The Golgi apparatus segregates from the lysosomal/acrosomal vesicle during rhesus spermiogenesis: structural alterations

The acrosome is an acidic secretory vesicle containing hydrolytic enzymes that are involved in the sperm's passage across the zona pellucida. Imaging of the acrosomal vesicle and the Golgi apparatus in live rhesus monkey spermatids was accomplished by using the vital fluorescent probe LysoTracker DND-26. Concurrently, the dynamics of living spermatid mitochondria was visualized using the specific probe MitoTracker CMTRos and LysoTracker DND-26 detected the acrosomal vesicle from its formation through spermatid differentiation. LysoTracker DND-26 also labeled the Golgi apparatus in spermatogenic cells. In spermatocytes the Golgi is spherical and, in round spermatids, it is localized over the acrosomal vesicle, as confirmed by using polyclonal antibodies against Golgin-95/GM130, Golgin-97, and Golgin-160. Using both live LysoTracker DND-26 imaging and Golgi antibodies, we found that the Golgi apparatus is cast off from the acrosomal vesicle and migrates toward the sperm tail in elongated spermatids. The Golgi is discarded in the cytoplasmic droplet and is undetectable in mature ejaculated spermatozoa. The combined utilization of three vital fluorescent probes (Hoechst 33342, LysoTracker DND-26, and MitoTracker CMTRos) permits the dynamic imaging of four organelles during primate spermiogenesis: the nucleus, the mitochondria, the acrosomal vesicle, and the Golgi apparatus.     

Stereoselective and multiple carrier‐mediated transport of cetirizine across Caco‐2 cell monolayers with potential drug interaction

The aim of this study was to explore potential transport mechanisms of cetirizine enantiomers across Caco‐2 cells. Cetirizine displayed polarized transport at concentrations ranging from 4.0 to 80.0 μM, with the permeability in the secretory direction being 1.4‐ to 4.0‐fold higher than that in the absorptive direction. Cetirizine enantiomers were transported distinctively different from each other. In the presence of inhibitors of P‐glycoprotein (P‐gp) and multidrug resistance‐associated protein (MRP), the absorptive transport was enhanced and secretory efflux was diminished. When verapamil, indomethacin, or probenecid were present, the difference in the absorptive permeability of R‐cetirizine and S‐cetirizine substantially intensified, whereas quinidine could eliminate. R‐cetirizine significantly increased the efflux ratio of rhodamine‐123 and doxorubicin in a fashion indicative of the upregulation of P‐gp and MRP activities. However, S‐cetirizine played a role of an inhibitor for P‐gp and MRP. Ranitidine modified the absorption of cetirizine enantiomers, suggesting that the potential drug–drug interaction would significantly change the cetirizine pharmacokinetics. In conclusion, the results indicated that there are several efflux transporters including P‐gp and MRP participating the absorption and efflux of cetirizine, which showed enantioselectivity in the transmembrane process. In addition, both P‐gp and MRP functions could be modulated by cetirizine in chiral discriminative ways. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Dynamic organelle localization and cytoskeletal reorganization during preimplantation mouse embryo development revealed by live imaging of genetically encoded fluorescent fusion proteins

Live imaging is one of the most powerful technologies for studying the behaviors of cells and molecules in living embryos. Previously, we established a series of reporter mouse lines in which specific organelles are labeled with various fluorescent proteins. In this study, we examined the localizations of fluorescent signals during preimplantation development of these mouse lines, as well as a newly established one, by time‐lapse imaging. Each organelle was specifically marked with fluorescent fusion proteins; fluorescent signals were clearly visible during the whole period of time‐lapse observation, and the expression of the reporters did not affect embryonic development. We found that some organelles dramatically change their sub‐cellular distributions during preimplantation stages. In addition, by crossing mouse lines carrying reporters of two distinct colors, we could simultaneously visualize two types of organelles. These results confirm that our reporter mouse lines can be valuable genetic tools for live imaging of embryonic development.     

