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.     

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.     

Signaling Processes for Initiating Smooth Muscle Contraction upon Neural Stimulation

Relationships among biochemical signaling processes involved in Ca²⁺/calmodulin (CaM)-dependent phosphorylation of smooth muscle myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) were determined. A genetically-encoded biosensor MLCK for measuring Ca²⁺-dependent CaM binding and activation was expressed in smooth muscles of transgenic mice. We performed real-time evaluations of the relationships among [Ca²⁺]_i, MLCK activation, and contraction in urinary bladder smooth muscle strips neurally stimulated for 3 s. Latencies for the onset of [Ca²⁺]_i and kinase activation were 55 ± 8 and 65 ± 6 ms, respectively. Both increased with RLC phosphorylation at 100 ms, whereas force latency was 109 ± 3 ms. [Ca²⁺]_i, kinase activation, and RLC phosphorylation responses were maximal by 1.2 s, whereas force increased more slowly to a maximal value at 3 s. A delayed temporal response between RLC phosphorylation and force is probably due to mechanical effects associated with elastic elements in the tissue. MLCK activation partially declined at 3 s of stimulation with no change in [Ca²⁺]_i and also declined more rapidly than [Ca²⁺]_i during relaxation. The apparent desensitization of MLCK to Ca²⁺ activation appears to be due to phosphorylation in its calmodulin binding segment. Phosphorylation of two myosin light chain phosphatase regulatory proteins (MYPT1 and CPI-17) or a protein implicated in strengthening membrane adhesion complexes for force transmission (paxillin) did not change during force development. Thus, neural stimulation leads to rapid increases in [Ca²⁺]_i, MLCK activation, and RLC phosphorylation in phasic smooth muscle, showing a tightly coupled Ca²⁺ signaling complex as an elementary mechanism initiating contraction.Increases in [Ca²⁺]_i3 in smooth muscle cells lead to Ca²⁺/CaM-dependent MLCK activation and RLC phosphorylation. Phosphorylation of RLC increases actin-activated myosin MgATPase activity leading to myosin cross-bridge cycling with force development (1–3).The activation of smooth muscle contraction may be affected by multiple cellular processes. Previous investigations show that free Ca²⁺/CaM is limiting for kinase activation despite the abundance of total CaM (4–6). The extent of RLC phosphorylation is balanced by the actions of MLCK and myosin light chain phosphatase, which is composed of three distinct protein subunits (7). The myosin phosphatase targeting subunit, MYPT1, in smooth muscle binds to myosin filaments, thus targeting the 37-kDa catalytic subunit (type 1 serine/threonine phosphatase, PP1c) to phosphorylated RLC. RLC phosphorylation and muscle force may be regulated by additional signaling pathways involving phosphorylation of RLC by Ca²⁺-independent kinase(s) and inhibition of myosin light chain phosphatase, processes that increase the contraction response at fixed [Ca²⁺]_i (Ca²⁺-sensitization) (8–14). Many studies indicate that agonist-mediated Ca²⁺-sensitization most often reflects decreased myosin light chain phosphatase activity involving two major pathways including MYPT1 phosphorylation by a Rho kinase pathway and phosphorylation of CPI-17 by PKC (8, 14–16). Additionally, phosphorylation of MLCK in its calmodulin-binding sequence by a Ca²⁺/calmodulin-dependent kinase pathway has been implicated in Ca²⁺ desensitization of RLC phosphorylation (17–19). How these signaling pathways intersect the responses of the primary Ca²⁺/CaM pathway during physiological neural stimulation is not known.There is also evidence that smooth muscle contraction requires the polymerization of submembranous cytoskeletal actin filaments to strengthen membrane adhesion complexes involved in transmitting force between actin-myosin filaments and external force-transmitting structures (20–23). In tracheal smooth muscle, paxillin at membrane adhesions undergoes tyrosine phosphorylation in response to contractile stimulation by an agonist, and this phosphorylation increases concurrently with force development in response to agonist. Expression of nonphosphorylatable paxillin mutants in tracheal muscle suppresses acetylcholine-induced tyrosine phosphorylation of paxillin, tension development, and actin polymerization without affecting RLC phosphorylation (24, 25). Thus, paxillin phosphorylation may play an important role in tension development in smooth muscle independently of RLC phosphorylation and cross-bridge cycling.Specific models relating signaling mechanisms in the smooth muscle cell to contraction dynamics are limited when cells in tissues are stimulated slowly and asynchronously by agonist diffusing into the preparation. Field stimulation leading to the rapid release of neurotransmitters from nerves embedded in the tissue avoids these problems associated with agonist diffusion (26, 27). In urinary bladder smooth muscle, phasic contractions are brought about by the parasympathetic nervous system. Upon activation, parasympathetic nerve varicosities release the two neurotransmitters, acetylcholine and ATP, that bind to muscarinic and purinergic receptors, respectively. They cause smooth muscle contraction by inducing Ca²⁺ transients as elementary signals in the process of nerve-smooth muscle communication (28–30). We recently reported the development of a genetically encoded, CaM-sensor for activation of MLCK. The CaM-sensor MLCK contains short smooth muscle MLCK fused to two fluorophores, enhanced cyan fluorescent protein and enhanced yellow fluorescent protein, linked by the MLCK calmodulin binding sequence (6, 14, 31). Upon dimerization, there is significant FRET from the donor enhanced cyan fluorescent protein to the acceptor enhanced yellow fluorescent protein. Ca²⁺/CaM binding dissociates the dimer resulting in a decrease in FRET intensity coincident with activation of kinase activity (31). Thus, CaM-sensor MLCK is capable of directly monitoring Ca²⁺/CaM binding and activation of the kinase in smooth muscle tissues where it is expressed specifically in smooth muscle cells of transgenic mice. We therefore combined neural stimulation with real-time measurements of [Ca²⁺]_i, MLCK activation, and force development in smooth muscle tissue from these mice. Additionally, RLC phosphorylation was measured precisely at specific times following neural stimulation in tissues frozen by a rapid-release electronic freezing device (26, 27). Results from these studies reveal that physiological stimulation of smooth muscle cells by neurotransmitter release leads to rapid increases in [Ca²⁺]_i, MLCK activation, and RLC phosphorylation at similar rates without the apparent activities of Ca²⁺-independent kinases, inhibition of myosin light chain phosphatase, or paxillin phosphorylation. Thus, the elemental processes for phasic smooth muscle contraction are represented by this tightly coupled Ca²⁺ signaling complex.     

