detail, providing a characteristic “fingerprint” culled from its ion selectivity, unitary conductance, and regulation by intra- and extracellular Ca²⁺ and pharmacological inhibitors (Parekh and Penner 1997; Prakriya et al. 2004).Among several currents that were described as store-operated in different cells, the CRAC current emerged as the prototype because of its extensive characterization and the weight of evidence showing that it could be activated by ER Ca²⁺ depletion independently of surface receptors or changes in cytosolic [Ca²⁺]. This included activation by intracellular Ca²⁺ chelators, SERCA inhibitors or ionomycin at constant intracellular [Ca²⁺]_i, and by the Ca²⁺ chelator TPEN loaded into the ER (Prakriya et al. 2004; Parekh and Putney 2005). In fact, the CRAC channel is the only store-operated channel whose input–output relation is known. This relation, first examined by Hofer et al. (1998) and later quantified by Luik et al. (2008) using an ER-targeted cameleon protein, shows that I_CRAC is a highly nonlinear function of [Ca²⁺]_ER, with a Hill coefficient of ∼4 and a K_1/2 of 170 µM. Given a resting [Ca²⁺]_ER of ∼400 µM, these results suggest that the ER must be depleted by ∼25% before I_CRAC begins to activate significantly.Over the two decades after Putney formalized the capacitative Ca²⁺ entry hypothesis, many mechanisms were proposed as the link between Ca²⁺ store depletion and SOCE. Among these, diffusible messengers released from the ER, insertion of CRAC channels into the plasma membrane, and conformational coupling of CRAC channels with IP₃ receptors in the ER were the most extensively studied, but in the absence of molecular substrates were difficult to establish (Prakriya et al. 2004; Parekh and Putney 2005). The CRAC channel fingerprint proved useful in ruling out a number of candidate genes for the CRAC channel itself, particularly members of the transient receptor potential (TRP) protein family, but its identity remained a mystery (Prakriya et al. 2004; Parekh and Putney 2005). The discoveries of the ER Ca²⁺ sensor STIM1 in 2005 and the CRAC channel protein Orai1 a year later marked an unmistakable turning point in the field, as they provided the first and most essential molecular tools with which to dissect the SOCE mechanism. The history of these discoveries and the early revelations they afforded have been reviewed extensively (Cahalan et al. 2007; Wu et al. 2007; Fahrner et al. 2009; Putney 2009; Várnai et al. 2009; Hogan et al. 2010). In this review, I will summarize our current understanding of how Ca²⁺ store depletion leads to Ca²⁺ entry at a molecular level, and the role of STIM oligomerization and additional proteins in this process. I will also describe how these discoveries and the ensuing studies have increased awareness of SOCE roles in physiology and disease, and have created entirely new directions for research. Throughout I will emphasize work on STIM1 and Orai1 mainly because they have been the most extensively studied isoforms, but will discuss other STIM and Orai isoforms to highlight important functional differences. For more information on these other isoforms, the reader is referred to the reviews cited above.     

Cadherin-mediated Intercellular Adhesion and Signaling Cascades Involving Small GTPases

Epithelia form physical barriers that separate the internal milieu of the body from its external environment. The biogenesis of functional epithelia requires the precise coordination of many cellular processes. One of the key events in epithelial biogenesis is the establishment of cadherin-dependent cell–cell contacts, which initiate morphological changes and the formation of other adhesive structures. Cadherin-mediated adhesions generate intracellular signals that control cytoskeletal reorganization, polarity, and vesicle trafficking. Among such signaling pathways, those involving small GTPases play critical roles in epithelial biogenesis. Assembly of E-cadherin activates several small GTPases and, in turn, the activated small GTPases control the effects of E-cadherin-mediated adhesions on epithelial biogenesis. Here, we focus on small GTPase signaling at E-cadherin-mediated epithelial junctions.Cell–cell adhesions are involved in a diverse range of physiological processes, including morphological changes during tissue development, cell scattering, wound healing, and synaptogenesis (Adams and Nelson 1998; Gumbiner 2000; Halbleib and Nelson 2006; Takeichi 1995; Tepass et al. 2000). In epithelial cells, cell–cell adhesions are classified into three kinds of adhesions: adherens junction, tight junction, and desmosome (for more details, see Meng and Takeichi 2009, Furuse 2009, and Delva et al. 2009, respectively). A key event in epithelial polarization and biogenesis is the establishment of cadherin-dependent cell–cell contacts. Cadherins belong to a large family of adhesion molecules that require Ca²⁺ for their homophilic interactions (Adams and Nelson 1998; Blanpain and Fuchs 2009; Gumbiner 2000; Hartsock and Nelson 2008; Takeichi 1995; Tepass et al. 2000). Cadherins form transinteraction on the surface of neighboring cells (for details, see Shapiro and Weis 2009). For the development of strong and rigid adhesions, cadherins are clustered concomitantly with changes in the organization of the actin cytoskeleton (Tsukita et al. 1992). Classical cadherins are required, but not sufficient, to initiate cell–cell contacts, and other adhesion protein complexes subsequently assemble (for details, see Green et al. 2009). These complexes include the tight junction, which controls paracellular permeability, and desmosomes, which support the structural continuum of epithelial cells. A fundamental problem is to understand how these diverse cellular processes are regulated and coordinated. Intracellular signals, generated when cells attach with one another, mediate these complicated processes.Several signaling pathways upstream or downstream of cadherin-mediated cell–cell adhesions have been identified (Perez-Moreno et al. 2003) (see also McCrea et al. 2009). Among these pathways, small GTPases including the Rho and Ras family GTPases play critical roles in epithelial biogenesis and have been studied extensively. Many key morphological and functional changes are induced when these small GTPases act at epithelial junctions, where they mediate an interplay between cell–cell adhesion molecules and fundamental cellular processes including cytoskeletal activity, polarity, and vesicle trafficking. In addition to these small GTPases, Ca²⁺ signaling and phosphorylation of cadherin complexes also play pivotal roles in the formation and maintenance of cadherin-mediated adhesions. Here, we focus on signaling pathways involving the small GTPases in E-cadherin-mediated cell–cell adhesions. Other signaling pathways are described in recent reviews (Braga 2002; Fukata and Kaibuchi 2001; Goldstein and Macara 2007; McLachlan et al. 2007; Tsukita et al. 2008; Yap and Kovacs 2003; see also McCrea et al. 2009).     

Molecular Bases of Cell–Cell Junctions Stability and Dynamics

Epithelial cell–cell junctions are formed by apical adherens junctions (AJs), which are composed of cadherin adhesion molecules interacting in a dynamic way with the cortical actin cytoskeleton. Regulation of cell–cell junction stability and dynamics is crucial to maintain tissue integrity and allow tissue remodeling throughout development. Actin filament turnover and organization are tightly controlled together with myosin-II activity to produce mechanical forces that drive the assembly, maintenance, and remodeling of AJs. In this review, we will discuss these three distinct stages in the lifespan of cell–cell junctions, using several developmental contexts, which illustrate how mechanical forces are generated and transmitted at junctions, and how they impact on the integrity and the remodeling of cell–cell junctions.Cell–cell junction formation and remodeling occur repeatedly throughout development. Epithelial cells are linked by apical adherens junctions (AJs) that rely on the cadherin-catenin-actin module. Cadherins, of which epithelial E-cadherin (E-cad) is the most studied, are Ca²⁺-dependent transmembrane adhesion proteins forming homophilic and heterophilic bonds in trans between adjacent cells. Cadherins and the actin cytoskeleton are mutually interdependent (Jaffe et al. 1990; Matsuzaki et al. 1990; Hirano et al. 1992; Oyama et al. 1994; Angres et al. 1996; Orsulic and Peifer 1996; Adams et al. 1998; Zhang et al. 2005; Pilot et al. 2006). This has long been attributed to direct physical interaction of E-cad with β-catenin (β-cat) and of α-catenin (α-cat) with actin filaments (for reviews, see Gumbiner 2005; Leckband and Prakasam 2006; Pokutta and Weis 2007). Recently, biochemical and protein dynamics analyses have shown that such a link may not exist and that instead, a constant shuttling of α-cat between cadherin/β-cat complexes and actin may be key to explain the dynamic aspect of cell–cell adhesion (Drees et al. 2005; Yamada et al. 2005). Regardless of the exact nature of this link, several studies show that AJs are indeed physically attached to actin and that cadherins transmit cortical forces exerted by junctional acto-myosin networks (Costa et al. 1998; Sako et al. 1998; Pettitt et al. 2003; Dawes-Hoang et al. 2005; Cavey et al. 2008; Martin et al. 2008; Rauzi et al. 2008). In addition, physical association depends in part on α-cat (Cavey et al. 2008) and additional intermediates have been proposed to represent alternative missing links (Abe and Takeichi 2008) (reviewed in Gates and Peifer 2005; Weis and Nelson 2006). Although further work is needed to address the molecular nature of cadherin/actin dynamic interactions, association with actin is crucial all throughout the lifespan of AJs. In this article, we will review our current understanding of the molecular mechanisms at work during three different developmental stages of AJs biology: assembly, stabilization, and remodeling, with special emphasis on the mechanical forces controlling AJs integrity and development.     

