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Highly regulated cardiomyocyte Ca 2+ fluxes drive heart contractions. Recent findings from multiple organisms demonstrate that the specific Ca 2+ transport mechanism known as store-operated Ca 2+ entry (SOCE) is essential in cardiomyocytes for proper heart function, and SOCE dysregulation results in cardiomyopathy. Mechanisms that regulate SOCE in cardiomyocytes are poorly understood. Here we tested the role of cytoskeletal septin proteins in cardiomyocyte SOCE regulation. Septins are essential SOCE modulators in other cell types, but septin functions in cardiomyocytes are nearly completely unexplored. We show using targeted genetics and intravital imaging of heart contractility in Drosophila that cardiomyocyte-specific depletion of septins 1, 2, and 4 results in heart dilation that phenocopies the effects of SOCE suppression. Heart dilation caused by septin 2 depletion was suppressed by SOCE upregulation, supporting the hypothesis that septin 2 is required in cardiomyocytes for sufficient SOCE function. A major function of SOCE is to support SERCA-dependent sarco/endoplasmic reticulum (S/ER) Ca 2+ stores, and augmenting S/ER store filling by SERCA overexpression also suppressed the septin 2 phenotype. We also ruled out several potential SOCE-independent septin functions, as septin 2 phenotypes were not due to septin function during development and septin 2 was not required for z-disk organization as defined by α-actinin labeling. These results demonstrate, for the first time, an essential role of septins in cardiomyocyte physiology and heart function that is due, at least in part, to septin regulation of SOCE function.

In this study, we examine the cause and progression of mitochondrial diseases linked to the loss of mtRNase P, a three-protein complex responsible for processing and cleaving mitochondrial transfer RNAs (tRNA) from their nascent transcripts. When mtRNase P function is missing, mature mitochondrial tRNA levels are decreased, resulting in mitochondrial dysfunction. mtRNase P is composed of Mitochondrial RNase P Protein (MRPP) 1, 2, and 3. MRPP1 and 2 have their own enzymatic activity separate from MRPP3, which is the endonuclease responsible for cleaving tRNA. Human mutations in all subunits cause mitochondrial disease. The loss of mitochondrial function can cause devastating, often multisystemic failures. When mitochondria do not provide enough energy and metabolites, the result can be skeletal muscle weakness, cardiomyopathy, and heart arrhythmias. These symptoms are complex and often difficult to interpret, making disease models useful for diagnosing disease onset and progression. Previously, we identified Drosophila orthologs of each mtRNase P subunit (Roswell/MRPP1, Scully/MRPP2, Mulder/MRPP3) and found that the loss of each subunit causes lethality and decreased mitochondrial tRNA processing in vivo . Here, we use Drosophila to model mtRNase P mitochondrial diseases by reducing the level of each subunit in skeletal and heart muscle using tissue-specific RNAi knockdown. We find that mtRNase P reduction in skeletal muscle decreases adult eclosion and causes reduced muscle mass and function. Adult flies exhibit significant age-progressive locomotor defects. Cardiac-specific mtRNase P knockdowns reduce fly lifespan for Roswell and Scully, but not Mulder. Using intravital imaging, we find that adult hearts have impaired contractility and exhibit substantial arrhythmia. This occurs for roswell and mulder knockdowns, but with little effect for scully . The phenotypes shown here are similar to those exhibited by patients with mitochondrial disease, including disease caused by mutations in MRPP1 and 2. These findings also suggest that skeletal and cardiac deficiencies induced by mtRNase P loss are differentially affected by the three subunits. These differences could have implications for disease progression in skeletal and heart muscle and shed light on how the enzyme complex functions in different tissues., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Saoji, Petersen, Sen, Tripoli, Smyth and Cox.)