Glycolytic pyruvate regulates P‐Glycoprotein expression in multicellular tumor spheroids via modulation of the intracellular redox state

ABC transporters like P‐glycoprotein (P‐gp/ABCB1) are membrane proteins responsible for the transport of toxic compounds out of non‐malignant cells and tumor tissue. Aim: To investigate the effect of glycolysis and the tissue redox state on P‐gp expression in multicellular tumor spheroids derived from prostate adenocarcinoma cells (DU‐145), glioma cells (Gli36), and the human cervix carcinoma cell line KB‐3‐1 transfected with a P‐gp–EGFP fusion gene that allows monitoring of P‐gp expression in living cells. During cell culture of DU‐145, Gli36, and KB‐3‐1 tumor spheroids P‐gp expression was observed as well as increased lactate and decreased pyruvate levels and expression of glycolytic enzymes. Inhibition of glycolysis for 24 h by either iodoacetate (IA) or 2‐deoxy‐D ‐glucose (2‐DDG) downregulated P‐gp expression which was reversed upon coincubation with the radical scavenger ebselen as shown by semi‐quantitative immunohistochemisty in DU‐145 and Gli36 tumor spheroids, and by EGFP fluorescence in KB‐3‐1 tumor spheroids. Consequently endogenous ROS generation in DU‐145 tumor spheroids was increased in the presence of either IA or 2‐DDG, which was abolished upon coincubation with ebselen. Exogenous addition of pyruvate significantly reduced ROS generation, increased P‐gp expression as well as efflux of the P‐gp substrate doxorubicin. Doxorubicin transport was significantly blunted by 2‐DDG and IA, indicating that inhibition of glycolysis reversed the multidrug resistance phenotype. In summary our data demonstrate that P‐gp expression in tumor spheroids is closely related to the glycolytic metabolism of tumor cells and can be downregulated by glycolysis inhibitors via mechanisms that involve changes in the cellular redox state. J. Cell. Biochem. 109: 434–446, 2010. © 2009 Wiley‐Liss, Inc.     

Characterization of 3-methoxy flavones for their interaction with ABCG2 as suggested by ATPase activity

Breast Cancer Resistance Protein (BCRP/ABCG2) belongs to the superfamily of ATP binding cassette (ABC) transporters. Characteristic of some of these transporter proteins is the transport of a variety of structurally unrelated substances against a concentration gradient by using the energy of ATP hydrolysis. ABCG2 has been found to confer multidrug resistance (MDR) in cancer cells. Several anticancer drugs have been identified as ABCG2 substrates including mitoxantrone, etoposide and topotecan. As inhibition of the transporter is one of the strategies to overcome MDR, we have synthesized and tested several 3-methoxy flavones and investigated them for their ABCG2 inhibition. Among these, pentamethyl quercetin (compound 4) and pentamethyl morin (compound 5) were found to be fluorescent and hence screened for their possible transport by ABCG2 using confocal microscopy. This study showed that pentamethyl quercetin was far less accumulated in ABCG2 overexpressing MDCK BCRP cells as compared to MDCK sensitive cells, suggesting possible efflux of this compound by ABCG2. Pentamethyl morin showed no visible difference in both cell lines. Based on this observation, we studied several other fluorescent 3-methoxy flavones for their accumulation in ABCG2 overexpressing cells. To confirm the substrate or inhibitor nature of the tested compounds, these compounds were further investigated by ATPase assay. If stimulation of the transporter ATPase activity is detected, one can conclude that the compound is probably a transported substrate. All compounds except pentamethyl morin (compound 5) and tetramethyl quercetin (compound 6) were found to stimulate ATPase activity pointing to possible substrates despite being potent inhibitors of ABCG2.     