Ca2+ Signaling During Mammalian Fertilization: Requirements,Players, and Adaptations

Changes in the intracellular concentration of calcium ([Ca²⁺]_i) represent a vital signaling mechanism enabling communication among cells and between cells and the environment. The initiation of embryo development depends on a [Ca²⁺]_i increase(s) in the egg, which is generally induced during fertilization. The [Ca²⁺]_i increase signals egg activation, which is the first stage in embryo development, and that consist of biochemical and structural changes that transform eggs into zygotes. The spatiotemporal patterns of [Ca²⁺]_i at fertilization show variability, most likely reflecting adaptations to fertilizing conditions and to the duration of embryonic cell cycles. In mammals, the focus of this review, the fertilization [Ca²⁺]_i signal displays unique properties in that it is initiated after gamete fusion by release of a sperm-derived factor and by periodic and extended [Ca²⁺]_i responses. Here, we will discuss the events of egg activation regulated by increases in [Ca²⁺]_i, the possible downstream targets that effect these egg activation events, and the property and identity of molecules both in sperm and eggs that underpin the initiation and persistence of the [Ca²⁺]_i responses in these species.An increase in the intracellular concentration of calcium ([Ca²⁺]_i) underlies the initiation, progression and/or completion of a wide variety of cellular processes, including fertilization, muscle contraction, secretion, cell division, and apoptosis (Berridge et al. 2000). To survive and proliferate, cells and organisms must communicate, and changes in [Ca²⁺]_i allow them to quickly respond to environmental, nutritional, or ligand challenges with responses that regulate cell fate and function. Cells devote significant amounts of their energy reserves to create and maintain ionic gradients between extracellular and intracellular milieus and also within the latter, thereby allowing brief alterations in these gradients to have profound signaling effects. In the case of Ca²⁺, myriad proteins have acquired the ability to bind Ca²⁺, which allows them to interpret and transform these elevations into cellular functions. This review will examine the cellular modifications induced by [Ca²⁺]_i changes during fertilization in mature mammalian oocytes, henceforth referred to as eggs.Oocytes during maturation ready themselves for fertilization and the initiation of embryogenesis. During this transition, oocytes undergo changes that include the resumption and progression of meiosis, the development of polyspermy-preventing mechanisms, the reorganization of the cytoskeleton with spindle formation and displacement to the cortex, and the translation, accumulation, and degradation of specific mRNAs and proteins involved in development (Horner and Wolfner 2008b). In most species, and in all mammals, a [Ca²⁺]_i signal is responsible for breaking the meiosis-imposed developmental pause, causing egg activation, which is the first stage of embryo development (Whitaker 2006; Horner and Wolfner 2008b). The egg activating [Ca²⁺]_i signal is generally associated with sperm-egg fusion, which occurs at different stages of meiosis depending on the species (Stricker 1999), although in insects, where fertilization is dissociated from activation and where embryos can develop parthenogenetically, the presumed [Ca²⁺]_i increase is thought to be induced by mechanical stimulation during ovulation/oviductal transport (Page and Orr-Weaver 1997; Horner and Wolfner 2008a).The [Ca²⁺]_i responses that underlie egg activation offer a great deal of diversity regarding their spatiotemporal configuration, reflecting both the plasticity of the Ca²⁺ signaling machinery as well as the dissimilar Ca²⁺ requirements for egg activation among species. Generally speaking, species can be categorized either as displaying a single [Ca²⁺]_i increase, which is the case of sea urchins, starfish, frogs, and fish, or showing multiple [Ca²⁺]_i changes, also known as oscillations, which is the case of nemertian worms, ascidians, and mammals (Stricker 1999; Miyazaki and Ito 2006). Elucidation of the signaling cascades and identification of the molecules/receptor(s) that initiate the Ca²⁺ signal at fertilization has proven elusive, and this review will not dwell on that literature; readers are referred to excellent recent reviews on the subject (Whitaker 2006; Parrington et al. 2007). Nonetheless, research has found that Src-family kinases (SFKs) and phospholipase Cγ (PLCγ) are involved in the activation of the phosphoinositide pathway and production of inositol 1,4,5-trisphosphate (IP₃) during fertilization in sea urchins, starfish, and frogs, which reflects the contribution of a plasma membrane receptor/signaling complex (Giusti et al. 1999; Sato et al. 2000). Remarkably, a receptor responsible for recruiting and activating SFKs during fertilization remains undiscovered (Mahbub Hasan et al. 2005). Similarly, it has proved difficult to uncover how the sperm initiates oscillations. Research now suggests that this may be accomplished by a novel mechanism whereby the signaling molecule/cargo, known as the sperm factor (SF), is released by the sperm into the ooplasm after fusion of the gametes. Importantly, the SF is not IP₃ or Ca²⁺ but rather it contains a protein moiety (Swann 1990; Wu et al. 1997; Kyozuka et al. 1998; Harada et al. 2007). To date, only the mammalian SF’s molecular identity has been resolved, and found to be another member of the PLC family, a novel sperm-specific isoform named PLCζ (Saunders et al. 2002). This review will examine the literature on mammalian PLCζs and will focus as well on the egg molecules that are required to initiate and sustain [Ca²⁺]_i oscillations in these species.     

Biology of the TAM Receptors

The TAM receptors—Tyro3, Axl, and Mer—comprise a unique family of receptor tyrosine kinases, in that as a group they play no essential role in embryonic development. Instead, they function as homeostatic regulators in adult tissues and organ systems that are subject to continuous challenge and renewal throughout life. Their regulatory roles are prominent in the mature immune, reproductive, hematopoietic, vascular, and nervous systems. The TAMs and their ligands—Gas6 and Protein S—are essential for the efficient phagocytosis of apoptotic cells and membranes in these tissues; and in the immune system, they act as pleiotropic inhibitors of the innate inflammatory response to pathogens. Deficiencies in TAM signaling are thought to contribute to chronic inflammatory and autoimmune disease in humans, and aberrantly elevated TAM signaling is strongly associated with cancer progression, metastasis, and resistance to targeted therapies.The name of the TAM family is derived from the first letter of its three constituents—Tyro3, Axl, and Mer (Prasad et al. 2006). As detailed in Figure 1, members of this receptor tyrosine kinase (RTK) family were independently identified by several different groups and appear in the early literature under multiple alternative names. However, Tyro3, Axl, and Mer (officially c-Mer or MerTK for the protein, Mertk for the gene) have now been adopted as the NCBI designations. The TAMs were first grouped into a distinct RTK family (the Tyro3/7/12 cluster) in 1991, through PCR cloning of their kinase domains (Lai and Lemke 1991). The isolation of full-length cDNAs for Axl (O''Bryan et al. 1991), Mer (Graham et al. 1994), and Tyro3 (Lai et al. 1994) confirmed their segregation into a structurally distinctive family of orphan RTKs (Manning et al. 2002b). The two ligands that bind and activate the TAMs—Gas6 and Protein S (Pros1)—were identified shortly thereafter (Ohashi et al. 1995; Stitt et al. 1995; Mark et al. 1996; Nagata et al. 1996).Open in a separate windowFigure 1.TAM receptors and ligands. The TAM receptors (red) are Tyro3 (Lai and Lemke 1991; Lai et al. 1994)—also designated Brt (Fujimoto and Yamamoto 1994), Dtk (Crosier et al. 1994), Rse (Mark et al. 1994), Sky (Ohashi et al. 1994), and Tif (Dai et al. 1994); Axl (O''Bryan et al. 1991)—also designated Ark (Rescigno et al. 1991), Tyro7 (Lai and Lemke 1991), and Ufo (Janssen et al. 1991); and Mer (Graham et al. 1994)—also designated Eyk (Jia and Hanafusa 1994), Nyk (Ling and Kung 1995), and Tyro12 (Lai and Lemke 1991). The TAMs are widely expressed by cells of the mature immune, nervous, vascular, and reproductive systems. The TAM ligands (blue) are Gas6 and Protein S (Pros1). The carboxy-terminal SHBG domains of the ligands bind to the immunoglobulin (Ig) domains of the receptors, induce dimerization, and activate the TAM tyrosine kinases. When γ-carboxylated in a vitamin-K-dependent reaction, the amino-terminal Gla domains of the dimeric ligands bind to the phospholipid phosphatidylserine expressed on the surface on an apposed apoptotic cell or enveloped virus. See text for details. (From Lemke and Burstyn-Cohen 2010; adapted, with permission, from the authors.)Subsequent progress on elucidating the biological roles of the TAM receptors was considerably slower and ultimately required the derivation of mouse loss-of-function mutants (Camenisch et al. 1999; Lu et al. 1999). The fact that Tyro3^−/−, Axl^−/−, and Mer^−/− mice are all viable and fertile permitted the generation of a complete TAM mutant series that included all possible double mutants and even triple mutants that lack all three receptors (Lu et al. 1999). Remarkably, these Tyro3^−/−Axl^−/−Mer^−/− triple knockouts (TAM TKOs) are viable, and for the first 2–3 wk after birth, superficially indistinguishable from their wild-type counterparts (Lu et al. 1999). Because many RTKs play essential roles in embryonic development, even single loss-of-function mutations in RTK genes often result in an embryonic-lethal phenotype (Gassmann et al. 1995; Lee et al. 1995; Soriano 1997; Arman et al. 1998). The postnatal viability of mice in which an entire RTK family is ablated completely—the TAM TKOs can survive for more than a year (Lu et al. 1999)—is therefore highly unusual. Their viability notwithstanding, the TAM mutants go on to develop a plethora of phenotypes, some of them debilitating (Camenisch et al. 1999; Lu et al. 1999; Lu and Lemke 2001; Scott et al. 2001; Duncan et al. 2003; Prasad et al. 2006). Almost without exception, these phenotypes are degenerative in nature and reflect the loss of TAM signaling activities in adult tissues that are subject to regular challenge, renewal, and remodeling. These activities are the subject of this review.     

