Mitochondria and Endoplastic Reticulum Targeting Strategy for Enhanced Phototherapy.

To ensure improved efficacy and minimized toxicity of therapeutic molecules, it is generally accepted that specifically delivering them to the subcellular site of their action will be attractive. Phototherapy has received considerable attention because of its noninvasiveness, high temporal-spatial resolution, and minimal drug resistance. As important functional organelles in cells, mitochondria and endoplasmic reticulum (ER) participate in fundamental cellular processes, which make them much more sensitive to reactive oxygen species (ROS) and hyperthermia. Thus, mitochondria- or ER-targeted phototherapy will be rational strategies for synergetic cancer therapy. In this review, we focus on the latest advances in molecules and nanomaterials currently used for mitochondria- and ER-targeted phototherapy.

Membrane Extracts from Plant Tissues.

The comparison of isolated plant cell membranous enclosures can be hampered if their extraction method differs, e.g., in regard to the utilized buffers, the tissue, or the developmental stage of the plant. Thus, for comparable results, different cellular compartments should be isolated synchronously in one procedure. Here, we devise a workflow to isolate different organelles from one tissue, which is applicable to different eudicots such as Medicago x varia and Solanum lycopersicum. We describe this method for the isolation of different organelles from one plant tissue for the example of Arabidopsis thaliana. All compartments are retrieved by utilizing differential centrifugation with organelle-specific parameters.

pH-dependent effects of procaine on equine gamete activation†.

Procaine directly triggers pH-dependent cytokinesis in equine oocytes and induces hypermotility in stallion spermatozoa, an important event during capacitation. However, procaine-induced hyperactivated motility is abolished when sperm is washed to remove the procaine prior to sperm-oocyte co-incubation. To understand how procaine exerts its effects, the external Ca2+ and Na+ and weak base activity dependency of procaine-induced hyperactivation in stallion spermatozoa was assessed using computer-assisted sperm analysis. Percoll-washed stallion spermatozoa exposed to Ca2+-depleted (+2 mM EGTA) procaine-supplemented capacitating medium (CM) still demonstrated hyperactivated motility, whereas CM without NaCl or Na+ did not. Both procaine and NH4Cl, another weak base, were shown to trigger a cytoplasmic pH increase (BCECF-acetoxymethyl (AM)), which is primarily induced by a pH rise in acidic cell organelles (Lysosensor green dnd-189), accompanied by hypermotility in stallion sperm. As for procaine, 25 mM NH4Cl also induced oocyte cytokinesis. Interestingly, hyperactivated motility was reliably induced by 2.5-10 mM procaine, whereas a significant cytoplasmic cAMP increase and tail-associated protein tyrosine phosphorylation were only observed at 10 mM. Moreover, 25 mM NH4Cl did not support the latter capacitation characteristics. Additionally, cAMP levels were more than 10× higher in boar than stallion sperm incubated under similar capacitating conditions. Finally, stallion sperm preincubated with 10 mM procaine did not fertilize equine oocytes. In conclusion, 10 mM procaine causes a cytoplasmic and acidic sperm cell organelle pH rise that simultaneously induces hyperactivated motility, increased levels of cAMP and tail-associated protein tyrosine phosphorylation in stallion spermatozoa. However, procaine-induced hypermotility is independent of the cAMP/protein tyrosine phosphorylation pathway.

Getting to know the neighborhood: using proximity-dependent biotinylation to characterize protein complexes and map organelles.

The use of proximity-dependent biotinylation approaches combined with mass spectrometry (e.g. BioID and APEX) has revolutionized the study of protein-protein interactions and organellar proteomics. These powerful techniques are based on the fusion of an enzyme (e.g. a biotin ligase or peroxidase) to a 'bait' protein of interest, which is then expressed in a relevant biological setting. Addition of enzyme substrate enables covalent biotin labeling of proteins in the vicinity of the bait in vivo. These approaches thus allow for the capture and identification of 'neighborhood' proteins in the context of a living cell, and provide data that are complementary to more established techniques such as fractionation or affinity purification. As compared to standard affinity-based purification approaches, proximity-dependent biotinylation (PDB) can help to: first, identify interactions with and amongst membrane proteins, and other polypeptide classes that are less amenable to study by standard pulldown techniques; second, enrich for transient and/or low affinity interactions that are not readily captured using affinity purification approaches; third, avoid post-lysis artefacts associated with standard biochemical purification experiments and; fourth, provide deep insight into the organization of membrane-less organelles and other subcellular structures that cannot be easily isolated or purified. Given the increasing use of these techniques to answer a variety of different types of biological questions, it is important to understand how best to design PDB-MS experiments, what type of data they generate, and how to analyze and interpret the results.

