Modeling Fusion/Fission‐Dependent Intracellular Transport of Fluid Phase Markers

A fundamental feature of eukaryotic cells is the presence of distinct membrane‐bound compartments having unique protein and lipid composition. These compartments are interconnected by active trafficking mechanisms that must direct macromolecules to defined locations, and at the same time maintain the protein and lipid composition of each organelle. It is well accepted that Rab proteins play a central role in intracellular transport regulating the recognition, fusion and fission of organelles. However, how the transport is achieved is not completely understood. We propose a model whereby a soluble component in the luminal compartment is transported along different Rab‐containing organelles that interact according to the following simple principles: (i) only organelles with the same or compatible Rab membrane domains can fuse; (ii) after fusion, an asymmetric fission occurs producing a tubule and a round‐shaped vesicle; and (iii) Rab membrane domains distribute asymmetrically between the two resulting organelles. When this model was tested in a simulation, efficient unidirectional transport was observed, while the compartment identity was preserved. All three principles were absolutely necessary for transport. The model is compatible with Rab association/dissociation dynamics and with Rab conversion. In simulations mimicking a simplified endocytic pathway, soluble and membrane‐associated markers were efficiently transported preserving the identity of the interacting compartments.     

Tracing of intracellular zinc(II) fluorescence flux to monitor cell apoptosis by using FluoZin‐3AM

Changes in the free zinc(II) concentration are closely related to cell proliferation and apoptosis, especially during the early apoptotic process. In the present paper, we demonstrated that zinc(II) probe FluoZin‐3AM owns sensitive properties to distinguish different stages of apoptotic cell (induced by an anticancer agent, etoposide) according to trace intracellular zinc(II) fluorescence flux. When apoptosis in HeLa or K562 cells was artificially induced, FluoZin‐3AM selectively and strongly stained apoptotic cells only at early and middle stages, which was attributed to significantly increased free zinc(II) flux during these stages. This conclusion was further verified by comparing it with the conventional apoptosis detector probe Annexin‐V‐FITC and PI. Furthermore, FluoZin‐3AM was found cell permeable to detect the intracellular zinc(II) fluorescence enhancement to threefolds within 120 s with low cytotoxicity when zinc(II) was incorporated into the cell by zinc(II) ionophore pyrithione. All the above implied that monitoring intracellular zinc fluorescence flux was an effective method to distinguish cell apoptosis from necrosis, and FluoZin‐3AM was found to be a suitable probe acting alone to fulfill the work. Copyright © 2009 John Wiley & Sons, Ltd.     

The regulation of endosome-to-Golgi retrograde transport by tethers and scaffolds

Retrograde transport between endosomes and the trans-Golgi network (TGN) is essential for the recycling of membrane proteins which are involved in a range of biological processes. A variety of machinery components have been identified at the TGN which regulate endosome-to-TGN transport, including small G proteins, SNAREs, tethering factors and scaffold molecules. The challenge is to understand how these regulatory components orchestrate not only the specific docking and fusion of retrograde membrane carriers with the TGN, but also maintain the integrity of this highly dynamic compartment to ensure efficient delivery and export of cargo. Here we review recent advances in defining the form and function of tethers and scaffolds in the regulation of the retrograde transport pathways.     

Rab12 Localizes to Shiga Toxin‐Induced Plasma Membrane Invaginations and Controls Toxin Transport

Several exogenous and endogenous cargo proteins are internalized independently of clathrin, including the bacterial Shiga toxin. The mechanisms underlying early steps of clathrin‐independent uptake remain largely unknown. In this study, we have designed a protocol to obtain gradient fractions containing Shiga toxin internalization intermediates. Using stable isotope labeling with amino acids in cell culture (SILAC) and quantitative mass spectrometry, Rab12 was found in association with these very early uptake carriers. The localization of the GTPase on Shiga toxin‐induced plasma membrane invaginations was shown by fluorescence microscopy in cells transfected with GFP‐Rab12. Furthermore, using a quantitative biochemical assay, it was found that the amount of receptor‐binding B‐subunit of Shiga toxin reaching the trans‐Golgi/TGN membranes was decreased in Rab12‐depleted cells, and that cells were partially protected against intoxication by Shiga‐like toxin 1 under these conditions. These findings demonstrate the functional importance of Rab12 for retrograde toxin trafficking. Among several other intracellular transport pathways, only the steady‐state localizations of TGN46 and cation‐independent mannose‐6‐phosphate receptor were affected. These data thus strongly suggest that Rab12 functions in the retrograde transport route.      