Origin and Evolution of the Self-Organizing Cytoskeleton in the Network of Eukaryotic Organelles

The eukaryotic cytoskeleton evolved from prokaryotic cytomotive filaments. Prokaryotic filament systems show bewildering structural and dynamic complexity and, in many aspects, prefigure the self-organizing properties of the eukaryotic cytoskeleton. Here, the dynamic properties of the prokaryotic and eukaryotic cytoskeleton are compared, and how these relate to function and evolution of organellar networks is discussed. The evolution of new aspects of filament dynamics in eukaryotes, including severing and branching, and the advent of molecular motors converted the eukaryotic cytoskeleton into a self-organizing “active gel,” the dynamics of which can only be described with computational models. Advances in modeling and comparative genomics hold promise of a better understanding of the evolution of the self-organizing cytoskeleton in early eukaryotes, and its role in the evolution of novel eukaryotic functions, such as amoeboid motility, mitosis, and ciliary swimming.The eukaryotic cytoskeleton organizes space on the cellular scale and this organization influences almost every process in the cell. Organization depends on the mechanochemical properties of the cytoskeleton that dynamically maintain cell shape, position organelles, and macromolecules by trafficking, and drive locomotion via actin-rich cellular protrusions, ciliary beating, or ciliary gliding. The eukaryotic cytoskeleton is best described as an “active gel,” a cross-linked network of polymers (gel) in which many of the links are active motors that can move the polymers relative to each other (Karsenti et al. 2006). Because prokaryotes have only cytoskeletal polymers but lack motor proteins, this “active gel” property clearly sets the eukaryotic cytoskeleton apart from prokaryotic filament systems.Prokaryotes contain elaborate systems of several cytomotive filaments (Löwe and Amos 2009) that share many structural and dynamic features with eukaryotic actin filaments and microtubules (Löwe and Amos 1998; van den Ent et al. 2001). Prokaryotic cytoskeletal filaments may trace back to the first cells and may have originated as higher-order assemblies of enzymes (Noree et al. 2010; Barry and Gitai 2011). These cytomotive filaments are required for the segregation of low copy number plasmids, cell rigidity and cell-wall synthesis, cell division, and occasionally the organization of membranous organelles (Komeili et al. 2006; Thanbichler and Shapiro 2008; Löwe and Amos 2009). These functions are performed by dynamic filament-forming systems that harness the energy from nucleotide hydrolysis to generate forces either via bending or polymerization (Löwe and Amos 2009; Pilhofer and Jensen 2013). Although the identification of actin and tubulin homologs in prokaryotes is a major breakthrough, we are far from understanding the origin of the structural and dynamic complexity of the eukaryotic cytoskeleton.Advances in genome sequencing and comparative genomics now allow a detailed reconstruction of the cytoskeletal components present in the last common ancestor of eukaryotes. These studies all point to an ancestrally complex cytoskeleton, with several families of motors (Wickstead and Gull 2007; Wickstead et al. 2010) and filament-associated proteins and other regulators in place (Jékely 2003; Richards and Cavalier-Smith 2005; Rivero and Cvrcková 2007; Chalkia et al. 2008; Eme et al. 2009; Fritz-Laylin et al. 2010; Eckert et al. 2011; Hammesfahr and Kollmar 2012). Genomic reconstructions and comparative cell biology of single-celled eukaryotes (Raikov 1994; Cavalier-Smith 2013) allow us to infer the cellular features of the ancestral eukaryote. These analyses indicate that amoeboid motility (Fritz-Laylin et al. 2010; although, see Cavalier-Smith 2013), cilia (Cavalier-Smith 2002; Mitchell 2004; Jékely and Arendt 2006; Satir et al. 2008), centrioles (Carvalho-Santos et al. 2010), phagocytosis (Cavalier-Smith 2002; Jékely 2007; Yutin et al. 2009), a midbody during cell division (Eme et al. 2009), mitosis (Raikov 1994), and meiosis (Ramesh et al. 2005) were all ancestral eukaryotic cellular features. The availability of functional information from organisms other than animals and yeasts (e.g., Chlamydomonas, Tetrahymena, Trypanosoma) also allow more reliable inferences about the ancestral functions of cytoskeletal components (i.e., not only their ancestral presence or absence) and their regulation (Demonchy et al. 2009; Lechtreck et al. 2009; Suryavanshi et al. 2010).The ancestral complexity of the cytoskeleton in eukaryotes leaves a huge gap between prokaryotes and the earliest eukaryote we can reconstruct (provided that our rooting of the tree is correct) (Cavalier-Smith 2013). Nevertheless, we can attempt to infer the series of events that happened along the stem lineage, leading to the last common ancestor of eukaryotes. Meaningful answers will require the use of a combination of gene family history reconstructions (Wickstead and Gull 2007; Wickstead et al. 2010), transition analyses (Cavalier-Smith 2002), and computer simulations relevant to cell evolution (Jékely 2008).     

Molecular Mechanisms of Fibroblast Growth Factor Signaling in Physiology and Pathology

