Flux In Mammalian Cells Research Articles

MON-2, a Golgi protein, mediates autophagy-dependent longevity in Caenorhabditis elegans.

The Golgi apparatus plays a central role in trafficking cargoes such as proteins and lipids. Defects in the Golgi apparatus lead to various diseases, but its role in organismal longevity is largely unknown. Using a quantitative proteomic approach, we found that a Golgi protein, MON-2, was up-regulated in long-lived Caenorhabditis elegans mutants with mitochondrial respiration defects and was required for their longevity. Similarly, we showed that DOP1/PAD-1, which acts with MON-2 to traffic macromolecules between the Golgi and endosome, contributed to the longevity of respiration mutants. Furthermore, we demonstrated that MON-2 was required for up-regulation of autophagy, a longevity-associated recycling process, by activating the Atg8 ortholog GABARAP/LGG-1 in C. elegans. Consistently, we showed that mammalian MON2 activated GABARAPL2 through physical interaction, which increased autophagic flux in mammalian cells. Thus, the evolutionarily conserved role of MON2 in trafficking between the Golgi and endosome is an integral part of autophagy-mediated longevity.

Evolution of tools and methods for monitoring autophagic flux in mammalian cells.

Autophagy is an evolutionarily conserved lysosome-mediated degradation and recycling process, which functions in cellular homeostasis and stress adaptation. The process is highly dynamic and involves autophagosome synthesis, cargo recognition and transport, autophagosome-lysosome fusion, and cargo degradation. The multistep nature of autophagy makes it challenging to quantify, and it is important to consider not only the number of autophagosomes within a cell but also the autophagic degradative activity. The rate at which cargos are recognized, segregated, and degraded through the autophagy pathway is defined as autophagic flux. In practice, methods to measure autophagic flux typically evaluate the lysosome-mediated cargo degradation step by leveraging known autophagy markers such as MAP1LC3B (microtubule-associated proteins 1A/1B light chain 3 beta) or lysosome-dependent fluorescent agents. In this review, we summarize the tools and methods used in mammalian cultured cells pertaining to these two approaches, and highlight innovations that have led to their evolution in recent years. We also discuss the potential limitations of these approaches and recommend using a combination of strategies and multiple different autophagy markers to reliably evaluate autophagic flux in mammalian cells.

Pharmacological Inhibition of Lysosomal Activity as a Method For Monitoring Thyroid Hormone-induced Autophagic Flux in Mammalian Cells In Vitro.

Autophagy is an evolutionarily conserved intracellular catabolic process that is essential for cellular housekeeping and nutrient homeostasis. Recently, we provided evidence that thyroid hormone (TH) is a major inducer of autophagy in mammalian cells. Here, we describe a method for detecting TH-induced autophagic flux in hepatic, muscle, and brown adipocyte cells using lysosomal inhibitor bafilomycin A1 (BafA1) and conventional Western blot techniques.

Cytofluorometric Assessment of Mitophagic Flux in Mammalian Cells and Tissues.

Mitophagy is the selective autophagic degradation of mitochondria inside lysosomes and is the only mechanism capable of eliminating whole mitochondria that are either damaged or no longer required. Mitochondrial dysfunction is a common hallmark in many pathological conditions and has been implicated in cellular damage in aged tissues. The limited availability of mitophagy research methods underscores the need for more robust, quantitative, and objective tools in order to better understand this process. Here we describe a flow cytometry-based method using MitoTracker Deep Red for mitophagy assessment that can be applied to cells and tissues. Moreover, we demonstrate that when used in conjunction with lysosomal inhibitors, our method provides a novel means of assessing mitophagic flux, which is the most reliable indicator of whether mitochondria are truly delivered to lysosomes for degradation.

Renilla Luciferase-LC3 Based Reporter Assay for Measuring Autophagic Flux.

