Showing papers on "Cellular compartment published in 2016"

Regulating Subcellular Metal Homeostasis: The Key to Crop Improvement.

Abstract: Iron (Fe), zinc (Zn) manganese (Mn), and copper (Cu) are essential micronutrient mineral elements for living organisms, as they regulate essential cellular processes, such as chlorophyll synthesis and photosynthesis (Fe, Cu, and Mn), respiration (Fe and Cu), and transcription (Zn). The storage and distribution of these minerals in various cellular organelles is strictly regulated to ensure optimal metabolic rates. Alteration of the balance in uptake, distribution, and/or storage of these minerals severely impairs cellular metabolism and significantly affects plant growth and development. Thus, any change in the metal profile of a cellular compartment significantly affects metabolism. Different subcellular compartments are suggested to be linked through complex retrograde signaling networks to regulate cellular metal homeostasis. Various genes regulating cellular and subcellular metal distribution have been identified and characterized. Understanding the role of these transporters is extremely important to elaborate the signaling between various subcellular compartments. Moreover, modulation of the proteins involved in cellular metal homeostasis may help in the regulation of metabolism, adaptability to a diverse range of environmental conditions, and biofortification. Here, we review progress in the understanding of different subcellular metal transport components in plants and discuss the prospects of regulating cellular metabolism and strategies to develop biofortified crop plants.

Flow-cytometric method for simultaneous analysis of mouse lung epithelial, endothelial, and hematopoietic lineage cells

Abstract: Flow cytometry is a powerful tool capable of simultaneously analyzing multiple parameters on a cell-by-cell basis. Lung tissue preparation for flow cytometry requires creation of a single-cell suspension, which often employs enzymatic and mechanical dissociation techniques. These practices may damage cells and cause cell death that is unrelated to the experimental conditions under study. We tested methods of lung tissue dissociation and sought to minimize cell death in the epithelial, endothelial, and hematopoietic lineage cellular compartments. A protocol that involved flushing the pulmonary circulation and inflating the lung with Dispase, a bacillus-derived neutral metalloprotease, at the time of tissue harvest followed by mincing, digestion in a DNase and collagenase solution, and filtration before staining with fluorescent reagents concurrently maximized viable yields of epithelial, endothelial, and hematopoietic lineage cells compared with a standard method that did not use enzymes at the time of tissue harvest. Flow cytometry identified each population-epithelial (CD326(+)CD31(-)CD45(-)), endothelial (CD326(-)CD31(+)CD45(-)), and hematopoietic lineage (CD326(-)CD31(-)CD45(+))-and measured cellular viability by 7-aminoactinomycin D (7-AAD) staining. The Dispase method permitted discrimination of epithelial vs. endothelial cell death in a systemic lipopolysaccharide model of increased pulmonary vascular permeability. We conclude that application of a dissociative enzyme solution directly to the cellular compartments of interest at the time of tissue harvest maximized viable cellular yields of those compartments. Investigators could employ this dissociation method to simultaneously harvest epithelial, endothelial, and hematopoietic lineage and other lineage-negative cells for flow-cytometric analysis.

Oxidative Stress in the Healthy and Wounded Hepatocyte: A Cellular Organelles Perspective

Abstract: Accurate control of the cell redox state is mandatory for maintaining the structural integrity and physiological functions. This control is achieved both by a fine-tuned balance between prooxidant and anti-oxidant molecules and by spatial and temporal confinement of the oxidative species. The diverse cellular compartments each, although structurally and functionally related, actively maintain their own redox balance, which is necessary to fulfill specialized tasks. Many fundamental cellular processes such as insulin signaling, cell proliferation and differentiation and cell migration and adhesion, rely on localized changes in the redox state of signal transducers, which is mainly mediated by hydrogen peroxide (H2O2). Therefore, oxidative stress can also occur long before direct structural damage to cellular components, by disruption of the redox circuits that regulate the cellular organelles homeostasis. The hepatocyte is a systemic hub integrating the whole body metabolic demand, iron homeostasis and detoxification processes, all of which are redox-regulated processes. Imbalance of the hepatocyte's organelles redox homeostasis underlies virtually any liver disease and is a field of intense research activity. This review recapitulates the evolving concept of oxidative stress in the diverse cellular compartments, highlighting the principle mechanisms of oxidative stress occurring in the healthy and wounded hepatocyte.