Fibroblast growth factors (FGFs) signal in a paracrine or endocrine fashion to mediate a myriad of biological activities, ranging from issuing developmental cues, maintaining tissue homeostasis, and regulating metabolic processes. FGFs carry out their diverse functions by binding and dimerizing FGF receptors (FGFRs) in a heparan sulfate (HS) cofactor- or Klotho coreceptor-assisted manner. The accumulated wealth of structural and biophysical data in the past decade has transformed our understanding of the mechanism of FGF signaling in human health and development, and has provided novel concepts in receptor tyrosine kinase (RTK) signaling. Among these contributions are the elucidation of HS-assisted receptor dimerization, delineation of the molecular determinants of ligand–receptor specificity, tyrosine kinase regulation, receptor cis-autoinhibition, and tyrosine trans-autophosphorylation. These structural studies have also revealed how disease-associated mutations highjack the physiological mechanisms of FGFR regulation to contribute to human diseases. In this paper, we will discuss the structurally and biophysically derived mechanisms of FGF signaling, and how the insights gained may guide the development of therapies for treatment of a diverse array of human diseases.Fibroblast growth factor (FGF) signaling fulfills essential roles in metazoan development and metabolism. A wealth of literature has documented the requirement for FGF signaling in multiple processes during embryogenesis, including implantation (Feldman et al. 1995), gastrulation (Sun et al. 1999), somitogenesis (Dubrulle and Pourquie 2004; Wahl et al. 2007; Lee et al. 2009; Naiche et al. 2011; Niwa et al. 2011), body plan formation (Martin 1998; Rodriguez Esteban et al. 1999; Tanaka et al. 2005; Mariani et al. 2008), morphogenesis (Metzger et al. 2008; Makarenkova et al. 2009), and organogenesis (Goldfarb 1996; Kato and Sekine 1999; Sekine et al. 1999; Sun et al. 1999; Colvin et al. 2001; Serls et al. 2005; Vega-Hernandez et al. 2011). Recent clinical and biochemical data have uncovered unexpected roles for FGF signaling in metabolic processes, including phosphate/vitamin D homeostasis (Consortium 2000; Razzaque and Lanske 2007; Nakatani et al. 2009; Gattineni et al. 2011; Kir et al. 2011), cholesterol/bile acid homeostasis (Yu et al. 2000a; Holt et al. 2003), and glucose/lipid metabolism (Fu et al. 2004; Moyers et al. 2007). Highlighting its diverse biology, deranged FGF signaling contributes to many human diseases, such as congenital craniosynostosis and dwarfism syndromes (Naski et al. 1996; Wilkie et al. 2002, 2005), Kallmann syndrome (Dode et al. 2003; Pitteloud et al. 2006a), hearing loss (Tekin et al. 2007, 2008), and renal phosphate wasting disorders (Shimada et al. 2001; White et al. 2001), as well as many acquired forms of cancers (Rand et al. 2005; Pollock et al. 2007; Gartside et al. 2009; di Martino et al. 2012). Endocrine FGFs have also been implicated in the progression of acquired metabolic disorders, including chronic kidney disease (Fliser et al. 2007), obesity (Inagaki et al. 2007; Moyers et al. 2007; Reinehr et al. 2012), and insulin resistance (Fu et al. 2004; Chen et al. 2008b; Chateau et al. 2010; Huang et al. 2011), giving rise to many opportunities for drug discovery in the field of FGF biology (Beenken and Mohammadi 2012).Based on sequence homology and phylogeny, the 18 mammalian FGFs are grouped into six subfamilies (Ornitz and Itoh 2001; Popovici et al. 2005; Itoh and Ornitz 2011). Five of these subfamilies act in a paracrine fashion, namely, the FGF1 subfamily (FGF1 and FGF2), the FGF4 subfamily (FGF4, FGF5, and FGF6), the FGF7 subfamily (FGF3, FGF7, FGF10, and FGF22), the FGF8 subfamily (FGF8, FGF17, and FGF18), and the FGF9 subfamily (FGF9, FGF16, and FGF20). In contrast, the FGF19 subfamily (FGF19, FGF21, and FGF23) signals in an endocrine manner (Beenken and Mohammadi 2012). FGFs exert their pleiotropic effects by binding and activating the FGF receptor (FGFR) subfamily of receptor tyrosine kinases that are coded by four genes (FGFR1, FGFR2, FGFR3, and FGFR4) in mammals (Johnson and Williams 1993; Mohammadi et al. 2005b). The extracellular domain of FGFRs consists of three immunoglobulin (Ig)-like domains (D1, D2, and D3), and the intracellular domain harbors the conserved tyrosine kinase domain flanked by the flexible amino-terminal juxtamembrane linker and carboxy-terminal tail (Lee et al. 1989; Dionne et al. 1991; Givol and Yayon 1992). A unique feature of FGFRs is the presence of a contiguous segment of glutamic and aspartic acids in the D1–D2 linker, termed the acid box (AB). The two-membrane proximal D2 and D3 and the intervening D2–D3 linker are necessary and sufficient for ligand binding/specificity (Dionne et al. 1990; Johnson et al. 1990), whereas D1 and the D1–D2 linker are implicated in receptor autoinhibition (Wang et al. 1995; Roghani and Moscatelli 2007; Kalinina et al. 2012). Alternative splicing and translational initiation further diversify both ligands and receptors. The amino-terminal regions of FGF8 and FGF17 can be differentially spliced to yield FGF8a, FGF8b, FGF8e, FGF8f (Gemel et al. 1996; Blunt et al. 1997), and FGF17a and FGF17b isoforms (Xu et al. 1999), whereas cytosine-thymine-guanine (CTG)-mediated translational initiation gives rise to multiple high molecular weight isoforms of FGF2 and FGF3 (Florkiewicz and Sommer 1989; Prats et al. 1989; Acland et al. 1990). The tissue-specific alternative splicing in D3 of FGFR1, FGFR2, and FGFR3 yields “b” and “c” receptor isoforms which, along with their temporal and spatial expression patterns, is the major regulator of FGF–FGFR specificity/promiscuity (Orr-Urtreger et al. 1993; Ornitz et al. 1996; Zhang et al. 2006). A large body of structural data on FGF–FGFR complexes has begun to reveal the intricate mechanisms by which different FGFs and FGFRs combine selectively to generate quantitatively and qualitatively different intracellular signals, culminating in distinct biological responses. In addition, these structural data have unveiled how pathogenic mutations hijack the normal physiological mechanisms of FGFR regulation to lead to pathogenesis. We will discuss the current state of the structural biology of the FGF–FGFR system, lessons learned from studying the mechanism of action of pathogenic mutations, and how the structural data are beginning to shape and advance the translational research.     

Studying Signal Transduction in Single Dendritic Spines

Many forms of synaptic plasticity are triggered by biochemical signaling that occurs in small postsynaptic compartments called dendritic spines, each of which typically houses the postsynaptic terminal associated with a single glutamatergic synapse. Recent advances in optical techniques allow investigators to monitor biochemical signaling in single dendritic spines and thus reveal the signaling mechanisms that link synaptic activity and the induction of synaptic plasticity. This is mostly in the study of Ca²⁺-dependent forms of synaptic plasticity for which many of the steps between Ca²⁺ influx and changes to the synapse are now known. This article introduces the new techniques used to investigate signaling in single dendritic spines and the neurobiological insights that they have produced.Each neuron typically receives 1000–10,000 synaptic inputs and sends information to an axon, which branches to produce a similar number of synaptic outputs. Most excitatory postsynaptic terminals are associated with dendritic spines, small protrusions emanating from the dendritic surface (Nimchinsky et al. 2002; Alvarez and Sabatini 2007). Each spine has a volume of ∼0.1 femtoliter, and connects to the parent dendrite through a narrow neck, which acts as a diffusion barrier and compartmentalizes biochemical reactions. Ca²⁺ influx into spines initiates a cascade of biochemical signals leading to various forms of synaptic plasticity including long-term potentiation (LTP).Because LTP in hippocampal CA1 pyramidal neurons is a cellular mechanism that may underlie long-term memory formation, the signal transduction underlying LTP has been extensively studied by pharmacological and genetic methods (Bliss and Collingridge 1993; Derkach et al. 2007). It is now well established that LTP is induced by Ca²⁺ influx into dendritic spines through NMDA-type glutamate receptors (NMDARs), which induces the insertion of AMPA-type glutamate receptors (AMPARs) into the synapse, thereby increasing the sensitivity of the postsynaptic terminal to glutamate (Derkach et al. 2007; Kessels and Malinow 2009). An increase of release probability during LTP has also been reported (Enoki et al. 2009), and thus both pre- and postsynaptic mechanisms may contribute to LTP (Lisman and Raghavachari 2006).Manipulations of signal transduction using specific pharmacological inhibitors or genetic perturbations have identified many signaling pathways that connect Ca²⁺ to LTP induction. For example, LTP requires the activation of many signaling proteins, including Ca²⁺/calmodulin-dependent kinase II (CaMKII), extracellular signal-related kinase (ERK), Phoshoinositide 3 kinase (PI3K), protein kinase A and C, and GTPases such as Ras, Rab, and Rho (Kennedy et al. 2005). The list is continually growing, and the hundreds of implicated proteins form a complex signaling network whose contribution to LTP is still unclear (Bromberg et al. 2008).Signaling dynamics in neurons have traditionally been measured using biochemical analyses (Bromberg et al. 2008). However, the spatiotemporal resolution of conventional biochemistry is limited, restricting analysis to the time scale of many minutes and requiring the homogenization of tissue containing millions of synapses and other cellular elements. Furthermore, resolving synaptically induced changes in signaling by biochemical analysis typically requires stimulating many synapses at the same time, which may produce unintended effects, for instance, excitotoxicity or homeostatic plasticity.The size of dendritic spines is similar to the resolution of an optical microscope, permitting the optical analysis of biochemical signaling in each dendritic spine (Svoboda and Yasuda 2006). In particular, the advent of two-photon-based FRET techniques and the development of appropriate fluorescent reporters of specific biochemical reactions (see below) have provided readouts for signal transduction with high spatiotemporal resolution in live brain tissue (Svoboda and Yasuda 2006; Yasuda 2006). This has provided detailed information about the dynamics of signal transduction in spines and dendrites, and insights into the molecular mechanisms of synaptic plasticity.     

