SUMMARY Store-operated Ca2+ channels activated by the depletion of Ca2+ from the endoplasmic reticulum (ER) are a major Ca2+ entry pathway in non-excitable cells and are essential for T cell activation and adaptive immunity. Following store depletion, the ER Ca2+ sensor STIM1 and the CRAC channel protein Orai1 redistribute to ER-plasma membrane (PM) junctions, but the fundamental issue of how STIM1 activates the CRAC channel at these sites is unresolved. Here we identify a minimal, highly conserved 107-aa CRAC activation domain (CAD) of STIM1 that binds directly to the N- and C-termini of Orai1 to open the CRAC channel. Purified CAD forms a tetramer that clusters CRAC channels, but analysis of STIM1 mutants reveals that channel clustering is not sufficient for channel activation. These studies establish a molecular mechanism for store-operated Ca2+ entry in which the direct binding of STIM1 to Orai1 drives the accumulation and the activation of CRAC channels at ER-PM junctions.

Cells comprising a tissue migrate as part of a collective. How collective processes are coordinated over large multi-cellular assemblies has remained unclear, however, because mechanical stresses exerted at cell-cell junctions have not been accessible experimentally. We report here maps of these stresses within and between cells comprising a monolayer. Within the cell sheet there arise unanticipated fluctuations of mechanical stress that are severe, emerge spontaneously, and ripple across the monolayer. This stress landscape becomes increasingly rugged, sluggish, and cooperative with increasing system density. Within that landscape, local cellular migrations follow local orientations of maximal principal stress. Migrations of both endothelial and epithelial monolayers conform to this behavior, as do breast cancer cell lines before but not after the epithelial-mesenchymal transition. Collective migration in these diverse systems is seen to be governed by a simple but unifying physiological principle: neighboring cells join forces to transmit appreciable normal stress across the cell-cell junction, but migrate along orientations of minimal intercellular shear stress.

Synapses are asymmetric cellular adhesions that are critical for nervous system development and function, but the mechanisms that induce their formation are not well understood. We have previously identified thrombospondin as an astrocyte-secreted protein that promotes central nervous system (CNS) synaptogenesis. Here, we identify the neuronal thrombospondin receptor involved in CNS synapse formation as alpha2delta-1, the receptor for the anti-epileptic and analgesic drug gabapentin. We show that the VWF-A domain of alpha2delta-1 interacts with the epidermal growth factor-like repeats common to all thrombospondins. alpha2delta-1 overexpression increases synaptogenesis in vitro and in vivo and is required postsynaptically for thrombospondin- and astrocyte-induced synapse formation in vitro. Gabapentin antagonizes thrombospondin binding to alpha2delta-1 and powerfully inhibits excitatory synapse formation in vitro and in vivo. These findings identify alpha2delta-1 as a receptor involved in excitatory synapse formation and suggest that gabapentin may function therapeutically by blocking new synapse formation.

From coffee beans flowing in a chute to cells remodelling in a living tissue, a wide variety of close-packed collective systems— both inert and living—have the potential to jam. The collective can sometimes flow like a fluid or jam and rigidify like a solid. The unjammed-to-jammed transition remains poorly understood, however, and structural properties characterizing these phases remain unknown. Using primary human bronchial epithelial cells, we show that the jamming transition in asthma is linked to cell shape, thus establishing in that system a structural criterion for cell jamming. Surprisingly, the collapse of critical scaling predicts a counter-intuitive relationship between jamming, cell shape and cell–cell adhesive stresses that is borne out by direct experimental observations. Cell shape thus provides a rigorous structural signature for classification and investigation of bronchial epithelial layer jamming in asthma, and potentially in any process in disease or development in which epithelial dynamics play a prominent role.

Every adherent eukaryotic cell exerts appreciable traction forces upon its substrate. Moreover, every resident cell within the heart, great vessels, bladder, gut or lung routinely experiences large periodic stretches. As an acute response to such stretches the cytoskeleton can stiffen, increase traction forces and reinforce, as reported by some, or can soften and fluidize, as reported more recently by our laboratory, but in any given circumstance it remains unknown which response might prevail or why. Using a novel nanotechnology, we show here that in loading conditions expected in most physiological circumstances the localized reinforcement response fails to scale up to the level of homogeneous cell stretch; fluidization trumps reinforcement. Whereas the reinforcement response is known to be mediated by upstream mechanosensing and downstream signaling, results presented here show the fluidization response to be altogether novel: it is a direct physical effect of mechanical force acting upon a structural lattice that is soft and fragile. Cytoskeletal softness and fragility, we argue, is consistent with early evolutionary adaptations of the eukaryotic cell to material properties of a soft inert microenvironment.

Voltage- and store-operated calcium (Ca(2+)) channels are the major routes of Ca(2+) entry in mammalian cells, but little is known about how cells coordinate the activity of these channels to generate coherent calcium signals. We found that STIM1 (stromal interaction molecule 1), the main activator of store-operated Ca(2+) channels, directly suppresses depolarization-induced opening of the voltage-gated Ca(2+) channel Ca(V)1.2. STIM1 binds to the C terminus of Ca(V)1.2 through its Ca(2+) release-activated Ca(2+) activation domain, acutely inhibits gating, and causes long-term internalization of the channel from the membrane. This establishes a previously unknown function for STIM1 and provides a molecular mechanism to explain the reciprocal regulation of these two channels in cells.