Membrane contact sites are critical junctures for organelle signaling and communication. Endoplasmic reticulum-plasma membrane (ER-PM) contact sites were the first membrane contact sites to be described; however, the protein composition and molecular function of these sites is still emerging. Here, we leverage yeast and Drosophila model systems to uncover a novel role for the Hobbit (Hob) proteins at ER-PM contact sites. We find that Hobbit localizes to ER-PM contact sites in both yeast cells and the Drosophila larval salivary glands, and this localization is mediated by an N-terminal ER membrane anchor and conserved C-terminal sequences. The C-terminus of Hobbit binds to plasma membrane phosphatidylinositols, and the distribution of these lipids is altered in hobbit mutant cells. Notably, the Hobbit protein is essential for viability in Drosophila, providing one of the first examples of a membrane contact site-localized lipid binding protein that is required for development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Heart failure is often preceded by pathological cardiac hypertrophy, a thickening of the heart musculature driven by complex gene regulatory and signaling processes. The Drosophila heart has great potential as a genetic model for deciphering the underlying mechanisms of cardiac hypertrophy. However, current methods for evaluating hypertrophy of the Drosophila heart are laborious and difficult to carry out reproducibly. Here, we demonstrate that microcomputerized tomography (microCT) is an accessible, highly reproducible method for nondestructive, quantitative analysis of Drosophila heart morphology and size. To validate our microCT approach for analyzing Drosophila cardiac hypertrophy, we show that expression of constitutively active Ras ( Ras85D V12 ), previously shown to cause hypertrophy of the fly heart, results in significant thickening of both adult and larval heart walls when measured from microCT images. We then show using microCT analysis that genetic upregulation of store-operated Ca 2+ entry (SOCE) driven by expression of constitutively active Stim (Stim CA ) or Orai (Orai CA ) proteins also results in significant hypertrophy of the Drosophila heart, through a process that specifically depends on Orai Ca 2+ influx channels. Intravital imaging of heart contractility revealed significantly reduced end-diastolic and end-systolic dimensions in Stim CA - and Orai CA -expressing hearts, consistent with the hypertrophic phenotype. These results demonstrate that increased SOCE activity is an important driver of hypertrophic cardiomyocyte growth, and demonstrate how microCT analysis combined with tractable genetic tools in Drosophila can be used to delineate molecular signaling processes that underlie cardiac hypertrophy and heart failure. NEW & NOTEWORTHY Genetic analysis of Drosophila cardiac hypertrophy holds immense potential for the discovery of new therapeutic targets to prevent and treat heart failure. This potential has been hindered by a lack of rapid and effective methods for analyzing heart size in flies. Here, we demonstrate that analysis of the Drosophila heart with microcomputerized tomography yields accurate and highly reproducible heart size measurements that can be used to analyze heart growth and cardiac hypertrophy in Drosophila .

We recently discovered that the histone deacetylase inhibitor, trichostatin A (TSA), increases expression of the sulfonylurea receptor 2 (SUR2; Abcc9) subunit of the ATP-sensitive K + (K ATP ) channel in HL-1 cardiomyocytes. Interestingly, the increase in SUR2 was abolished with exogenous cholesterol, suggesting that cholesterol may regulate channel expression. In the present study, we tested the hypothesis that TSA increases SUR2 by depleting cholesterol and activating the sterol response element binding protein (SREBP) family of transcription factors. Treatment of HL-1 cardiomyocytes with TSA (30 ng/ml) caused a time-dependent increase in SUR2 mRNA expression that correlates with the time course of cholesterol depletion assessed by filipin staining. Consistent with the cholesterol-dependent regulation of SREBP increasing SUR2 mRNA expression, we observe a significant increase in SREBP cleavage and translocation to the nucleus following TSA treatment that is inhibited by exogenous cholesterol. Further supporting the role of SREBP in mediating the effect of TSA on K ATP subunit expression, SREBP1 significantly increased luciferase reporter gene expression driven by the upstream SUR2 promoter. Lastly, HL-1 cardiomyocytes treated with the SREBP inhibitor PF429242 significantly suppresses the effect of TSA on SUR2 gene expression. These results demonstrate that SREBP is an important regulator of K ATP channel expression and suggest a novel method by which hypercholesterolemia may exert negative effects on the cardiovascular system, namely, by suppressing expression of the K ATP channel., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes' large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by loss of alpha motor neurons and skeletal muscle atrophy. The disease is caused by mutations of the SMN1 gene that result in reduced functional expression of survival motor neuron (SMN) protein. SMN is ubiquitously expressed, and there have been reports of cardiovascular dysfunction in the most severe SMA patients and animal models of the disease. In this study, we directly assessed the function of cardiomyocytes isolated from a severe SMA model mouse and cardiomyocytes generated from patient-derived IPSCs. Consistent with impaired cardiovascular function at the very early disease stages in mice, heart failure markers such as brain natriuretic peptide were significantly elevated. Functionally, cardiomyocyte relaxation kinetics were markedly slowed and the T 50 for Ca 2+ sequestration increased to 146 ± 4 ms in SMN-deficient cardiomyocytes from 126 ± 4 ms in wild type cells. Reducing SMN levels in cardiomyocytes from control patient IPSCs slowed calcium reuptake similar to SMA patent-derived cardiac cells. Importantly, restoring SMN increased calcium reuptake rate. Taken together, these results indicate that SMN deficiency impairs cardiomyocyte function at least partially through intracellular Ca 2+ cycling dysregulation.