Macroautophagy (autophagy) is a dynamic intracellular degradation pathway. Monitoring the flux through the autophagy pathway is experimentally challenging but obviously a prerequisite for the proper investigation of the process. Here, we present an indirect autophagy flux assay based on monitoring the degradation of an autophagosome-associated fusion protein Rluc-LC3 by luminescence detection. The method is suitable for screening purposes with a high number of parallel samples and can be used for measurements in cell lysates as well as in living cells. The Rluc-LC3 assay has proven useful for the identification of genes, miRNAs, and small molecules that regulate autophagy flux in mammalian cells.

The GATOR2 Component Wdr24 Regulates TORC1 Activity and Lysosome Function.

TORC1 is a master regulator of metabolism in eukaryotes that responds to multiple upstream signaling pathways. The GATOR complex is a newly defined upstream regulator of TORC1 that contains two sub-complexes, GATOR1, which inhibits TORC1 activity in response to amino acid starvation and GATOR2, which opposes the activity of GATOR1. While the GATOR1 complex has been implicated in a wide array of human pathologies including cancer and hereditary forms of epilepsy, the in vivo relevance of the GATOR2 complex remains poorly understood in metazoans. Here we define the in vivo role of the GATOR2 component Wdr24 in Drosophila. Using a combination of genetic, biochemical, and cell biological techniques we demonstrate that Wdr24 has both TORC1 dependent and independent functions in the regulation of cellular metabolism. Through the characterization of a null allele, we show that Wdr24 is a critical effector of the GATOR2 complex that promotes the robust activation of TORC1 and cellular growth in a broad array of Drosophila tissues. Additionally, epistasis analysis between wdr24 and genes that encode components of the GATOR1 complex revealed that Wdr24 has a second critical function, the TORC1 independent regulation of lysosome dynamics and autophagic flux. Notably, we find that two additional members of the GATOR2 complex, Mio and Seh1, also have a TORC1 independent role in the regulation of lysosome function. These findings represent a surprising and previously unrecognized function of GATOR2 complex components in the regulation of lysosomes. Consistent with our findings in Drosophila, through the characterization of a wdr24-/- knockout HeLa cell line we determined that Wdr24 promotes lysosome acidification and autophagic flux in mammalian cells. Taken together our data support the model that Wdr24 is a key effector of the GATOR2 complex, required for both TORC1 activation and the TORC1 independent regulation of lysosomes.

Proteomic Mapping and Optogenetic Control of ER-PM Junctions in Living Cells

Specialized junctional sites that connect the plasma membrane (PM) and endoplasmic reticulum (ER) play critical roles in controlling lipid metabolism and calcium signaling. Store-operated calcium entry mediated by dynamic STIM1-ORAI1 coupling constitutes a classical molecular event occurring at ER-PM junctions, but the protein composition and how previously-unrecognized protein regulators facilitate calcium influx at this particular subcellular compartment remain poorly defined. By taking advantage of a spatially-restricted biotin-labeling technique coupled with mass spectrometry, we systematically mapped the proteome of intact ER-PM junctions in living cells while maintaining their architectural integrity. Using a secondary screen based on bimolecular fluorescence complementation, we further discovered a novel multi-transmembrane protein that we call STIMATE (STIM-activating enhancer, encoded by TMEM110) as a positive regulator of calcium influx in vertebrates. STIMATE colocalizes with STIM1 to the ER and promotes STIM1 conformational switch through physical contact with the STIM1 juxtamembrane coiled coil domain. This represents a novel regulatory mechanism to modulate STIM1-dependent calcium flux in mammalian cells. Genetic depletion of STIMATE substantially reduces STIM1 puncta formation at ER-PM junctions and suppresses the calcium-NFAT signaling. To noninvasively manipulate membrane contact sites, we also created a novel tool to reversibly control the formation of ER-PM junctions by harnessing the power of light. This optogenetic tool makes it possible for future screening of protein/chemical regulators that are involved in maintaining ER-PM junctions. Our findings not only afford an initial view on the protein composition of intact ER-PM junctions under physiological conditions, but also provide a solid framework to uncover more uncharted functions of hitherto underexplored ER-PM junctions. Increasing our comprehension of knowledge on this elusive and intriguing subcellular structure will lead to better design of therapeutic strategies to treat human disorders associated with aberrant lipid metabolism and calcium signaling.