Ca 2؉ -dependent inactivation (CDI) is a key regulator and hallmark of the Ca 2؉ release-activated Ca 2؉ (CRAC) channel, a prototypic store-operated Ca 2؉ channel. Although the roles of the endoplasmic reticulum Ca 2؉ sensor STIM1 and the channel subunit Orai1 in CRAC channel activation are becoming well understood, the molecular basis of CDI remains unclear. Recently, we defined a minimal CRAC activation domain (CAD; residues 342-448) that binds directly to Orai1 to activate the channel. Surprisingly, CADinduced CRAC currents lack fast inactivation, revealing a critical role for STIM1 in this gating process. Through truncations of full-length STIM1, we identified a short domain (residues 470 -491) C-terminal to CAD that is required for CDI. This domain contains a cluster of 7 acidic amino acids between residues 475 and 483. Neutralization of aspartate or glutamate pairs in this region either reduced or enhanced CDI, whereas the combined neutralization of six acidic residues eliminated inactivation entirely. Based on bioinformatics predictions of a calmodulin (CaM) binding site on Orai1, we also investigated a role for CaM in CDI. We identified a membrane-proximal N-terminal domain of Orai1 (residues 68 -91) that binds CaM in a Ca 2؉ -dependent manner and mutations that eliminate CaM binding abrogate CDI. These studies identify novel structural elements of STIM1 and Orai1 that are required for CDI and support a model in which CaM acts in concert with STIM1 and the N terminus of Orai1 to evoke rapid CRAC channel inactivation.calcium ͉ ion channel gating ͉ store-operated calcium entry ͉ patch-clamp ͉ calcium-binding proteins S tore-operated Ca 2ϩ channels provide a major route for receptor-stimulated Ca 2ϩ entry in nonexcitable cells (1). The Ca 2ϩ -release activated Ca 2ϩ (CRAC) channel, the bestcharacterized store-operated channel, is essential for generating Ca 2ϩ signals that drive the activation of T lymphocytes, mast cells, and platelets (2-4). CRAC channel activity is shaped by the combination of store-dependent activation and Ca 2ϩ -dependent inactivation (CDI) processes. The mechanisms linking depletion of Ca 2ϩ from the endoplasmic reticulum (ER) to activation of the CRAC channel are becoming well understood: reduction of ER luminal Ca 2ϩ causes the ER Ca 2ϩ sensor STIM1 (5, 6) to oligomerize (7), enabling its accumulation at ER-plasma membrane junctions (3,8,9), where it binds directly to the CRAC channel subunit to open the channel (13,14).Compared with activation, much less is known about the mechanisms underlying CDI, one of the hallmark characteristics of the CRAC current (I CRAC ) in mammalian cells. Initial studies in mast cells and T cells revealed that Ca 2ϩ influx at hyperpolarized potentials drives rapid inactivation on a time scale of tens of milliseconds through the binding of Ca 2ϩ to sites located several nanometers from the intracellular mouth of the pore (15, 16). Subsequent studies have suggested that calmodulin (CaM) and STIM1 both may contribute to this process. In a rat liver cell l...

The Ca 21-binding protein calmodulin mediates cellular Ca 21 signals in response to a wide array of stimuli in higher eukaryotes. Plants express numerous CaM isoforms. Transcription of one soybean (Glycine max) CaM isoform, SCaM-4, is dramatically induced within 30 min of pathogen or NaCl stresses. To characterize the cis-acting element(s) of this gene, we isolated an approximately 2-kb promoter sequence of the gene. Deletion analysis of the promoter revealed that a 130-bp region located between nucleotide positions 2858 and 2728 is required for the stressors to induce expression of SCaM-4. A hexameric DNA sequence within this region, GAAAAA (GT-1 cis-element), was identified as a core cis-acting element for the induction of the SCaM-4 gene. The GT-1 cis-element interacts with an Arabidopsis GT-1-like transcription factor, AtGT-3b, in vitro and in a yeast selection system. Transcription of AtGT-3b is also rapidly induced within 30 min after pathogen and NaCl treatment. These results suggest that an interaction between a GT-1 cis-element and a GT-1-like transcription factor plays a role in pathogen-and salt-induced SCaM-4 gene expression in both soybean and Arabidopsis.Plant cells, like animal cells, elevate their cytosolic free-calcium levels ([Ca 21 ] cyt ) with varying amplitude, frequency, and duration in response to a variety of external stimuli (Thomas et al., 1996; Berridge, 1997;McAinsh and Hetherington, 1998 -bound CaM transduces the signals into many cellular processes through modulation of a variety of CaM-binding proteins, including enzymes such as kinases, phosphatases, and nitric-oxide synthase, as well as receptors, ion channels, G-proteins, and transcription factors (Liao et al., 1996;Snedden and Fromm, 1998;Lee et al., 1999a;Zuhlke et al., 1999).In plant cells, in contrast to mammalian cells, multiple CaM genes code for a number of CaM isoforms. This has been shown in wheat (Triticum aestivum; Yang et al., 1996), potato (Solanum tuberosum; Takezawa et al., 1995;Poovaiah et al., 1996), and soybean (Glycine max; Lee et al., 1995a), among others. Over 30 genes encoding CaM isoforms are found in the Arabidopsis genome (The Arabidopsis Genome Initiative, 2000). We have recently cloned five CaM isoforms from soybean (SCaM-1-5). Although SCaM-1-3 are more than 90% identical to mammalian CaM, SCaM-4 and SCaM-5 exhibit only a 78% homology with SCaM-1 and are therefore the most divergent isoforms reported thus far in the plant and animal kingdoms. SCaM-4 is considered to be a bona fide CaM isoform based on the following characteristics. In its primary protein structure, SCaM-4 has four conserved putative EF-hands and a central linker region, hallmark structural features of CaM (Lee et al., 1995a). In addition, most of the nonconsensus amino acids occur outside the EF-hands, and the total number of Article, publication date, and citation information can be found at www.plantphysiol.org/cgi