Online Fluorescent Method to Assess BCRP/ABCG2 Activity in Suspension Cells

An online method was developed to monitor BCRP mediated efflux of fluorescent substrates in suspension cells. To this end, a 2‐compartment system consisting of a transwell cup and a cuvette was used. In this system we were able to observe differences in efflux kinetics between BCRP overexpressing RPMI 8226/MR cells and parental myeloid RPMI 8226(s) cells using only 50,000 cells per experiment. 8226/MR cells displayed a larger cellular efflux rate of the BCRP substrate Hoechst 33342, as compared to the wildtype cells. This difference in efflux rate was completely decreased in the presence of the BCRP inhibitor Ko143.     

Effects of mitochondria-selective fluorescent probes on mitochondrial movement in Arabidopsis mesophyll cells evaluated by using the quantification

Mitochondria-selective fluorescent probes such as MitoTracker are often used for mitochondria imaging in various plants. Although some of the probes are reported to induce mitochondria dysfunction in animal cells, the effect on plant cells remains to be determined. In the present study, we applied quantitative methods to analyze mitochondrial movement, speed frequency, and speed-angle changes, based on trajectory analysis of mitochondria in mesophyll protoplast cells of Arabidopsis thaliana expressing the mitochondria-localized fluorescent protein. Using the quantitative method, we assessed whether MitoTracker Red (FM and CMXRos) induce mitochondria dysfunction in A. thaliana. Although both the fluorescent probes well-stained mitochondria, the CMXRos probe, not the FM probe, gave a severe effect on mitochondrial movement at the low concentration (10 nM), indicating a MitoTracker-induced mitochondria dysfunction in A. thaliana. These results revealed that our quantitative method based on mitochondrial movement can be used to determine the appropriate concentrations of mitochondria-selective fluorescent probes in plants.     

Phospholipid Synthesis and Transport in Mammalian Cells

Membranes of mammalian subcellular organelles contain defined amounts of specific phospholipids that are required for normal functioning of proteins in the membrane. Despite the wide distribution of most phospholipid classes throughout organelle membranes, the site of synthesis of each phospholipid class is usually restricted to one organelle, commonly the endoplasmic reticulum (ER). Thus, phospholipids must be transported from their sites of synthesis to the membranes of other organelles. In this article, pathways and subcellular sites of phospholipid synthesis in mammalian cells are summarized. A single, unifying mechanism does not explain the inter‐organelle transport of all phospholipids. Thus, mechanisms of phospholipid transport between organelles of mammalian cells via spontaneous membrane diffusion, via cytosolic phospholipid transfer proteins, via vesicles and via membrane contact sites are discussed. As an example of the latter mechanism, phosphatidylserine (PS) is synthesized on a region of the ER (mitochondria‐associated membranes, MAM) and decarboxylated to phosphatidylethanolamine in mitochondria. Some evidence is presented suggesting that PS import into mitochondria occurs via membrane contact sites between MAM and mitochondria. Recent studies suggest that protein complexes can form tethers that link two types of organelles thereby promoting lipid transfer. However, many questions remain about mechanisms of inter‐organelle phospholipid transport in mammalian cells.     

Inhibition of P‐Glycoprotein‐Induced Multidrug Resistance by a Clerodane‐Type Diterpenoid from Sindora sumatrana

The aim of the present study was to investigate the effects of di‐ and sesquiterpenoids isolated from the pods of Sindora sumatrana Miq. (Leguminosae) on P‐glycoprotein (P‐gp) function in an adriamycin‐resistant human breast cancer cell line, MCF‐7/ADR. Over‐expression of P‐gp is known to be one of the mechanisms involved in multidrug resistance (MDR), which is a major obstacle in clinical cancer treatment. Among six di‐ and sesquiterpenoids extracted from S. sumatrana, (+)‐7β‐acetoxy‐15,16‐epoxycleroda‐3,13(16),14‐trien‐18‐oic acid ( 1 ) showed a strong P‐gp inhibitory effect, as great as that of verapamil, a representative P‐gp inhibitor. Compound 1 enhanced daunomycin accumulation more than fourfold and significantly decreased daunomycin efflux compared with control, resulting in a decrease in the IC₅₀ value for daunomycin. These results suggest that compound 1 inhibits the functioning of P‐gp and, therefore, can be developed as an MDR‐reversing agent.     