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).     

Fluorescence Resonance Energy Transfer-Sensitized Emission of Yellow Cameleon 3.60 Reveals Root Zone-Specific Calcium Signatures in Arabidopsis in Response to Aluminum and Other Trivalent Cations

Fluorescence resonance energy transfer-sensitized emission of the yellow cameleon 3.60 was used to study the dynamics of cytoplasmic calcium ([Ca²⁺]_cyt) in different zones of living Arabidopsis (Arabidopsis thaliana) roots. Transient elevations of [Ca²⁺]_cyt were observed in response to glutamic acid (Glu), ATP, and aluminum (Al³⁺). Each chemical induced a [Ca²⁺]_cyt signature that differed among the three treatments in regard to the onset, duration, and shape of the response. Glu and ATP triggered patterns of [Ca²⁺]_cyt increases that were similar among the different root zones, whereas Al³⁺ evoked [Ca²⁺]_cyt transients that had monophasic and biphasic shapes, most notably in the root transition zone. The Al³⁺-induced [Ca²⁺]_cyt increases generally started in the maturation zone and propagated toward the cap, while the earliest [Ca²⁺]_cyt response after Glu or ATP treatment occurred in an area that encompassed the meristem and elongation zone. The biphasic [Ca²⁺]_cyt signature resulting from Al³⁺ treatment originated mostly from cortical cells located at 300 to 500 μ m from the root tip, which could be triggered in part through ligand-gated Glu receptors. Lanthanum and gadolinium, cations commonly used as Ca²⁺ channel blockers, elicited [Ca²⁺]_cyt responses similar to those induced by Al³⁺. The trivalent ion-induced [Ca²⁺]_cyt signatures in roots of an Al³⁺-resistant and an Al³⁺-sensitive mutant were similar to those of wild-type plants, indicating that the early [Ca²⁺]_cyt changes we report here may not be tightly linked to Al³⁺ toxicity but rather to a general response to trivalent cations.The role of calcium ions (Ca²⁺) as a ubiquitous cellular messenger in animal and plant cells is well established (Berridge et al., 2000; Sanders et al., 2002; Ng and McAinsh, 2003). Cellular signal transduction pathways are elicited as a result of fluctuations of free Ca²⁺ in the cytoplasm ([Ca²⁺]_cyt) in response to external and intracellular signals. These changes in [Ca²⁺]_cyt influence numerous cellular processes, including vesicle trafficking, cell metabolism, cell proliferation and elongation, stomatal opening and closure, seed and pollen grain germination, fertilization, ion transport, and cytoskeletal organization (Hepler, 2005). [Ca²⁺]_cyt fluctuations occur because cells have a Ca²⁺ signaling “toolkit” (Berridge et al., 2000) composed of on/off switches and a multitude of Ca²⁺-binding proteins. The on switches depend on membrane-localized Ca²⁺ channels that control the entry of Ca²⁺ into the cytosol (Piñeros and Tester, 1995, 1997; Thion et al., 1998; Kiegle et al., 2000a; White et al., 2000; Demidchik et al., 2002; Miedema et al., 2008). On the other hand, the off switches consist of a family of Ca²⁺-ATPases and Ca²⁺/H⁺ exchangers in the plasma membrane or endomembrane that remove Ca²⁺ from the cytosol, bringing the [Ca²⁺]_cyt down to the initial resting level (Lee et al., 2007; Li et al., 2008).The numerous cellular processes regulated by Ca²⁺ have led investigators to ask how specificity in Ca²⁺ signaling is maintained. It has been proposed that specificity in Ca²⁺ signaling is achieved because a particular stimulus elicits a distinct Ca²⁺ signature, which is defined by the timing, magnitude, and frequency of [Ca²⁺]_cyt changes. For instance, tip-growing plant cells such as root hairs and pollen tubes exhibit oscillatory elevations in [Ca²⁺]_cyt that partly mirror the oscillatory nature of growth in these cell types (Cárdenas et al., 2008; Monshausen et al., 2008). Another example is nuclear Ca²⁺ spiking in root hairs of legumes exposed to NOD factors (Oldroyd and Downie, 2006; Peiter et al., 2007). Recently, it was shown that mechanical forces applied to an Arabidopsis (Arabidopsis thaliana) root can trigger a stimulus-specific [Ca²⁺]_cyt response (Monshausen et al., 2009). Translating the Ca²⁺ signature into a defined cellular response is governed by a number of Ca²⁺-binding proteins such as calreticulin that act as [Ca²⁺]_cyt buffers, which shape both the amplitude and duration of the Ca²⁺ signal or Ca²⁺ sensors such as calmodulin that impact other downstream cellular effectors (Berridge et al., 2000; White and Broadley, 2003; Hepler, 2005).A deeper understanding of Ca²⁺ signaling mechanisms in plants has been driven in large part by our ability to monitor