Origin of Microglia: Current Concepts and Past Controversies

Microglia are the resident macrophages of the central nervous system (CNS), which sit in close proximity to neural structures and are intimately involved in brain homeostasis. The microglial population also plays fundamental roles during neuronal expansion and differentiation, as well as in the perinatal establishment of synaptic circuits. Any change in the normal brain environment results in microglial activation, which can be detrimental if not appropriately regulated. Aberrant microglial function has been linked to the development of several neurological and psychiatric diseases. However, microglia also possess potent immunoregulatory and regenerative capacities, making them attractive targets for therapeutic manipulation. Such rationale manipulations will, however, require in-depth knowledge of their origins and the molecular mechanisms underlying their homeostasis. Here, we discuss the latest advances in our understanding of the origin, differentiation, and homeostasis of microglial cells and their myelomonocytic relatives in the CNS.Microglia are the resident macrophages of the central nervous system (CNS), which are uniformly distributed throughout the brain and spinal cord with increased densities in neuronal nuclei, including the Substantia nigra in the midbrain (Lawson et al. 1990; Perry 1998). They belong to the nonneuronal glial cell compartment and their function is crucial to maintenance of the CNS in both health and disease (Ransohoff and Perry 2009; Perry et al. 2010; Ransohoff and Cardona 2010; Prinz and Priller 2014).Two key functional features define microglia: immune defense and maintenance of CNS homeostasis. As part of the innate immune system, microglia constantly sample their environment, scanning and surveying for signals of external danger (Davalos et al. 2005; Nimmerjahn et al. 2005; Lehnardt 2010), such as those from invading pathogens, or internal danger signals generated locally by damaged or dying cells (Bessis et al. 2007; Hanisch and Kettenmann 2007). Detection of such signals initiates a program of microglial responses that aim to resolve the injury, protect the CNS from the effects of the inflammation, and support tissue repair and remodeling (Minghetti and Levi 1998; Goldmann and Prinz 2013).Microglia are also emerging as crucial contributors to brain homeostasis through control of neuronal proliferation and differentiation, as well as influencing formation of synaptic connections (Lawson et al. 1990; Perry 1998; Hughes 2012; Blank and Prinz 2013). Recent imaging studies revealed dynamic interactions between microglia and synaptic connections in the healthy brain, which contributed to the modification and elimination of synaptic structures (Perry et al. 2010; Tremblay et al. 2010; Bialas and Stevens 2013). In the prenatal brain, microglia regulate the wiring of forebrain circuits, controlling the growth of dopaminergic axons in the forebrain and the laminar positioning of subsets of neocortical interneurons (Squarzoni et al. 2014). In the postnatal brain, microglia-mediated synaptic pruning is similarly required for the remodeling of neural circuits (Paolicelli et al. 2011; Schafer et al. 2012). In summary, microglia occupy a central position in defense and maintenance of the CNS and, as a consequence, are a key target for the treatment of neurological and psychiatric disorders.Although microglia have been studied for decades, a long history of experimental misinterpretation meant that their true origins remained debated until recently. Although we knew that microglial progenitors invaded the brain rudiment at very early stages of embryonic development (Alliot et al. 1999; Ransohoff and Perry 2009), it has now been established that microglia arise from yolk sac (YS)-primitive macrophages, which persist in the CNS into adulthood (Davalos et al. 2005; Nimmerjahn et al. 2005; Ginhoux et al. 2010, 2013; Kierdorf and Prinz 2013; Kierdorf et al. 2013a). Moreover, early embryonic brain colonization by microglia is conserved across vertebrate species, implying that it is essential for early brain development (Herbomel et al. 2001; Bessis et al. 2007; Hanisch and Kettenmann 2007; Verney et al. 2010; Schlegelmilch et al. 2011; Swinnen et al. 2013). In this review, we will present the latest findings in the field of microglial ontogeny, which provide new insights into their roles in health and disease.     

A Ca2+ Release-activated Ca2+ (CRAC) Modulatory Domain (CMD) within STIM1 Mediates Fast Ca2+-dependent Inactivation of ORAI1 Channels

STIM1 and ORAI1, the two limiting components in the Ca²⁺ release-activated Ca²⁺ (CRAC) signaling cascade, have been reported to interact upon store depletion, culminating in CRAC current activation. We have recently identified a modulatory domain between amino acids 474 and 485 in the cytosolic part of STIM1 that comprises 7 negatively charged residues. A STIM1 C-terminal fragment lacking this domain exhibits enhanced interaction with ORAI1 and 2–3-fold higher ORAI1/CRAC current densities. Here we focused on the role of this CRAC modulatory domain (CMD) in the fast inactivation of ORAI1/CRAC channels, utilizing the whole-cell patch clamp technique. STIM1 mutants either with C-terminal deletions including CMD or with 7 alanines replacing the negative amino acids within CMD gave rise to ORAI1 currents that displayed significantly reduced or even abolished inactivation when compared with STIM1 mutants with preserved CMD. Consistent results were obtained with cytosolic C-terminal fragments of STIM1, both in ORAI1-expressing HEK 293 cells and in RBL-2H3 mast cells containing endogenous CRAC channels. Inactivation of the latter, however, was much more pronounced than that of ORAI1. The extent of inactivation of ORAI3 channels, which is also considerably more prominent than that of ORAI1, was also substantially reduced by co-expression of STIM1 constructs missing CMD. Regarding the dependence of inactivation on Ca²⁺, a decrease in intracellular Ca²⁺ chelator concentrations promoted ORAI1 current fast inactivation, whereas Ba²⁺ substitution for extracellular Ca²⁺ completely abrogated it. In summary, CMD within the STIM1 cytosolic part provides a negative feedback signal to Ca²⁺ entry by triggering fast Ca²⁺-dependent inactivation of ORAI/CRAC channels.The Ca²⁺ release-activated Ca²⁺ (CRAC)⁵ channel is one of the best characterized store-operated entry pathways (1–7). Substantial efforts have led to identification of two key components of the CRAC channel machinery: the stromal interaction molecule 1 (STIM1), which is located in the endoplasmic reticulum and acts as a Ca²⁺ sensor (8–10), and ORAI1/CRACM1, the pore-forming subunit of the CRAC channel (11–13). Besides ORAI1, two further homologues named ORAI2 and ORAI3 belong to the ORAI channel family (12, 14).STIM1 senses endoplasmic reticulum store depletion primarily by its luminal EF-hand in its N terminus (8, 15), redistributes close to the plasma membrane, where it forms puncta-like structures, and co-clusters with ORAI1, leading to inward Ca²⁺ currents (12, 16–19). The STIM1 C terminus, located in the cytosol, contains two coiled-coil regions overlapping with an ezrin-radixin-moesin (ERM)-like domain followed by a serine/proline- and a lysine-rich region (2, 8, 20–22). Three recent studies have described the essential ORAI-activating region within the ERM domain, termed SOAR (Stim ORAI-activating region) (23), OASF (ORAI-activating small fragment) (24), and CAD (CRAC-activating domain) (25), including the second coiled coil domain and the following ∼55 amino acids. We and others have provided evidence that store depletion leads to a dynamic coupling of STIM1 to ORAI1 (26–28) that is mediated by a direct interaction of the STIM1 C terminus with ORAI1 C terminus probably involving the putative coiled-coil domain in the latter (27).Furthermore, different groups have proven that the C terminus of STIM1 is sufficient to activate CRAC as well as ORAI1 channels independent of store depletion (22–25, 27, 29). We have identified that OASF-(233–474) or shorter fragments exhibit further enhanced coupling to ORAI1 resulting in 3-fold increased constitutive Ca²⁺ currents. A STIM1 fragment containing an additional cluster of anionic amino acids C-terminal to position 474 displays weaker interaction with ORAI1 as well as reduced Ca²⁺ current comparable with that mediated by wild-type STIM1 C terminus. Hence, we have suggested that these 11 amino acids (474–485) act in a modulatory manner onto ORAI1; however, their detailed mechanistic impact within the STIM1/ORAI1 signaling machinery has remained so far unclear.In this study, we focused on the impact of this negative cluster on fast inactivation of STIM1-mediated ORAI Ca²⁺ currents. Lis et al. (30) have shown that all three ORAI homologues display distinct inactivation profiles, where ORAI2 and ORAI3 show a much more pronounced fast inactivation than ORAI1. Moreover, it has been reported (31) that different expression levels of STIM1 to ORAI1 affect the properties of CRAC current inactivation. Yamashita et al. (32) have demonstrated a linkage between the selectivity filter of ORAI1 and its Ca²⁺-dependent fast inactivation. Here we provide evidence that a cluster of acidic residues within the C terminus of STIM1 is involved in the fast inactivation of ORAI1 and further promotes that of ORAI3 and native CRAC currents.     

