共查询到20条相似文献,搜索用时 31 毫秒
1.
Miao Xu Ke Liu Manju Swaroop Forbes D. Porter Rohini Sidhu Sally Finkes Daniel S. Ory Juan J. Marugan Jingbo Xiao Noel Southall William J. Pavan Cristin Davidson Steven U. Walkley Alan T. Remaley Ulrich Baxa Wei Sun John C. McKew Christopher P. Austin Wei Zheng 《The Journal of biological chemistry》2012,287(47):39349-39360
Niemann-Pick disease type C (NPC) and Wolman disease are two members of a family of storage disorders caused by mutations of genes encoding lysosomal proteins. Deficiency in function of either the NPC1 or NPC2 protein in NPC disease or lysosomal acid lipase in Wolman disease results in defective cellular cholesterol trafficking. Lysosomal accumulation of cholesterol and enlarged lysosomes are shared phenotypic characteristics of both NPC and Wolman cells. Utilizing a phenotypic screen of an approved drug collection, we found that δ-tocopherol effectively reduced lysosomal cholesterol accumulation, decreased lysosomal volume, increased cholesterol efflux, and alleviated pathological phenotypes in both NPC1 and Wolman fibroblasts. Reduction of these abnormalities may be mediated by a δ-tocopherol-induced intracellular Ca2+ response and subsequent enhancement of lysosomal exocytosis. Consistent with a general mechanism for reduction of lysosomal lipid accumulation, we also found that δ-tocopherol reduces pathological phenotypes in patient fibroblasts from other lysosomal storage diseases, including NPC2, Batten (ceroid lipofuscinosis, neuronal 2, CLN2), Fabry, Farber, Niemann-Pick disease type A, Sanfilippo type B (mucopolysaccharidosis type IIIB, MPSIIIB), and Tay-Sachs. Our data suggest that regulated exocytosis may represent a potential therapeutic target for reduction of lysosomal storage in this class of diseases. 相似文献
2.
《Cell calcium》2017
Lysosomes and lysosome-related organelles are emerging as intracellular Ca2+ stores and play important roles in a variety of membrane trafficking processes, including endocytosis, exocytosis, phagocytosis and autophagy. Impairment of lysosomal Ca2+ homeostasis and membrane trafficking has been implicated in many human diseases such as lysosomal storage diseases (LSDs), neurodegeneration, myopathy and cancer. Lysosomal membrane proteins, in particular ion channels, are crucial for lysosomal Ca2+ signaling. Compared with ion channels in the plasma membrane, lysosomal ion channels and their roles in lysosomal Ca2+ signaling are less understood, largely due to their intracellular localization and the lack of feasible functional assays directly applied to the native environment. Recent advances in biomedical methodology have made it possible to directly investigate ion channels in the lysosomal membrane. In this review, we provide a summary of the newly developed methods for monitoring lysosomal Ca2+ and ion channels, as well as the recent discovery of lysosomal ion channels and their significances in intracellular Ca2+ signaling. These new techniques will expand our research scope and our understanding of the nature of lysosomes and lysosome-related diseases. 相似文献
3.
Mitochondria-induced oxidative stress and flawed autophagy are common features of neurodegenerative and lysosomal storage diseases (LSDs). Although defective autophagy is particularly prominent in Pompe disease, mitochondrial function has escaped examination in this typical LSD. We have found multiple mitochondrial defects in mouse and human models of Pompe disease, a life-threatening cardiac and skeletal muscle myopathy: a profound dysregulation of Ca2+ homeostasis, mitochondrial Ca2+ overload, an increase in reactive oxygen species, a decrease in mitochondrial membrane potential, an increase in caspase-independent apoptosis, as well as a decreased oxygen consumption and ATP production of mitochondria. In addition, gene expression studies revealed a striking upregulation of the β 1 subunit of L-type Ca2+ channel in Pompe muscle cells. This study provides strong evidence that disturbance of Ca2+ homeostasis and mitochondrial abnormalities in Pompe disease represent early changes in a complex pathogenetic cascade leading from a deficiency of a single lysosomal enzyme to severe and hard-to-treat autophagic myopathy. Remarkably, L-type Ca2+channel blockers, commonly used to treat other maladies, reversed these defects, indicating that a similar approach can be beneficial to the plethora of lysosomal and neurodegenerative disorders. 相似文献
4.
