12/15-Lipoxygenase (LOX) enzymatically generates oxidized phospholipids in monocytes and macrophages. Herein, we show that cells deficient in 12/15-LOX contain defective mitochondria and numerous cytoplasmic vacuoles containing electron dense material, indicating defects in autophagy or membrane processing, However, both LC3 expression and lipidation were normal both basally and on chloroquine treatment. A LOX-derived oxidized phospholipid, 12-hydroxyeicosatetraenoic acid-phosphatidylethanolamine (12-HETE-PE) was found to be a preferred substrate for yeast Atg8 lipidation, versus native PE, while both native and oxidized PE were effective substrates for LC3 lipidation. Last, phospholipidomics demonstrated altered levels of several phospholipid classes. Thus, we show that oxidized phospholipids generated by 12/15-LOX can act as substrates for key proteins required for effective autophagy and that cells deficient in this enzyme show evidence of autophagic dysfunction. The data functionally link phospholipid oxidation with autophagy for the first time.
•12/15-Lipoxygenase-deficient macrophages show evidence of autophagic dysfunction.•12-HETE-PE is a substrate for LC2 and Atg8 lipidation.•Macrophages deficient in 12/15-lipoxygenase show altered phospholipid content.
Lipid; Autophagy; Lipoxygenase; Mitochondria
Cystic fibrosis (CF) is a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Initially, Cl− conductance in the sweat duct was discovered to be impaired in CF1, a finding that has been extended to all CFTR-expressing cells2–4. Subsequent cloning of the gene5,6 showed that CFTR functions as a cyclic-AMP-regulated Cl− channel7; and some CF-causing mutations inhibit CFTR Cl− channel activity2–4,8. The identification of additional CF-causing mutants with normal Cl− channel activity indicates, however, that other CFTR-dependent processes contribute to the disease. Indeed, CFTR regulates other transporters3,4, including Cl−-coupled
HCO3- transport9,10. Alkaline fluids are secreted by normal tissues, whereas acidic fluids are secreted by mutant CFTR-expressing tissues11, indicating the importance of this activity.
HCO3- and pH affect mucin viscosity12,13 and bacterial binding14,15. We have examined Cl−-coupled
HCO3- transport by CFTR mutants that retain substantial or normal Cl− channel activity. Here we show that mutants reported to be associated with CF with pancreatic insufficiency do not support
HCO3- transport, and those associated with pancreatic sufficiency show reduced
HCO3- transport. Our findings demonstrate the importance of
HCO3- transport in the function of secretory epithelia and in CF.
Regulation of organellar fusion and fission by Ca2+ has emerged as a central paradigm in intracellular membrane traffic. Originally formulated for Ca2+-driven SNARE-mediated exocytosis in the presynaptic terminals, it was later expanded to explain membrane traffic in other exocytic events within the endo-lysosomal system. The list of processes and conditions that depend on the intracellular membrane traffic includes aging, antigen and lipid processing, growth factor signaling and enzyme secretion. Characterization of the ion channels that regulate intracellular membrane fusion and fission promises novel pharmacological approaches in these processes when their function becomes aberrant. The recent identification of Ca2+ permeability through the intracellular ion channels comprising the mucolipin (TRPMLs) and the two-pore channels (TPCs) families pinpoints the candidates for the Ca2+ channel that drive intracellular membrane traffic. The present review summarizes the recent developments and the current questions relevant to this topic.
organelles; TRPMLs; TPCs; calcium; membrane traffic; fusion; fission
Zn is critical for a multitude of cellular processes, including gene expression, secretion and enzymatic activities. Cellular Zn is controlled by Zn-chelating proteins and by Zn transporters. The recent identification of Zn permeability of the lysosomal ion channel TRPML1, and the evidence of abnormal Zn levels in cells deficient in TRPML1, suggested a role for TRPML1 in Zn transport. Here we provide new evidence for such a role and identify additional cellular components responsible for it. In agreement with the previously published data, an acute siRNA-driven TRPML1 knockdown (KD) leads to the buildup of large cytoplasmic vesicles positive for Lysotracker and Zn staining, when cells are exposed to high concentrations of Zn. We now show that lysosomal enlargement and Zn buildup in TRPML1-KD cells exposed to Zn are ameliorated by KD of the Zn-sensitive transcription factor MTF-1 or Zn transporter ZnT4. TRPML1 KD is associated with a buildup of cytoplasmic Zn and with enhanced transcriptional response of mRNA for metallothionein 2a (MT2a). TRPML1 KD did not suppress lysosomal secretion, but it did delay Zn leak from the lysosomes into the cytoplasm. These data underscore a role for TRPML1 in Zn metabolism. Furthermore, they suggest that TRPML1 works in concert with ZnT4 to regulate Zn translocation between the cytoplasm and lysosomes.