Comprehensive characterization of bioactive peptides from Buffalo (Bubalus bubalis) colostrum and milk fat globule membrane proteins.

Milk fat is dispersed in milk as small spherical globules stabilized in the form of emulsion by its surrounding membrane, often referred to as fat globule membrane (FGM). Buffalo, a major milking mammal of Asia and second most milking mammal across the globe presents physicochemical features different from that of other ruminant species containing higher content of lipids and proteins. The present study describes characterization of FGM proteins isolated from both buffalo milk and colostrum. A detailed proteomic analysis of peptides generated by in vitro gastrointestinal simulation digestion of buffalo milk and colostrum FGM fractions was performed by nLC-ESI MS/MS. The peptide based clustering of FGM proteins unravelled association of membrane proteins in fat transport, enzymatic activity, general transport, defence, cell signalling, membrane/protein trafficking protein synthesis/binding/folding including unknown functions. Gene annotation, STRING and YLoc analyses provided putative insights into major secretory pathways in milk and colostrum FGM peptides, interactive protein networks including their sub cellular localization. The peptides of milk and colostrum FGM offered cellular protection as powerful antioxidants indicated their promising perspectives in commercial formulations and nutraceuticals.

Tapetal oleosins play an essential role in tapetosome formation and protein relocation to the pollen coat.

The Arabidopsis pollen grain is covered by a lipidic pollen coat representing select constituents released upon the programmed cell death of the anther secretory tapetum. These constituents originate primarily from two specialized tapetal organelles, elaioplasts and tapetosomes. Tapetosomes are distinctive Brassicaceae organelles derived from the endoplasmic reticulum that store triacylglycerols, flavonoids, alkanes, and proteins. The tapetosome triacylglycerols are found within lipid droplets surrounded by the highly variable tapetal oleosins that eventually generate the most abundant proteins of the pollen coat. Many questions remain regarding the sub-cellular targeting of tapetal oleosins as well as their role in tapetosome formation. Translational fusions of different tapetal oleosins or their derived domains to marker proteins were introduced into Arabidopsis thaliana to investigate their localization, processing and function. Arabidopsis tapetal oleosins were shown to be proteolytically cleaved following tapetum degeneration and different protein domains were targeted to the pollen coat despite vast differences in composition and size. Importantly, specific fusions were discovered to affect distinct aspects of tapetosome formation. This report not only highlighted the critical role of individual tapetal oleosin domains in Arabidopsis tapetosome formation, but revealed translational fusions to be a valuable tool in deciphering this evidently complex developmental process.

Real-time mapping of salt glands on the leaf surface of Cynodon dactylon L. using scanning electrochemical microscopy.

Salt glands are specialized organelles present in the leaf tissues of halophytes, which impart salt-tolerance capability to the plant species. These glands are usually identified only by their morphology using conventional staining procedures coupled with optical microscopy. In this work, we have employed scanning electrochemical microscopy to identify the salt glands not only by their morphology but also by their salt excretion behavior. Bermuda grass (Cynodon dactylon L.) species was chosen for the study as they are known to be salt-tolerant and contain salt glands on leaf surfaces. Scanning electrochemical microscopy performed in sodium chloride medium in the presence and absence of potassium ferrocyanide as redox mediator, reveals the identity of salt glands. More insight into the ion expulsion behavior of these glands was obtained by mapping lateral and vertical variations in ion concentrations using surface impedance measurements which indicated five times higher resistance over the salt glands compared to the surrounding tissues and bulk solution. The protocol could be used to understand the developmental processes in plants grown in different soil/water conditions in order to improve salt tolerance of food crops by genetic engineering and hence improve their agricultural productivity.