《Cell calcium》2018
Adenosine triphosphate (ATP) is stored as lysosomal vesicles in marginal cells of the stria vascular in neonatal rats, but the mechanisms of ATP release are unclear. Primary cultures of marginal cells from 1-day-old Sprague–Dawley rats were established. P2Y2 receptor and inositol 1,4,5-trisphosphate (IP3) receptor were immunolabelled in marginal cells of the stria vascular. We found that 30 μM ATP and 30 μM uridine triphosphate (UTP) evoked comparable significant increases in the intracellular Ca2+ concentration ([Ca2+]i) in the absence of extracellular Ca2+, whereas the response was suppressed by 100 μM suramin, 10 μM 1-(6-(17β-3-methoxyester-1,3,5(10)-trien-17-yl)amino)-hexyl)-1H-pyrrole-2,5-dione(U-73122), 100 μM 2-aminoethoxydiphenyl borate (2-APB) and 5 μM thapsigargin (TG), thus indicating that ATP coupled with the P2Y2R-PLC-IP3 pathway to evoke Ca2+ release from the endoplasmic reticulum (ER). Incubation with 200 μM Gly-Phe-β-naphthylamide (GPN) selectively disrupted lysosomes and caused significant increases in [Ca2+]I; this effect was partly inhibited by P2Y2R-PLC-IP3 pathway antagonists. After pre-treatment with 5 μM TG, [Ca2+]i was significantly lower than that after treatment with P2Y2R-PLC-IP3 pathway antagonists under the same conditions, thus indicating that lysosomal Ca2+ triggers Ca2+ release from ER Ca2+ stores. Baseline [Ca2+]i declined after treatment with the Ca2+ chelator 50 μM bis-(aminophenolxy) ethane-N,N,Nʹ,Nʹ-tetra-acetic acid acetoxyme-thyl ester (BAPTA-AM) and 4 IU/ml apyrase. 30 μM ATP decrease of the number of quinacrine-positive vesicles via lysosome exocytosis, whereas the number of lysosomes did not change. However, lysosome exocytosis was significantly suppressed by pre-treatment with 5 μM vacuolin-1. Release of ATP and β-hexosaminidase both increased after treatment with 200 μM GPN and 5 μM TG, but decreased after incubation with 50 μM BAPTA-AM, 4 IU/ml apyrase and 5 μM vacuolin-1. We suggest that ATP triggers Ca2+ release from the ER, thereby contributing to secretion of lysosomal ATP via lysosomal exocytosis. Lysosomal stored Ca2+ triggers Ca2+ release from the ER directly though the IP3 receptors, and lysosomal ATP evokes Ca2+ signals indirectly via the P2Y2R-PLC-IP3 pathway. 相似文献
5.
Changes in the concentration of cytosolic Ca2+ form the basis of a ubiquitous signal transduction pathway. Accumulating evidence implicates acidic organelles in the control of Ca2+ dynamics in organisms across phyla. In this special issue, we discuss Ca2+ signalling by these “acidic Ca2+ stores” which include acidocalcisomes, vacuoles, the endo-lysosomal system, lysosome-related organelles, secretory vesicles and the Golgi complex. Ca2+ release from these morphologically very different organelles is mediated by members of the TRP channel superfamily and two-pore channels. Inositol trisphosphate and ryanodine receptors which are traditionally viewed as endoplasmic reticulum Ca2+ release channels can also mobilize acidic Ca2+ stores. Ca2+ uptake into acidic Ca2+ stores is driven by Ca2+ ATPases and Ca2+/H+ exchangers. In animal cells, the Ca2+-mobilizing messenger NAADP plays a central role in mediating Ca2+ signals from acidic Ca2+ stores through activation of two-pore channels. These signals are important for several physiological processes including muscle contraction and differentiation. Dysfunctional acidic Ca2+ stores have been implicated in diseases such as acute pancreatitis and lysosomal storage disorders. Acidic Ca2+ stores are therefore emerging as essential components of the Ca2+ signalling network and merit extensive further study. 相似文献
6.
Uma Chandrachud Mathew W. Walker Alexandra M. Simas Sasja Heetveld Anton Petcherski Madeleine Klein Hyejin Oh Pavlina Wolf Wen-Ning Zhao Stephanie Norton Stephen J. Haggarty Emyr Lloyd-Evans Susan L. Cotman 《The Journal of biological chemistry》2015,290(23):14361-14380
Abnormal accumulation of undigested macromolecules, often disease-specific, is a major feature of lysosomal and neurodegenerative disease and is frequently attributed to defective autophagy. The mechanistic underpinnings of the autophagy defects are the subject of intense research, which is aided by genetic disease models. To gain an improved understanding of the pathways regulating defective autophagy specifically in juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), a neurodegenerative disease of childhood, we developed and piloted a GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) screening assay to identify, in an unbiased fashion, genotype-sensitive small molecule autophagy modifiers, employing a JNCL neuronal cell model bearing the most common disease mutation in CLN3. Thapsigargin, a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) Ca2+ pump inhibitor, reproducibly displayed significantly more activity in the mouse JNCL cells, an effect that was also observed in human-induced pluripotent stem cell-derived JNCL neural progenitor cells. The mechanism of thapsigargin sensitivity was Ca2+-mediated, and autophagosome accumulation in JNCL cells could be reversed by Ca2+ chelation. Interrogation of intracellular Ca2+ handling highlighted alterations in endoplasmic reticulum, mitochondrial, and lysosomal Ca2+ pools and in store-operated Ca2+ uptake in JNCL cells. These results further support an important role for the CLN3 protein in intracellular Ca2+ handling and in autophagic pathway flux and establish a powerful new platform for therapeutic screening. 相似文献
7.