Improved proteostasis in the secretory pathway rescues Alzheimer's disease in the mouse.

Abstract: The aberrant accumulation of toxic protein aggregates is a key feature of many neurodegenerative diseases, including Huntington's disease, amyotrophic lateral sclerosis and Alzheimer's disease. As such, improving normal proteostatic mechanisms is an active target for biomedical research. Although they share common pathological features, protein aggregates form in different subcellular locations. Ne-lysine acetylation in the lumen of the endoplasmic reticulum has recently emerged as a new mechanism to regulate the induction of autophagy. The endoplasmic reticulum acetylation machinery includes AT-1/SLC33A1, a membrane transporter that translocates acetyl-CoA from the cytosol into the endoplasmic reticulum lumen, and ATase1 and ATase2, two acetyltransferases that acetylate endoplasmic reticulum cargo proteins. Here, we used a mutant form of α-synuclein to show that inhibition of the endoplasmic reticulum acetylation machinery specifically improves autophagy-mediated disposal of toxic protein aggregates that form within the secretory pathway, but not those that form in the cytosol. Consequently, haploinsufficiency of AT-1/SLC33A1 in the mouse rescued Alzheimer's disease, but not Huntington's disease or amyotrophic lateral sclerosis. In fact, intracellular toxic protein aggregates in Alzheimer's disease form within the secretory pathway while in Huntington's disease and amyotrophic lateral sclerosis they form in different cellular compartments. Furthermore, biochemical inhibition of ATase1 and ATase2 was also able to rescue the Alzheimer's disease phenotype in a mouse model of the disease. Specifically, we observed reduced levels of soluble amyloid-β aggregates, reduced amyloid-β pathology, reduced phosphorylation of tau, improved synaptic plasticity, and increased lifespan of the animals. In conclusion, our results indicate that Ne-lysine acetylation in the endoplasmic reticulum lumen regulates normal proteostasis of the secretory pathway; they also support therapies targeting endoplasmic reticulum acetyltransferases, ATase1 and ATase2, for a subset of chronic degenerative diseases.

Crosstalk between cellular compartments protects against proteotoxicity and extends lifespan

Abstract: In cells living under optimal conditions, protein folding defects are usually prevented by the action of chaperones. Here, we investigate the cell-wide consequences of loss of chaperone function in cytosol, mitochondria or the endoplasmic reticulum (ER) in budding yeast. We find that the decline in chaperone activity in each compartment results in loss of respiration, demonstrating the dependence of mitochondrial activity on cell-wide proteostasis. Furthermore, each chaperone deficiency triggers a response, presumably via the communication among the folding environments of distinct cellular compartments, termed here the cross-organelle stress response (CORE). The proposed CORE pathway encompasses activation of protein conformational maintenance machineries, antioxidant enzymes, and metabolic changes simultaneously in the cytosol, mitochondria, and the ER. CORE induction extends replicative and chronological lifespan in budding yeast, highlighting its protective role against moderate proteotoxicity and its consequences such as the decline in respiration. Our findings accentuate that organelles do not function in isolation, but are integrated in a functional crosstalk, while also highlighting the importance of organelle communication in aging and age-related diseases.

Alterations in Mitochondrial and Endoplasmic Reticulum Signaling by p53 Mutants

Abstract: The p53 protein is probably the most important tumor suppressor, acting as a nuclear transcription factor primarily through the modulation of cell death. However, currently, it is well accepted that p53 can also exert important transcription-independent pro-cell death actions. Indeed, cytosolic localization of endogenous wild-type or trans-activation-deficient p53 is necessary and sufficient for the induction of apoptosis and autophagy. Here, we present the extra-nuclear activities of p53 associated with the mitochondria and the endoplasmic reticulum (ER), highlighting the activities of the p53 mutants on these compartments. These two intracellular organelles play crucial roles in the regulation of cell death, and it is now well established that they also represent sites where p53 can accumulate.