dynamic changes in [Ca²⁺]_cyt in the cell. Such measurements have been conducted using Ca²⁺-sensitive fluorescent indicator dyes (e.g. Indo and Fura), the luminescent protein aequorin (Knight et al., 1991, 1996; Legué et al., 1997; Wymer et al., 1997; Cárdenas et al., 2008), and more recently the yellow cameleon (YC) Ca²⁺ sensor, a chimeric protein that relies on fluorescence resonance energy transfer (FRET) as an indicator of [Ca²⁺]_cyt changes in the cell (Allen et al., 1999; Miwa et al., 2006; Qi et al., 2006; Tang et al., 2007; Haruta et al., 2008). The YC reporter is composed of cyan fluorescent protein (CFP), the C terminus of calmodulin (CaM), a Gly-Gly linker, the CaM-binding domain of myosin light chain kinase (M13), and a yellow fluorescent protein (YFP; Miyawaki et al., 1997, 1999). The increased interaction between M13 and CaM upon binding of Ca²⁺ to CaM triggers a conformational change in the protein that brings the CFP and YFP in close proximity, resulting in enhanced FRET efficiency between the two fluorophores (Miyawaki, 2003). Thus, changes in FRET efficiency between CFP and YFP in the cameleon reporter are correlated with changes in [Ca²⁺]_cyt.Since it was first introduced, improved versions of the cameleon reporter have been selected to more accurately report [Ca²⁺]_cyt levels in the cell. For instance, the YC3.60 version was selected because of its resistance to cytoplasmic acidification and its higher dynamic range compared with the earlier cameleons. The higher dynamic range of YC3.60 is due to the use of a circularly permutated YFP called Venus (cpVenus) that is capable of absorbing a greater amount of energy from CFP (Nagai et al., 2004). Recently, the utility of YC3.60 for monitoring [Ca²⁺]_cyt was demonstrated in Arabidopsis roots and pollen tubes using ratiometric imaging approaches (Monshausen et al., 2007, 2008, 2009; Haruta et al., 2008; Iwano et al., 2009). Here, we further evaluated YC3.60 as a [Ca²⁺]_cyt sensor in plants using confocal microscopy and FRET-sensitized emission imaging. Unlike the direct ratiometric measurement of cpVenus and CFP reported in previous studies using YC3.60-expressing plants (Monshausen et al., 2008, 2009), the sensitized FRET approach we describe here involves the use of donor-only (CFP) and acceptor-only (YFP) controls, allowing us to correct for bleed-through and background signals from the FRET specimen (van Rheenen et al., 2004; Feige et al., 2005).For this study, we focused on monitoring [Ca²⁺]_cyt changes in Arabidopsis seedling roots after aluminum (Al³⁺) exposure. Although Ca²⁺ signaling has long been implicated in mediating Al³⁺ responses in plants (Rengel and Zhang, 2003), the [Ca²⁺]_cyt changes evoked by Al³⁺ reported in the literature have been inconsistent, and as such, the significance of these [Ca²⁺]_cyt responses to mechanisms of Al³⁺ toxicity are not very clear. For instance, some studies reported that Al³⁺ caused a decrease in [Ca²⁺]_cyt in plants (Jones et al., 1998b; Kawano et al., 2004), and others demonstrated elevated [Ca²⁺]_cyt upon Al³⁺ treatment (Nichol and Oliveira, 1995; Lindberg and Strid, 1997; Jones et al., 1998a; Zhang and Rengel, 1999; Ma et al., 2002; Bhuja et al., 2004).Here, we report that Arabidopsis roots expressing the YC3.60 reporter exhibited transient elevations in [Ca²⁺]_cyt within seconds of Al³⁺ exposure. The general pattern of [Ca²⁺]_cyt changes observed after Al³⁺ treatment were distinct from those elicited by ATP or Glu, reinforcing the concept of specificity in [Ca²⁺]_cyt signaling. We also observed root zone-dependent variations in the [Ca²⁺]_cyt signatures evoked by Al³⁺ in regard to the shape, duration, and timing of the [Ca²⁺]_cyt response. Other trivalent ions such as lanthanum (La³⁺) and gadolinium (Ga³⁺), which have been widely used as Ca²⁺ channel blockers (Monshausen et al., 2009), also induced a rapid rise in [Ca²⁺]_cyt in root cells that were similar to those elicited by Al³⁺. Al³⁺, La³⁺, and Gd³⁺ elicited similar [Ca²⁺]_cyt signatures in the Al³⁺-tolerant mutant alr104 (Larsen et al., 1998) and the Al³⁺-sensitive mutant als3-1 (Larsen et al., 2005), indicating that the early [Ca²⁺]_cyt increases we report here may not be tightly linked to mechanisms of Al³⁺ toxicity but rather to a general trivalent cation response. Our study further shows that FRET-sensitized emission imaging of Arabidopsis roots expressing YC3.60 provides a robust method for documenting [Ca²⁺]_cyt signatures in different root developmental zones that should be useful for future studies on Ca²⁺ signaling mechanisms in plants.     

