The hemicholinium-3 sensitive high affinity choline transporter is internalized by clathrin-mediated endocytosis and is present in endosomes and synaptic vesicles

Synthesis of acetylcholine depends on the plasma membrane uptake of choline by a high affinity choline transporter (CHT1). Choline uptake is regulated by nerve impulses and trafficking of an intracellular pool of CHT1 to the plasma membrane may be important for this regulation. We have generated a hemagglutinin (HA) epitope tagged CHT1 to investigate the organelles involved with intracellular trafficking of this protein. Expression of CHT1-HA in HEK 293 cells establishes Na+-dependent, hemicholinium-3 sensitive high-affinity choline transport activity. Confocal microscopy reveals that CHT1-HA is found predominantly in intracellular organelles in three different cell lines. Importantly, CHT1-HA seems to be continuously cycling between the plasma membrane and endocytic organelles via a constitutive clathrin-mediated endocytic pathway. In a neuronal cell line, CHT1-HA colocalizes with the early endocytic marker green fluorescent protein (GFP)-Rab 5 and with two markers of synaptic-like vesicles, VAMP-myc and GFP-VAChT, suggesting that in cultured cells CHT1 is present mainly in organelles of endocytic origin. Subcellular fractionation and immunoisolation of organelles from rat brain indicate that CHT1 is present in synaptic vesicles. We propose that intracellular CHT1 can be recruited during stimulation to increase choline uptake in nerve terminals.     

Neural cell adhesion molecule promotes accumulation of TGN organelles at sites of neuron-to-neuron contacts

Transformation of a contact between axon and dendrite into a synapse is accompanied by accumulation of the synaptic machinery at this site, being delivered in intracellular organelles mainly of TGN origin. Here, we report that in cultured hippocampal neurons, TGN organelles are linked via spectrin to clusters of the neural cell adhesion molecule (NCAM) in the plasma membrane. These complexes are translocated along neurites and trapped at sites of initial neurite-to-neurite contacts within several minutes after initial contact formation. The accumulation of TGN organelles at contacts with NCAM-deficient neurons is reduced when compared with wild-type cells, suggesting that NCAM mediates the anchoring of intracellular organelles in nascent synapses.     

Coherent phase microscopy in cell biology: visualization of metabolic states

Visualization of functional properties of individual cells and intracellular organelles still remains an experimental challenge in cell biology. The coherent phase microscopy (CPM) provides a convenient and non-invasive tool for imaging cells and intracellular organelles. In this work, we report results of statistical analysis of CPM images of cyanobacterial cells (Synechocystis sp. PCC 6803) and spores (Bacillus licheniformis). It has been shown that CPM images of cyanobacterial cells and spores are sensitive to variations of their metabolic states. We found a correlation between one of optical parameters of the CPM image ('phase thicknesses' Deltah) and cell energization. It was demonstrated that the phase thickness Deltah decreased after cell treatment with the uncoupler CCCP or inhibitors of electron transport (KCN or DCMU). Statistical analysis of distributions of parameter Deltah and cell diameter d demonstrated that a decrease in the phase thickness Deltah could not be attributed entirely to a decrease in geometrical sizes of cells. This finding demonstrates that the CPM technique may be a convenient tool for fast and non-invasive diagnosis of metabolic states of individual cells and intracellular organelles.     

Timing cell-fate determination during asymmetric cell divisions

From invertebrates to mammals, cell-cycle progression during an asymmetric cell division is accompanied by precisely timed redistribution of cell-fate determinants so that they segregate asymmetrically to enable the two daughter cells to choose different fates. Interestingly, studies on how cell fates are specified in such divisions reveal that the same fate determinants can be reiteratively used to specify a variety of cell types through multiple rounds of cell divisions or to exert seemingly contradictory effects on cell proliferation and differentiation. Here I summarize the molecular mechanisms governing asymmetric cell division and review recent findings pointing to a novel mechanism for coupling intracellular signaling and cell-cycle progression. This mechanism uses changes in the morphology, subcellular distribution, and molecular composition of cellular organelles like the Golgi apparatus and centrosomes, which not only accompany the progression of cell cycle to activate but also temporally constrain the activity of fate determinants during asymmetric cell divisions.     