Organelle extensions in plant cells

The life strategy of plants includes their ability to respond quickly at the cellular level to changes in their environment. The use of targeted fluorescent protein probes and imaging of living cells has revealed several rapidly induced organelle responses that create the efficient sub-cellular machinery for maintaining homeostasis in the plant cell. Several organelles, including plastids, mitochondria, and peroxisomes, extend and retract thin tubules that have been named stromules, matrixules, and peroxules, respectively. Here, I combine all these thin tubular forms under the common head of organelle extensions. All extensions change shape continuously and in their elongated form considerably increase organelle outreach into the surrounding cytoplasm. Their pleomorphy reflects their interactions with the dynamic endoplasmic reticulum and cytoskeletal elements. Here, using foundational images and time-lapse movies, and providing salient information on some molecular and biochemically characterized mutants with increased organelle extensions, I draw attention to their common role in maintaining homeostasis in plant cells.

Dynamic tubules extended from different organelles are integral components of the rapid subcellular response machinery involved in maintaining optimal working conditions within plant cells.     

Down‐regulation of c‐fos by shRNA sensitizes adriamycin‐resistant MCF‐7/ADR cells to chemotherapeutic agents via P‐glycoprotein inhibition and apoptosis augmentation

Multidrug resistance (MDR) is a major hurdle in the treatment of cancer. Research indicated that the main mechanisms of most cancers included so‐called “pump” (P‐glycoprotein, P‐gp) and “non‐pump” (apoptosis) resistance. Identification of novel signaling molecules associated with both P‐gp and apoptosis will facilitate the development of more effective strategies to overcome MDR in tumor cells. Since the proto‐oncogene c‐fos has been implicated in cell adaptation to environmental changes, we analyzed its role in mediating “pump” and “non‐pump” resistance in MCF‐7/ADR, an adriamycin (ADR)‐selected human breast cancer cell line with the MDR phenotype. Elevated expression of c‐fos in MCF‐7/ADR cells and induction of c‐fos by ADR in the parental drug‐sensitive MCF‐7 cells suggested a link between c‐fos and MDR phenotype. Down‐regulation of c‐fos expression via shRNA resulted in sensitization of MCF‐7/ADR cells to chemotherapeutic agents, including both P‐gp and non‐P‐gp substrates. Further results proved that c‐fos down‐regulation in MCF‐7/ADR cells resulted in decreased P‐gp expression and activity, enhanced apoptosis, and altered expression of apoptosis‐associated proteins (i.e., Bax, Bcl‐2, p53, and PUMA). All above facts indicate that c‐fos is involved in both P‐gp‐ and anti‐apoptosis‐mediated MDR of MCF‐7/ADR cells. Based on these results, we propose that c‐fos may represent a potential molecular target for resistant cancer therapy, and suppressing c‐fos gene expression may therefore be an effective means to temper breast cancer cell's MDR to cytotoxic chemotherapy. J. Cell. Biochem. 114: 1890–1900, 2013. © 2013 Wiley Periodicals, Inc.     

Targeting of mitochondria-endoplasmic reticulum by fluorescent macrocyclic compounds

Background

Useful probes of the intracellular environment that target a specific organelle in order to allow direct observation of the changes in these regions is of high current interest. Macrocyclic ligands have already revealed themselves as important selective hosts in some biological applications, forming stable and specific complexes. Therefore, in this paper, several macrocyclic ligands are evaluated as potential molecular probes.