8.
Drosophila TRPML Forms PI(3,5)P2-activated Cation Channels in Both Endolysosomes and Plasma Membrane
Xinghua Feng Yu Huang Yungang Lu Jian Xiong Ching-On Wong Pu Yang Jintang Xia De Chen Guangwei Du Kartik Venkatachalam Xuefeng Xia Michael X. Zhu 《The Journal of biological chemistry》2014,289(7):4262-4272
Transient Receptor Potential mucolipin (TRPML) channels are implicated in endolysosomal trafficking, lysosomal Ca2+ and Fe2+ release, lysosomal biogenesis, and autophagy. Mutations in human TRPML1 cause the lysosome storage disease, mucolipidosis type IV (MLIV). Unlike vertebrates, which express three TRPML genes, TRPML1–3, the Drosophila genome encodes a single trpml gene. Although the trpml-deficient flies exhibit cellular defects similar to those in mammalian TRPML1 mutants, the biophysical properties of Drosophila TRPML channel remained uncharacterized. Here, we show that transgenic expression of human TRPML1 in the neurons of Drosophila trpml mutants partially suppressed the pupal lethality phenotype. When expressed in HEK293 cells, Drosophila TRPML was localized in both endolysosomes and plasma membrane and was activated by phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) applied to the cytoplasmic side in whole lysosomes and inside-out patches excised from plasma membrane. The PI(3,5)P2-evoked currents were blocked by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), but not other phosphoinositides. Using TRPML A487P, which mimics the varitint-waddler (Va) mutant of mouse TRPML3 with constitutive whole-cell currents, we show that TRPML is biphasically regulated by extracytosolic pH, with an optimal pH about 0.6 pH unit higher than that of human TRPML1. In addition to monovalent cations, TRPML exhibits high permeability to Ca2+, Mn2+, and Fe2+, but not Fe3+. The TRPML currents were inhibited by trivalent cations Fe3+, La3+, and Gd3+. These features resemble more closely to mammalian TRPML1 than TRPML2 and TRPML3, but with some obvious differences. Together, our data support the use of Drosophila for assessing functional significance of TRPML1 in cell physiology. 相似文献
9.
Muscle uses Ca2+ as a messenger to control contraction and relies on ATP to maintain the intracellular Ca2+ homeostasis. Mitochondria are the major sub-cellular organelle of ATP production. With a negative inner membrane potential, mitochondria take up Ca2+ from their surroundings, a process called mitochondrial Ca2+ uptake. Under physiological conditions, Ca2+ uptake into mitochondria promotes ATP production. Excessive uptake causes mitochondrial Ca2+ overload, which activates downstream adverse responses leading to cell dysfunction. Moreover, mitochondrial Ca2+ uptake could shape spatio-temporal patterns of intracellular Ca2+ signaling. Malfunction of mitochondrial Ca2+ uptake is implicated in muscle degeneration. Unlike non-excitable cells, mitochondria in muscle cells experience dramatic changes of intracellular Ca2+ levels. Besides the sudden elevation of Ca2+ level induced by action potentials, Ca2+ transients in muscle cells can be as short as a few milliseconds during a single twitch or as long as minutes during tetanic contraction, which raises the question whether mitochondrial Ca2+ uptake is fast and big enough to shape intracellular Ca2+ signaling during excitation-contraction coupling and creates technical challenges for quantification of the dynamic changes of Ca2+ inside mitochondria. This review focuses on characterization of mitochondrial Ca2+ uptake in skeletal muscle and its role in muscle physiology and diseases. 相似文献
10.