Impact of esterified bacteriochlorophylls on the biogenesis of chlorosomes in Chloroflexus aurantiacus.

A chlorosome is an antenna complex located on the cytoplasmic side of the inner membrane in green photosynthetic bacteria that contains tens of thousands of self-assembled bacteriochlorophylls (BChls). Green bacteria are known to incorporate various esterifying alcohols at the C-17 propionate position of BChls in the chlorosome. The effect of these functional substitutions on the biogenesis of the chlorosome has not yet been fully explored. In this report, we address this question by investigating various esterified bacteriochlorophyll c (BChl c) homologs in the thermophilic green non-sulfur bacterium Chloroflexus aurantiacus. Cultures were supplemented with exogenous long-chain alcohols at 52 °C (an optimal growth temperature) and 44 °C (a suboptimal growth temperature), and the morphology, optical properties and exciton transfer characteristics of chlorosomes were investigated. Our studies indicate that at 44 °C Cfl. aurantiacus synthesizes more carotenoids, incorporates more BChl c homologs with unsaturated and rigid polyisoprenoid esterifying alcohols and produces more heterogeneous BChl c homologs in chlorosomes. Substitution of phytol for stearyl alcohol of BChl c maintains similar morphology of the intact chlorosome and enhances energy transfer from the chlorosome to the membrane-bound photosynthetic apparatus. Different morphologies of the intact chlorosome versus in vitro BChl aggregates are suggested by small-angle neutron scattering. Additionally, phytol cultures and 44 °C cultures exhibit slow assembly of the chlorosome. These results suggest that the esterifying alcohol of BChl c contributes to long-range organization of BChls, and that interactions between BChls with other components are important to the assembly of the chlorosome. Possible mechanisms for how esterifying alcohols affect the biogenesis of the chlorosome are discussed.

Oxidative stability of soybean oil in oleosomes as affected by pH and iron.

The oxidative stability of oil in soybean oleosomes, isolated using the Enzyme-Assisted Aqueous Extraction Process (EAEP), was evaluated. The effects of ferric chloride, at two concentration levels (100 and 500 µM), on lipid oxidation, was examined under pH 2 and 7. The peroxide value (PV) and thiobarbituric acid-reactive substance (TBARS) value of oil, in oleosome suspensions stored at 60 °C, were measured over a 12 day period. The presence of ferric chloride significantly (P<0.05) affected the oxidative stability of oil in the isolated oleosome, as measured by the PV and TBARS. Greater lipid oxidation occurred under an acidic pH. In the pH 7 samples, the positively charged transition metals were strongly attracted to the negatively charged droplets. However, the low ζ-potential and the high creaming rate at this pH, may have limited the oxidation. Freezing, freeze-drying or heating of oleosomes have an insignificant impact on the oxidative stability of oil in isolated soybean oleosomes. Manufacturers should be cautious when adding oleosomes as ingredients in food systems containing transition metal ions.

Influence of cell integrity on textural properties of raw, high pressure, and thermally processed onions.

UNLABELLED: The integrity of onion cells and its impact on tissue texture after high pressure and thermal processing was studied. The contribution of cell membranes and the pectic component of cell walls on the texture properties of onion tissue were analyzed. Neutral red (NR) staining of onion parenchyma cell vacuoles was used for the evaluation of cell membrane integrity and microscopic image analysis was used for its quantification. The content of methanol in tissue as a result of pectin methylesterase activity was used to evaluate the pectin component of the middle lamella and cell walls and the hardening effect on the tissue after processing. High pressure treatments consisted of 5-min holding times at 50, 100, 200, 300, or 600 MPa. Thermal treatments consisted of 30-min water bath exposure to 40, 50, 60, 70, or 90 °C. In the high pressure treatments, loss of membrane integrity commenced at 200 MPa and total loss of membrane integrity occurred at 300 MPa and above. In the thermal treatments, membrane integrity was lost between 50 and 60 °C. The texture of onions was influenced by the state of the membranes and texture profiles were abruptly modified once membrane integrity was lost. Hardening of the tissue corresponded with pressure and temperature PME activation and occurred after membrane integrity loss. PRACTICAL APPLICATION: The texture of vegetables is an important quality attribute that affects consumer preference. Loss of textural integrity also indicates that other biochemical reactions that affect color, flavor, and nutrient content may occur more rapidly. In this study, we analyzed changes in the texture of onions after preservation with heat and high pressure.