Intracellular viscosity: Methods of measurement and role in metabolism

This review is devoted to the study of intracellular viscosity. Methods of intracellular viscosity measurement in cell populations and single cells are characterized and critically evaluated. Examples of intracellular viscosity assessment in a number of various cell types and intracellular organelles are presented. The main results of the in vitro and in vivo studies on the role of viscosity in metabolism are discussed.     

Invasion factors are coupled to key signalling events leading to the establishment of infection in apicomplexan parasites

Invasion of host cells by apicomplexan parasites is initiated when specialized secretory organelles called micronemes discharge protein complexes onto the parasite surface in response to a rise in parasite intracellular calcium levels. The microneme proteins establish interactions with host cell receptors, engaging the parasite with the host cell surface, and signal for the immediate exocytosis of another set of secretory organelles named the rhoptries. The rhoptry proteins reprogram the invaded host cell and participate in the formation of the parasitophorous vacuole in which the intracellular parasite resides and replicates. Disengagement of the invading parasite from the host cell receptors involves the action of at least one parasite plasma membrane rhomboid protease, which is concomitantly implicated in a checkpoint that signals the parasite to switch from an invasive to a replicative mode.     

Intracellular Selection, Conversion Bias, and the Expected Substitution Rate of Organelle Genes

A key step in the substitution of a new organelle mutant throughout a population is the generation of germ-line cells homoplasmic for that mutant. Given that each cell typically contains multiple copies of organelles, each of which in turn contains multiple copies of the organelle genome, processes akin to drift and selection in a population are responsible for producing homoplasmic cells. This paper examines the expected substitution rate of new mutants by obtaining the probability that a new mutant is fixed throughout a cell, allowing for arbitrary rates of genome turnover within an organelle and organelle turnover within the cell, as well as (possibly biased) gene conversion and genetic differences in genome and/or organelle replication rates. Analysis is based on a variation of Moran's model for drift in a haploid population. One interesting result is that if the rate of unbiased conversion is sufficiently strong, it creates enough intracellular drift to overcome even strong differences in the replication rates of wild-type and mutant genomes. Thus, organelles with very high conversion rates are more resistant to intracellular selection based on differences in genome replication and/or degradation rates. It is found that the amount of genetic exchange between organelles within the cell greatly influences the probability of fixation. In the absence of exchange, biased gene conversion and/or differences in genome replication rates do not influence the probability of fixation beyond the initial fixation within a single organelle. With exchange, both these processes influence the probability of fixation throughout the entire cell. Generally speaking, exchange between organelles accentuates the effects of directional intracellular forces.(ABSTRACT TRUNCATED AT 250 WORDS)     

Internalization of 125I-human choriogonadotropin in bovine luteal slices. A biochemical study

Various intracellular organelles as well as outer cell membranes of bovine corpora lutea intrinsically contain gonadotropin receptors (Rao et al., J biol chem 256 (1981) 2628 [5]). In order to investigate whether exogenously added human choriogonadotropin (hCG) can internalize and bind to the intracellular sites, bovine luteal slices that had been carefully checked with respect to structural and functional integrity were incubated with 0.1 nM 125I-hCG. Following incubation, specific radioactivity was found to be associated with various intracellular organelles, but not with cytosol. The order of radioactivity uptake by subcellular organelles following a 2-h incubation was: Golgi medium greater than Golgi heavy greater than Golgi light greater than plasma membranes = rough endoplasmic reticulum greater than mitochondria-lysosomes- greater than nuclei. The 5'-nucleotidase activity and electron microscopic examination of the fractions revealed that the presence of radioactivity in the intracellular organelles cannot be attributed solely to plasma membrane contamination. The internalization and intracellular binding of 125I-hCG was time and temperature-dependent. Only excess unlabeled hCG and hLH (but not hCG subunits, FSH and PRL) competed with 125I-hCG for internalization in luteal slices. Very little or no 125I-hCG added was internalized in liver or kidney slices; luteal, liver and kidney slices accumulated neither 125I-BSA nor 125I. The radioactivity eluted from various luteal subcellular organelles was able to rebind to fresh corresponding organelles and came off Sepharose 6B columns in a position corresponding to native 125I-hCG. The gel filtration profile of detergent-solubilized radioactivity revealed that 125I-hCG was macromolecular bound. The degraded and altered 125I-hCG was found in the incubation media.     