A.A. Sánchez-Tusie S.R. Vasudevan G.C. Churchill T. Nishigaki C.L. Treviño 《Biochemical and biophysical research communications》2014
Ca2+ signaling in spermatozoa plays a crucial role during processes such as capacitation and release of the acrosome, but the underlying molecular mechanisms still remain unclear. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+-releasing second messenger in a variety of cellular processes. The presence of a NAADP synthesizing enzyme in sea urchin sperm has been previously reported, suggesting a possible role of NAADP in sperm Ca2+ signaling. In this work we used in vitro enzyme assays to show the presence of a novel NAADP synthesizing enzyme in human sperm, and to characterize its sensitivity to Ca2+ and pH. Ca2+ fluorescence imaging studies demonstrated that the permeable form of NAADP (NAADP-AM) induces intracellular [Ca2+] increases in human sperm even in the absence of extracellular Ca2+. Using LysoTracker®, a fluorescent probe that selectively accumulates in acidic compartments, we identified two such stores in human sperm cells. Their acidic nature was further confirmed by the reduction in staining intensity observed upon inhibition of the endo-lysosomal proton pump with Bafilomycin, or after lysosomal bursting with glycyl-l-phenylalanine-2-naphthylamide. The selective fluorescent NAADP analog, Ned-19, stained the same subcellular regions as LysoTracker®, suggesting that these stores are the targets of NAADP action. 相似文献
11.
Barry Boland David A. Smith Declan Mooney Sonia S. Jung Dominic M. Walsh Frances M. Platt 《The Journal of biological chemistry》2010,285(48):37415-37426
Alterations in the metabolism of amyloid precursor protein (APP) are believed to
play a central role in Alzheimer disease pathogenesis. Burgeoning data indicate
that APP is proteolytically processed in endosomal-autophagic-lysosomal
compartments. In this study, we used both in vivo and
in vitro paradigms to determine whether alterations in
macroautophagy affect APP metabolism. Three mouse models of glycosphingolipid
storage diseases, namely Niemann-Pick type C1, GM1 gangliosidosis, and Sandhoff
disease, had mTOR-independent increases in the autophagic vacuole
(AV)-associated protein, LC3-II, indicative of impaired lysosomal flux. APP
C-terminal fragments (APP-CTFs) were also increased in brains of the three mouse
models; however, discrepancies between LC3-II and APP-CTFs were seen between
primary (GM1 gangliosidosis and Sandhoff disease) and secondary (Niemann-Pick
type C1) lysosomal storage models. APP-CTFs were proportionately higher than
LC3-II in cerebellar regions of GM1 gangliosidosis and Sandhoff disease,
although LC3-II increased before APP-CTFs in brains of NPC1 mice. Endogenous
murine Aβ40 from RIPA-soluble extracts was increased in brains of all
three mice. The in vivo relationship between AV and APP-CTF
accumulation was also seen in cultured neurons treated with agents that impair
primary (chloroquine and leupeptin + pepstatin) and secondary (U18666A
and vinblastine) lysosomal flux. However, Aβ secretion was unaffected by
agents that induced autophagy (rapamycin) or impaired AV clearance, and
LC3-II-positive AVs predominantly co-localized with degradative LAMP-1-positive
lysosomes. These data suggest that neuronal macroautophagy does not directly
regulate APP metabolism but highlights the important anti-amyloidogenic role of
lysosomal proteolysis in post-secretase APP-CTF catabolism. 相似文献
12.
Emyr Lloyd-Evans 《The Journal of cell biology》2016,212(7):755-757
Acidic Ca2+ stores are important sources of Ca2+ during cell signaling but little is known about how Ca2+ enters these stores. In this issue, Melchionda et al. (2016. J. Cell Biol.