Methodology

Four polyammonium macrocycles and one macrotricyclic bearing pyridine and phenanthroline chromophores have been synthesised and evaluated as molecular probes. The cytotoxicity of the compounds has been analyzed using human breast cancer cells (MCF-7), non-cancerous human dermal fibroblasts (NHDF) and human adult dermal skin fibroblasts from a breast cancer patient (P14). All the compounds showed low toxicity at concentrations ranging from 10 nM to 10 µM, except for [32]phen₂N₄ which proved to be highly cytotoxic for MCF-7 cells. Flow cytometry studies evidenced that the percentage of apoptotic and necrotic MCF-7 and NHDF cells induced by the compounds is considerably low. Also, flow cytometry analysis showed that some compounds seem to modify the mitochondrial membrane potential (MMP) of the cells. Fluorescence microscopy evidenced that compounds easily cross the plasma membrane (5 min) and accumulated into the mitochondria, as confirmed by co-localization with MitoTracker Green™. The fluorescence images also evidenced an intact mitochondria structure after 48 h. Moreover, reticular staining suggestive of endoplasmic reticulum (ER) localization, in addition to the mitochondrial one, has been found by confocal microscopy.

Conclusion

Our study reveals that compounds Me₂[28]py₂N₆, cryptphen, [16]phenN₂, [30]phen₂N₆, have low toxicity and localize in mitochondria and ER. The ability of these compounds for translocating the cellular membrane (5 min) without special conditioning of the cells or derivatization of the probe, the time-dependent localization (48 h) and the cellular viability provide a proof-of-concept towards their use as promising probes towards biomedical studies.     

Mitochondria of a human multidrug‐resistant hepatocellular carcinoma cell line constitutively express inducible nitric oxide synthase in the inner membrane

Mitochondria play a crucial role in pathways of stress conditions. They can be transported from one cell to another, bringing their features to the cell where they are transported. It has been shown in cancer cells overexpressing multidrug resistance (MDR) that mitochondria express proteins involved in drug resistance such as P‐glycoprotein (P‐gp), breast cancer resistant protein and multiple resistance protein‐1. The MDR phenotype is associated with the constitutive expression of COX‐2 and iNOS, whereas celecoxib, a specific inhibitor of COX‐2 activity, reverses drug resistance of MDR cells by releasing cytochrome c from mitochondria. It is possible that COX‐2 and iNOS are also expressed in mitochondria of cancer cells overexpressing the MDR phenotype. This study involved experiments using the human HCC PLC/PRF/5 cell line with and without MDR phenotype and melanoma A375 cells that do not express the MDR1 phenotype but they do iNOS. Western blot analysis, confocal immunofluorescence and immune electron microscopy showed that iNOS is localized in mitochondria of MDR1‐positive cells, whereas COX‐2 is not. Low and moderate concentrations of celecoxib modulate the expression of iNOS and P‐gp in mitochondria of MDR cancer cells independently from inhibition of COX‐2 activity. However, A375 cells that express iNOS also in mitochondria, were not MDR1 positive. In conclusion, iNOS can be localized in mitochondria of HCC cells overexpressing MDR1 phenotype, however this phenomenon appears independent from the MDR1 phenotype occurrence. The presence of iNOS in mitochondria of human HCC cells phenotype probably concurs to a more aggressive behaviour of cancer cells.     

Acidic vesicles in living hyphae of an arbuscular mycorrhizal fungus, Gigaspora margarita

Localization and movement of organelles in living hyphae of an arbuscular mycorrhizal fungus, Gigaspora margarita, were observed using a combination of fluorescent probes and laser-scanning confocal microscopy. Dense, evenly distributed acidic vesicles were visible in germ tubes and extraradical hyphae using DIC with the fluorescent acidotropic probe LysoTracker. These vesicles were distinct from both tubular vacuoles stained with DFFDA and lipid bodies stained with BODIPY 493/503 or Nile Red. Tubular vacuole bundles appeared to be influenced by the bidirectional cytoplasmic streaming of acidic vesicles and lipid bodies. Movement of the acidic vesicles occurred bidrectionally at different rates. The size and distribution of lipid bodies were variable. Based on our observations, the function of these organelles is discussed in relation to nutrient translocation in arbuscular mycorrhizas. Abbreviations: AM – arbuscular mycorrhiza; DAPI – 4′,6-diamidino-2-phenylindole; DIC – differential interference contrast; BODIPY 493/503 – 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene; DMSO – dimethyl sulfoxide; FITC – fluorescein isothiocynate; caboxy-DFFDA – Oregon Green 488 carboxylic acid diacetate.     