A novel intracellular compartment with unusual secretory properties in human neutrophils

Human neutrophils contain a novel intracellular compartment that is distinct from the previously characterized azurophil and specific granules. This compartment is distinguished by the presence of cytochemically detectable alkaline phosphatase activity. The alkaline phosphatase-containing compartments are short rod-shaped organelles that rapidly undergo a dramatic reorganization upon cell stimulation with either a chemoattractant or an active phorbol ester. Biochemical analysis shows that in unstimulated neutrophils the majority of the alkaline phosphatase activity is intracellular, but after stimulation essentially all of this activity becomes associated with the cell surface. The exocytotic pathway is unusual in that these small organelles fuse to form elongated tubular structures before their association with the plasmalemma.     

Seeing is believing: recent trends in the measurement of Ca2+ in subcellular domains and intracellular organelles

The role of Ca2+ in the regulation of the cell cycle has been investigated mostly in studies assessing global cytosolic free Ca2+. Recent studies, however, have used unique techniques to assess Ca2+ in subcellular organelles, such as mitochondria, and in discrete regions of the cytoplasm. These studies have used advanced fluorescence digital imaging techniques and Ca2+-sensitive fluorescence probes, and/or targeting of Ca2+-sensitive proteins to intracellular organelles. The present review describes the results of some of these studies and the techniques used. The novel techniques used to measure Ca2+ in microdomains and intracellular organelles are likely to be of great use in future investigations assessing Ca2+ homeostasis during the cell cycle.     

山溪鲵卵黄发生的显微与超微结构

用光镜和透射电镜观察了山溪鲵（Batrachuperus pinchonii)不同发育时期卵母细胞的显微与超微结构,特别注意了与卵黄发生相关的细胞器变化。结果表明,类核周体为线粒体、高尔基体、内质网等膜性细胞器聚集的场所;线粒体自身也可演变成卵黄前颗粒,参与构成卵黄小板的成分;吞饮泡和髓样小体是卵母细胞利用外源卵黄物质的中间媒介。经过与其他动物卵黄发生过程相比较,认为非哺乳脊椎动物卵黄发生是卵母细胞在多种细胞器参与下整体活动的结果,不是经由单一的模式或途径形成,因此呈现发生上的多元化;不同物种在卵黄发生中分别采取与各自相适应的模式或途径。     

Functional study of intracellular P-gp- and MRP1-mediated pumping of free cytosolic pirarubicin into acidic organelles in intrinsic resistant SiHa cells

We sought to determine the efficiency of the intracellular functional P-gp- and MRP1-mediated pumping of THP into acidic organelles in SiHa cells and etoposide-resistant SiHa/VP16 cells. The expression of both MDR1 and MRP1 genes of SiHa and SiHa/VP16 cells was clearly shown by using RT-PCR. The functional studies of both intracellular functional P-gp- and MRP1-mediated pumping were performed by using THP in a conventional spectrofluorometer, and they demonstrated that SiHa and SiHa/VP16 cells are good models to illustrate the functional role of intracellular P-gp and MRP1 in the transport of free cytosolic drug into acidic organelles. The functional P-gp and MRP1 proteins were identified both on plasma membranes and on intracellular vesicle membranes. Within the limit of experimental error, similar efficiencies in THP transport were observed in the two proteins at both locations in SiHa and SiHa/VP16 cells. The P-gp- and MRP1-mediated pump coefficient (k v a), Michealis-Menten's constant (K V m), and maximal pumping rate (V V max) values of those located on vesicular membranes were 1.87 +/- 0.30 pL x cell-1 x s-1, 1.63 +/- 0.21 microM, and 4.95 +/- 0.45 nM x s-1, respectively. Drug retention inside acidic organelles (C mon V) of SiHa cells was significantly higher than that of SiHa/VP16 cells, perhaps a consequence of slower movement of recycling endosomes and (or) lysosomes to the cell membrane of SiHa cells, leading to distended organelles and cell death. Our results suggest that intracellular P-gp and MRP1 proteins play an important role in the transport of free drug from cytosol to cytoplasmic acidic organelles.     