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.     

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.     

Plasma Membrane-associated Annexin A6 Reduces Ca2+ Entry by Stabilizing the Cortical Actin Cytoskeleton

The annexins are a family of Ca²⁺- and phospholipid-binding proteins, which interact with membranes upon increase of [Ca²⁺]_i or during cytoplasmic acidification. The transient nature of the membrane binding of annexins complicates the study of their influence on intracellular processes. To address the function of annexins at the plasma membrane (PM), we fused fluorescent protein-tagged annexins A6, A1, and A2 with H- and K-Ras membrane anchors. Stable PM localization of membrane-anchored annexin A6 significantly decreased the store-operated Ca²⁺ entry (SOCE), but did not influence the rates of Ca²⁺ extrusion. This attenuation was specific for annexin A6 because PM-anchored annexins A1 and A2 did not alter SOCE. Membrane association of annexin A6 was necessary for a measurable decrease of SOCE, because cytoplasmic annexin A6 had no effect on Ca²⁺ entry as long as [Ca²⁺]_i was below the threshold of annexin A6-membrane translocation. However, when [Ca²⁺]_i reached the levels necessary for the Ca²⁺-dependent PM association of ectopically expressed wild-type annexin A6, SOCE was also inhibited. Conversely, knockdown of the endogenous annexin A6 in HEK293 cells resulted in an elevated Ca²⁺ entry. Constitutive PM localization of annexin A6 caused a rearrangement and accumulation of F-actin at the PM, indicating a stabilized cortical cytoskeleton. Consistent with these findings, disruption of the actin cytoskeleton using latrunculin A abolished the inhibitory effect of PM-anchored annexin A6 on SOCE. In agreement with the inhibitory effect of annexin A6 on SOCE, constitutive PM localization of annexin A6 inhibited cell proliferation. Taken together, our results implicate annexin A6 in the actin-dependent regulation of Ca²⁺ entry, with consequences for the rates of cell proliferation.Calcium entry into cells either through voltage- or receptor-operated channels, or following the depletion of intracellular stores is a major factor in maintaining intracellular Ca²⁺ homeostasis. Resting [Ca²⁺]_i is low (∼100 nm compared with extracellular [Ca²⁺]_ex of 1.2 mm) and can be rapidly increased by inositol triphosphate-mediated release from the intracellular Ca²⁺ stores (mostly endoplasmic reticulum (ER)³), or by channel-mediated influx across the plasma membrane (PM). Store-operated calcium entry (SOCE) has been proposed as the main process controlling Ca²⁺ entry in non-excitable cells (1), and the recent discovery of Orai1 and STIM provided the missing link between the Ca²⁺-release activated current (I_CRAC) and the ER Ca²⁺ sensor (2–4). Translocation of STIM within the ER, accumulation in punctae at the sites of contact with PM and activation of Ca²⁺ channels have been proposed as a model of its regulation of Orai1 activity (5, 6). However, many details of the functional STIM-Orai1 protein complex and its regulation remain to be elucidated. The actin cytoskeleton plays a major role in the regulation of SOCE, possibly by influencing the function of ion channels or by interfering with the interaction between STIM and Orai1 (7–9). However, the proteins connecting the actin cytoskeleton and SOCE activity at the PM have yet to be identified.The annexins are a multigene family of Ca²⁺- and phospholipid-binding proteins, which have been implicated in many Ca²⁺-regulated processes. Their C-terminal core is evolutionarily conserved and contains Ca²⁺-binding sites, their N-terminal tails are unique and enable the protein to interact with distinct cytoplasmic partners. At low [Ca²⁺]_i, annexins are diffusely distributed throughout the cytosol, however, after stimulation resulting in the increase of [Ca²⁺]_i, annexins are targeted to distinct subcellular membrane locations, such as the PM, endosomes, or secretory vesicles (10). Annexins are