http://dx.doi.org/10.1083/jcb.201510019) identify a Ca2+/H+ exchanger (CAX) that is required for Ca2+ uptake and cell migration in vertebrates.Intracellular Ca2+ signaling is of fundamental importance in processes such as cell migration but we do not fully understand the contribution made by different intracellular Ca2+ stores to this particular function. Elevation of cytosolic Ca2+ by 10- to 100-fold the normal resting levels can occur by entry of external Ca2+ across the plasma membrane and release of Ca2+ from intracellular organelles such as the ER. Ca2+ ions are transported across membranes by ligand-gated ion channels, energy-dependent pumps, and transporters (Berridge et al., 2003; Lloyd-Evans et al., 2010). Intracellular Ca2+ levels are regulated in this manner from simple organisms, such as yeast, through to complex multicellular organisms, suggesting a degree of conservation across the taxonomic kingdoms (Patel and Cai, 2015). Recent evidence has indicated that “acidic Ca2+ stores” such as lysosomes in mammalian cells are a key intracellular Ca2+ signaling store, like the ER (Lloyd-Evans and Platt, 2011; Patel and Muallem, 2011). The Ca2+ concentration of the lysosome (500 µM) is similar to the ER (Christensen et al., 2002; Lloyd-Evans et al., 2008) but lysosomes are smaller in volume and their impact on cellular Ca2+ signaling seems localized to events that regulate endocytosis, vesicular fusion, and recycling (Ruas et al., 2010; López-Sanjurjo et al., 2013). However, there is a significant amount of evidence emerging that lysosomes are capable of triggering much larger changes in cytosolic Ca2+ during signaling via the induction of Ca2+ release from the ER. This effect appears to be mediated by the most potent intracellular Ca2+–releasing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP), which triggers Ca2+ release from lysosomes via two-pore channels (Brailoiu et al., 2009; Calcraft et al., 2009). In addition to two-pore channels, acidic stores also express other Ca2+-permeable channels (summarized in Fig. 1).Open in a separate windowFigure 1.Lysosomal Ca2+ transporters and channels. Our current understanding of lysosomal Ca2+ transport and the proteins that regulate the transport of Ca2+ into and out of the lysosome is heavily stacked in favor of Ca2+ release channels. To date, voltage-gated (CaV2.1/CACNA1A), ligand-gated (TRPML1 and TRPM2), and nucleotide-gated (TPC1, TPC2, and P2X4) channels have all been identified or implicated in lysosomal Ca2+ release (Patel and Cai, 2015). Much less is known about the mechanisms of Ca2+ entry into lysosomes. In lower order organisms, CAX mediates lysosomal Ca2+ entry against the proton gradient. In this issue, Melchionda et al. (2016) provide the first evidence for a mammalian lysosomal Ca2+ uptake mechanism in nonplacental mammals. These findings provide further support for the key role of the lysosome as an intracellular Ca2+ store.Despite recent advances in our knowledge of lysosomal Ca2+ release channels, we have so far failed to identify the transport proteins that fill the lysosome with Ca2+. Ca2+ entering the cell by endocytosis is removed by the early endosome after the initiation of endosomal acidification by the vATPase; therefore, it is likely that lysosomes have their own transporters or pumps to take up Ca2+ (Gerasimenko et al., 1998; Christensen et al., 2002). Although there have been studies suggesting the presence of ATPases and putative ion exchangers on mammalian cells (Styrt et al., 1988), the identity of the proteins that mediate lysosomal Ca2+ uptake remains elusive. In this issue, Melchionda et al. describe the first lysosomal CAX in nonplacental mammals and link lysosomal Ca2+ import via CAX to the maintenance of normal cellular migration during development.To identify novel regulators of Ca2+ transport in vertebrates, Melchionda et al. (2016) searched gene databases for homologues of the CAX proteins, which are known to use the proton gradient across the vacuole to drive Ca2+ uptake in plant and yeast cells (Dunn et al., 1994). They identified putative CAX genes in many species, from sea urchins and frogs to reptiles and birds. The CAX homologues discovered in the genomes of the platypus and Tasmanian devil are the first lysosomal Ca2+ exchangers to be identified in any mammalian species. This new work is a significant finding as it suggests that these mechanisms do clearly exist in some mammalian cells and are required for lysosomal Ca2+ store filling. To examine the regulation of lysosomal Ca2+ uptake by vertebrate CAX transporters, the authors cloned full-length CAX from the frog and found that expression of frog CAX could rescue Ca2+ transport in yeast lacking their own CAX. Furthermore, the authors show that the frog CAX channels correctly localize to lysosomes when expressed in human cell lines and that these CAX are capable of manipulating lysosomal and cytosolic Ca2+ levels (in a manner perhaps comparable to plasma membrane Ca2+ ATPases). The findings reported in Melchionda et al. (2016) also have significance for researchers who are using simpler model organisms to characterize mechanisms regulating acidic store Ca2+. A study by Churchill et al. (2002) that used acidic stores purified from sea urchin egg homogenate to monitor acidic store Ca2+ entry concluded that vanadate-sensitive Ca2+ pumps were absent and suggested instead the presence of a CAX. This now appears to be the case through the reported cloning of sea urchin CAX. The findings