Variable subcellular localization of glycosphingolipids

Although most glycosphingolipids (GSLs) are thought to be locatedin the outer leaflet of the plasma membrane, recent evidenceindicates that GSLs are also associated with intracellular organelles.We now report that the subcellular localization of GSLs variesdepending on the GSL structure and cell type. GSL localizationwas determined by indirect immunofluorescence microscopy offixed permeabilized cells. A single GSL exhibited variable subcellularlocalization in different cells. For example, antibody to GalCeris localized primarily to the plasma membrane of HaCaT II-3keratinocytes, but to intracellular organelies in other epithelialcells. GalCer is localized to small vesicles and tubulovesicularstructures in MDCK cells, and to the surface of phase-denselipid droplets in HepG2 hepatoma cells. Furthermore, withina single cell type, individual GSLs were found to exhibit differentpatterns of subcellular localization. In HepG2 cells, LacCerwas associated with small vesicles, which differed from thephase-dense vesicles stained by anti-GalCer, and Gb4Cer wasassociated with the intermediate filaments of the cytoskeleton.Both anti-GalCer and monoclonal antibody A2B5, which binds polysialogangliosides,localized to mitochondria. The distinct subcellular localizationpatterns of GSLs raise interesting questions about their functionsin different organelles. Together with published data on theenrichment of GSLs in specific organelles and in apical plasmamembrane, these findings indicate the existence of specificsorting mechanisms that regulate the intracellular transportand localization of GSLs. cytoskeleton glycosphingolipid intracellular organelles mitochondria subcellular localization     

The F(1)F(0) ATP synthase and mitochondrial respiratory chain complexes are present on the plasma membrane of an osteosarcoma cell line: An immunocytochemical study

F(1)F(0) ATP synthase is ectopically expressed on the surface of several cell types, including endothelium and cancer cells. This study uses immunocytochemical detection methods via highly specific monoclonal antibodies to explore the possibility of plasma membrane localization of other mitochondrial proteins using an osteosarcoma cell line in which the location of the mitochondrial reticulum can be clearly traced by green fluorescent protein tagging of the organelle. We found that subunits of three of the four respiratory chain complexes were present on the surface of these cells. Additionally, we show for the first time that F(0) subunits d and OSCP of the ATP synthase are ectopically expressed. In all cases the OXPHOS proteins show a punctate distribution, consistent with data from proteome analysis of isolated lipid rafts that place the various mitochondrial proteins in plasma membrane microdomains. We also examined the cell surface for marker membrane proteins from several other intracellular organelles including ER, golgi and nuclear envelope. They were not found on the surface of the osteosarcoma cells. We conclude that mitochondrial membrane proteins are ectopically expressed, but not proteins from other cellular organelles. A specific mechanism by which the mitochondrion and plasma membrane fuse to deliver organellar proteins is suggested.     

Caspase activation in the terminal differentiation of human epidermal keratinocytes

The epidermis is a multilayered squamous epithelium in which dividing basal cells withdraw from the cell cycle and progressively differentiate as they are displaced toward the skin surface. Eventually, the cells lose their nucleus and other organelles to become flattened squames, which are finally shed from the surface as bags of cross-linked keratin filaments enclosed in a cornified envelope [1]. Although keratinocytes can undergo apoptosis when stimulated by a variety of agents [2], it is not known whether their normal differentiation programme uses any components of the apoptotic biochemical machinery to produce the cornified cell. Differentiating keratinocytes have been reported to share some features with apoptotic cells, such as DNA fragmentation, but these features have not been seen consistently [3]. Apoptosis involves an intracellular proteolytic cascade, mainly mediated by members of the caspase family of cysteine proteases, which cleave one another and various key intracellular target proteins to kill the cell neatly and quickly [4]. Here, we show for the first time that caspases are activated during normal human keratinocyte differentiation and that this activation is apparently required for the normal loss of the nucleus.     

Localization of the pathway of the penicillin biosynthesis in Penicillium chrysogenum.

The localization of the enzymes involved in penicillin biosynthesis in Penicillium chrysogenum hyphae has been studied by immunological detection methods in combination with electron microscopy and cell fractionation. The results suggest a complicated pathway involving different intracellular locations. The enzyme delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase was found to be associated with membranes or small organelles. The next enzyme isopenicillin N-synthetase appeared to be a cytosolic enzyme. The enzyme which is involved in the last step of penicillin biosynthesis, acyltransferase, was located in organelles with a diameter of 200-800 nm. These organelles, most probably, are microbodies. A positive correlation was found between the capacity for penicillin production and the number of organelles per cell when comparing different P. chrysogenum strains.     