Quality Control of Mitochondrial Proteostasis

A decline in mitochondrial activity has been associated with aging and is a hallmark of many neurological diseases. Surveillance mechanisms acting at the molecular, organellar, and cellular level monitor mitochondrial integrity and ensure the maintenance of mitochondrial proteostasis. Here we will review the central role of mitochondrial chaperones and proteases, the cytosolic ubiquitin-proteasome system, and the mitochondrial unfolded response in this interconnected quality control network, highlighting the dual function of some proteases in protein quality control within the organelle and for the regulation of mitochondrial fusion and mitophagy.In all cellular compartments, correct protein folding is critical to maintain cellular homeostasis. In cases where proteins become misfolded or damaged, it is imperative that they are turned over and removed to prevent the formation of toxic folding intermediates or the accumulation of aggregates to levels that can be deleterious for the cell. Several neurodegenerative diseases share a common pathogenic mechanism, which involves the formation of fibrillar aggregates of a particular protein that can accumulate in the cytosol, the nucleus, or the mitochondria. Examples of this include accumulation of the amyloid-β peptide in Alzheimer’s disease (Kayed et al. 2003; Tanzi and Bertram 2005), accumulation of α-synuclein in Parkinson’s disease (Spillantini et al. 1997; Zarranz et al. 2004), and aggregation of a mutant form of the huntingtin protein caused by extended polyglutamine stretches in Huntington’s disease (DiFiglia et al. 1997). Although the exact mechanism of pathogenesis for these diseases remains unresolved, mitochondrial dysfunction is implicated in their progression, which may in turn be responsible for the loss of neurological cell populations because of their sensitivity and requirement for functional mitochondria (Rodolfo et al. 2010).The evolution of mitochondria began approximately 1.5 billion years ago after an α-proteobacterium was engulfed by a preeukaryotic cell (Gray et al. 1999). Since that time, mitochondria have retained two phospholipid bilayers that segregate two aqueous compartments, the mitochondrial intermembrane space (IMS) and the mitochondrial matrix (Palade 1953). Mitochondria are found in essentially all eukaryotic cells and play integral roles in a number of the cell''s metabolic pathways. For example, mitochondria are the key players in cellular ATP production through an elaborate respiratory chain network found in the organelles inner membrane (IM) (Mitchell 1961; Leonard and Schapira 2000). Mitochondria are also required for the β-oxidation of fatty acids, Fe-S biosynthesis, and Ca²⁺ homeostasis (Pinton et al. 1998; Rizzuto et al. 2000; Lill 2009; Modre-Osprian et al. 2009). Moreover, mitochondria are key regulators of programmed cell death and they participate in developmental processes as well as aging (Singh 2004; Green 2005).In contrast to early depictions of mitochondria as singular kidney bean shaped entities, it is now well established that mitochondria form elaborate, reticular networks in many tissues (Bereiter-Hahn 1990). The ability of mitochondria to form such networks arises from two major factors: (1) Specialized machineries in the mitochondrial outer membrane (OM) and the IM allow mitochondria to fuse and divide and (2) mitochondria are able to be shuttled along cytoskeletal elements (Anesti and Scorrano 2006; Hoppins et al. 2007). This plasticity of mitochondria ensures that they are able to respond to different cellular cues, which is potentially important for their numerous functions. In different cell types, mitochondria adopt varying morphologies (Kuznetsov et al. 2009). For example, in cultured fibroblasts mitochondria form extensive reticular networks, whereas in neuronal cells, mitochondria can be found enriched at areas of high-energy demand, including presynaptic termini, axon initial segments, and growth cones. Furthermore, in muscle cells, mitochondria adopt a very uniform intermyofibrillar conformation (Vendelin et al. 2005). The dynamic nature of mitochondria provides an explanation as to how they adopt varying organizations in different cell populations. The importance of mitochondrial networks is highlighted by the fact that mutations in components involved in maintaining mitochondrial dynamics results in neurodegenerative diseases (Chan 2006; Olichon et al. 2006; Knott et al. 2008; Martinelli and Rugarli 2010; Winklhofer and Haass 2010).     

The Diversity of Calcium Sensor Proteins in the Regulation of Neuronal Function

Calcium signaling in neurons as in other cell types mediates changes in gene expression, cell growth, development, survival, and cell death. However, neuronal Ca²⁺ signaling processes have become adapted to modulate the function of other important pathways including axon outgrowth and changes in synaptic strength. Ca²⁺ plays a key role as the trigger for fast neurotransmitter release. The ubiquitous Ca²⁺ sensor calmodulin is involved in various aspects of neuronal regulation. The mechanisms by which changes in intracellular Ca²⁺ concentration in neurons can bring about such diverse responses has, however, become a topic of widespread interest that has recently focused on the roles of specialized neuronal Ca²⁺ sensors. In this article, we summarize synaptotagmins in neurotransmitter release, the neuronal roles of calmodulin, and the functional significance of the NCS and the CaBP/calneuron protein families of neuronal Ca²⁺ sensors.Calcium signaling in many cell types can mediate changes in gene expression, cell growth, development, survival, and cell death. However, neuronal calcium signaling processes have become adapted to modulate the function of important pathways in the brain, including neuronal survival, axon outgrowth, and changes in synaptic strength. Changes in the concentration of intracellular free Ca²⁺ ([Ca²⁺]i) are essential for the transmission of information through the nervous system as the trigger for neurotransmitter release at synapses. In addition, alterations in [Ca²⁺]i can lead to a wide range of different physiological changes that can modify neuronal functions over time scales of milliseconds through tens of minutes to days or longer (Berridge 1998). Many of these processes have been shown to be dependent upon the particular route of Ca²⁺ entry into the cell. It has long been known that the physiological outcome from a change in [Ca²⁺]i depends on its location, amplitude, and duration. The importance of location becomes even more pronounced in neurons because of their complex and extended morphologies. [Ca²⁺]i also regulates neuronal development and neuronal survival (Spitzer 2006). In addition, modifications to Ca²⁺ signaling pathways have been suggested to underlie various neuropathological disorders (Braunewell 2005; Berridge 2010).Highly localized Ca²⁺ elevations (Augustine et al. 2003) formed following Ca²⁺ entry though voltage-gated Ca²⁺ channels (VGCCs) lead to synaptic vesicle fusion with the presynaptic membrane and thereby allow neurotransmitter release within less than a millisecond. Differently localized and timed Ca²⁺ signals can, for example, result in changes to the properties of the VGCCs (Catterall and Few 2008) or lead to changes in gene expression (Bito et al. 1997). Postsynaptic Ca²⁺ signals arising from activation of NMDA receptors give rise to two important processes in synaptic plasticity, long term potentiation (LTP) and long term depression (LTD). LTP and LTD are examples of the way synaptic transmission can change synaptic efficacy and are thought to be important in modulating learning and memory. Importantly, the Ca²⁺ signals that bring about either LTP or LTD differ only in their timing and duration. LTP is triggered by Ca²⁺ signals on the micromolar scale for shorter durations, whereas LTD is triggered by changes in [Ca²⁺]i on the nanomolar scale for longer durations (Yang et al. 1999). Specific Ca²⁺ signals are likely to be decoded by different Ca²⁺ sensor proteins. These are proteins that undergo a conformational change on Ca²⁺ binding and then interact with and regulate various target proteins. Among those Ca²⁺ sensors that are important for neuronal function are the synaptotagmins that control neurotransmitter release (Chapman 2008), the ubiquitous EF-hand containing sensor calmodulin that has many neuronal roles, and the more recently discovered neuronal EF-hand containing proteins, including the neuronal calcium sensor (NCS) protein (Burgoyne 2007) and the calcium-binding protein (CaBP)/calneuron (Haeseleer et al. 2002) families. We will briefly review synaptotagmins and the neuronal functions of calmodulin but concentrate on the NCS and CaBP families of Ca²⁺ sensors.     

Functional Ryanodine Receptors in the Plasma Membrane of RINm5F Pancreatic ��-Cells