TPC2 Is a Novel NAADP-sensitive Ca2+ Release Channel, Operating as a Dual Sensor of Luminal pH and Ca2+

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a molecule capable of initiating the release of intracellular Ca2+ required for many essential cellular processes. Recent evidence links two-pore channels (TPCs) with NAADP-induced release of Ca2+ from lysosome-like acidic organelles; however, there has been no direct demonstration that TPCs can act as NAADP-sensitive Ca2+ release channels. Controversial evidence also proposes ryanodine receptors as the primary target of NAADP. We show that TPC2, the major lysosomal targeted isoform, is a cation channel with selectivity for Ca2+ that will enable it to act as a Ca2+ release channel in the cellular environment. NAADP opens TPC2 channels in a concentration-dependent manner, binding to high affinity activation and low affinity inhibition sites. At the core of this process is the luminal environment of the channel. The sensitivity of TPC2 to NAADP is steeply dependent on the luminal [Ca2+] allowing extremely low levels of NAADP to open the channel. In parallel, luminal pH controls NAADP affinity for TPC2 by switching from reversible activation of TPC2 at low pH to irreversible activation at neutral pH. Further evidence earmarking TPCs as the likely pathway for NAADP-induced intracellular Ca2+ release is obtained from the use of Ned-19, the selective blocker of cellular NAADP-induced Ca2+ release. Ned-19 antagonizes NAADP-activation of TPC2 in a non-competitive manner at 1 μm but potentiates NAADP activation at nanomolar concentrations. This single-channel study provides a long awaited molecular basis for the peculiar mechanistic features of NAADP signaling and a framework for understanding how NAADP can mediate key physiological events.

From biomarkers to integrated network responses

The ability to quantify carbon fluxes in mammalian cells is a step toward elucidating the dynamic metabolic networks associated with health and disease.

Going with the flux

Researchers at Princeton and University of Rochester have developed a new way to measure metabolic fluxes in mammalian cells and they have determined that fatty acid biosynthesis may play a critical role in CMV and influenza A infection. Although other researchers suggest the approach does not advance far beyond other profiling methods, Princeton is looking to protect the IP around its application in viral settings.

Macroautophagy-dependent, intralysosomal cleavage of a betaine homocysteine methyltransferase fusion protein requires stable multimerization

Cargo-based assays have proven invaluable in the study of macroautophagy in yeast and mammalian cells. Proteomic analysis of autolysosomes identified the metabolic enzyme, betaine homocysteine methyltransferase (BHMT), as a potential cargo-based, end-point marker for mammalian macroautophagy. To test whether degradation of BHMT can be used to measure macroautophagic flux in mammalian cells, we created a BHMT fusion protein (GST-BHMT) that demonstrates starvation-induced, site-specific fragmentation in a variety of cell lines. Subcellular fractionation studies show that the GST-BHMT fragment co-fractionates with vesicles containing lysosomal and autolysosomal markers. Furthermore, both pharmacological inhibitors of macroautophagy and depletion of macroautophagy-specific proteins reduce accumulation of the fragment. In the course of these studies, we observed that fragmentation of GST-BHMT did not occur in forms of the reporter with truncation or point mutations that destabilize oligomerization. Since stable oligomerization of BHMT is essential for its catalytic activity, a point mutation known to ablate BHMT activity was tested. We show that accumulation of the GST-BHMT fragment is not impaired in a catalytically inactive mutant, indicating that selective proteolysis of GST-BHMT requires stable quaternary structure independent of effects on activity. Also, the loss of fragmentation observed in the oligomerization deficient mutants does not seem to be due to a defect of sequestration and lysosomal loading, suggesting that disruption of stable quaternary structure affects the ability of a lysosomal protease to cleave the newly-delivered cargo. Finally, we propose that the cargo-based GST-BHMT assay will be a valuable addition to existing macroautophagy assays in mammalian cells.