In Cellulo Mapping of Subcellular Localized Bilirubin

Abstract: Bilirubin (BR) is a de novo synthesized metabolite of human cells. However, subcellular localization of BR in the different organelles of human cells has been largely unknown. Here, utilizing UnaG as a genetically encoded fluorescent BR sensor, we report the existence of relatively BR-enriched and BR-depleted microspaces in various cellular organelles of live cells. Our studies indicate that (i) the cytoplasmic facing membrane of the endoplasmic reticulum (ER) and the nucleus are relatively BR-enriched spaces and (ii) mitochondrial intermembrane space and the ER lumen are relatively BR-depleted spaces. Thus, we demonstrate a relationship between such asymmetrical BR distribution in the ER membrane and the BR metabolic pathway. Furthermore, our results suggest plausible BR-transport and BR-regulating machineries in other cellular compartments, including the nucleus and mitochondria.

The Drosophila photoreceptor as a model system for studying signalling at membrane contact sites.

Abstract: Several recent studies have demonstrated the existence of membrane contact sites (MCS) between intracellular organelles in eukaryotic cells. Recent exciting studies have also demonstrated the existence of biomolecular interactions at these contact sites in mediating changes in the membrane composition of the cellular compartments. However, the role of such contact sites in regulating organelle function and physiological processes remains less clear. In this review we discuss the existence of a contact site between the plasma membrane (PM) and the endoplasmic reticulum (ER) inDrosophilaphotoreceptors. Further, we discuss the role of specific proteins present at this location in regulating phospholipid turnover and its impact in regulating a physiological process, namely phototransduction.

A Phosphatidic Acid (PA) conveyor system of continuous intracellular transport from cell membrane to nucleus maintains EGF receptor homeostasis

Abstract: // Karen M. Henkels 2 , Taylor E. Miller 2 , Ramya Ganesan 2 , Brandon A. Wilkins 2 , Kristen Fite 2 and Julian Gomez-Cambronero 1,2 1 Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women Hospital and Harvard Medical School, Boston, MA, USA 2 Department of Biochemistry and Molecular Biology, Wright State University Boonshoft School of Medicine, Dayton, Ohio, USA Correspondence to: Julian Gomez-Cambronero, email: // Keywords : mammalian cells, breast cancer, gene expression, cell signaling, phospholipids Received : April 14, 2016 Accepted : May 14, 2016 Published : May 31, 2016 Abstract The intracellular concentration of the mitogen phosphatidic acid (PA) must be maintained at low levels until the need arises for cell proliferation. How temporal and spatial trafficking of PA affects its target proteins in the different cellular compartments is not fully understood. We report that in cancer cells, PA cycles back and forth from the cellular membrane to the nucleus, affecting the function of epidermal growth factor (EGF), in a process that involves PPARα/LXRα signaling. Upon binding to its ligand, EGF receptor (EGFR)-initiated activation of phospholipase D (PLD) causes a spike in intracellular PA production that forms vesicles transporting EGFR from early endosomes (EEA1 marker) and prolonged internalization in late endosomes and Golgi (RCAS marker). Cells incubated with fluorescent-labeled PA (NBD-PA) show PA in “diffuse” locations throughout the cytoplasm, punctae (small, 0.5 μm) vesicles that co-localize with EGFR. We also report that PPARα/LXRα form heterodimers that bind to new Responsive Elements (RE) in the EGFR promoter. Nuclear PA enhances EGFR expression, a role compatible with the mitogenic ability of the phospholipid. Newly made EGFR is packaged into PA recycling vesicles (Rab11 marker) and transported back to the cytoplasm and plasma membrane. However, a PLD+PA combination impedes binding of PPARα/LXRα to the EGFR promoter. Thus, if PA levels inside the nucleus reach a certain threshold (>100 nM) PA outcompetes the nuclear receptors and transcription is inhibited. This new signaling function of PLD-PA targeting EGFR trafficking and biphasically modulating its transcription, could explain cell proliferation initiation and its maintenance in cancer cells.