Nanoporous Polytetrafluoroethylene/Silica Composite Separator as a High‐Performance All‐Vanadium Redox Flow Battery Membrane

A novel low‐cost nanoporous polytetrafluoroethylene (PTFE)/silica composite separator has been prepared and evaluated for its use in an all‐vanadium redox flow battery (VRB). The separator consists of silica particles enmeshed in a PTFE fibril matrix. It possesses unique nanoporous structures with an average pore size of 38 nm and a porosity of 48%. These pores function as the ion transport channels during redox flow battery operation. This separator provides excellent electrochemical performance in the mixed‐acid VRB system. The VRB using this separator delivers impressive energy efficiency, rate capability, and temperature tolerance. In additon, the flow cell using the novel separator also demonstrates an exceptional capacity retention capability over extended cycling, thus offering excellent stability for long‐term operation. The characteristics of low cost, excellent electrochemical performance and proven chemical stability afford the PTFE/silica nanoporous separator great potential as a substitute for the Nafion membrane used in VRB applications.     

Segregation of the Qb‐SNAREs GS27 and GS28 into Golgi Vesicles Regulates Intra‐Golgi Transport

The Golgi apparatus is the main glycosylation and sorting station along the secretory pathway. Its structure includes the Golgi vesicles, which are depleted of anterograde cargo, and also of at least some Golgi‐resident proteins. The role of Golgi vesicles remains unclear. Here, we show that Golgi vesicles are enriched in the Qb‐SNAREs GS27 (membrin) and GS28 (GOS‐28), and depleted of nucleotide sugar transporters. A block of intra‐Golgi transport leads to accumulation of Golgi vesicles and partitioning of GS27 and GS28 into these vesicles. Conversely, active intra‐Golgi transport induces fusion of these vesicles with the Golgi cisternae, delivering GS27 and GS28 to these cisternae. In an in vitro assay based on a donor compartment that lacks UDP‐galactose translocase (a sugar transporter), the segregation of Golgi vesicles from isolated Golgi membranes inhibits intra‐Golgi transport; re‐addition of isolated Golgi vesicles devoid of UDP‐galactose translocase obtained from normal cells restores intra‐Golgi transport. We conclude that this activity is due to the presence of GS27 and GS28 in the Golgi vesicles, rather than the sugar transporter. Furthermore, there is an inverse correlation between the number of Golgi vesicles and the number of inter‐cisternal connections under different experimental conditions. Finally, a rapid block of the formation of vesicles via COPI through degradation of ϵCOP accelerates the cis‐to‐trans delivery of VSVG. These data suggest that Golgi vesicles, presumably with COPI, serve to inhibit intra‐Golgi transport by the extraction of GS27 and GS28 from the Golgi cisternae, which blocks the formation of inter‐cisternal connections .     

Improved Phycocatalysis of Carotene Production by Flow Cytometry and Cell Sorting

Using flow cytometry, strains of Dunaliella salina that produce large amounts of beta-carotene were sorted from mixed populations. When grown in large outdoor bioreactors, one carotene-producing strain thrived and was phenotypically stable throughout the three year trial. The use of this high-yield strain in industrial-scale processes doubled beta-carotene productivity. Flow cytometry provided a rapid and precise method to identify and isolate algal strains of potential commercial value.     

Improved Phycocatalysis of Carotene Production by Flow Cytometry and Cell Sorting

Using flow cytometry, strains of Dunaliella salina that produce large amounts of beta-carotene were sorted from mixed populations. When grown in large outdoor bioreactors, one carotene-producing strain thrived and was phenotypically stable throughout the three year trial. The use of this high-yield strain in industrial-scale processes doubled beta-carotene productivity. Flow cytometry provided a rapid and precise method to identify and isolate algal strains of potential commercial value.     

Trafficking of Acetyl‐C16‐Ceramide‐NBD with Long‐Term Stability and No Cytotoxicity into the Golgi Complex

The Golgi complex plays a prominent role in the modification and sorting of lipids and proteins, and is a highly dynamic organelle that is dispersed and rearranged before and after mitosis. Several reagents including 4‐nitrobenzo‐2‐oxa‐1,3‐diazole‐labeled C6‐ceramide (NBD‐C6‐ceramide, a ceramide having an NBD‐bound C6‐N‐acyl chain) and Golgi‐specific proteins that emit fluorescence are used as Golgi markers. In the present study, we synthesized a new ceramide analog, acetyl‐C16‐ceramide‐NBD (a ceramide having an acetylated C‐1 hydroxyl group, C16‐N‐acyl chain, and NBD‐bound C15‐sphingosine), and showed that it preferentially accumulated in the Golgi complex without cytotoxicity for over 24 h. Pathways for cellular uptake and interorganelle trafficking of acetyl‐C16‐ceramide‐NBD were investigated. Acetyl‐C16‐ceramide‐NBD was transported to the Golgi complex via ceramide transport proteins. In contrast to NBD‐C6‐ceramide, acetyl‐C16‐ceramide‐NBD was resistant to ceramide metabolic enzymes such as sphingomyelin synthase and glucosylceramide synthase. Because of its weaker cytotoxicity and resistance to ceramide metabolic enzymes, the localization of the Golgi complex could be observed in acetyl‐C16‐ceramide‐NBD‐labeled cells before and after mitosis.      