of Melchionda et al. (2016) are a step forward in unraveling the molecular mechanisms of Ca2+ handling in model animals.Ca2+ signaling plays an important role in development, particularly for cellular migration, where localized elevations in intracellular Ca2+ drive rearrangement of the cytoskeleton, cellular contraction, and adhesion (Wei et al., 2009; Sumoza-Toledo et al., 2011; Praitis et al., 2013). A concentration gradient of Ca2+ exists across the migrating cell, with higher levels at the rear that contribute to cellular detachment and contraction (Praitis et al., 2013). Recent evidence has highlighted the presence of Ca2+ flickers at the leading edge of the migrating cell that have been shown to underlie changes in direction (Wei et al., 2009). Despite the clear importance of Ca2+ in mediating cellular migration events and the emergent role of lysosomes in maintaining intracellular Ca2+ signaling, very little is known about the roles of lysosomal Ca2+ stores in cellular migration. ER Ca2+ channels including the inositol 1,4,5-trisphosphate receptors and ryanodine receptors as well as the secretory pathway Ca2+ ATPase and lysosomal TRPM2 have all been implicated in regulating changes in intracellular Ca2+ to mediate cellular migration, but to date no lysosomal transporters have been implicated in this process (Wei et al., 2009; Sumoza-Toledo et al., 2011; Praitis et al., 2013). Melchionda et al. (2016) investigated the migration of neural crest cells during frog development to find out whether or not CAX transporters control cell motility. CAX proteins are expressed in the neural crest of developing frogs and morpholino-mediated knockdown of CAX expression increased cytosolic Ca2+ levels and impeded neural crest cell migration. Confocal imaging of neural crest tissue in vitro revealed the dynamic recruitment of CAX-containing vesicles to the protrusions that contain focal adhesion complexes at the leading edge of the migrating neural cells. Loss of CAX protein expression reduced the ability of neural crest cells to form stable focal adhesions and undergo the initial cell spreading required for migration. The work presented by Melchionda et al. (2016) is a significant discovery providing evidence that lysosomal Ca2+ uptake is involved in cell migration and that lower organisms are useful model systems to investigate the role of acidic store Ca2+ in this critical cellular function during embryo development.Melchionda et al. (2016) have made a significant step forward in our understanding of the mechanisms that regulate lysosomal/vacuolar Ca2+ entry. However, we remain in the dark about the identity of the transporters that pump Ca2+ into the lysosomes of placental mammals. What led to the loss of CAX genes in these organisms is as much a mystery as the identity of the transporters that have replaced CAX. Evidence from a study using purified mammalian lysosomes to observe Ca2+ uptake indicates that the process is ATP-dependent (Styrt et al., 1988). Placental mammals may have completely different ATP-dependent mechanisms governing lysosomal Ca2+ uptake compared with lower order organisms and nonplacental mammals. Interestingly, defects in lysosomal Ca2+ uptake are associated with two human diseases, Niemann-Pick type C and Chediak-Higashi syndrome (CHS; Styrt et al., 1988; Lloyd-Evans et al., 2008). The lysosomal accumulation of sphingosine, a Ca2+ ATPase inhibitor (Lloyd-Evans and Platt, 2011), leads to reduced lysosomal Ca2+ levels in Niemann-Pick type C disease cells and defects in NAADP-mediated lysosomal Ca2+ release (Lloyd-Evans et al., 2008). In CHS, there have been reports of enhanced lysosomal Ca2+ ATPase transporter activity in neutrophils (Styrt et al., 1988). Interestingly, CHS leukocytes show alterations in chemotaxis with a reduced response to chemotactic factors (Clark and Kimball, 1971), which is supportive of the findings of Melchionda et al. (2016). Much remains to be elucidated about the enigma of mammalian lysosomal Ca2+ uptake, but the work of Melchionda et al. (2016) begins to pick this mystery apart. 相似文献
13.
14.
Inositol 1,4,5-trisphosphate (IP3) evokes release of Ca2+ from the endoplasmic reticulum (ER), but the resulting Ca2+ signals are shaped by interactions with additional intracellular organelles. Bafilomycin A1, which prevents lysosomal Ca2+ uptake by inhibiting H+ pumping into lysosomes, increased the amplitude of the initial Ca2+ signals evoked by carbachol in human embryonic kidney (HEK) cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH) evoke Ca2+ release from distinct IP3-sensitive Ca2+ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A1 similarly exaggerated the Ca2+ signals evoked by carbachol or carbachol with PTH, indicating that Ca2+ released from distinct IP3-sensitive Ca2+ stores is sequestered by lysosomes. The Ca2+ signals resulting from store-operated Ca2+ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A1. Using Gd3+ (1 mM) to inhibit both Ca2+ entry and Ca2+ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca2+ recycling to the ER after IP3-evoked Ca2+ release. Blocking lysosomal Ca2+ uptake with bafilomycin A1 increased the amplitude of each carbachol-evoked Ca2+ signal without affecting the rate of Ca2+ recycling to the ER. This suggests that Ca2+ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca2+ released by IP3 receptors residing within distinct Ca2+ stores, but not the Ca2+ entering cells via receptor-regulated, store-operated Ca2+ entry pathways. 相似文献
15.