Ryanodine receptors (RyR) are Ca²⁺ channels that mediate Ca²⁺ release from intracellular stores in response to diverse intracellular signals. In RINm5F insulinoma cells, caffeine, and 4-chloro-m-cresol (4CmC), agonists of RyR, stimulated Ca²⁺ entry that was independent of store-operated Ca²⁺ entry, and blocked by prior incubation with a concentration of ryanodine that inactivates RyR. Patch-clamp recording identified small numbers of large-conductance (γ_K = 169 pS) cation channels that were activated by caffeine, 4CmC or low concentrations of ryanodine. Similar channels were detected in rat pancreatic β-cells. In RINm5F cells, the channels were blocked by cytosolic, but not extracellular, ruthenium red. Subcellular fractionation showed that type 3 IP₃ receptors (IP₃R3) were expressed predominantly in endoplasmic reticulum, whereas RyR2 were present also in plasma membrane fractions. Using RNAi selectively to reduce expression of RyR1, RyR2, or IP₃R3, we showed that RyR2 mediates both the Ca²⁺ entry and the plasma membrane currents evoked by agonists of RyR. We conclude that small numbers of RyR2 are selectively expressed in the plasma membrane of RINm5F pancreatic β-cells, where they mediate Ca²⁺ entry.Ryanodine receptors (RyR)³ and inositol 1,4,5-trisphosphate receptors (IP₃R) (1, 2) are the archetypal intracellular Ca²⁺ channels. Both are widely expressed, although RyR are more restricted in their expression than IP₃R (3, 4). In common with many cells, pancreatic β-cells and insulin-secreting cell lines express both IP₃R (predominantly IP₃R3) (5, 6) and RyR (predominantly RyR2) (7). Both RyR and IP₃R are expressed mostly within membranes of the endoplasmic (ER), where they mediate release of Ca²⁺. Functional RyR are also expressed in the secretory vesicles (8, 9) or, and perhaps more likely, in the endosomes of β-cells (10). Despite earlier suggestions (11), IP₃R are probably not present in the secretory vesicles of β-cells (8, 12, 13).All three subtypes of IP₃R are stimulated by IP₃ with Ca²⁺ (1), and the three subtypes of RyR are each directly regulated by Ca²⁺. However, RyR differ in whether their most important physiological stimulus is depolarization of the plasma membrane (RyR1), Ca²⁺ (RyR2) or additional intracellular messengers like cyclic ADP-ribose. The latter stimulates both Ca²⁺ release and insulin secretion in β-cells (8, 14). The activities of both families of intracellular Ca²⁺ channels are also modulated by many additional signals that act directly or via phosphorylation (15, 16). Although they commonly mediate release of Ca²⁺ from the ER, both IP₃R and RyR select rather poorly between Ca²⁺ and other cations (permeability ratio, P_Ca/P_K ∼7) (1, 17). This may allow electrogenic Ca²⁺ release from the ER to be rapidly compensated by uptake of K⁺ (18), and where RyR or IP₃R are expressed in other membranes it may allow them to affect membrane potential.Both Ca²⁺ entry and release of Ca²⁺ from intracellular stores contribute to the oscillatory increases in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) that stimulate exocytosis of insulin-containing vesicles in pancreatic β-cells (7). Glucose rapidly equilibrates across the plasma membrane (PM) of β-cells and its oxidative metabolism by mitochondria increases the cytosolic ATP/ADP ratio, causing K_ATP channels to close (19). This allows an unidentified leak current to depolarize the PM (20) and activate voltage-gated Ca²⁺ channels, predominantly L-type Ca²⁺ channels (21). The resulting Ca²⁺ entry is amplified by Ca²⁺-induced Ca²⁺ release from intracellular stores (7), triggering exocytotic release of insulin-containing dense-core vesicles (22). The importance of this sequence is clear from the widespread use of sulfonylurea drugs, which close K_ATP channels, in the treatment of type 2 diabetes. Ca²⁺ uptake by mitochondria beneath the PM further stimulates ATP production, amplifying the initial response to glucose and perhaps thereby contributing to the sustained phase of insulin release (23). However, neither the increase in [Ca²⁺]_i nor the insulin release evoked by glucose or other nutrients is entirely dependent on Ca²⁺ entry (7, 24) or closure of K_ATP channels (25). This suggests that glucose metabolism may also more directly activate RyR (7, 26) and/or IP₃R (27) to cause release of Ca²⁺ from intracellular stores. A change in the ATP/ADP ratio is one means whereby nutrient metabolism may be linked to opening of intracellular Ca²⁺ channels because both RyR (28) and IP₃R (1) are stimulated by ATP.The other major physiological regulators of insulin release are the incretins: glucagon-like peptide-1 and glucose-dependent insulinotropic hormone (29). These hormones, released by cells in the small intestine, stimulate synthesis of cAMP in β-cells and thereby potentiate glucose-evoked insulin release (30). These pathways are also targets of drugs used successfully to treat type 2 diabetes (29). The responses of β-cells to cAMP involve both cAMP-dependent protein kinase and epacs (exchange factors activated by cAMP) (31, 32). The effects of the latter are, at least partly, due to release of Ca²⁺ from intracellular stores via RyR (33–35) and perhaps also via IP₃R (36). The interplays between Ca²⁺ and cAMP signaling generate oscillatory changes in the concentrations of both messengers (37). RyR and IP₃R are thus implicated in mediating responses to each of the major physiological regulators of insulin secretion: glucose and incretins.Here we report that in addition to expression in intracellular stores, which probably include both the ER and secretory vesicles and/or endosomes, functional RyR2 are also expressed in small numbers in the PM of RINm5F insulinoma cells and rat pancreatic β-cells.     

Glial Cell Development and Function in Zebrafish

The zebrafish is a premier vertebrate model system that offers many experimental advantages for in vivo imaging and genetic studies. This review provides an overview of glial cell types in the central and peripheral nervous system of zebrafish. We highlight some recent work that exploited the strengths of the zebrafish system to increase the understanding of the role of Gpr126 in Schwann cell myelination and illuminate the mechanisms controlling oligodendrocyte development and myelination. We also summarize similarities and differences between zebrafish radial glia and mammalian astrocytes and consider the possibility that their distinct characteristics may represent extremes in a continuum of cell identity. Finally, we focus on the emergence of zebrafish as a model for elucidating the development and function of microglia. These recent studies have highlighted the power of the zebrafish system for analyzing important aspects of glial development and function.Following the pioneering work of George Streisinger in the early 1980s, the zebrafish has emerged as a premier vertebrate model system (Streisinger et al. 1981). A key strength of the zebrafish is that the embryos and early larvae are transparent, allowing exquisite cellular analysis of many dynamic processes, including cell migration, axonal pathfinding, and myelination, among many others (e.g., Gilmour et al. 2002; Lyons et al. 2005; Czopka et al. 2013). The zebrafish also has many advantages for large-scale genetic studies, including relatively small size and rapid development, high fecundity, and the ability to manipulate the ploidy of gametes and early embryos (Kimmel 1989). Through the 1980s and early 1990s, insightful studies of several interesting mutations elegantly exploited these experimental advantages (e.g., Kimmel et al. 1989; Ho and Kane 1990; Hatta et al. 1991; Grunwald and Eisen 2002), attracting many researchers from other fields to the zebrafish system. Following the explosion of interest in the zebrafish in the 1990s, advances in many areas have added to the strengths of the system, including large-scale screens that identified thousands of new mutations (Driever et al. 1996; Haffter et al. 1996), rapid transgenesis (Kawakami et al. 2004), new methods for imaging and tracking all cells during development (Huisken 2012), genetic mapping and sequencing to identify genes and mutated loci (Postlethwait et al. 1994; Howe et al. 2013), optogenetic methods to control neural activity (Portugues et al. 2013), the advent of targeted nucleases to create mutations in genes of interest (Huang et al. 2011; Sander et al. 2011; Bedell et al. 2012; Chang et al. 2013; Hwang et al. 2013), and small molecule screening approaches to isolate compounds with novel biological activities in vivo (Peterson and Fishman 2011).Many fundamental similarities in physiology and body plan unite the zebrafish and other vertebrates (Kimmel 1989). In addition, analysis of genes and genomes has revealed that sequence, expression, and function of many genes are conserved among zebrafish and other vertebrates (Postlethwait and Talbot 1997; Howe et al. 2013). Thus, insights from studies in zebrafish will apply broadly to other vertebrates, including humans. On the other hand, there are important genetic, genomic, and physiological differences among vertebrates. It is, therefore, important to keep possible differences in mind and to recognize that analyzing the diversity among different species may enhance overall understanding of important processes. For example, zebrafish and other teleosts have a much more extensive regenerative ability than mammals, so that studies of fin, heart, and spinal cord regeneration in zebrafish may suggest avenues toward new therapeutic approaches in humans (Gemberling et al. 2013; Becker and Becker 2014).In this review, we provide an overview of different types of glia in the zebrafish, with a focus on some recent studies that highlight the power of the zebrafish system to analyze different aspects of glial development and function.     