Isolation of Microsomal Membrane Proteins from Arabidopsis thaliana.

Abstract: Cellular membranes define the boundaries between organelles and the cytosol or the extracellular environment, thus providing functional separation between subcellular compartments. In addition, membranes assist in a diverse range of cellular functions, including serving as signaling platforms, mediating transport of molecules, and facilitating trafficking of cargo between cellular compartments. Because membrane functionality is largely defined by protein composition, exploring the roles of membrane proteins is of interest to many researchers. This article focuses on the subcellular fractionation of microsomes, which are membrane-derived vesicles formed during cell lysis. In plants, microsomes mainly consist of the plasma membrane and membranes derived from the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, and tonoplast. The article describes the different steps involved in enriching for and solubilizing microsomal membrane proteins from Arabidopsis thaliana seedlings and cultured cells by differential centrifugation. Solubilized microsomal proteins can be used for subsequent immunoblot analysis, co-immunoprecipitation, or proteomic studies. © 2016 by John Wiley & Sons, Inc. Keywords: Arabidopsis thaliana; cellular fractionation; differential centrifugation; microsomes; solubilization of microsomal proteins

Prohibitin (PHB) roles in granulosa cell physiology

Abstract: Ovarian granulosa cells (GC) play an important role in the growth and development of the follicle in the process known as folliculogenesis. In the present review, we focus on recent developments in prohibitin (PHB) research in relation to GC physiological functions. PHB is a member of a highly conserved eukaryotic protein family containing the repressor of estrogen activity (REA)/stomatin/PHB/flotillin/HflK/C (SPFH) domain (also known as the PHB domain) found in diverse species from prokaryotes to eukaryotes. PHB is ubiquitously expressed in a circulating free form or is present in multiple cellular compartments including mitochondria, nucleus and plasma membrane. In mitochondria, PHB is anchored to the mitochondrial inner membrane and forms complexes with the ATPases associated with proteases having diverse cellular activities. PHB continuously shuttles between the mitochondria, cytosol and nucleus. In the nucleus, PHB interacts with various transcription factors and modulates transcriptional activity directly or through interactions with chromatin remodeling proteins. Many functions have been attributed to the mitochondrial and nuclear PHB complexes such as cellular differentiation, anti-proliferation, morphogenesis and maintenance of the functional integrity of the mitochondria. However, to date, the regulation of PHB expression patterns and GC physiological functions are not completely understood.

Development of redox-sensitive red fluorescent proteins for imaging redox dynamics in cellular compartments.

Abstract: We recently reported a redox-sensitive red fluorescent protein, rxRFP1, which is one of the first genetically encoded red-fluorescent probes for general redox states in living cells. As individual cellular compartments have different basal redox potentials, we hereby describe a group of rxRFP1 mutants, showing different midpoint redox potentials for detection of redox dynamics in various subcellular domains, such as mitochondria, the cell nucleus, and endoplasmic reticulum (ER). When these redox probes were expressed and subcellularly localized in human embryonic kidney (HEK) 293 T cells, they responded to membrane-permeable oxidants and reductants. In addition, a mitochondrially localized rxRFP1 mutant, Mito-rxRFP1.1, was used to detect mitochondrial oxidative stress induced by doxorubicin-a widely used cancer chemotherapy drug. Our work has expanded the fluorescent protein toolkit with new research tools for studying compartmentalized redox dynamics and oxidative stress under various pathophysiological conditions.

Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

Abstract: Golgi phosphoprotein 3 (GOLPH3) has been implicated in the development of carcinomas in many human tissues, and is currently considered a bona fide oncoprotein. Importantly, several tumor types show overexpression of GOLPH3, which is associated with tumor progress and poor prognosis. However, the underlying molecular mechanisms that connect GOLPH3 function with tumorigenicity are poorly understood. Experimental evidence shows that depletion of GOLPH3 abolishes transformation and proliferation of tumor cells in GOLPH3-overexpressing cell lines. Conversely, GOLPH3 overexpression drives transformation of primary cell lines and enhances mouse xenograft tumor growth in vivo. This evidence suggests that overexpression of GOLPH3 could result in distinct features of GOLPH3 in tumor cells compared to that of non-tumorigenic cells. GOLPH3 is a peripheral membrane protein mostly localized at the trans-Golgi network, and its association with Golgi membranes depends on binding to phosphatidylinositol-4-phosphate. GOLPH3 is also contained in a large cytosolic pool that rapidly exchanges with Golgi-associated pools. GOLPH3 has also been observed associated with vesicles and tubules arising from the Golgi, as well as other cellular compartments, and hence it has been implicated in several membrane trafficking events. Whether these and other features are typical to all different types of cells is unknown. Moreover, it remains undetermined how GOLPH3 acts as an oncoprotein at the Golgi. Therefore, to better understand the roles of GOLPH3 in cancer cells, we sought to compare some of its biochemical and cellular properties in the human breast cancer cell lines MCF7 and MDA-MB-231 with that of the non-tumorigenic breast human cell line MCF 10A. We found unexpected differences that support the notion that in different cancer cells, overexpression of GOLPH3 functions in diverse fashions, which may influence specific tumorigenic phenotypes.

The Fluorescence Methods to Study Neurotransmitters (Biomediators) in Plant Cells

Abstract: Fluorescence as a parameter for analysis of intracellular binding and localization of neurotransmitters also named biomediators (acetylcholine and biogenic amines such as catecholamines, serotonin, histamine) as well as their receptors in plant cells has been estimated basing on several world publications and own experiments of the author. The subjects of the consideration were 1. application of reagents forming fluorescent products (for catecholamines - glyoxylic acid, for histamine - formaldehyde or ortho-phthalic aldehyde) to show the presence and binding of the compounds in cells, 2. binding of their fluorescent agonists and antagonists with cell, 3. effects of the compounds, their agonists and antagonists on autofluorescence, 4. action of external factors on the accumulation of the compounds in cells. How neurotransmitters can bind to certain cellular compartments has been shown on intact individual cells (vegetative microspores, pollens, secretory cells) and isolated organelles. The staining with reagents on biogenic amines leads to the appearance blue or blue-green emission on the surface and excretions of intact cells as well in some DNA-containing organelles within cells. The difference between autofluorescence and histochemically induced fluorescence may reflect the occurrence and amount of biogenic amines in the cells studied. Ozone and salinity as external factors can regulate the emission of intact cells related to biogenic amines. After the treatment of isolated cellular organelles with glyoxylic acid blue emission with maximum 460–475 nm was seen in nuclei and chloroplasts (in control variants in this spectral region the noticeable emission was absent) and very expressive fluorescence (more than twenty times as compared to control) in the vacuoles. After exposure to ortho-phthalic aldehyde blue emission was more noticeable in nuclei and chloroplasts. Fluorescent agonists (muscarine, 6,7-diOHATN, BODIPY-dopamine or BODIPY-5HT) or antagonists (d-tubocurarine for acetylcholine, yohimbine for dopamine and norepinephrine, inmecarb for serotonin) of neurotransmitters that bound with animal receptors fluorescent in blue (460–480 nm) or blue-green (490–530 nm) and usually are bound with the plasmatic membrane of intact cells or with membrane of the isolated organelles studied. In some model cells autofluorescence (belonging to chlorophyll or not, for example secondary metabolites) may be stimulated by exogenous biogenic amines or their agonists and, on the contrary, be inhibited by certain antagonists. The fluorescence data may be applied for the testing in ecological monitoring, medicine and pharmacology.

Expression of genes involved in lipid droplet formation (BSCL2, SNAP23 and COPA) during porcine in vitro adipogenesis.