Store-operated Ca 2+ entry (SOCE) is an essential Ca 2+ signaling mechanism present in most animal cells. SOCE refers to Ca 2+ influx that is activated by depletion of sarco/endoplasmic reticulum (S/ER) Ca 2+ stores. The main components of SOCE are STIM and Orai. STIM proteins function as S/ER Ca 2+ sensors, and upon S/ER Ca 2+ depletion STIM rearranges to S/ER-plasma membrane junctions and activates Orai Ca 2+ influx channels. Studies have implicated SOCE in cardiac hypertrophy pathogenesis, but SOCE's role in normal heart physiology remains poorly understood. We therefore analyzed heart-specific SOCE function in Drosophila , a powerful animal model of cardiac physiology. We show that heart-specific suppression of Stim and Orai in larvae and adults resulted in reduced contractility consistent with dilated cardiomyopathy. Myofibers were also highly disorganized in Stim and Orai RNAi hearts, reflecting possible decompensation or upregulated stress signaling. Furthermore, we show that reduced heart function due to SOCE suppression adversely affected animal viability, as heart specific Stim and Orai RNAi animals exhibited significant delays in post-embryonic development and adults died earlier than controls. Collectively, our results demonstrate that SOCE is essential for physiological heart function, and establish Drosophila as an important model for understanding the role of SOCE in cardiac pathophysiology., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

In dividing animal cells the endoplasmic reticulum (ER) concentrates around the poles of the spindle apparatus by associating with astral microtubules (MTs), and this association is essential for proper ER partitioning to progeny cells. The mechanisms that associate the ER with astral MTs are unknown. Because astral MT minus-ends are anchored by centrosomes at spindle poles, we hypothesized that the MT minus-end motor dynein mediates ER concentration around spindle poles. Live in vivo imaging of Drosophila spermatocytes revealed that dynein is required for ER concentration around centrosomes during late interphase. In marked contrast, however, dynein suppression had no effect on ER association with astral MTs and concentration around spindle poles in early M-phase. In fact, there was a sudden onset of ER association with astral MTs in dynein RNAi cells, revealing activation of an M-phase specific mechanism of ER-MT association. ER redistribution to spindle poles also did not require non-claret disjunctional (ncd), the other known Drosophila MT minus-end motor, nor Klp61F, a MT plus-end motor that generates spindle poleward forces. Collectively, our results suggest that a novel, M-phase specific mechanism of ER-MT association that is independent of MT minus-end motors is required for proper ER partitioning in dividing cells.

Mechanisms that regulate partitioning of the endoplasmic reticulum (ER) during cell division are largely unknown. Previous studies have mostly addressed ER partitioning in cultured cells, which may not recapitulate physiological processes that are critical in developing, intact tissues. We have addressed this by analysing ER partitioning in asymmetrically dividing stem cells, in which precise segregation of cellular components is essential for proper development and tissue architecture. We show that in Drosophila neural stem cells, called neuroblasts, the ER asymmetrically partitioned to centrosomes early in mitosis. This correlated closely with the asymmetric nucleation of astral microtubules (MTs) by centrosomes, suggesting that astral MT association may be required for ER partitioning by centrosomes. Consistent with this, the ER also associated with astral MTs in meiotic Drosophila spermatocytes and during syncytial embryonic divisions. Disruption of centrosomes in each of these cell types led to improper ER partitioning, demonstrating the critical role for centrosomes and associated astral MTs in this process. Importantly, we show that the ER also associated with astral MTs in cultured human cells, suggesting that this centrosome/astral MT-based partitioning mechanism is conserved across animal species., (© 2015 The Authors.)

During cellular division, centrosomes are tasked with building the bipolar mitotic spindle, which partitions the cellular contents into two daughter cells. While every cell will receive an equal complement of chromosomes, not every organelle is symmetrically passaged to the two progeny in many cell types. In this review, we highlight the conservation of nonrandom centrosome segregation in asymmetrically dividing stem cells, and we discuss how the asymmetric function of centrosomes could mediate nonrandom segregation of organelles and mRNA. We propose that such a mechanism is critical for insuring proper cell fitness, function, and fate.

The endoplasmic reticulum (ER) undergoes significant reorganization between interphase and mitosis, but the underlying mechanisms are unknown. Stromal interaction molecule 1 (STIM1) is an ER Ca(2+) sensor that activates store-operated Ca(2+) entry (SOCE) and also functions in ER morphogenesis through its interaction with the microtubule +TIP protein end binding 1 (EB1). We previously demonstrated that phosphorylation of STIM1 during mitosis suppresses SOCE. We now show that STIM1 phosphorylation is a major regulatory mechanism that excludes ER from the mitotic spindle. In mitotic HeLa cells, the ER forms concentric sheets largely excluded from the mitotic spindle. We show that STIM1 dissociates from EB1 in mitosis and localizes to the concentric ER sheets. However, a nonphosphorylatable STIM1 mutant (STIM1(10A)) colocalized extensively with EB1 and drove ER mislocalization by pulling ER tubules into the spindle. This effect was rescued by mutating the EB1 interaction site of STIM1(10A), demonstrating that aberrant association of STIM1(10A) with EB1 is responsible for the ER mislocalization. A STIM1 phosphomimetic exhibited significantly impaired +TIP tracking in interphase but was ineffective at inhibiting SOCE, suggesting different mechanisms of regulation of these two STIM1 functions by phosphorylation. Thus, ER spindle exclusion and ER-dependent Ca(2+) signaling during mitosis require multimodal STIM1 regulation by phosphorylation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Store-operate Ca2+ channels gate Ca2+ entry into the cytoplasm in response to the depletion of Ca2+ from endoplasmic reticulum Ca2+ stores. The major molecular components of store-operated Ca2+ entry are STIM (stromal-interacting molecule) 1 (and in some instances STIM2) that serves as the endoplasmic reticulum Ca2+ sensor, and Orai (Orai1, Orai2 and Orai3) which function as pore-forming subunits of the store-operated channel. It has been known for some time that store-operated Ca2+ entry is shut down during cell division. Recent work has revealed complex mechanisms regulating the functions and locations of both STIM1 and Orai1 in dividing cells.