Fatty acid metabolism in adipocytes: functional analysis of fatty acid transport proteins 1 and 4

The role of fatty acid transport protein 1 (FATP1) and FATP4 in facilitating adipocyte fatty acid metabolism was investigated using stable FATP1 or FATP4 knockdown (kd) 3T3-L1 cell lines derived from retrovirus-delivered short hairpin RNA (shRNA). Decreased expression of FATP1 or FATP4 did not affect preadipocyte differentiation or the expression of FATP1 (in FATP4 kd), FATP4 (in FATP1 kd), fatty acid translocase, acyl-coenzyme A synthetase 1, and adipocyte fatty acid binding protein but did lead to increased levels of peroxisome proliferator-activated receptor gamma and CCAAT/enhancer binding protein alpha. Both FATP1 and FATP4 kd adipocytes exhibited reduced triacylglycerol deposition and corresponding reductions in diacylglycerol and monoacylglycerol levels compared with control cells. FATP1 kd adipocytes displayed an approximately 25% reduction in basal (3)H-labeled fatty acid uptake and a complete loss of insulin-stimulated (3)H-labeled fatty acid uptake compared with control adipocytes. In contrast, FATP4 kd adipocytes as well as HEK-293 cells overexpressing FATP4 did not display any changes in fatty acid influx. FATP4 kd cells exhibited increased basal lipolysis, whereas FATP1 kd cells exhibited no change in lipolytic capacity. Consistent with reduced triacylglycerol accumulation, FATP1 and FATP4 kd adipocytes exhibited enhanced 2-deoxyglucose uptake compared with control adipocytes. These findings define unique and distinct roles for FATP1 and FATP4 in adipose fatty acid metabolism.

Microtubule Flux: What Is It Good for?

During mitosis in a eukaryotic cell, microtubule subunits continuously move towards spindle poles. A new study has revealed that inhibiting this microtubule flux in mammalian cells has no major effects on chromosome movements; it does, however, increase the probability of erroneous chromosome segregation.

Nicotinic acid-adenine dinucleotide phosphate (NAADP) elicits specific microsomal Ca2+ release from mammalian cells.

Nicotinic acid-adenine dinucleotide phosphate (NAADP), a molecule derived from beta-NADP, has been shown to promote intracellular calcium release in sea urchin eggs. However, there is little information regarding the role of NAADP in the regulation of intracellular calcium fluxes in mammalian cells. We found recently that several mammalian tissues have a high capacity for NAADP synthesis, as assessed by sea urchin egg bioassay. To determine the functional significance of NAADP production by mammalian tissues, we sought to determine whether NAADP is capable of inducing calcium release from microsomes prepared from cultured cells. We found that NAADP, but not beta-NADP, activates a specific microsomal calcium release system in mesangial cells isolated from rat kidney; NAADP was without effect in renal tubular epithelial cells. NAADP-induced calcium release is not affected by inhibitors of the inositol 1,4,5-trisphosphate or ryanodine channels. However, NAADP-elicited calcium release was inhibited by L-type calcium channel blockers and by alkaline phosphatase treatment of NAADP. NAADP also promotes specific microsomal calcium release in rat vascular smooth muscle cells, cardiac myocytes, fibroblasts and a human leukaemia cell line, indicating that the capacity for NAADP-induced calcium release is widespread in mammalian cells. We propose that NAADP may be an important regulator of intracellular calcium in many mammalian tissues.