ToolBox: Live Imaging of intracellular organelle transport in induced pluripotent stem cell‐derived neurons

Induced pluripotent stem cells (iPSCs) hold promise to revolutionize studies of intracellular transport in live human neurons and to shed new light on the role of dysfunctional transport in neurodegenerative disorders. Here, we describe an approach for live imaging of axonal and dendritic transport in iPSC‐derived cortical neurons. We use transfection and transient expression of genetically‐encoded fluorescent markers to characterize the motility of Rab‐positive vesicles, including early, late and recycling endosomes, as well as autophagosomes and mitochondria in iPSC‐derived neurons. Comparing transport parameters of these organelles with data from primary rat hippocampal neurons, we uncover remarkable similarities. In addition, we generated lysosomal‐associated membrane protein 1 (LAMP1)‐enhanced green fluorescent protein (EGFP) knock‐in iPSCs and show that knock‐in neurons can be used to study the transport of endogenously labeled vesicles, as a parallel approach to the transient overexpression of fluorescently labeled organelle markers.     

Single organelle dynamics linked to 3D structure by correlative live‐cell imaging and 3D electron microscopy

Live‐cell correlative light‐electron microscopy (live‐cell‐CLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3‐dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB‐SEM) in a modular live‐cell‐CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal‐associated membrane protein 1‐green fluorescent protein (LAMP‐1‐GFP), analyzed the dynamics of individual GFP‐positive spots, and correlated these to their corresponding fine‐architecture and immediate cellular environment. By FIB‐SEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIB‐SEM, which significantly reduces time required for image acquisition and data processing.      

SR‐BI,CD36, and caveolin‐1 contribute positively to cholesterol efflux in hepatic cells

In non‐hepatic cells, scavenger receptor class B type I (SR‐BI), cluster of differentiation 36 (CD36), and caveolin‐1 were described as mediators of cholesterol efflux, the first step of reverse cholesterol transport (RCT). Stable transformants of HepG2 cells overexpressing SR‐BI, CD36, or caveolin‐1 were generated, as well as cells overexpressing both caveolin‐1 and SR‐BI or caveolin‐1 and CD36 in order to address the effect of caveolin‐1 on both receptor activities. These cells were analyzed for their ability to efflux cholesterol to HDL₃. Our results show that overexpressing SR‐BI, CD36, or caveolin‐1 increases cholesterol efflux by 106, 92, and 48%, respectively. Moreover, the dual overexpressions of caveolin‐1 and SR‐BI or caveolin‐1 and CD36 lead to a more prominent increase in cholesterol efflux. Studies were also conducted with primary cultures of SR‐BI knockout (KO), CD36 KO, and SR‐BI/CD36 double‐KO (dKO) mice. SR‐BI KO and SR‐BI/CD36 dKO hepatic cells show 41 and 56% less cholesterol efflux, respectively, than normal hepatic cells. No significant difference was observed between the efflux of normal and CD36 KO cells. The difference between the role of human and murine CD36 correlated with the absence of CD36 dimers in mouse caveolae/rafts. Overall, our results show that SR‐BI is clearly involved in cholesterol efflux in mouse and human hepatic cells, while CD36 plays a significant role in human cells. Copyright © 2010 John Wiley & Sons, Ltd.     