Abstract: Adipogenesis is a complex process of fat cells development driven by the expression of numerous genes. Differentiation of progenitor cells into mature adipocytes is accompanied by changes in cell shape, as a result of lipid accumulation. In the present study, expression of three genes involved in lipid droplet formation (SNAP23, BSCL2 and COPA) was evaluated during porcine adipogenesis. It was found that mRNA levels of BSCL2 and SNAP23, but not COPA, increased during differentiation. Redistribution of SNAP23 protein to different cellular compartments was observed when comparing undifferentiated mesenchymal stem cells and differentiated adipocytes. The BSCL2 protein was found to be highly specific to cells with accumulated lipids, while COPA protein coated the lipid droplets. Obtained results indicated that the studied genes may be considered as candidates for fatness traits in pigs. Moreover, this study has shown that the porcine in vitro adipogenesis system provides a useful tool for the characterisation of novel genes involved in adipose tissue accumulation.

Inorganic Polyphosphates Regulate Hexokinase Activity and Reactive Oxygen Species Generation in Mitochondria of Rhipicephalus (Boophilus) microplus Embryos

Abstract: The present study focused on the tick Rhipicephalus microplus, wich is an important cattle ectoparasite in South and Central Americas, Asia, Africa and Oceania. Polyphosphate (poly P) metabolism in eukaryotics cells shows specific peculiarieties for differnt cellular compartments and the physiological roles of poly Ps recently found arthorpd mitochondria remais obscure. Here, the possible involvement of poly P with reactive oxygen generation in mitochondria of Rhipicephalus microplus embryos was investigated. Mitochondria was isolated by differential centrifugation, mitochondria hexokinase and scavenger antioxidant enzimes, such as superoxide dismutase, catalase and glutathione reductase were assayed spectrophotometrically. The poly P inhibited hexokinase activity by up to 90% and, interestingly, the mitochondrial membrane exopolyphosphatase activity was stimulated by the hexokinase reaction product, glucose - 6 - phosphate. Poly P increased hydrogen peroxide generation in mitochondria in a situation where mitochondria hexokinase is also active. All of scavenger oxidant enzymes were stimulated by poly P. Altogether, our results indicate that inorganic polyphosphate and mitochondrial membrane exopolyphosphatase regulation can be correlated with the generation of reactive oxygen species in the mitochondria of R. microplus embryos.

Extracellular vesicles released from cells exposed to reactive oxygen species increase annexin A2 expression and survival of target cells exposed to the same conditions.

Abstract: Annexin A2 (AnxA2) is present in multiple cellular compartments and interacts with numerous ligands including calcium, proteins, cholesterol, negatively charged phospholipids and RNA. These interactions are tightly regulated by its post-translational modifications. The levels of AnxA2 and its Tyr23 phosphorylated form (pTyr23AnxA2) are increased in many cancers and the protein is involved in malignant cell transformation, metastasis and angiogenesis. Our previous studies of rat pheochromocytoma (PC12) cells showed that reactive oxygen species (ROS) induce rapid, simultaneous and transient dephosphorylation of nuclear AnxA2, most likely associating with PML bodies, while AnxA2 associated with F-actin at the cell cortex undergoes Tyr23 phosphorylation. The pTyr23AnxA2 in the periphery of the cells is incorporated into intraluminal vesicles of multivesicular endosomes and subsequently released to the extracellular space. We show here that extracellular vesicles (EVs) from cells exposed to ROS prime u...

Phospholipase C-η2 interacts with nuclear and cytoplasmic LIMK-1 during retinoic acid-stimulated neurite growth

Abstract: Neurite growth is central to the formation and differentiation of functional neurons, and recently, an essential role for phospholipase C-η2 (PLCη2) in neuritogenesis was revealed. Here we investigate the function of PLCη2 in neuritogenesis using Neuro2A cells, which upon stimulation with retinoic acid differentiate and form neurites. We first investigated the role of the PLCη2 calcium-binding EF-hand domain, a domain that is known to be required for PLCη2 activation. To do this, we quantified neurite outgrowth in Neuro2A cells, stably overexpressing wild-type PLCη2 and D256A (EF-hand) and H460Q (active site) PLCη2 mutants. Retinoic acid-induced neuritogenesis was highly dependent on PLCη2 activity, with the H460Q mutant exhibiting a strong dominant-negative effect. Expression of the D256A mutant had little effect on neurite growth relative to the control, suggesting that calcium-directed activation of PLCη2 is not essential to this process. We next investigated which cellular compartments contain endogenous PLCη2 by comparing immunoelectron microscopy signals over control and knockdown cell lines. When signals were analyzed to reveal specific labeling for PLCη2, it was found to be localized predominantly over the nucleus and cytosol. Furthermore in these compartments (and also in growing neurites), a proximity ligand assay revealed that PLCη2 specifically interacts with LIMK-1 in Neuro2A cells. Taken together, these data emphasize the importance of the PLCη2 EF-hand domain and articulation of PLCη2 with LIMK-1 in regulating neuritogenesis.