The pollen organelle membrane proteome reveals highly spatial-temporal dynamics during germination and tube growth of lily pollen.

As a first step in understanding the membrane-related dynamics during pollen grain germination and subsequent tube growth, the changes in protein abundance of membrane and membrane-associated proteins of 5 different membrane/organelle fractions were studied at physiologically important stages (0, 10, 30, 60, and 240 min) of Lilium longiflorum pollen in vitro culture. Proteins of each fraction and time point were identified by 'shot-gun' proteomics (LC-MS/MS). Analysis of more than 270 identified proteins revealed an increase in the abundance of proteins involved in cytoskeleton, carbohydrate and energy metabolism, as well as ion transport before pollen grain germination (10-30 min), whereas proteins involved in membrane/protein trafficking, signal transduction, stress response and protein biosynthesis decreased in abundance during this time. Proteins of amino acids and lipids/steroids metabolism, proteolysis, transcription, cell wall biosynthesis as well as nutrient transport showed a time-independent abundance profile. These spatiotemporal patterns were confirmed by immunodetection of specific proteins of the cellular processes membrane/protein trafficking and ion transport. Our results reveal major protein rearrangements at endomembranes and the plasma membrane before and as the pollen grains start tube growth. The spatiotemporal protein abundance changes correlate with the underlying developmental and physiological processes of the germinating pollen grain.

Stability of artificial oil bodies constituted with recombinant caleosins.

Caleosin is a unique calcium binding protein anchoring to the surface of seed oil bodies by its central hydrophobic domain composed of an amphiphatic alpha-helix and a proline-knot subdomain. Stable artificial oil bodies were successfully constituted with recombinant caleosin overexpressed in Escherichia coli. The stability of artificial oil bodies was slightly or severely reduced when the amphiphatic alpha-helix or proline-knot subdomain in the hydrophobic domain of caleosin was truncated. Deletion of the entire central hydrophobic domain substantially increased the solubility of the recombinant caleosin, leading to a complete loss of its capability to stabilize these oil bodies. A recombinant protein engineered with the hydrophobic domain of caleosin replaced by that of oleosin, the abundant structural protein of seed oil bodies, could stabilize the artificial oil bodies, in terms of thermo- and structural stability, as effectively as caleosin or oleosin.

Acidocalcisomes in Apicomplexan parasites.

Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to man. They posses an acidic matrix that contains several cations bound to phosphates, mainly present in the form of short and long polyphosphate chains. Their matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. The calcium uptake occurs through a Ca2+/H+ counter transporting ATPase located in the membrane of the organelle. Acidocalcisomes have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in Toxoplasma gondii. In this paper, we review the structural, biochemical and physiological aspects of acidocalcisomes in Apicomplexan parasites and discuss their functional roles in the maintenance of intracellular ion homeostasis.

Mitochondrial steps of arginine biosynthesis are conserved in the hydrogenosomes of the chytridiomycete Neocallimastix frontalis.

Arginine biosynthesis in eukaryotes is divided between the mitochondria and the cytosol. The anaerobic chytridiomycete Neocallimastix frontalis contains highly reduced, anaerobic modifications of mitochondria, the hydrogenosomes. Hydrogenosomes also occur in the microaerophilic flagellate Trichomonas vaginalis, which does not produce arginine but uses one of the mitochondrial enzymes, ornithine transcarbamoylase, in a cytosolic arginine dihydrolase pathway for ATP generation. EST sequencing and analysis of the hydrogenosomal proteome of N. frontalis provided evidence for two mitochondrial enzymes of arginine biosynthesis, carbamoylphosphate synthase and ornithine transcarbamoylase, while activities of the arginine dehydrolase pathway enzymes were not detectable in this fungus.