Nicotinic acid–adenine dinucleotide phosphate (NAADP) elicits specific microsomal Ca2+ release from mammalian cells

Nicotinic acid–adenine dinucleotide phosphate (NAADP), a molecule derived from β-NADP, has been shown to promote intracellular calcium release in sea urchin eggs. However, there is little information regarding the role of NAADP in the regulation of intracellular calcium fluxes in mammalian cells. We found recently that several mammalian tissues have a high capacity for NAADP synthesis, as assessed by sea urchin egg bioassay. To determine the functional significance of NAADP production by mammalian tissues, we sought to determine whether NAADP is capable of inducing calcium release from microsomes prepared from cultured cells. We found that NAADP, but not β-NADP, activates a specific microsomal calcium release system in mesangial cells isolated from rat kidney; NAADP was without effect in renal tubular epithelial cells. NAADP-induced calcium release is not affected by inhibitors of the inositol 1,4,5-trisphosphate or ryanodine channels. However, NAADP-elicited calcium release was inhibited by L-type calcium channel blockers and by alkaline phosphatase treatment of NAADP. NAADP also promotes specific microsomal calcium release in rat vascular smooth muscle cells, cardiac myocytes, fibroblasts and a human leukaemia cell line, indicating that the capacity for NAADP-induced calcium release is widespread in mammalian cells. We propose that NAADP may be an important regulator of intracellular calcium in many mammalian tissues.

Regulation of ATP-induced calcium release in COS-7 cells by calcineurin

Experiments were conducted to examine the role of calcineurin in regulating Ca2+ fluxes in mammalian cells. In COS-7 cells, increasing concentrations (1-10 μM) of ATP triggered intracellular Ca2+ release in a dose-dependent manner. Treatment of the cells with calcineurin inhibitors such as cyclosporin A (CsA), deltamethrin and FK506 resulted in an enhancement of ATP-induced intracellular Ca2+ release. Measurement of calcineurin-specific phosphatase activity in vitro demonstrated a high level of endogenous calcineurin activities in COS-7 cells, which was effectively inhibited by the addition of deltamethrin or CsA. The expression of constitutively active calcineurin (CnA∆CaMAI) inhibited the ATP-induced increase in intracellular Ca2+ concentration ([Ca2+]i), in both the presence and the absence of extracellular Ca2+. These results suggest that the constitutively active calcineurin prevented Ca2+ release from the intracellular stores. In the calcineurin-transfected cells, treatment with CsA restored the calcineurin-mediated inhibition of intracellular Ca2+ release. Protein kinase C-mediated phosphorylation of Ins(1,4,5)P3 receptor [Ins(1,4,5)P3R] was partly inhibited by the extracts prepared from the vector-transfected cells and completely inhibited by those from cells co-transfected with CnA∆CaMAI and calcineurin B. On the addition of 10 μM CsA, the inhibited phosphorylation of Ins(1,4,5)P3R was restored in both the vector-transfected cells and the calcineurin-transfected cells. These results show direct evidence that Ca2+ release through Ins(1,4,5)P3R in COS-7 cells is regulated by calcineurin-mediated dephosphorylation.

Regulation of ATP-induced calcium release in COS-7 cells by calcineurin

Experiments were conducted to examine the role of calcineurin in regulating Ca(2+) fluxes in mammalian cells. In COS-7 cells, increasing concentrations (1-10 microM) of ATP triggered intracellular Ca(2+) release in a dose-dependent manner. Treatment of the cells with calcineurin inhibitors such as cyclosporin A (CsA), deltamethrin and FK506 resulted in an enhancement of ATP-induced intracellular Ca(2+) release. Measurement of calcineurin-specific phosphatase activity in vitro demonstrated a high level of endogenous calcineurin activities in COS-7 cells, which was effectively inhibited by the addition of deltamethrin or CsA. The expression of constitutively active calcineurin (CnADeltaCaMAI) inhibited the ATP-induced increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), in both the presence and the absence of extracellular Ca(2+). These results suggest that the constitutively active calcineurin prevented Ca(2+) release from the intracellular stores. In the calcineurin-transfected cells, treatment with CsA restored the calcineurin-mediated inhibition of intracellular Ca(2+) release. Protein kinase C-mediated phosphorylation of Ins(1,4,5)P(3) receptor [Ins(1,4,5)P(3)R] was partly inhibited by the extracts prepared from the vector-transfected cells and completely inhibited by those from cells co-transfected with CnADeltaCaMAI and calcineurin B. On the addition of 10 microM CsA, the inhibited phosphorylation of Ins(1,4,5)P(3)R was restored in both the vector-transfected cells and the calcineurin-transfected cells. These results show direct evidence that Ca(2+) release through Ins(1, 4,5)P(3)R in COS-7 cells is regulated by calcineurin-mediated dephosphorylation.