Changes in Leydig cell ultrastructure and function during pubertal development in the boar

Changes in the ultrastructure of Leydig cells during pubertal development in the boar (40 to 250 days of age) were assessed using quantitative morphometric procedures, and the results were compared to the in vitro steroid-producing capacity and gonadotropin sensitivity of testicular tissue obtained from the same boars. Volume of individual Leydig cells declined through 100 days of age, increased rapidly to a peak at 130-160 days (i.e., puberty), and then declined to intermediate levels by 220-250 days of age. The pattern of change in the number of intracellular organelles per Leydig cell was very similar to the change that occurred in Leydig cell volume. Changes in the total intracellular volume occupied by each type of organelle were highly correlated with changes in Leydig cell volume (r = 0.40-0.99, p less than 0.01), and this was particularly true for the nucleus (r = 0.63), mitochondria (r = 0.88), smooth endoplasmic reticulum (SER; r = 0.97), and total cytoplasm (r = 0.99) of the boar Leydig cell. In vitro production of testosterone and estradiol, expressed per Leydig cell, also peaked at 130-160 days, and was highly correlated to average Leydig cell volume, volume of SER, and number and total volume of mitochondria (r = 0.63-0.84; p less than 0.01). Observations in the present study indicated that onset of puberty in boars coincides with a dramatic increase in average Leydig cell size and SER volume per Leydig cell, accompanied by an increase in number of other intracellular organelles, including mitochondria, lysosomes, and lipid droplets, and a peak in the steroid-producing capacity per Leydig cell. A decline in Leydig cell size, intracellular organelles, and sensitivity to gonadotropin stimulation occurred postpubertally.     

Coherent phase microscopy in cell biology: visualization of metabolic states

Visualization of functional properties of individual cells and intracellular organelles still remains an experimental challenge in cell biology. The coherent phase microscopy (CPM) provides a convenient and non-invasive tool for imaging cells and intracellular organelles. In this work, we report results of statistical analysis of CPM images of cyanobacterial cells (Synechocystis sp. PCC 6803) and spores (Bacillus licheniformis). It has been shown that CPM images of cyanobacterial cells and spores are sensitive to variations of their metabolic states. We found a correlation between one of optical parameters of the CPM image (‘phase thicknesses’ Δh) and cell energization. It was demonstrated that the phase thickness Δh decreased after cell treatment with the uncoupler CCCP or inhibitors of electron transport (KCN or DCMU). Statistical analysis of distributions of parameter Δh and cell diameter d demonstrated that a decrease in the phase thickness Δh could not be attributed entirely to a decrease in geometrical sizes of cells. This finding demonstrates that the CPM technique may be a convenient tool for fast and non-invasive diagnosis of metabolic states of individual cells and intracellular organelles.     

Evolutionary genomics: is Buchnera a bacterium or an organelle?

The first genome sequence of an intracellular bacterial symbiont of a eukaryotic cell has been determined. The Buchnera genome shares features with the genomes of both intracellular pathogenic bacteria and eukaryotic organelles, and it may represent an intermediate between the two.     

Intracellular trafficking of pigeon beta-very low density lipoprotein and low density lipoprotein at low and high concentrations in pigeon macrophages

Foam cell formation occurs in vitro at lipoprotein concentrations above 50 microgram/ml in pigeon macrophages. Hypothetically, intracellular trafficking of lipoproteins at higher concentrations may differ from uptake of lipoproteins associated with low concentrations, revealing a separate atherogenic endocytic pathway. Macrophage intracellular trafficking of pigeon beta-very low density lipoprotein (beta-VLDL) and low density lipoprotein (LDL) at low concentrations (12 microgram/ml) near the saturation of high affinity binding sites and high lipoprotein concentrations (50-150 microgram/ml) used to induce foam cell formation were examined. Pigeon beta-VLDL and LDL, differentially labeled with colloidal gold, were added simultaneously to contrast trafficking of beta-VLDL, which causes in vitro foam cell formation, with LDL, which does not. The binding of lipoproteins to cell surface structures, distribution of lipoproteins in endocytic organelles, and the extent of colabeling in the endocytic organelles were determined by thin-section transmission electron microscopy.At low concentrations, the intracellular trafficking of pigeon LDL and beta-VLDL was identical. At high concentrations, LDL was removed more rapidly from the plasma membrane and reached lysosomes more quickly than beta-VLDL. No separate endocytic route was present at high concentrations of beta-VLDL; rather, an increased residence on the plasma membrane, association with nonmicrovillar portions of the plasma membrane, and slower trafficking in organelles of coated-pit endocytosis reflected a more atherogenic trafficking pattern.