The process of store-operated Ca(2+) entry (SOCE), whereby Ca(2+) influx across the plasma membrane is activated in response to depletion of intracellular Ca(2+) stores in the endoplasmic reticulum (ER), has been under investigation for greater than 25 years; however, only in the past 5 years have we come to understand this mechanism at the molecular level. A surge of recent experimentation indicates that STIM molecules function as Ca(2+) sensors within the ER that, upon Ca(2+) store depletion, rearrange to sites very near to the plasma membrane. At these plasma membrane-ER junctions, STIM interacts with and activates SOCE channels of the Orai family. The molecular and biophysical data that have led to these findings are discussed in this review, as are several controversies within this rapidly expanding field., (No claim to original US government works Journal compilation © 2010 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Store-operated Ca(2+) entry (SOCE) and Ca(2+) release-activated Ca(2+) currents (I(crac)) are strongly suppressed during cell division, the only known physiological situation in which Ca(2+) store depletion is uncoupled from the activation of Ca(2+) influx [corrected]. We found that the endoplasmic reticulum (ER) Ca(2+) sensor STIM1 failed to rearrange into near-plasma membrane puncta in mitotic cells, a critical step in the SOCE-activation pathway. We also found that STIM1 from mitotic cells is recognized by the phospho-specific MPM-2 antibody, suggesting that STIM1 is phosphorylated during mitosis. Removal of ten MPM-2 recognition sites by truncation at amino acid 482 abolished MPM-2 recognition of mitotic STIM1, and significantly rescued STIM1 rearrangement and SOCE response in mitosis. We identified Ser 486 and Ser 668 as mitosis-specific phosphorylation sites, and STIM1 containing mutations of these sites to alanine also significantly rescued mitotic SOCE. Therefore, phosphorylation of STIM1 at Ser 486 and Ser 668, and possibly other sites, underlies suppression of SOCE during mitosis.

When cells are activated by calcium-mobilizing agonists at low, physiological concentrations, the resulting calcium signals generally take the form of repetitive regenerative discharges of stored calcium, termed calcium oscillations [1]. These intracellular calcium oscillations have long fascinated biologists as a mode of digitized intracellular signaling. Recent work has highlighted the role of calcium influx as an essential component of calcium oscillations [2]. This influx occurs through a process known as store-operated calcium entry, which is initiated by calcium sensor proteins, STIM1 and STIM2, in the endoplasmic reticulum [3]. STIM2 is activated by changes in endoplasmic reticulum calcium near the resting level, whereas a threshold of calcium depletion is required for STIM1 activation [4]. Here we show that, surprisingly, it is STIM1 and not STIM2 that is exclusively involved in calcium entry during calcium oscillations. The implication is that each oscillation produces a transient drop in endoplasmic reticulum calcium and that this drop is sufficient to transiently activate STIM1. This transient activation of STIM1 can be observed in some cells by total internal reflection fluorescence microscopy. This arrangement nicely provides a clearly defined and unambiguous signaling system, translating a digital calcium release signal into calcium influx that can signal to downstream effectors.