An In Vitro Assay to Study Targeting of Membrane Proteins to the Inner Nuclear Membrane.

Abstract: Newly synthesized membrane proteins are inserted into the endoplasmic reticulum (ER) from where they are constantly sorted to various cellular compartments. To analyze and visualize sorting of membrane proteins to the inner nuclear membrane (INM), we developed a trap-release system that uncouples membrane integration into the ER from transport. This assay allows the simultaneous release of a large pool of an INM-destined membrane protein from the ER and microscopy-based monitoring of targeting to the INM. The use of semi-permeabilized HeLa cells further enables the identification and characterization of essential requirements of the targeting process. This protocol provides a detailed description of reporter construction, in vitro reconstitution, and visualization of trafficking.

High-Pressure Freezing Electron Microscopy of Zebrafish Oocytes.

Abstract: Oogenesis is an essential cellular and developmental process to prepare the oocyte for propagation of a species after fertilization. Oocytes of oviparous animals are enormous cells endowed with many, big cellular compartments, which are interconnected through active intracellular transport. The dynamic transport pathways and the big organelles of the oocyte provide the opportunity to study cellular trafficking with outstanding resolution. Hence, oocytes were classically used to investigate cellular compartments. Though many novel regulators of vesicle trafficking have been discovered in yeast, tissue culture cells and invertebrates, recent forward genetic screens in invertebrate and vertebrate oocytes isolated novel control proteins specific to multicellular organisms. Zebrafish is a widely used vertebrate model to study cellular and developmental processes in an entire animal. The transparency of zebrafish embryos allows following cellular events during early development with in vivo imaging. Unfortunately, the active endocytosis of the oocyte also represents a drawback for imaging. The massive amounts of yolk globules prevent the penetration of light-beams and currently make in vivo microscopy a challenge. As a consequence, electron microscopy (EM) still provides the highest resolution to analyze the ultra-structural details of compartments and organelles and the mechanisms controlling many cellular pathways of the oocyte. Among different fixation approaches for EM, High Pressure Freezing (HPF) in combination with freeze substitution significantly improves the samples preservation closest to their natural status. Here, we describe the HPF with freeze substitution embedding method for analyzing cellular processes in zebrafish oocytes using electron microscopy.

Presence of an isoform of H+-pyrophosphatase located in the alveolar sacs of a scuticociliate parasite of turbot: physiological consequences.

Abstract: H+-pyrophosphatases (H+-PPases) are integral membrane proteins that couple pyrophosphate energy to an electrochemical gradient across biological membranes and promote the acidification of cellular compartments. Eukaryotic organisms, essentially plants and protozoan parasites, contain various types of H+-PPases associated with vacuoles, plasma membrane and acidic Ca+2 storage organelles called acidocalcisomes. We used Lysotracker Red DND-99 staining to identify two acidic cellular compartments in trophozoites of the marine scuticociliate parasite Philasterides dicentrarchi: the phagocytic vacuoles and the alveolar sacs. The membranes of these compartments also contain H+-PPase, which may promote acidification of these cell structures. We also demonstrated for the first time that the P. dicentrarchi H+-PPase has two isoforms: H+-PPase 1 and 2. Isoform 2, which is probably generated by splicing, is located in the membranes of the alveolar sacs and has an amino acid motif recognized by the H+-PPase-specific antibody PABHK. The amino acid sequences of different isolates of this ciliate are highly conserved. Gene and protein expression in this isoform are significantly regulated by variations in salinity, indicating a possible physiological role of this enzyme and the alveolar sacs in osmoregulation and salt tolerance in P. dicentrarchi.