抑癌基因PTEN在鼻咽癌细胞株中表达的研究

目的:检测人鼻咽癌细胞株中PTEN表达情况,探讨鼻咽癌细胞中PTEN表达与细胞分化程度的关系。方法:进行细胞株的培养,采用流式细胞仪和共聚焦显微镜检测方法对细胞中PTEN的表达进行定位定量检测。结果:两种细胞株均有PTEN的表达,表达强度和分布与分化程度有关,分化越好,表达越高,流式细胞仪检测PTEN在细胞株中表达强弱顺序为CNE1>CNE2,阳性表达细胞数CNE1>CNE2差异有统计学意义(P<0.01);激光共聚焦扫描显微镜检测PTEN主要表达在细胞核和细胞浆,分布与分化程度有关,细胞核表达强度CNE1相似文献   

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) α and β loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders^1-4. T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the αβTCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the αβTCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC⁵.Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events⁶. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes^7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCRα chain at the DN stage, resulting in premature negative selection. Our lab has developed the HY^cd4 model, in which the transgenic HY TCRα is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice¹⁰.Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HY^cd4 mouse model. While negative selection in HY^cd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.     

Regulation of endosomal clathrin and retromer‐mediated endosome to Golgi retrograde transport by the J‐domain protein RME‐8

After endocytosis, most cargo enters the pleiomorphic early endosomes in which sorting occurs. As endosomes mature, transmembrane cargo can be sequestered into inwardly budding vesicles for degradation, or can exit the endosome in membrane tubules for recycling to the plasma membrane, the recycling endosome, or the Golgi apparatus. Endosome to Golgi transport requires the retromer complex. Without retromer, recycling cargo such as the MIG‐14/Wntless protein aberrantly enters the degradative pathway and is depleted from the Golgi. Endosome‐associated clathrin also affects the recycling of retrograde cargo and has been shown to function in the formation of endosomal subdomains. Here, we find that the Caemorhabditis elegans endosomal J‐domain protein RME‐8 associates with the retromer component SNX‐1. Loss of SNX‐1, RME‐8, or the clathrin chaperone Hsc70/HSP‐1 leads to over‐accumulation of endosomal clathrin, reduced clathrin dynamics, and missorting of MIG‐14 to the lysosome. Our results indicate a mechanism, whereby retromer can regulate endosomal clathrin dynamics through RME‐8 and Hsc70, promoting the sorting of recycling cargo into the retrograde pathway.     

Silencing of Mammalian Sar1 Isoforms Reveals COPII‐Independent Protein Sorting and Transport

The Sar1 GTPase coordinates the assembly of coat protein complex‐II (COPII) at specific sites of the endoplasmic reticulum (ER). COPII is required for ER‐to‐Golgi transport, as it provides a structural and functional framework to ship out protein cargoes produced in the ER. To investigate the requirement of COPII‐mediated transport in mammalian cells, we used small interfering RNA (siRNA)‐mediated depletion of Sar1A and Sar1B. We report that depletion of these two mammalian forms of Sar1 disrupts COPII assembly and the cells fail to organize transitional elements that coordinate classical ER‐to‐Golgi protein transfer. Under these conditions, minimal Golgi stacks are seen in proximity to juxtanuclear ER membranes that contain elements of the intermediate compartment, and from which these stacks coordinate biosynthetic transport of protein cargo, such as the vesicular stomatitis virus G protein and albumin. Here, transport of procollagen‐I is inhibited. These data provide proof‐of‐principle for the contribution of alternative mechanisms that support biosynthetic trafficking in mammalian cells, providing evidence of a functional boundary associated with a bypass of COPII .     