Recent studies have defined roles for STIM1 and Orai1 as calcium sensor and calcium channel, respectively, for Ca(2+)-release activated Ca(2+) (CRAC) channels, channels underlying store-operated Ca(2+) entry (SOCE). In addition, these proteins have been suggested to function in signalling and constructing other channels with biophysical properties distinct from the CRAC channels. Using the human kidney cell line, HEK293, we examined the hypothesis that STIM1 can interact with and regulate members of a family of non-selective cation channels (TRPC) which have been suggested to also function in SOCE pathways under certain conditions. Our data reveal no role for either STIM1 or Orai1 in signalling of TRPC channels. Specifically, Ca(2+) entry seen after carbachol treatment in cells transiently expressing TRPC1, TRPC3, TRPC5 or TRPC6 was not enhanced by the co-expression of STIM1. Further, knockdown of STIM1 in cells expressing TRPC5 did not reduce TRPC5 activity, in contrast to one published report. We previously reported in stable TRPC7 cells a Ca(2+) entry which was dependent on TRPC7 and appeared store-operated. However, we show here that this TRPC7-mediated entry was also not dependent on either STIM1 or Orai1, as determined by RNA interference (RNAi) and expression of a constitutively active mutant of STIM1. Further, we determined that this entry was not actually store-operated, but instead TRPC7 activity which appears to be regulated by SERCA. Importantly, endogenous TRPC activity was also not regulated by STIM1. In vascular smooth muscle cells, arginine-vasopressin (AVP) activated non-selective cation currents associated with TRPC6 activity were not affected by RNAi knockdown of STIM1, while SOCE was largely inhibited. Finally, disruption of lipid rafts significantly attenuated TRPC3 activity, while having no effect on STIM1 localization or the development of I(CRAC). Also, STIM1 punctae were found to localize in regions distinct from lipid rafts. This suggests that TRPC signalling and STIM1/Orai1 signalling occur in distinct plasma membrane domains. Thus, TRPC channels appear to be activated by mechanisms dependent on phospholipase C which do not involve the Ca(2+) sensor, STIM1.

Activation of surface membrane receptors coupled to phospholipase C results in the generation of cytoplasmic Ca2+ signals comprised of both intracellular Ca2+ release, and enhanced entry of Ca2+ across the plasma membrane. A primary mechanism for this Ca2+ entry process is attributed to store-operated Ca2+ entry, a process that is activated by depletion of Ca2+ ions from an intracellular store by inositol 1,4,5-trisphosphate. Our understanding of the mechanisms underlying both Ca2+ release and store-operated Ca2+ entry have evolved from experimental approaches that include the use of fluorescent Ca2+ indicators and electrophysiological techniques. Pharmacological manipulation of this Ca2+ signaling process has been somewhat limited; but recent identification of key molecular players, STIM and Orai family proteins, has provided new approaches. Here we describe practical methods involving fluorescent Ca2+ indicators and electrophysiological approaches for dissecting the observed intracellular Ca2+ signal to reveal characteristics of store-operated Ca2+ entry, highlighting the advantages, and limitations, of these approaches.

Store-operated Ca2+ entry (SOCE) is likely the most common mode of regulated influx of Ca2+ into cells. However, only a limited number of pharmacological agents have been shown to modulate this process. 2-Aminoethyldiphenyl borate (2-APB) is a widely used experimental tool that activates and then inhibits SOCE and the underlying calcium release-activated Ca2+ current (I CRAC). The mechanism by which depleted stores activates SOCE involves complex cellular movements of an endoplasmic reticulum Ca2+ sensor, STIM1, which redistributes to puncta near the plasma membrane and, in some manner, activates plasma membrane channels comprising Orai1, -2, and -3 subunits. We show here that 2-APB blocks puncta formation of fluorescently tagged STIM1 in HEK293 cells. Accordingly, 2-APB also inhibited SOCE and I(CRAC)-like currents in cells co-expressing STIM1 with the CRAC channel subunit, Orai1, with similar potency. However, 2-APB inhibited STIM1 puncta formation less well in cells co-expressing Orai1, indicating that the inhibitory effects of 2-APB are not solely dependent upon STIM1 reversal. Further, 2-APB only partially inhibited SOCE and current in cells co-expressing STIM1 and Orai2 and activated sustained currents in HEK293 cells expressing Orai3 and STIM1. Interestingly, the Orai3-dependent currents activated by 2-APB showed large outward currents at potentials greater than +50 mV. Finally, Orai3, and to a lesser extent Orai1, could be directly activated by 2-APB, independently of internal Ca2+ stores and STIM1. These data reveal novel and complex actions of 2-APB effects on SOCE that can be attributed to effects on both STIM1 as well as Orai channel subunits.

Stim1 responds to depletion of ER Ca2+ stores by rearranging from tubular structures throughout the ER into punctate structures near the plasma membrane, where it activates Orai store-operated Ca2+ entry (SOCE) channels. However, the mechanism and structural determinants of the localization and reversal of Stim1 puncta formation are poorly understood. Using HEK293 cells expressing Stim1 tagged with enhanced yellow fluorescent protein (EYFP-Stim1), we show that the basis for SOCE termination is the reversal of the punctate Stim1 localization, which absolutely depends on SOCE-dependent store refilling. We also describe rapid, store-independent reversal of EYFP-Stim1 punctae by the ML-9 inhibitor of myosin-light-chain kinase (MLCK). ML-9 similarly inhibited SOCE and the Ca2+-release-activated Ca2+ (CRAC) current. Reversal by ML-9 resulted in full re-establishment of the tubular EYFP-Stim1 localization. A constitutively active EF-hand mutant of EYFP-Stim1 was also reversed by ML-9, regardless of the Ca2+ store content. Inhibition by ML-9 was not due to MLCK inhibition as other inhibitors of MLCK had no effect. Finally, we provide evidence that EYFP-Stim1 punctae form in specific predetermined cellular loci. We conclude that SOCE is tightly coupled to formation of Stim1 puncta, and both SOCE and puncta formation involve a dynamic, reversible signaling complex that probably consists of components in addition to Stim1 and Orai channels.