Heat flux and the calorimetric-respirometric ratio as measures of catabolic flux in mammalian cells

Abstract It is advocated that cellular heat flow rate ( O = d Q d t , where Q is heat) be expressed as an intensive quantity specific to cell size (X) and termed heat flux ( J O X ). It has been the practice to cite such data on a ‘per cell’ basis, but it would be preferable to use biomass (cellular volume or mass). This quantity is shown to be a measure of metabolic activity and, more accurately, catabolic rate coupled to the demand for ATP in anabolic processes and work in the cell. Recent developments in flow microcalorimetry and dielectric spectroscopy reveal that heat flux can be measured on-line, with the potential of industrial use as a control variable in the growth of hybridoma and genetically engineered cells. This is because the enthalpy change of growth can be regarded as a unique kind of stoichiometric coefficient directly related to the mass coefficients in the growth reaction. This can be verified by an enthalpy balance comparing data for material fluxes of catabolites with the value for heat flux. Information revealed by the stoichiometric growth equation can be used to improve medium design. The ratio of heat flux to oxygen consumption (flux) is known as the calorimetric-respirometric (CR) ratio. It detects anaerobic processes when the value is more negative than −450 (±5%) kJ mol−1 O2. These processes are found in cells growing under fully aerobic conditions, because glycolysis provides biosynthetic precursors with lactate as the byproduct. It is suggested that the CR ratio would be a powerful on-line control variable for the growth of animal cells in bioreactors.

Spodoptera frugiperda (Sf9) cells express novel purine- and pyrimidine-selective nucleoside transporters

Nucleoside fluxes in mammalian cells are mediated by a family of plasma membrane transporters that function by equilibrative or concentrative mechanisms. Members of this family can be identified on the basis of their permeant selectivities and sensitivities to various inhibitors. This study was initiated to characterize nucleoside transport processes of cultured armyworm ovary (Sf9) cells in anticipation of using Sf9 cells for the functional expression of recombinant mammalian nucleoside transporter proteins. Kinetic analysis of zero- trans influx of 3H-nucleosides in Sf9 cells revealed the presence of high ( K m; 10–40 μM) and low ( K m; ⩾0.4 mM) affinity transport processes. Influx of [ 3H]adenosine at 1 μM (high-affinity process) was inhibited only by purine nucleosides (adenosine > 3′-deoxyadenosine > formycin B > guanosine > inosine), and that of [ 3H]uridine was inhibited only by pyrimidine nucleosides (uridine > 3′-deoxyuridine > thymidine). By contrast, fluxes of [ 3H]adenosine and [ 3H]uridine at 100 μM (low-affinity process) were inhibited by both pyrimidine and purine nucleosides. These results suggested the presence of at least two high-affinity transport processes with selectivity for either purine or pyrimidine nucleosides and a low-affinity process with selectivity for both. None of the transport processes were sodium-dependent since uptake of adenosine and uridine was unaffected by elimination of the sodium gradient, and assays that employed formycin B, a non-metabolized permeant of the high-affinity purine-selecive nucleoside transport process, revealed that transport was equilibrative. Nitrobenzylthioinosine, dipyridamole, and dilazep, potent inhibitors of equilibrative nucleoside transport processes in mammalian cells, were poor inhibitors of adenosine and uridine fluxes in Sf9 cells.