Tapetosomes in Brassica tapetum accumulate endoplasmic reticulum-derived flavonoids and alkanes for delivery to the pollen surface.

Tapetosomes are abundant organelles in tapetum cells during the active stage of pollen maturation in Brassicaceae species. They possess endoplasmic reticulum (ER)-derived vesicles and oleosin-coated lipid droplets, but their overall composition and function have not been established. In situ localization analyses of developing Brassica napus anthers revealed flavonoids present exclusively in tapetum cells, first in an ER network along with flavonoid-3'-hydroxylase and then in ER-derived tapetosomes. Flavonoids were absent in the cytosol, elaioplasts, vacuoles, and nuclei. Subcellular fractionation of developing anthers localized both flavonoids and alkanes in tapetosomes. Subtapetosome fractionation localized flavonoids in ER-derived vesicles, and alkanes and oleosins in lipid droplets. After tapetum cell death, flavonoids, alkanes, and oleosins were located on mature pollen. In the Arabidopsis thaliana mutants tt12 and tt19 devoid of a flavonoid transporter, flavonoids were present in the cytosol in reduced amounts but absent in tapetosomes and were subsequently located on mature pollen. tt4, tt12, and tt19 pollen was more susceptible than wild-type pollen to UV-B irradiation on subsequent germination. Thus, tapetosomes accumulate ER-derived flavonoids, alkanes, and oleosins for discharge to the pollen surface upon cell death. This tapetosome-originated pollen coat protects the haploidic pollen from UV light damage and water loss and aids water uptake.

Protein composition of oil bodies in Arabidopsis thaliana ecotype WS.

Till now, only scattered data are available in the literature, which describes the protein content of plant oil bodies. Especially, the proteins closely associated with the model plant Arabidopsis thaliana oil bodies have never been previously purified and characterized. Oil bodies have been purified using flotation techniques, combined with incubations under high salt concentration, in the presence of detergents and urea in order to remove non-specifically trapped proteins. The identity and integrity of the oil bodies have been characterized. Oil bodies exhibited hydrodynamic diameters close to 2.6 microm, and a ratio fatty acid-protein content near 20. The proteins composing these organelles were extracted, separated by SDS-PAGE, digested by trypsin, and their peptides were subsequently analyzed by nano-chromatography-mass spectrometry (nano-LC-MS/MS). This led to the identification of a limited number of proteins: four different oleosins, ATS1, a protein homologous to calcium binding protein, a 11-beta-hydroxysteroid dehydrogenase-like protein, a probable aquaporin and a glycosylphosphatidylinositol-anchored protein with no known function. The two last proteins were till now never identified in plant oil bodies. Structural proteins (oleosins) represented up to 79% of oil body proteins and the 18.5 kDa oleosin was the most abundant among them.

Acidocalcisomes of trypanosomatids have species-specific elemental composition.

The elemental composition and stoichiometric profile of elements present in acidocalcisomes of different genera of the Trypanosomatidae family (insect, plant, and mammalian parasites) submitted to parallel cultivation conditions were studied. X-ray microanalysis using transmission electron microscopy in conjunction with a morphometric approach was used to investigate the elemental content, number, distribution, and volumetric density of acidocalcisomes of different species. Microanalytical data showed that the different parasites possess the same elemental composition (oxygen, sodium, magnesium, phosphorus, calcium, iron, and zinc) in their acidocalcisomes. However, the relative concentrations of the elements varied among species, but not within acidocalcisomes of individual species. Iron was detected in acidocalcisomes of all species analyzed, characterizing this element as a constituent of these organelles. Taken together, the results strongly indicate a species-specific composition of acidocalcisomes in trypanosomatid parasites.

Temporal association of Ca(2+)-dependent protein kinase with oil bodies during seed development in Santalum album L.: its biochemical characterization and significance.