Functional Differentiation of Adult-Born Neurons along the Septotemporal Axis of the Dentate Gyrus

Over the past several decades, the proliferation and integration of adult-born neurons into existing hippocampal circuitry has been implicated in a wide range of behaviors, including novelty recognition, pattern separation, spatial learning, anxiety behaviors, and antidepressant response. In this review, we suggest that the diversity in behavioral requirements for new neurons may be partly caused by separate functional roles of individual neurogenic niches. Growing evidence shows that the hippocampal formation can be compartmentalized not only along the classic trisynaptic circuit, but also along a longitudinal septotemporal axis. We suggest that subpopulations of hippocampal adult-born neurons may be specialized for distinct mnemonic- or mood-related behavioral tasks. We will examine the literature supporting a functional and anatomical dissociation of the hippocampus along the longitudinal axis and discuss techniques to functionally dissect the roles of adult-born hippocampal neurons in these distinct subregions.Since the presence of dividing cells in the mostly postmitotic adult brain was first described (Altman and Das 1965), the generation of new neurons in adulthood has been proposed to be involved in a variety of behaviors (Doetsch and Hen 2005; Becker and Wojtowicz 2007; Sahay and Hen 2007; Deng et al. 2010; Ming and Song 2011; Miller and Hen 2014). Adult neurogenesis in the healthy mammalian brain is consistently seen in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG). Recent studies have implicated hippocampal neurogenesis in learning- and memory-related tasks, such as contextual discrimination and spatial navigation and, specifically, in behavioral pattern separation (Clelland et al. 2009; Sahay et al. 2011; Nakashiba et al. 2012; Niibori et al. 2012; see also reviews in Deng et al. 2010; Ming and Song 2011; Marin-Burgin and Schinder 2012), but also in some behavioral effects of antidepressants (Santarelli et al. 2003; see also reviews in Sahay and Hen 2007; Kheirbek et al. 2012; Tanti and Belzung 2013). However, the exact role of adult hippocampal neurogenesis in some of these behaviors has been debated as some studies have shown no effects of altering adult neurogenesis on spatial navigation or antidepressant response. Proposed explanations have included differences in the behavioral tasks used to measure cognition or emotion, motivational state of subjects, species differences, or in how neurogenesis is defined, either as proliferation, survival, or differentiation (see reviews in Zhao et al. 2008; Aimone et al. 2011; Petrik et al. 2012b; Miller and Hen 2014).It must also be noted, however, that these hippocampal neurons are not born into a singular structure. Work in the past several decades has shown that the hippocampus can be divided, not only along the classic trisynaptic loop, but also longitudinally along a septotemporal axis. The septal (dorsal in rodents; posterior in primates) and temporal (ventral in rodents; anterior in primates) poles, as well as potential intermediate zones of the hippocampus, have different anatomic connections and electrophysiological properties, express a gradient of molecular markers, and play different functional roles, such as performance in spatial learning tasks and stress responses (see reviews in Moser and Moser 1998; Fanselow and Dong 2010). Consequently, adult-born neurons in the hippocampal DG may also be segregated along this longitudinal axis, and conflicting functional roles for neurogenesis may be a result of attempting to examine hippocampal neurogenesis as a unitary phenomenon. It is possible that there are intrinsic, cell-autonomous differences in adult-born neurons generated at opposite poles of the DG. An alternative, although not mutually exclusive, hypothesis is that progenitor cells are initially identical, but differentiate in a dissimilar manner as a result of integration into distinct network circuitry. We will, therefore, first discuss heterogeneity of the hippocampus along its longitudinal axis before reviewing differences in neurogenesis between the septal and temporal poles of the DG. As these topics have been reviewed extensively elsewhere (Moser and Moser 1998; Deng et al. 2010; Fanselow and Dong 2010; Koehl and Abrous 2011; Samuels and Hen 2011; Kheirbek et al. 2012; Petrik et al. 2012b), we will not try to exhaustively cover all the current literature. Rather, we attempt to gather key studies examining a septotemporal gradient of the hippocampus and hippocampal neurogenesis. We will then suggest possible approaches to examine neurogenesis in specific subregions of the hippocampal DG. Finally, a short section will examine segregation of the DG along its transverse axis.     

Molecular Determinants of the Coupling between STIM1 and Orai Channels: DIFFERENTIAL ACTIVATION OF Orai1�C3 CHANNELS BY A STIM1 COILED-COIL MUTANT*

STIM1 and Orai1 have been reported to interact upon store depletion culminating in Ca²⁺ release-activated Ca²⁺ current activation. Recently, the essential region has been identified within the STIM1 C terminus that includes the second coiled-coil domain C-terminally extended by ∼50 amino acids and exhibits a strong binding to the Orai1 C terminus. Based on the homology within the Orai family, an analogous scenario might be assumed for Orai2 as well as Orai3 channels as both are activated in a similar STIM1-dependent manner. A combined approach of electrophysiology and Foerster resonance energy transfer microscopy uncovered a general mechanism in the communication of STIM1 with Orai proteins that involved the conserved putative coiled-coil domains in the respective Orai C terminus and the second coiled-coil motif in the STIM1 C terminus. A coiled-coil single mutation in the Orai1 C terminus abrogated communication with the STIM1 C terminus, whereas an analogous mutation in Orai2 and Orai3 still allowed for their moderate activation. However, increasing coiled-coil probability by a gain of function deletion in Orai1 or by generating an Orai1-Orai3 chimera containing the Orai3 C terminus recovered stimulation to a similar extent as with Orai2/3. At the level of STIM1, decreasing probability of the second coiled-coil domain by a single mutation within the STIM1 C terminus abolished activation of Orai1 but still enabled partial stimulation of Orai2/3 channels. A double mutation within the second coiled-coil motif of the STIM1 C terminus fully disrupted communication with all three Orai channels. In aggregate, the impairment in the overall communication between STIM1 and Orai channels upon decreasing probabilities of either one of the putative coiled-coil domains in the C termini might be compatible with the concept of their functional, heteromeric interaction.Store-operated Ca²⁺ entry is a key to cellular regulation of short term responses such as contraction and secretion as well as long term processes like proliferation and cell growth (1). The prototypic and best characterized store-operated channel is the Ca²⁺ release-activated Ca²⁺ (CRAC)⁵ channel (2–6). However, its molecular components have remained elusive until 4 years ago; the STIM1 (stromal interacting molecule 1) (7, 8) and later on Orai1 (9–11) have been identified as the two limiting components for CRAC activation. STIM1 is an ER-located Ca²⁺ sensor, and store depletion triggers its aggregation into punctae close to the plasma membrane, resulting in stimulation of CRAC currents (12, 13). Its N terminus is located in the ER lumen and contains an EF-hand Ca²⁺-binding motif, which senses the ER Ca²⁺ level, and a sterile α-motif, which is suggested to mediate homomeric STIM1 aggregation (14–16). In the cytosolic STIM1 C terminus, two coiled-coil regions overlapping with the ezrin-radixin-moesin-like domain and a lysine-rich region are essential for CRAC activation (14, 17, 18). Three recent studies have independently identified the ezrin-radixin-moesin domain as the essential Orai activating domain, named SOAR (STIM1 Orai-activating region) (20) which represents so far the shortest active fragment, OASF (Orai-activating small fragment) (21) or CAD (CRAC-activating domain) (22), which includes the second, more C terminally located coiled-coil domain and the following ∼55 amino acids. The latter amino acids are suggested to contain an additional cytosolic homomerization domain indispensable for OASF homomerization and Orai activation (21).The Orai family includes three highly Ca²⁺-selective ion channels (Orai1–3) that locate to the plasma membrane, and each protein contains four predicted transmembrane segments with cytosolic N and C termini (10). All three Orai proteins possess a conserved putative coiled-coil domain in the C terminus (23, 24), whereas only the N terminus of Orai1 consists of a proline/arginine-rich region (25). Orai1 has been assumed to act in concert with STIM1 (10, 27)-activating inward Ca²⁺ currents after store depletion. The two other members of the Orai family, Orai2 and Orai3, display similar but smaller store-operated inward Ca²⁺ currents when co-expressed with STIM1 with distinct inactivation profiles, permeability properties, and 2-aminoethoxydiphenyl borate sensitivity (28–32). Recently, we have provided evidence for a store depletion-induced, dynamic coupling of STIM1 to Orai1 that involves the putative coiled-coil domain in the C terminus of Orai1 (33). Furthermore, the C terminus of STIM1, in particular the essential cytosolic region 344–442 as narrowed down by SOAR, OASF, and CAD (20–22), has been established as the key fragment for CRAC as well as Orai1 activation, because its expression alone, without the necessity to deplete ER store, is sufficient for constitutive current activation (18, 32, 33). These fragments SOAR, OASF, and CAD when co-expressed with Orai1 (20–22) exhibit enhanced plasma membrane localization in comparison with the complete STIM1 C terminus in the presence of Orai1. Specificity of interaction of SOAR to the Orai1 C terminus has been shown by its disruption (20) employing the Orai1 L273S mutant (33). Park et al. (22) have provided additional, conclusive evidence for a direct binding by combining multiple biochemical approaches demonstrating CAD interaction with Orai1.This study focused specifically on the role of the putative coiled-coil domains of STIM1 as well as Orai proteins in their coupling. Coiled-coils generally function as protein-protein interaction sites with the ability of dynamic protein assembly and disassembly (35–37). We suggest the C-terminal, putative coiled-coil domains in all three Orai proteins and the second coiled-coil motif of STIM1 as essential for STIM1/Orai communication. Moreover, the single point coiled-coil STIM1 L373S mutant allowed for differential activation of Orai channels partially stimulating Orai2 as well as Orai3 but not Orai1.