involved in the processes of vesicle trafficking, cell division, apoptosis, calcium signaling, and growth regulation (11), and frequent changes in expression levels of annexins are observed in disease (12, 13). Previously, using biochemical methods and imaging of fluorescent protein-tagged annexins in live cells, we demonstrated that annexins A1, A2, A4, and A6 interacted with the PM as well as with internal membrane systems in a highly coordinated manner (10, 14). In addition, there is evidence of Ca²⁺-independent membrane association of several annexins, including annexin A6 (15–19); some of which point to the existence of pH-dependent binding mechanisms (20–22). Given the fact that several annexins are present within any one cell, it is likely that they form a [Ca²⁺] and pH sensing system, with a regulatory influence on other signaling pathways.The role of annexins as regulators of ion channel activity has been addressed previously (23–25). In particular, annexin A6 has been implicated in regulation of the sarcoplasmic reticulum ryanodine-sensitive Ca²⁺ channel (25), the neuronal K⁺ and Ca²⁺ channels (26), and the cardiac Na⁺/Ca²⁺ exchanger (27). Cardiac-specific overexpression of annexin A6 resulted in lower basal [Ca²⁺], a depression of [Ca²⁺]_i transients and impaired cardiomyocyte contractility (28). In contrast, the cardiomyocytes from the annexin A6 null-mutant mice showed increased contractility and accelerated Ca²⁺ clearance (29). Consistent with its role in mediating the intracellular Ca²⁺ signals, especially Ca²⁺ influx, ectopic overexpression of annexin A6 in A431 cells, which lack endogenous annexin A6, resulted in inhibition of EGF-dependent Ca²⁺ entry (30).The difficulty of investigating the influence of annexins on signaling events occurring at the PM lies in the transient and reversible nature of their Ca²⁺ and pH-dependent lipid binding. Although the intracellular Ca²⁺ increase following receptor activation or Ca²⁺ influx promotes the association of the Ca²⁺-sensitive annexins A2 and A6 with the PM, the proteins quickly resume their cytoplasmic localization upon restoration of the basal [Ca²⁺]_i (14). Therefore, to investigate the effects of membrane-associated annexins on Ca²⁺ homeostasis and the cell signaling machinery, we aimed to develop a model system allowing for a constitutive membrane association of annexins. Here we used the PM-anchoring sequences of the H- and K-Ras proteins to target annexins A6 and A1 to the PM independently of [Ca²⁺]. The Ras GTPases are resident at the inner leaflet of the PM and function as molecular switches (31). The C-terminal 9 amino acids of H- and N-Ras and the C-terminal 14 amino acids of K-Ras comprise the signal sequences for membrane anchoring of Ras isoforms (32). Although the palmitoylation and farnesylation of the C terminus of H-Ras (tH) serves as a targeting signal for predominantly cholesterol-rich membrane microdomains at the PM (lipid rafts/caveolae) (33), the polybasic group and the lipid anchor of K-Ras (tK) ensures the association of K-Ras with cholesterol-poor PM membrane domains. Importantly, these minimal C-terminal amino acid sequences are sufficient to target heterologous proteins, for example GFP, to different microdomains at the PM and influence their trafficking (34).In the present study we fused annexins A6, A2, and A1 with fluorescent proteins and introduced the PM-anchoring sequences of either H-Ras (annexin-tH) or K-Ras (annexin-tK) at the C termini of the fusion constructs. We demonstrate that the constitutive PM localization of annexin A6 results in down-regulation of store-operated Ca²⁺ entry. Expression of membrane-anchored annexin A6 causes an accumulation of the cortical F-actin, and cytoskeletal destabilization with latrunculin A abolishes the inhibitory effect of PM-anchored annexin A6 on SOCE. Taken together, our results implicate annexin A6 in the maintenance of intracellular Ca²⁺ homeostasis via actin-dependent regulation of Ca²⁺ entry.     