Mechanisms Controlling the Activity of Localization Signal Sequences

Abstract: All eukaryotic cells contain numerous membrane-enclosed compartments, such as nucleus, endoplasmic reticulum, Golgi complex, mitochondria, endosomes, lysosomes, and peroxisomes. The function of each compartment is assured by a specific set of proteins. Since protein synthesis occurs primarily in the cytosol, accurate translocation, and protein trafficking from the cytosol to their final destinations is essential to maintain proper cellular function and activity. Indeed, it has been estimated that at least half of the intracellular proteins have to be transported to their functional destination. To be at the right place at the right time, proteins use specific localization signal sequences to interact with the molecular partners deputed to guarantee their correct localization. For many years the function of these localization signals was considered as a housekeeping function, but a number of studies have revealed that fine molecular mechanisms may control the activity of signal sequences. Interestingly, many proteins do not have a single subcellular localization but are present in more cellular compartments. For some of them, multiple localizations are due to the generation of different mRNA forms by alternative splicing. However, most of the time, proteins can occupy multiple cellular locations without any change in the amino acid sequence, indicating the existence of regulatory mechanisms based on post-translational events. These events include post-translational modifications and signal exposing/masking switching mechanisms that regulate the interaction between signals and transport proteins. A number of evidences showing how the function of signal sequences can be controlled is illustrated and discussed in this article.

Prohibitin Deficiency Causes Opposing Lipid Metabolism between 3T3-L1 Adipocytes and Clone 9 Hepatocytes

Abstract: Prohibitin (PHB) is a highly conserved protein in eukaryotic cells that are present in multiple cellular compartments and has potential roles as a tumor suppressor, an anti-proliferative protein, a regulator of cell-cycle progression and in apoptosis. In the present study, we generated PHB-deficient 3T3-L1 adipocytes and Clone 9 (C9) hepatocytes by oligonucleotide siRNA and investigated whether PHB affect lipid metabolism. It was revealed that PHB deficiency caused opposing lipid metabolism between the two cell models. PHB deficiency increased expression of adipogenic, lipogenic, and other lipid metabolic proteins in 3T3-L1 adipocytes, whereas significantly decreased the levels of those proteins in C9 cells. Collectively, PHB deficiency promoted lipid metabolism in 3T3-L1 adipocytes while it aggravated lipid metabolism in C9 hepatocytes.

Fluorescence of neurotransmitters and their reception in plant cell

Abstract: The use of fluorescent reagents for the histochemical detection of catecholamines or histamine, as well as luminescent antagonists of the intracellular neurotransmitters revealed that they can bind to certain cellular compartments. After the treatment with glyoxylic acid (a reagent used for the detection of catecholamines), blue fluorescence with maximum at 460–475 nm was visualized in nuclei and chloroplasts (in control preparations no emission in this spectral region was recorded), as well as an intense fluorescence, exceeding the control level, in the vacuoles. After the exposure to ortho-phthalic aldehyde (a reagent used for the histamine detection), blue emission was more noticeable in nuclei and chloroplasts, which correlates with previously observed effects on intact cells, such as pollen and vegetative microspores. A comparison of the intensities of the biogenic amine-related emission in various organelles showed that the greatest emission was in vacuoles and the weakest, in chloroplasts. Thus, on the surface, and possibly within the organelles, fluorescence could demonstrate the presence of biogenic amines. Antagonists of the neurotransmitters (dtubocurarine for acetylcholine; yohimbine for dopamine; norepinephrine and inmecarb for serotonin), which fluoresce in the blue and blue-green region and usually bind with the plasmalemma of intact cells, also interacted with the membranes of the organelles studied. Fluorescence intensity depended on the object; most prominent it was for yohimbine in the outer membrane of the nucleus, vacuoles, and chloroplasts.