Replication Timing of Human Telomeric DNA and Other Repetitive Sequences Analyzed by Fluorescence in Situ Hybridization and Flow Cytometry

The replication timing of telomeres seems to differ between species. Yeast telomeres are late replicating, whereas limited data from very few human cell lines have indicated telomere replication throughout S phase. In the present study a series of permanent cell lines and patient samples was investigated using a flow cytometric approach for telomere length determination based on in situ hybridization using peptide nucleic acid probes and DNA staining. This method permits selective analysis of cells in specific phases of the cell cycle without perturbation of the cell cycle machinery. The timing of replication of telomeric C(3)TA(2) and T(2)AG(3) repeats was found to differ between individual samples and could precede or be concomitant with the replication of bulk DNA. Replication of the T(2)AG(3) strand seemed to occur somewhat later than that of the C(3)TA(2) strand in some samples. (GTG)(n) and other repetitive sequences generally showed a replication pattern similar to that of the bulk of DNA with slightly individual differences, whereas centromeric DNA repeats consistently replicated within a short time frame in late S phase. The apparent variability in replication timing seen for telomeric DNA might suggest individual differences in firing of replication origins.     

Simultaneous Measurement of DAPI-Sulforhodamine 101 Stained Nuclear DNA and Protein in Higher Plants by Flow Cytometry

A 2-step staining procedure is presented for simultaneous measurement of nuclear DNA and protein content in higher plants by flow cytometry. To release nuclei, plant tissues were chopped and stirred in the presence of the DNA specific fluorochrome 4', 6-diamidino-2-phenylindole (DAPI) and the nonionic detergent Triton X-100. Plant protoplasts were stirred in the DAPI dye solution with the detergent. After a short incubation period a second dye solution containing DAPI and the protein fluorochrome sulforhodamine 101 (SR 101) without detergent was added. Following another incubation, and after filtration through nylon gauze, the highly fluorescent nuclei were analyzed with an impulse cytophotometer. Accurate bivariate DNA-protein histograms were obtained with CV-values of about 2% or less for the 2C-peak of the univariate DNA parameter. The method presented here can be used for basic and applied cytogenetic studies of higher plants, for characterization of subcompartments of the cell cycle phases, or for examination of heterogeneity in plant tissues.     

Organelle‐specific probe JC‐1 identifies membrane potential differences in the mitochondrial function of bovine sperm

The mitochondrial probe 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazolyl‐carbocyanine iodide (JC‐1) not only identifies mitochondria exhibiting low membrane potentials by the emission of green fluorescence (range, 510–520 nm) but also differentiates these from mitochondria exhibiting relatively high membrane potentials. This discrimination occurs because JC‐1 forms aggregates at high membrane potentials. These J‐aggregates emit a bright red‐orange fluorescence at 590 nm. In this study, JC‐1 was combined with the classical dead cell stain, propidium iodide (PI), to identify a spectrum of functional sperm along with degenerate sperm. Flow cytometric analysis of bull sperm showed that the aggregate:monomer ratio differed among bulls before cryopreservation (P < 0.001) but not afterwards (P > 0.05). The effects of stain equilibration time, sperm concentration, and live:dead ratios were examined. The addition of SYBR‐14 to the JC‐1 and PI combination enhanced the distinction between the red PI‐stained and red‐orange JC‐1–stained populations. This discrimination between J‐aggregates and the PI‐stained sperm was affected by sperm concentration. These studies show that JC‐1 can be useful in monitoring mitochondrial function in bovine sperm. Mol. Reprod. Dev. 53:222–229, 1999. © 1999 Wiley‐Liss, Inc.     

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca²⁺. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSMα operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.     

A Single Conserved Leucine Residue on the First Intracellular Loop Regulates ER Export of G Protein‐Coupled Receptors

The intrinsic structural determinants for export trafficking of G protein‐coupled receptors (GPCRs) have been mainly identified in the termini of the receptors. In this report, we determined the role of the first intracellular loop (ICL1) in the transport from the endoplasmic reticulum (ER) to the cell surface of GPCRs. The α_2B‐adrenergic receptor (AR) mutant lacking the ICL1 is unable to traffic to the cell surface and to initiate signaling measured as ERK1/2 activation. Mutagenesis studies identify a single Leu48 residue in the ICL1 modulates α_2B‐AR export from the ER. The ER export function of the Leu48 residue can be substituted by Phe, but not Ile, Val, Tyr and Trp, and is unlikely involved in correct folding or dimerization of α_2B‐AR in the ER. Importantly, the isolated Leu residue is remarkably conserved in the center of the ICL1s among the family A GPCRs and is also required for the export to the cell surface of β₂‐AR, α_1B‐AR and angiotensin II type 1 receptor. These data indicate a crucial role for a single Leu residue within the ICL1 in ER export of GPCRs.