NHERF3 (PDZK1) Contributes to Basal and Calcium Inhibition of NHE3 Activity in Caco-2BBe Cells

Elevated intracellular Ca²⁺ ([Ca²⁺]_i) inhibition of NHE3 is reconstituted by NHERF2, but not NHERF1, by a mechanism involving the formation of multiprotein signaling complexes. To further evaluate the specificity of the NHERF family in calcium regulation of NHE3 activity, the current study determined whether NHERF3 reconstitutes elevated [Ca²⁺]_i regulation of NHE3. In vitro, NHERF3 bound the NHE3 C terminus between amino acids 588 and 667. In vivo, NHE3 and NHERF3 associate under basal conditions as indicated by co-immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer. Treatment of PS120/NHE3/NHERF3 cells, but not PS120/NHE3 cells, with the Ca²⁺ ionophore, 4-bromo-{"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (0.5 μm): 1) inhibited NHE3 V_max activity; 2) decreased NHE3 surface amount; 3) dissociated NHE3 and NHERF3 at the plasma membrane by confocal immunofluorescence and fluorescence resonance energy transfer. Similarly, in Caco-2BBe cells, NHERF3 and NHE3 colocalized in the BB under basal conditions but after elevation of [Ca²⁺]_i by carbachol, this overlap was abolished. NHERF3 short hairpin RNA knockdown (>50%) in Caco-2BBe cells significantly reduced basal NHE3 activity by decreasing BB NHE3 amount. Also, carbachol-mediated inhibition of NHE3 activity was abolished in Caco-2BBe cells in which NHERF3 protein expression was significantly reduced. In summary: 1) NHERF3 colocalizes and directly binds NHE3 at the plasma membrane under basal conditions; 2) NHERF3 reconstitutes [Ca²⁺]_i inhibition of NHE3 activity and dissociates from NHE3 in fibroblasts and polarized intestinal epithelial cells with elevated [Ca²⁺]_i; 3) NHERF3 short hairpin RNA significantly reduced NHE3 basal activity and brush border expression in Caco-2BBe cells. These results demonstrate that NHERF3 reconstitutes calcium inhibition of NHE3 activity by anchoring NHE3 basally and releasing it with elevated Ca²⁺.In normal digestive physiology, the brush border (BB)² Na⁺/H⁺ exchanger, NHE3, mediates the majority of the NaCl and NaHCO₃ absorption in the ileum (1). Sequential inhibition and stimulation of NHE3 occur as part of digestive physiology. Short-term regulation of NHE3 activity is achieved through a variety of factors that affect NHE3 turnover number and/or surface expression and often involve a role for the cytoskeleton and accessory proteins, including the multi-PDZ domain containing proteins, NHERF1 and NHERF2 (1, 2). However, many details of this regulation are not understood.The NHERF (Na⁺/H⁺ exchanger regulatory factor) family of multi-PDZ domain containing proteins consists of four evolutionarily related members, all of which are expressed in epithelial cells of the mammalian small intestine (2). NHERF1 and NHERF2 have been previously shown to contribute to acute NHE3 stimulation and inhibition (3–10). Recently, two additional PDZ domain containing proteins, termed NHERF3/PDZK1 and NHERF4/PDZK2/IKEPP, have been demonstrated to possess sequence homology with NHERF1 and NHERF2 (11–14). However, unlike NHERF1 and NHERF2, which are comprised of two tandem PDZ domains flanked by a C-terminal ezrin/radixin/moesin binding domain, NHERF3 and NHERF4 consist of four PDZ domains but no other protein-protein interacting domains (12).NHERF3 was initially identified by a yeast two-hybrid screen from a human kidney cDNA library using the membrane-associated protein MAP17, as bait (12). NHERF3 is expressed in the brush border of epithelial cells of the kidney proximal tubule and the small intestine (12). NHERF3 associates with and, in a few cases, has been shown to regulate the activity of multiple apical membrane ion transporters including the cystic fibrosis transmembrane regulator (CFTR), urate anion exchanger 1 (URAT1), sodium-phosphate cotransporter type IIa (NaP_iIIa), proton-coupled peptide transporter (PEPT2), and organic cation/carnitine cotransporter (OCTN2) (15–19). Furthermore, NHERF3 directly binds the C terminus of NHE3 (20). Recent studies have begun evaluating the effect of NHERF3 on mouse intestinal Na⁺ and Cl⁻ transport. Basal electroneutral sodium absorption was decreased by >40% in the NHERF3 null mouse jejunum (21) and by >80% in the colon (22). In addition, Cinar et al. (22) demonstrated that cAMP and [Ca²⁺]_i inhibition of NHE3 activity was abolished in the NHERF3 null mouse colon. However, the mechanism by which NHERF3 regulates NHE3 activity was not resolved.Several physiological and pathophysiological agonists, acting through [Ca²⁺]_i-induced second messenger systems, are known to inhibit electroneutral NaCl absorption in the small intestine (1, 23). Elevation of [Ca²⁺]_i has previously been demonstrated to inhibit NHE3 activity in a NHERF2-, but not NHERF1-dependent manner (5). NHERF2 regulation of NHE3 involves the formation of multiprotein complexes at the plasma membrane that include NHE3, NHERF2, α-actinin-4, and PKCα, which induce endocytic removal of NHE3 from the plasma membrane by a PKC-dependent mechanism (5, 24). Because multiple PDZ proteins exist in the apical pole of epithelial cells (2), the current study was designed to determine whether NHERF3 could reconstitute Ca²⁺ regulation of NHE3 activity and to define how that occurred.     