Exocytosis of acid sphingomyelinase by wounded cells promotes endocytosis and plasma membrane repair
Christina Tam Vincent Idone Cecilia Devlin Maria Cecilia Fernandes Andrew Flannery Xingxuan He Edward Schuchman Ira Tabas Norma W. Andrews 《The Journal of cell biology》2010,189(6):1027-1038
Rapid plasma membrane resealing is essential for cellular survival. Earlier studies showed that plasma membrane repair requires Ca2+-dependent exocytosis of lysosomes and a rapid form of endocytosis that removes membrane lesions. However, the functional relationship between lysosomal exocytosis and the rapid endocytosis that follows membrane injury is unknown. In this study, we show that the lysosomal enzyme acid sphingomyelinase (ASM) is released extracellularly when cells are wounded in the presence of Ca2+. ASM-deficient cells, including human cells from Niemann-Pick type A (NPA) patients, undergo lysosomal exocytosis after wounding but are defective in injury-dependent endocytosis and plasma membrane repair. Exogenously added recombinant human ASM restores endocytosis and resealing in ASM-depleted cells, suggesting that conversion of plasma membrane sphingomyelin to ceramide by this lysosomal enzyme promotes lesion internalization. These findings reveal a molecular mechanism for restoration of plasma membrane integrity through exocytosis of lysosomes and identify defective plasma membrane repair as a possible component of the severe pathology observed in NPA patients. 相似文献
16.
Abasha Lewis Teruo Hayashi Tsung-Ping Su Michael J. Betenbaugh 《Journal of bioenergetics and biomembranes》2014,46(1):1-15
Bcl-2 family proteins, known for their apoptosis functioning at the mitochondria, have been shown to localize to other cellular compartments to mediate calcium (Ca2+) signals. Since the proper supply of Ca2+ in cells serves as an important mechanism for cellular survival and bioenergetics, we propose an integrating role for Bcl-2 family proteins in modulating Ca2+ signaling. The endoplasmic reticulum (ER) is the main Ca2+ storage for the cell and Bcl-2 family proteins competitively regulate its Ca2+ concentration. Bcl-2 family proteins also regulate the flux of Ca2+ from the ER by physically interacting with inositol 1,4,5-trisphosphate receptors (IP3Rs) to mediate their opening. Type 1 IP3Rs reside at the bulk ER to coordinate cytosolic Ca2+ signals, while type 3 IP3Rs reside at mitochondria-associated ER membrane (MAM) to facilitate mitochondrial Ca2+ uptake. In healthy cells, mitochondrial Ca2+ drives pyruvate into the citric acid (TCA) cycle to facilitate ATP production, while a continuous accumulation of Ca2+ can trigger the release of cytochrome c, thus initiating apoptosis. Since multiple organelles and Bcl-2 family proteins are involved in Ca2+ signaling, we aim to clarify the role that Bcl-2 family proteins play in facilitating Ca2+ signaling and how mitochondrial Ca2+ is relevant in both bioenergetics and apoptosis. We also explore how these insights could be useful in controlling bioenergetics in apoptosis-resistant cell lines. 相似文献
17.
Convergent regulation of the lysosomal two‐pore channel‐2 by Mg2+, NAADP,PI(3,5)P2 and multiple protein kinases
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Archana Jha Malini Ahuja Sandip Patel Eugen Brailoiu Shmuel Muallem 《The EMBO journal》2014,33(5):501-511
Lysosomal Ca2+ homeostasis is implicated in disease and controls many lysosomal functions. A key in understanding lysosomal Ca2+ signaling was the discovery of the two‐pore channels (TPCs) and their potential activation by NAADP. Recent work concluded that the TPCs function as a PI(3,5)P2 activated channels regulated by mTORC1, but not by NAADP. Here, we identified Mg2+ and the MAPKs, JNK and P38 as novel regulators of TPC2. Cytoplasmic Mg2+ specifically inhibited TPC2 outward current, whereas lysosomal Mg2+ partially inhibited both outward and inward currents in a lysosomal lumen pH‐dependent manner. Under controlled Mg2+, TPC2 is readily activated by NAADP with channel properties identical to those in response to PI(3,5)P2. Moreover, TPC2 is robustly regulated by P38 and JNK. Notably, NAADP‐mediated Ca2+ release in intact cells is regulated by Mg2+, PI(3,5)P2, and P38/JNK kinases, thus paralleling regulation of TPC2 currents. Our data affirm a key role for TPC2 in NAADP‐mediated Ca2+ signaling and link this pathway to Mg2+ homeostasis and MAP kinases, pointing to roles for lysosomal Ca2+ in cell growth, inflammation and cancer. 相似文献
18.