Stromal interaction molecule 1 (STIM1) is a transmembrane protein that is essential for store-operated Ca(2+) entry, a process of extracellular Ca(2+) influx in response to the depletion of Ca(2+) stores in the endoplasmic reticulum (ER) (reviewed in [1-4]). STIM1 localizes predominantly to the ER; upon Ca(2+) release from the ER, STIM1 translocates to the ER-plasma membrane junctions and activates Ca(2+) channels (reviewed in [1-4]). Here, we show that STIM1 directly binds to the microtubule-plus-end-tracking protein EB1 and forms EB1-dependent comet-like accumulations at the sites where polymerizing microtubule ends come in contact with the ER network. Therefore, the previously observed tubulovesicular motility of GFP-STIM1 [5] is not a motor-based movement but a traveling wave of diffusion-dependent STIM1 concentration in the ER membrane. STIM1 overexpression strongly stimulates ER extension occurring through the microtubule "tip attachment complex" (TAC) mechanism [6, 7], a process whereby an ER tubule attaches to and elongates together with the EB1-positive end of a growing microtubule. Depletion of STIM1 and EB1 decreases TAC-dependent ER protrusion, indicating that microtubule growth-dependent concentration of STIM1 in the ER membrane plays a role in ER remodeling.

We examined the role of the microtubule cytoskeleton in the localization and store-operated Ca(2+) entry (SOCE) function of the endoplasmic reticulum (ER) Ca(2+) sensor stromal interaction molecule 1 (STIM1) in HEK 293 cells. STIM1 tagged with an enhanced yellow fluorescent protein (EYFP-STIM1) exhibited a fibrillar localization that colocalized with endogenous alpha-tubulin. Depolymerization of microtubules with nocodazole caused a change from a fibrillar EYFP-STIM1 localization to one that was similar to that of the ER. Treatment of HEK 293 cells with nocodazole had a detrimental impact on SOCE and the associated Ca(2+) release-activated Ca(2+) current (I(CRAC)). This inhibition was significantly reversed in cells overexpressing EYFP-STIM1, implying that the primary inhibitory effect of nocodazole is related to STIM1 function. Surprisingly, nocodazole treatment alone induced significant SOCE and I(CRAC) in cells expressing EYFP-STIM1, and this was accompanied by an increase in EYFP-STIM1 fluorescence near the plasma membrane. We conclude that microtubules play a facilitative role in the SOCE signaling pathway by optimizing the localization of STIM1.

The recent discoveries of Stim1 and Orai proteins have shed light on the molecular makeup of both the endoplasmic reticulum Ca(2+) sensor and the calcium release-activated calcium (CRAC) channel, respectively. In this study, we investigated the regulation of CRAC channel function by extracellular Ca(2+) for channels composed primarily of Orai1, Orai2, and Orai3, by co-expressing these proteins together with Stim1, as well as the endogenous channels in HEK293 cells. As reported previously, Orai1 or Orai2 resulted in a substantial increase in CRAC current (I(crac)), but Orai3 failed to produce any detectable Ca(2+)-selective currents. However, sodium currents measured in the Orai3-expressing HEK293 cells were significantly larger in current density than Stim1-expressing cells. Moreover, upon switching to divalent free external solutions, Orai3 currents were considerably more stable than Orai1 or Orai2, indicating that Orai3 channels undergo a lesser degree of depotentiation. Additionally, the difference between depotentiation from Ca(2+) and Ba(2+) or Mg(2+) solutions was significantly less for Orai3 than for Orai1 or -2. Nonetheless, the Na(+) currents through Orai1, Orai2, and Orai3, as well as the endogenous store-operated Na(+) currents in HEK293 cells, were all inhibited by extracellular Ca(2+) with a half-maximal concentration of approximately 20 mum. We conclude that Orai1, -2, and -3 channels are similarly inhibited by extracellular Ca(2+), indicating similar affinities for Ca(2+) within the selectivity filter. Orai3 channels appeared to differ from Orai1 and -2 in being somewhat resistant to the process of Ca(2+) depotentiation.