Zinc; lysosomal storage disease; TRP channels; MTF-1; Slc30a4; ZnT4
Key aspects of lysosomal function are affected by the ionic content of the lysosomal lumen and, therefore, by the ion permeability in the lysosomal membrane. Such functions include regulation of lysosomal acidification, a critical process in delivery and activation of the lysosomal enzymes, release of metals from lysosomes into the cytoplasm and the Ca2+-dependent component of membrane fusion events in the endocytic pathway. While the basic mechanisms of lysosomal acidification have been largely defined, the lysosomal metal transport system is not well understood. TRPML1 is a lysosomal ion channel whose malfunction is implicated in the lysosomal storage disease Mucolipidosis Type IV. Recent evidence suggests that TRPML1 is involved in Fe2+, Ca2+ and Zn2+ transport across the lysosomal membrane, ascribing novel physiological roles to this ion channel, and perhaps to its relatives TRPML2 and TRPML3 and illuminating poorly understood aspects of lysosomal function. Further, alterations in metal transport by the TRPMLs due to mutations or environmental factors may contribute to their role in the disease phenotype and cell death.
Lysosomal storage diseases (LSDs) are caused by inability of cells to process the material captured during endocytosis. While they are essentially diseases of cellular “indigestion”, LSDs affect large number of cellular activities and, as such, they teach us about the integrative function of the cell, as well as about the gaps in our knowledge of the endocytic pathway and membrane transport. The present review summarizes recent findings on Ca2+ handling in LSDs and attempts to identify the key questions on alterations inCa2+ signaling and membrane transport in this group of diseases, answers to which may lie in delineating the cellular pathogeneses of LSDs.
Disorazoles are polyene macrodiolides isolated from a myxobacterium fermentation broth. Disorazole C1 was newly synthesized and found to depolymerize microtubules and cause mitotic arrest. Here we examined the cellular responses to disorazole C1 in both non-cancer and cancer cells and compared our results to vinblastine and taxol.
In non-cancer cells, disorazole C1 induced a prolonged mitotic arrest, followed by mitotic slippage, as confirmed by live cell imaging and cell cycle analysis. This mitotic slippage was associated with cyclin B degradation, but did not require p53. Four assays for apoptosis, including western blotting for poly(ADP-ribose) polymerase cleavage, microscopic analyses for cytochrome C release and annexin V staining, and gel electrophoresis examination for DNA laddering, were conducted and demonstrated little induction of apoptosis in non-cancer cells treated with disorazole C1. On the contrary, we observed an activated apoptotic pathway in cancer cells, suggesting that normal and malignant cells respond differently to disorazole C1.
Our studies demonstrate that non-cancer cells undergo mitotic slippage in a cyclin B-dependent and p53-independent manner after prolonged mitotic arrest caused by disorazole C1. In contrast, cancer cells induce the apoptotic pathway after disorazole C1 treatment, indicating a possibly significant therapeutic window for this compound.
Lysosomal storage diseases (LSDs) are debilitating genetic conditions that frequently manifest as neurodegenerative disorders. They severely affect eye, motor and cognitive functions and, in most cases, abbreviate the lifespan. Postmitotic cells such as neurons and mononuclear phagocytes rich in lysosomes are most often affected by the accumulation of undegraded material. Cell death is well documented in parts of the brain and in other cells of LSD patients and animal models, although little is known about mechanisms by which death pathways are activated in these diseases, and not all cells exhibiting increased storage material are affected by cell death. Lysosomes are essential for maturation and completion of autophagy-initiated protein and organelle degradation. Moreover, accumulation of effete mitochondria has been documented in postmitotic cells whose lysosomal function is suppressed or in aging cells with lipofuscin accumulation. Based upon observations in the literature and our own data showing similar mitochondrial abnormalities in several LSDs, we propose a new model of cell death in LSDs. We suggest that the lysosomal deficiencies in LSDs inhibit autophagic maturation, leading to a condition of autophagic stress. The resulting accumulation of dysfunctional mitochondria showing impaired Ca2+ buffering increases the vulnerability of the cells to pro-apoptotic signals.
autophagy; mitochondrial homeostasis; lysosome; calcium; caspase; programmed cell death; mucolipidosis; neurodegeneration; Niemann-Pick; neuronal ceroid lipofuscinosis
At the 2008 Society for Neuroscience annual meeting a minisymposium entitled, “TRP channels contribute to disease processes” included talks from six heads of newlyestablished laboratories, each with a unique research focus, model system, and set of experimental tools. Among the questions addressed in these talks were: What is the role of TRP channels in pain perception? How do normally-functioning TRP channels contribute to cell death pathways? What are the characteristics of TRPpathies, disease states that result from over-active or under-active TRP channels? How are TRP channels regulated by signal transduction cascades? This review summarizes recent results from those laboratories and provides six perspectives on the subject of TRP channels and disease.