Calcium-dependent protein kinase (CDPK) is expressed in sandalwood (Santalum album L.) seeds under developmental regulation, and it is localized with spherical storage organelles in the endosperm [Anil et al. (2000) Plant Physiol. 122: 1035]. This study identifies these storage organelles as oil bodies. A 55 kDa protein associated with isolated oil bodies, showed Ca(2+)-dependent autophosphorylation and also cross-reacted with anti-soybean CDPK. The CDPK activity detected in the oil body-protein fraction was calmodulin-independent and sensitive to W7 (N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide) inhibition. Differences in Michaelis Menton kinetics, rate of histone phosphorylation and sensitivity to W7 inhibition between a soluble CDPK from embryos and the oil body-associated CDPK of endosperm suggest that these are tissue-specific isozymes. The association of CDPK with oil bodies of endosperm was found to show a temporal pattern during seed development. CDPK protein and activity, and the in vivo phosphorylation of Ser and Thr residues were detected strongly in the oil bodies of endosperm from maturing seed. Since oil body formation occurs during seed maturation, the observations indicate that CDPK and Ca(2+) may have a regulatory role during oil accumulation/oil body biogenesis. The detection of CDPK-protein and activity in oil bodies of groundnut, sesame, cotton, sunflower, soybean and safflower suggests the ubiquity of the association of CDPKs with oil bodies.

Acidocalcisomes are functionally linked to the contractile vacuole of Dictyostelium discoideum.

The mass-dense granules of Dictyostelium discoideum were shown to contain large amounts of phosphorus, magnesium, and calcium, as determined by x-ray microanalysis, either in situ or when purified using iodixanol gradient centrifugation. The high phosphorus content was due to the presence of pyrophosphate and polyphosphate, which were also present in the contractile vacuoles. Both organelles also possessed a vacuolar H(+)-ATPase, an H(+)-pyrophosphatase, and a Ca(2+)-ATPase, as determined by biochemical methods or by immunofluorescence microscopy. The H(+)-pyrophosphatase activity of isolated mass-dense granules was stimulated by potassium ions and inhibited by the pyrophosphate analogs aminomethylenediphosphonate and imidodiphosphate and by KF and N-ethylmaleimide in a dose-dependent manner. The mass-dense granules and the contractile vacuole appeared to contact each other when the cells were submitted to hyposmotic stress. Acetazolamide inhibited the carbonic anhydrase activity of the contractile vacuoles and prolonged their contraction cycle in a dose-dependent manner. Similar effects were observed with the anion exchanger inhibitor 4,4' -diisothiocyanatodihydrostilbene-2, 2' -disulfonic acid and the vacuolar H(+)-ATPase inhibitor bafilomycin A(1). Together, these results suggest that the mass-dense granules of D. discoideum are homologous to the acidocalcisomes described in protozoan parasites and are linked to the function of the contractile vacuole.

Squid p196, a new member of the myosin-V class of motor proteins, is associated with motile axoplasmic organelles.

Axoplasmic organelles obtained from the squid giant axon move on actin filaments at an average velocity of 1 microm/s [Nature 356 (1992) 722]. The unconventional myosins, in particular, the myosin-V class of motor proteins, represent the most likely candidates to have a role in this motility. Experiments were performed to determine whether a member of the myosin-V class of unconventional myosins is present in axoplasm and optic lobes. Western blots of axoplasm probed with an affinity purified antibody to chicken brain myosin-V (CBM-V) showed cross-reactivity with a protein of Mr 196 kD (p196) which was subsequently purified from squid optic lobes using a modification of a protocol for the purification of CBM-V [Methods Enzymol. 298 (1998) 3; Cell 75 (1993) 215]. Western blots of CBM-V probed with an alpha-p196 polyclonal IgG showed cross-reactivity with CBM-V. Purified p196 has been found to be a calmodulin (CaM) binding protein that possesses calcium-stimulated actin-activated ATPase activity. Equilibrium density fractionation of motile axoplasmic organelle preparations has revealed that p196 cosedimented with the peak organelle fraction into Percoll gradients in the presence of cytochalasin B and ATP. Based on this evidence, we conclude that the p196 present in axoplasm and purified from optic lobes is a squid homolog of CBM-V and functions as a motor for fast transport of membranous organelles on actin filaments in neurons.