The canonical transient receptor potential (TRPC) cation channels are mammalian homologs of the photoreceptor channel TRP in Drosophila melanogaster. All seven TRPCs (TRPC1 through TRPC7) can be activated through Gq/11 receptors or receptor tyrosine kinase (RTK) by mechanisms downstream of phospholipase C. The last decade saw a rapidly growing interest in understanding the role of TRPC channels in calcium entry pathways as well as in understanding the signal(s) responsible for TRPC activation. TRPC channels have been proposed to be activated by a variety of signals including store depletion, membrane lipids, and vesicular insertion into the plasma membrane. Here we discuss recent developments in the mode of activation as well as the pharmacological and electrophysiological properties of this important and ubiquitous family of cation channels.

Depletion of intracellular Ca2+ stores induces Ca2+ influx across the plasma membrane through store-operated channels (SOCs). This store-operated Ca2+ influx is important for the replenishment of the Ca2+ stores, and is also involved in many signaling processes by virtue of the ability of intracellular Ca2+ to act as a second messenger. For many years, the molecular identities of particular SOCs, as well as the signaling mechanisms by which these channels are activated, have been elusive. Recently, however, the mammalian proteins STIM1 and Orai1 were shown to be necessary for the activation of store-operated Ca2+ entry in a variety of mammalian cells. Here we present molecular, pharmacological, and electrophysiological properties of SOCs, with particular focus on the roles that STIM1 and Orai1 may play in the signaling processes that regulate various pathways of store-operated entry.

The molecular nature of store-operated Ca(2+)-selective channels has remained an enigma, due largely to the continued inability to convincingly demonstrate Ca(2+)-selective store-operated currents resulting from exogenous expression of known genes. Recent findings have implicated two proteins, Stim1 and Orai1, as having essential roles in store-operated Ca(2+) entry across the plasma membrane. However, transient overexpression of these proteins on their own results in little or no increase in store-operated entry. Here we demonstrate dramatic synergism between these two mediators; co-transfection of HEK293 cells with Stim1 and Orai1 results in an approximate 20-fold increase in store-operated Ca(2+) entry and Ca(2+)-selective current. This demonstrates that these two proteins are limiting for both the signaling and permeation mechanisms for Ca(2+)-selective store-operated Ca(2+) entry. There are three mammalian homologs of Orai1, and in expression experiments they all produced or augmented store-operated Ca(2+) entry with efficacies in the order Orai1 > Orai2 > Orai3. Stim1 apparently initiates the signaling process by acting as a Ca(2+) sensor in the endoplasmic reticulum. This results in rearrangement of Stim1 within the cell and migration toward the plasma membrane to regulate in some manner Orai1 located in the plasma membrane. However, we demonstrate that Stim1 does not incorporate in the surface membrane, and thus likely regulates or interacts with Orai1 at sites of close apposition between the plasma membrane and an intracellular Stim1-containing organelle.

Regulated translocation of canonical transient receptor potential (TRPC) proteins to the plasma membrane has been proposed as a mechanism of their activation. By using total internal reflection fluorescence microscopy (TIRFM), we monitored green fluorescent protein-labeled TRPC3 (TRPC3-GFP) movement to the plasma membrane in HEK293 cells stably expressing this fusion protein. We observed no increase in TRPC3-GFP TIRFM in response to the muscarinic receptor agonist methacholine or the synthetic diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol, despite activation of TRPC3 by these agents. We did, however, observe a TIRFM response to epidermal growth factor (EGF). This TIRFM response to EGF was accompanied by increased Ba2+ entry and TRPC3 currents. However, 1-oleoyl-2-acetyl-sn-glycerol-induced TRPC3 activity was not increased. TIRFM also increased in response to Gd3+, a competitive inhibitor of TRPC3 channels. This may be indicative of constitutive trafficking of TRPC3, with Gd3+ acting to "trap" cycling TRPC3 molecules in the plasma membrane. Consistent with this interpretation, TRPC3-expressing cells exhibited large variance in membrane capacitance, and this variance was decreased by both Gd3+ and EGF. These results indicate the following: (i) trafficking of TRPC3 may play a role in regulating the concentration of channels in the plasma membrane but is not involved in activation through the phospholipase C pathway; (ii) TRPC3 undergoes constitutive cyclical trafficking in the plasma membrane, and the mechanism by which growth factors increase the number of plasma membrane channels may involve stabilizing them in the plasma membrane.

TRPC3 is a nonselective cation channel member of the "canonical" transient receptor potential (TRPC) family whose members are activated by phospholipase C-coupled receptors. TRPC3 can be activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) in a protein kinase C-independent manner. On the other hand, phorbol 12-myristate 13-acetate (PMA) inhibits OAG-mediated TRPC3 channel activation, suggesting that phosphorylation of TRPC3 by protein kinase C is a mechanism of receptor-mediated negative feedback. Here, we show PMA-induced phosphorylation of TRPC3 channels in vivo. We demonstrate by site-directed mutagenesis that a single site containing Ser(712) and conserved among all members of the TRPC family is essential for protein kinase C-mediated negative regulation of TRPC3. In human embryonic kidney 293 cells expressing a TRPC3 mutant in which Ser(712) was replaced by alanine (S712A), PMA failed to block channel activation, whereas wild-type TRPC3 activity was completely inhibited. Phosphorylation of the S712A TRPC3 mutant was not stimulated in response to PMA treatment. Furthermore, S712A TRPC3 mutant-mediated Ca(2+) entry after methacholine activation was significantly greater than that of wild-type TRPC3. These findings demonstrate a dual role for phospholipase C-generated diacylglycerol, which serves as a signal for TRPC3 activation as well as a signal for negative feedback via protein kinase C-mediated phosphorylation.