《Cell calcium》2015,57(6):457-466
Mitochondrial Ca2+ plays a critical physiological role in cellular energy metabolism and signaling, and its overload contributes to various pathological conditions including neuronal apoptotic death in neurological diseases. Live cell mitochondrial Ca2+ imaging is an important approach to understand mitochondrial Ca2+ dynamics. Recently developed GCaMP genetically-encoded Ca2+ indicators provide unique opportunity for high sensitivity/resolution and cell type-specific mitochondrial Ca2+ imaging. In the current study, we implemented cell-specific mitochondrial targeting of GCaMP5G/6s (mito-GCaMP5G/6s) and used two-photon microscopy to image astrocytic and neuronal mitochondrial Ca2+ dynamics in culture, revealing Ca2+ uptake mechanism by these organelles in response to cell stimulation. Using these mitochondrial Ca2+ indicators, our results show that mitochondrial Ca2+ uptake in individual mitochondria in cultured astrocytes and neurons can be seen after stimulations by ATP and glutamate, respectively. We further studied the dependence of mitochondrial Ca2+ dynamics on cytosolic Ca2+ changes following ATP stimulation in cultured astrocytes by simultaneously imaging mitochondrial and cytosolic Ca2+ increase using mito-GCaMP5G and a synthetic organic Ca2+ indicator, x-Rhod-1, respectively. Combined with molecular intervention in Ca2+ signaling pathway, our results demonstrated that the mitochondrial Ca2+ uptake is tightly coupled with inositol 1,4,5-trisphosphate receptor-mediated Ca2+ release from the endoplasmic reticulum and the activation of G protein-coupled receptors. The current study provides a novel approach to image mitochondrial Ca2+ dynamics as well as Ca2+ interplay between the endoplasmic reticulum and mitochondria, which is relevant for neuronal and astrocytic functions in health and disease. 相似文献
19.
Antje Grotemeier Sebastian Alers Simon G. Pfisterer Florian Paasch Merle Daubrawa Alexandra Dieterle Benoit Viollet Sebastian Wesselborg Tassula Proikas-Cezanne Björn Stork 《Cellular signalling》2010,22(6):914-925
Autophagy is a eukaryotic lysosomal bulk degradation system initiated by cytosolic cargo sequestration in autophagosomes. The Ser/Thr kinase mTOR has been shown to constitute a central role in controlling the initiation of autophagy by integrating multiple nutrient-dependent signaling pathways that crucially involves the activity of PI3K class III to generate the phosphoinositide PI(3)P. Recent reports demonstrate that the increase in cytosolic Ca2+ can induce autophagy by inhibition of mTOR via the CaMKK-α/β-mediated activation of AMPK. Here we demonstrate that Ca2+ signaling can additionally induce autophagy independently of the Ca2+-mediated activation of AMPK. First, by LC3-II protein monitoring in the absence or presence of lysosomal inhibitors we confirm that the elevation of cytosolic Ca2+ induces autophagosome generation and does not merely block autophagosome degradation. Further, we demonstrate that Ca2+-chelation strongly inhibits autophagy in human, mouse and chicken cells. Strikingly, we found that the PI(3)P-binding protein WIPI-1 (Atg18) responds to the increase of cytosolic Ca2+ by localizing to autophagosomal membranes (WIPI-1 puncta) and that Ca2+-chelation inhibits WIPI-1 puncta formation, although PI(3)P-generation is not generally affected by these Ca2+ flux modifications. Importantly, using AMPK-α1?/?α2?/? MEFs we show that thapsigargin application triggers autophagy in the absence of AMPK and does not involve complete mTOR inhibition, as detected by p70S6K phosphorylation. In addition, STO-609-mediated CaMKK-α/β inhibition decreased the level of thapsigargin-induced autophagy only in AMPK-positive cells. We suggest that apart from reported AMPK-dependent regulation of autophagic degradation, an AMPK-independent pathway triggers Ca2+-mediated autophagy, involving the PI(3)P-effector protein WIPI-1 and LC3. 相似文献
20.
Palmitoyl-protein thioesterase-1 (PPT1) deficiency causes infantile neuronal ceroid lipofuscinosis (INCL), a devastating childhood neurodegenerative storage disorder. We previously reported that neuronal apoptosis in INCL is mediated by endoplasmic reticulum-stress. ER-stress disrupts Ca2+-homeostasis and stimulates the expression of Ca2+-binding proteins. We report here that in the PPT1-deficient human and mouse brain the levels of S100B, a Ca2+-binding protein, and its receptor, RAGE (receptor for advanced glycation end-products) are elevated. We further demonstrate that activation of RAGE signaling in astroglial cells mediates pro-inflammatory cytokine production, which is inhibited by SiRNA-mediated suppression of RAGE expression. We propose that RAGE signaling contributes to neuroinflammation in INCL. 相似文献