Lysosomal storage diseases (LSDs) are a class of genetic disorders in which proteins responsible for digestion or absorption of endocytosed material do not function or do not localize properly. The resulting cellular “indigestion” causes buildup of intracellular storage inclusions that contain unprocessed lipids and proteins that form macromolecular complexes. The buildup of storage material is associated with degenerative processes that are observed in all LSDs, albeit the correlation between the amount of storage inclusions and the severity of the degenerative processes is not always evident. The latter suggests that a specific mechanism set in motion by aberrant lysosomal function drives the degenerative processes in LSDs. It is becoming increasingly clear that in addition to their function in degrading endocytosed material, lysosomes are essential housekeeping organelles responsible for maintaining healthy population of intracellular organelles, in particular mitochondria. The present review surveys the current knowledge on the lysosomal-mitochondrial axis and its possible role as a contributing factor to mitochondrial Ca2+ homeostasis and to cell death in LSDs.
Research in autophagy continues to accelerate,1 and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.2,3 There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
autolysosome; autophagosome; flux; lysosome; phagophore; stress; vacuole
Biochemical assessment of channel structure/function is incredibly challenging. Developing computational tools that provide these data would enable translational research, accelerating mechanistic experimentation for the bench scientist studying ion channels. Starting with the premise that protein sequence encodes information about structure, function and evolution (SF&E), we developed a unified framework for inferring SF&E from sequence information using a knowledge-based approach. The Gestalt Domain Detection Algorithm-Basic Local Alignment Tool (GDDA-BLAST) provides phylogenetic profiles that can model, ab initio, SF&E relationships of biological sequences at the whole protein, single domain and single-amino acid level.1,2 In our recent paper,4 we have applied GDDA-BLAST analysis to study canonical TRP (TRPC) channels1 and empirically validated predicted lipid-binding and trafficking activities contained within the TRPC3 TRP_2 domain of unknown function. Overall, our in silico, in vitro, and in vivo experiments support a model in which TRPC3 has signal-sensing antennae which are adorned with lipid-binding, trafficking and calmodulin regulatory domains. In this Addendum, we correlate our functional domain analysis with the cryo-EM structure of TRPC3.3 In addition, we synthesize recent studies with our new findings to provide a refined model on the mechanism(s) of TRPC3 activation/deactivation.
transient receptor potential channel; lipid; GDDA-BLAST; phylogenetic profile; diacylglycerol; SNARE; TRP_2 domain
The lysosomal storage disorder mucolipidosis type IV (MLIV) is caused by mutations in the transient receptor potential–mucolipin-1 (TRP-ML1) ion channel. The “biogenesis” model for MLIV pathogenesis suggests that TRP-ML1 modulates postendocytic delivery to lysosomes by regulating interactions between late endosomes and lysosomes. This model is based on observed lipid trafficking delays in MLIV patient fibroblasts. Because membrane traffic aberrations may be secondary to lipid buildup in chronically TRP-ML1–deficient cells, we depleted TRP-ML1 in HeLa cells using small interfering RNA and examined the effects on cell morphology and postendocytic traffic. TRP-ML1 knockdown induced gradual accumulation of membranous inclusions and, thus, represents a good model in which to examine the direct effects of acute TRP-ML1 deficiency on membrane traffic. Ratiometric imaging revealed decreased lysosomal pH in TRP-ML1–deficient cells, suggesting a disruption in lysosomal function. Nevertheless, we found no effect of TRP-ML1 knockdown on the kinetics of protein or lipid delivery to lysosomes. In contrast, by comparing degradation kinetics of low density lipoprotein constituents, we confirmed a selective defect in cholesterol but not apolipoprotein B hydrolysis in MLIV fibroblasts. We hypothesize that the effects of TRP-ML1 loss on hydrolytic activity have a cumulative effect on lysosome function, resulting in a lag between TRP-ML1 loss and full manifestation of MLIV.
Homers are scaffolding proteins that bind Ca2+ signaling proteins in cellular microdomains. The Homers participate in targeting and localization of Ca2+ signaling proteins in signaling complexes. However, recent work showed that the Homers are not passive scaffolding proteins, but rather they regulate the activity of several proteins within the Ca2+ signaling complex in an isoform specific manner. Homer2 increases the GAP activity of RGS proteins and PLCβ that accelerate the GTPase activity of Gα subunits. Homer1 gates the activity of TRPC channels, controls the rates of their translocation and retrieval from the plasma membrane and mediates the conformational coupling between TRPC channels and IP3Rs. Homer1 stimulates the activity of the cardiac and neuronal L-type Ca2+ channels Cav1.2 and Cav1.3. Homer1 also mediates the communication between the cardiac and smooth muscle ryanodine receptor RyR2 and Cav1.2 to regulate E–C coupling. In many cases the Homers function as a buffer to reduce the intensity of Ca2+ signaling and create a negative bias that can be reversed by the immediate early gene form of Homer 1. Hence, the Homers should be viewed as the buffers of Ca2+ signaling that ensure a high spatial and temporal fidelity of the Ca2+ signaling and activation of downstream effects.