KN-93, a Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor, concentration-dependently and reversibly inhibited inositol 1,4,5-trisphosphate receptor (IP(3)R)-mediated [Ca(2+)](i) signaling in mouse eggs and permeabilized A7r5 smooth muscle cells, two cell types predominantly expressing type-1 IP(3)R (IP(3)R-1). KN-92, an inactive analog, was ineffective. The inhibitory action of KN-93 on Ca(2+) signaling depended neither on effects on IP(3) metabolism nor on the filling grade of Ca(2+) stores, suggesting a direct action on the IP(3)R. Inhibition was independent of CaMKII, since in identical conditions other CaMKII inhibitors (KN-62, peptide 281-309, and autocamtide-related inhibitory peptide) were ineffective and since CaMKII activation was precluded in permeabilized cells. Moreover, KN-93 was most effective in the absence of Ca(2+). Analysis of Ca(2+) release in A7r5 cells at varying [IP(3)], of IP(3)R-1 degradation in eggs, and of [(3)H]IP(3) binding in Sf9 microsomes all indicated that KN-93 did not affect IP(3) binding. Comparison of the inhibition of Ca(2+) release and of [(3)H]IP(3) binding by KN-93 and calmodulin (CaM), either separately or combined, was compatible with a specific interaction of KN-93 with a CaM-binding site on IP(3)R-1. This was also consistent with the much smaller effect of KN-93 in permeabilized 16HBE14o(-) cells that predominantly express type 3 IP(3)R, which lacks the high affinity CaM-binding site. These findings indicate that KN-93 inhibits IP(3)R-1 directly and may therefore be a useful tool in the study of IP(3)R functional regulation.

Subclinical inflammatory activity may be one of the factors which influences the variable natural history of farmers' lung. Serum lysozyme (LYS) and angiotensin converting enzyme (ACE) have been measured in 52 farmers with a previous history of farmers' lung and in 51 healthy control farmers. The group with farmers' lung assessed during the winter, although having had no recent acute symptoms, had significantly higher levels of LYS and ACE compared to both healthy control farmers seen in winter and farmers' lung subjects seen in summer. There was a mild but significant negative correlation between LYS and farm size in the farmers' lung subjects assessed in winter. The results suggest that active inflammation may be present in subjects with farmers' lung in the absence of acute symptoms during the season when dust exposure occurs as the result of feeding cattle.

Surveys were carried out on random samples of the farming population in Devon and Wales in order to estimate the prevalence of respiratory symptoms and of positive precipitin reactions to thermophilic fungi. Bronchitis, as defined, was common among the Welsh hill farmers, and the proportion of positive serological tests was higher in both the areas surveyed in Wales as compared with Devon. All three surveys confirmed a previous finding that the proportion of positive precipitin tests was higher among non-smokers than smokers. Although the numbers were small there was some indication that measurement of peak expiratory flow showed different relationships with age in non-smokers according to the presence or absence of positive precipitin tests. The difficulty of determining prevalence rates for farmer's lung is discussed, but the results suggest a rate not dissimilar to those found in two areas of Scotland which were more than 20 times higher than any figure previously reported in Britain.

Farmer's lung is a cause of disability to agricultural workers in Devon and there is no evidence that the incidence is falling. A survey of known cases was made to assess the degree of disability in relation to the clinical history, the presence of farmer's lung precipitins, tests of lung function, and radiographic changes. Information was obtained about 200 patients diagnosed between 1939 and 1971. A survey of 148 of these patients showed that the disease was most commonly diagnosed in men aged 40 to 50 years and the most important symptom at diagnosis was dyspnoea related to occupational exposure to hay or grain. The onset was often insidious and only 67 patients (45%) were diagnosed during the first year of the disorder. Disability was severe in about one-third of the cases. The degree of disability did not seem to be related to the serological changes recorded either at diagnosis or at the time of our survey. Disability was commonly associated with restriction and reduced gas transfer factor and with airways obstruction in more severe cases. Many individuals reporting significant disability had only slightly abnormal ventilatory function tests at rest. Radiographic changes were found at survey in about one-third of the subjects reporting disability. Many farmers had not used an efficient mask. Treatment is unsatisfactory but steroid therapy is effective in acute episodes.