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).     

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.     

Fibronectins,Their Fibrillogenesis,and In Vivo Functions

Fibronectin (FN) is a multidomain protein with the ability to bind simultaneously to cell surface receptors, collagen, proteoglycans, and other FN molecules. Many of these domains and interactions are also involved in the assembly of FN dimers into a multimeric fibrillar matrix. When, where, and how FN binds to its various partners must be controlled and coordinated during fibrillogenesis. Steps in the process of FN fibrillogenesis including FN self-association, receptor activities, and intracellular pathways have been under intense investigation for years. In this review, the domain organization of FN including the extra domains and variable region that are controlled by alternative splicing are described. We discuss how FN–FN and cell–FN interactions play essential roles in the initiation and progression of matrix assembly using complementary results from cell culture and embryonic model systems that have enhanced our understanding of this process.As a ubiquitous component of the extracellular matrix (ECM), fibronectin (FN) provides essential connections to cells through integrins and other receptors and regulates cell adhesion, migration, and differentiation. FN is secreted as a large dimeric glycoprotein with subunits that range in size from 230 kDa to 270 kDa (Mosher 1989; Hynes 1990). Variation in subunit size depends primarily on alternative splicing. FN was first isolated from blood more than 60 years ago (Edsall 1978), and this form is called plasma FN. The other major form, called cellular FN, is abundant in the fibrillar matrices of most tissues. Although FN is probably best known for promoting attachment of cells to surfaces, this multidomain protein has many interesting structural features and functional roles beyond cell adhesion.FN is composed of three different types of modules termed type I, II, and III repeats (Fig. 1) (Petersen et al. 1983; Hynes 1990). These repeats have distinct structures. Although the conformations of type I and type II repeats are maintained by pairs of intramodule disulfide bonds, the type III repeat is a 7-stranded β-barrel structure that lacks disulfide bonds (Main et al. 1992; Leahy et al. 1996, 1992) and, therefore, can undergo conformational changes. FN type III repeats are widely distributed among animal, bacterial, and plant proteins and are found in both extracellular and intracellular proteins (Bork and Doolittle 1992; Tsyguelnaia and Doolittle 1998).Open in a separate windowFigure 1.FN domain organization and isoforms. Each FN monomer has a modular structure consisting of 12 type I repeats (cylinders), 2 type II repeats (diamonds), and 15 constitutive type III repeats (hexagons). Two additional type III repeats (EIIIA and EIIIB, green) are included or omitted by alternative splicing. The third region of alternative splicing, the V region (green box), is included (V120), excluded (V0), or partially included (V95, V64, V89). Sets of modules comprise domains for binding to other extracellular molecules as indicated. Domains required for fibrillogenesis are in red: the assembly domain (repeats I_1-5) binds FN, III_9-10 contains the RGD and synergy sequences for integrin binding, and the carboxy-terminal cysteines form the disulfide-bonded FN dimer (‖). The III_1-2 domain (light red) has two FN binding sites that are important for fibrillogenesis. The amino-terminal 70-kDa fragment contains assembly and gelatin-binding domains and is routinely used in FN binding and matrix assembly studies.Sets of adjacent modules form binding domains for a variety of proteins and carbohydrates (Fig. 1). ECM proteins, including FN, bind to cells via integrin receptors, αβ heterodimers with two transmembrane subunits (Hynes 2002). FN-binding integrins have specificity for one of the two cell-binding sites within FN, either the RGD-dependent cell-binding domain in III₁₀ (Pierschbacher and Ruoslahti 1984) or the CS1 segment of the alternatively spliced V region (IIICS) (Wayner et al. 1989; Guan and Hynes 1990). Some integrins require a synergy sequence in repeat III₉ for maximal interactions with FN (Aota et al. 1994; Bowditch et al. 1994). Another family of cell surface receptors is the syndecans, single-chain transmembrane proteoglycans (Couchman 2010). Syndecans use their glycosaminoglycan (GAG) chains to interact with FN at its carboxy-terminal heparin-binding (HepII) domain (Fig. 1) (Saunders and Bernfield 1988; Woods et al. 2000), which binds to heparin, heparan sulfate, and chondroitin sulfate GAGs (Hynes 1990; Barkalow and Schwarzbauer 1994). Syndecan binding to the HepII domain enhances integrin-mediated cell spreading and intracellular signaling, suggesting that syndecans act as coreceptors with integrins in cell–FN binding (Woods and Couchman 1998; Morgan et al. 2007).A major site for FN self-association is within the amino-terminal assembly domain spanning the first five type I repeats (I_1-5) (Fig. 1) (McKeown-Longo and Mosher 1985; McDonald et al. 1987; Schwarzbauer 1991b; Sottile et al. 1991). This domain plays an essential role in FN fibrillogenesis. As a major blood protein, FN interacts with fibrin during blood coagulation, also using the I_1-5 domain (Mosher 1989; Hynes 1990). As fibrin polymerizes, factor XIII transglutaminase covalently cross-links glutamine residues near the amino terminus of FN to fibrin α chains (Mosher 1975; Corbett et al. 1997). The amino-terminal domain has multiple binding partners in addition to FN and fibrin; these include heparin, S. aureus, and other bacteria, thrombospondin-1, and tenascin-C (Hynes 1990; Ingham et al. 2004; Schwarz-Linek et al. 2006). Adjacent to this domain is the gelatin/collagen-binding domain composed of type I and type II modules (Ingham et al. 1988). This domain also binds to tissue transglutaminase (Radek et al. 1993) and fibrillin-1 (Sabatier et al. 2009). Within the 15 type III repeats reside several FN binding sites that interact with the amino-terminal assembly domain as well as three sites of alternative splicing that generate multiple isoforms. At the carboxyl terminus is a pair of cysteine residues that form the FN dimer through antiparallel disulfide bonds (Hynes 1990). This dimerization may be facilitated by disulfide isomerase activity located in the last set of type I repeats (Langenbach and Sottile 1999).The diverse set of binding domains provides FN with the ability to interact simultaneously with other FN molecules, other ECM components (e.g., collagens and proteoglycans), cell surface receptors, and extracellular enzymes (Pankov and Yamada 2002; Fogelgren et al. 2005; Hynes 2009; Singh et al. 2010). Multitasking by FN probably underlies its essential role during embryogenesis (George et al. 1993). Furthermore, FN''s interactions can be modulated by exposure or sequestration of its binding sites within matrix fibrils, through the presence of ECM proteins that bind to FN, or through variation in structure by alternative splicing.     