Tracker Dyes to Probe Mitochondrial Autophagy (Mitophagy) in Rat Hepatocytes

Mitochondria become targets for autophagic degradation after nutrient deprivation, a process also termed mitophagy. In this study, we used LysoTracker Red (LTR) and MitoTracker Green to characterize the kinetics of autophagosomal proliferation and mitophagy in cultured rat hepatocytes. Autophagy induced by nutrient deprivation plus glucagon increased LTR uptake assessed with a fluorescence plate reader and the number of LTR-labeled acidic organelles assessed with confocal microscopy in individual hepatocytes both by 4- to 6-fold. Serial imaging of hepatocytes co-loaded with MitoTracker Green (MTG) revealed an average mitochondrial digestion time of 7.5 min after autophagic induction. In the presence of protease inhibitors, digestion time more than doubled, and the total number of LTR-labeled organelles increased about 40%, but the proportion of the LTR-labeled acidic organelles containing MTG fluorescence remained constant at about 75%. Autophagy inhibitors, 3-methyladenine, wortmannin and LY204002, suppressed the increase of LTR uptake after nutrient deprivation by up to 85%, confirming that increased LTR uptake reflected autophagy induction. Cyclosporin A and NIM811, specific inhibitors of the mitochondrial permeability transition (MPT), alsodecreased LTR uptake, whereas tacrolimus, an immunosuppressive reagent that does not inhibit the MPT, was without effect. In addition, the c-Jun N-terminal kinase (JNK) inhibitors, SCP25041 and SP600125, blocked LTR uptake by 47% and 61%, respectively, but ERK1, p38 and caspase inhibitors had no effect. The results show that mitochondria once selected for mitophagy are rapidly digested and support the concept that mitochondrial autophagy involves the MPT and signaling through PI3 kinase and possibly JNK.     

Tracker dyes to probe mitochondrial autophagy (mitophagy) in rat hepatocytes

Mitochondria become targets for autophagic degradation after nutrient deprivation, a process also termed mitophagy. In this study, we used LysoTracker Red (LTR) and MitoTracker Green to characterize the kinetics of autophagosomal proliferation and mitophagy in cultured rat hepatocytes. Autophagy induced by nutrient deprivation plus glucagon increased LTR uptake assessed with a fluorescence plate reader and the number of LTR-labeled acidic organelles assessed with confocal microscopy in individual hepatocytes both by 4- to 6-fold. Serial imaging of hepatocytes coloaded with MitoTracker Green (MTG) revealed an average mitochondrial digestion time of 7.5 min after autophagic induction. In the presence of protease inhibitors, digestion time more than doubled, and the total number of LTR-labeled organelles increased about 40%, but the proportion of the LTR-labeled acidic organelles containing MTG fluorescence remained constant at about 75%. Autophagy inhibitors, 3-methyladenine, wortmannin and LY204002, suppressed the increase of LTR uptake after nutrient deprivation by up to 85%, confirming that increased LTR uptake reflected autophagy induction. Cyclosporin A and NIM811, specific inhibitors of the mitochondrial permeability transition (MPT), also decreased LTR uptake, whereas tacrolimus, an immunosuppressive reagent that does not inhibit the MPT, was without effect. In addition, the c-Jun N-terminal kinase (JNK) inhibitors, SCP25041 and SP600125, blocked LTR uptake by 47% and 61%, respectively, but ERK1, p38 and caspase inhibitors had no effect. The results show that mitochondria once selected for mitophagy are rapidly digested and support the concept that mitochondrial autophagy involves the MPT and signaling through PI3 kinase and possibly JNK.     

Design,synthesis and evaluation of a series of 5-methoxy-2,3-naphthalimide derivatives as AcrB inhibitors for the reversal of bacterial resistance

A series of novel 5-methoxy-2,3-naphthalimide derivatives were designed, synthesized and evaluated for their biological activities. In particular, the ability of the compounds to synergize with antimicrobials, to inhibit Nile Red efflux, and to target AcrB was assayed. The results showed that the most of the tested compounds more sensitized the Escherichia coli BW25113 to the antibiotics than the parent compounds 7c and 15, which were able to inhibit Nile Red efflux. Significantly, compound A5 possessed the most potent antibacterial synergizing activity in combination with levofloxacin by 4 times and 16 times at the concentration of 8 and 16?µg/mL, respectively, whilst A5 could effectively abolish Nile Red efflux at 100?μM. Additionally, target effect of A5 was confirmed in the outer- or inner membrane permeabilization assays. Therefore, A5 is an excellent lead compound for further structural optimization.