Dynamics of ATP-induced Calcium Signaling in Single Mouse Thymocytes

Extracellular ATP (ATP_o) elicits a robust change in the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) in fura-2–loaded mouse thymocytes. Most thymocytes (60%) exposed to ATP_o exhibited a biphasic rise in [Ca²⁺]_i; [Ca²⁺]_i rose slowly at first to a mean value of 260 nM after 163 s and then increased rapidly to a peak level of 735 nM. In many cells, a declining plateau, which lasted for more than 10 min, followed the crest in [Ca²⁺]_i. Experiments performed in the absence of extracellular [Ca²⁺]_o abolished the rise in thymocyte [Ca²⁺]_i, indicating that Ca²⁺ influx, rather than the release of stored Ca²⁺, is stimulated by ATP_o. ATP_o- mediated Ca²⁺ influx was potentiated as the [Mg²⁺]_o was reduced, confirming that ATP⁴⁻ is the active agonist form. In the absence of Mg²⁺ _o, 3′-O-(4-benzoyl)benzoyl-ATP (BzATP) proved to be the most effective agonist of those tested. The rank order of potency for adenine nucleotides was BzATP⁴⁻>ATP⁴⁻>MgATP²⁻>ADP³⁻, suggesting purinoreceptors of the P2X₇/P2Z class mediate the ATP_o response. Phenotyping experiments illustrate that both immature (CD4⁻CD8⁻, CD4⁺CD8⁺) and mature (CD4⁺CD8⁻, CD4⁻CD8⁺) thymocyte populations respond to ATP. Further separation of the double-positive population by size revealed that the ATP_o-mediated [Ca²⁺]_i response was much more pronounced in large (actively dividing) than in small (terminally differentiated) CD4⁺CD8⁺ thymocytes. We conclude that thymocytes vary in sensitivity to ATP_o depending upon the degree of maturation and suggest that ATP_o may be involved in processes that control cellular differentiation within the thymus.Extracellular ATP (ATP_o)¹ and its metabolic products evoke physiological responses in virtually all tissues and cell types from central nervous to peripheral organ systems (for review see Dubyak and El-Moatassim, 1993; Harden et al., 1995). Tissues and isolated cells vary in sensitivity to purine agonists. Nucleotides (ATP, ADP, and AMP) and adenosine, the nucleoside product of ATP catabolism, elicit distinct responses in target cells by triggering P2 and P1 purinergic receptors, respectively (Burnstock, 1978). P2 purinoceptors can be further separated into two broad categories. The first group, divided into P2Y and P2U subtypes, couples nucleotide binding to effector molecules via G proteins. The second P2 category is comprised of nucleotide-sensitive ion channels and pores. ATP-gated P2 purinoceptors, designated P2X₁ through P2X₆ (cation channels) and P2X₇ (a dual function cation channel/pore), display extensive sequence identity (North, 1996) but disparate tissue distribution, biophysical properties, agonist profiles, and pharmacology (P2X₁, Valera et al., 1994; P2X₂-P2X₆, Collo et al., 1996; P2X₇, Surprenant et al., 1996). Moreover, P2X receptors functionally resemble acetylcholine- and serotonin-gated channels with respect to gating and ionic permeability but are structurally unique. Thus, nucleotides, together with acetylcholine, glutamate, GABA, glycine, and serotonin, are included in a small group of compounds that function as agonists for a structurally diverse set of ligand-gated ion channels and pores, as well as G protein-coupled receptors.ATP_o elicits a broad spectrum of physiological changes in cells of the immune system. In mast cells, ATP release has been shown to mediate cell-to-cell signaling (Osipchuk and Cahalan, 1992). In lymphocytes, ATP_o triggers cellular depolarization, greater permeability to small organic molecules (<400 D; Wiley et al., 1993; Chused et al., 1996), and a rise in the concentration of intracellular Ca²⁺ ([Ca²⁺]_i; El-Moatassim et al., 1987; Wiley and Dubyak, 1989). The ATP_o-mediated rise in [Ca²⁺]_i modifies the functional properties of thymocytes via DNA synthesis (Gregory and Kern, 1978, 1981; Ikehara et al., 1981) and blastogenesis (El-Moatassim et al., 1987). Moreover, an increase in [Ca²⁺]_i has been linked to programmed cell death in thymocyte populations; Ca²⁺ release from intracellular stores evoked by thapsigargin, a microsomal Ca²⁺-ATPase inhibitor, triggers the DNA fragmentation correlated with thymocyte apoptosis (Jiang et al., 1994; Zhivotovsky et al., 1994).Based upon a sensitivity profile for purine agonists and pharmacological agents, lymphocytes are not believed to possess G protein-linked purinoceptors (El-Moatassim et al., 1989b ). Rather, lymphocytes and related cell lines express purinoceptors of the ion channel/pore subtype (P2X₇). This ATP-gated pathway, originally termed P2Z (Gordon, 1986), has been characterized in mast cells (Cockcroft and Gomperts, 1979a ; Tatham and Lindau, 1990), transformed 3T3 fibroblasts (Heppel et al., 1985), macrophages (Buisman et al., 1988), parotid acinar cells (Soltoff et al., 1992), and phagocytic cells of the thymic reticulum (Coutinho-Silva et al., 1996). During whole cell patch–clamp experiments, putative P2Z channels in human B lymphocytes (Bretschneider et al., 1995) and rat peritoneal macrophages (Naumov et al., 1995) exhibit rapid activation kinetics when exposed to ATP_o. The ATP_o response depends critically upon extracellular divalent cations (Mg²⁺ and Ca²⁺), such that cellular depolarization and membrane permeability are greatest in divalent-free media. The ability of Mg²⁺- and Ca²⁺–ATP complexes to reduce receptor occupancy by lowering the concentration of ATP⁴⁻, the effective form of the nucleotide agonist, is a hallmark of P2X₇/P2Z purinoceptor physiology (Cockcroft and Gomperts, 1979b ).In this study, we examined the dynamics of [Ca²⁺]_i changes elicited by ATP_o at the single-cell level in fura-2– loaded thymocytes. To our surprise, we found that the ATP_o-mediated [Ca²⁺]_i increase varies significantly between individual cells. Moreover, the kinetics of the rise in [Ca²⁺]_i at the single-cell level is characterized by a biphasic time course that is not detectable in average profiles. To correlate stages of thymocyte development with the degree of sensitivity to ATP_o, we measured the surface expression of specific T-lymphocyte markers, CD4 and CD8, before performing Ca²⁺-imaging experiments. Our data illustrate that thymocytes vary in sensitivity to ATP_o depending upon level of maturation and degree of blastogenesis. Small, terminally differentiated, CD4⁺CD8⁺ thymocytes were least sensitive to ATP_o, while 90% of the single-positive (CD4⁺CD8⁻ or CD4⁻CD8⁺) cells, believed to be the immediate precursors of mature peripheral T-lymphocytes, exhibited a robust, ATP_o-dependent rise in [Ca²⁺]_i. The in vitro data we have gathered suggest that ATP_o may drive thymocyte differentiation in the intact thymus.