Regulation of endosome fusion.

Homotypic fusion between early endosomes can be reconstituted in vitro. By using wortmannin and LY294002, inhibitors of phosphatidylinositol (Pl) 3-kinase, a requirement for this activity has been established in order for fusion to proceed efficiently. It has been shown that Pl 3-kinase activity is required downstream of rab5 activation, although a large excess of activated rab5 can overcome wortmannin inhibition. A series of experiments have also been performed which indicate a role for early endosomal autoantigen 1 (EEA1) in determining fusion efficiency. EEA1 dissociates from membranes following wortmannin treatment. It is proposed that the requirement of endosome fusion for Pl 3-kinase activity is to promote the association of EEA1 with endosomes.     

基于pH值敏感的荧光染料分析腺病毒诱导T淋巴细胞胞内体膜的裂解北大核心CSCD

【目的】探索基于pH值敏感的荧光染料分析腺病毒裂解T淋巴细胞胞内体膜的实验方法。【方法】本文以Jurkat细胞(T淋巴瘤细胞)为靶细胞,将pH值敏感的荧光染料pHrodo dextran与5型腺病毒(Ad5)共同孵育Jurkat细胞,对pHrodo dextran孵育的浓度与时间进行了优化,利用激光共聚焦显微镜分析胞内相对平均荧光强度百分比随时间的变化情况,反映Ad5诱导胞内体膜裂解情况。【结果】研究结果表明,在pHrodo dextran终浓度为80μg/m L,孵育时间为10 min条件下,在病毒感染后的30 min,相对平均荧光强度百分比出现显著下降;利用巴佛洛酶素A1抑制胞内体膜质子泵活性后,相对平均荧光强度百分比出现轻微下降。【结论】建立了基于pHrodo dextran分析腺病毒诱导T细胞胞内体膜裂解的新方法。     

Differential cytotoxic activity of potassium dichromate on nucleoside uptake in BHK fibroblasts.

In cultures of hamster fibroblasts (BHK cell line) treated with potassium dichromate (K2Cr2O7) nucleic acid and protein syntheses are differentially inhibited, and nucleoside uptake into the intracellular pool is characterized by a stimulation phase followed by an inhibition phase. Different patterns are observed for the uptake of each ribo- and deoxyribonucleoside, pyrimidine nucleoside (particularly deoxycytidine) uptake reaching the highest stimulation level. Kinetics of thymidine and deoxycytidine initial uptake at different exogenous nucleoside concentrations show that K2Cr2O7 affects both simple and facilitated diffusion of nucleosides. The time course of thymidine and deoxycytidine pool saturation suggests however that the effects of K2Cr2O7 on plasma membrane permeability are partially counterbalanced by modifications of pool size deriving from the concomitant alteration of steps of nucleoside metabolism separate from nucleoside uptake.     

Cleavage of rabaptin-5 blocks endosome fusion during apoptosis.

Cells undergoing apoptosis exhibit striking changes in membrane organization, including plasma membrane blebbing and invagination, vacuolation and fragmentation of organelles, and alterations in the surface expression of receptors. The underlying mechanisms for these changes are unknown, though alterations in vesicular fusion are likely to play a role. Using a cell-free system based on Xenopus laevis egg extracts we have found that endosome fusion is blocked during apoptosis. Inhibition of fusion is prevented by Bcl-2 or Bcl-xL, two negative regulators of apoptosis, or by specific inhibitors of members of the caspase family of apoptotic proteases. Selective cleavage of Rabaptin-5, an essential and rate-limiting component of endosome fusion, is responsible for the loss of fusion activity. Cleavage of Rabaptin-5 also occurs in cellular models for apoptosis. These results suggest that inactivation of Rabaptin-5 and inhibition of vesicle transport lead to fragmentation of endosomes and inhibition of the endocytic pathway during the execution phase of apoptosis. We propose that parallel changes to other membrane transport pathways would give rise to general membrane fragmentation in apoptotic cells. These changes are likely to play an important role in the generation of apoptotic bodies and their recognition by phagocytosing cells.     

A novel serum assay for breast epithelial antigen using a fusion protein.

Serum levels of breast epithelial antigens (BrE-Ags) are presently used in the follow-up of breast cancer patients. Available assays do not have optimal sensitivity and rely on reagents that could vary in their source and purity. A novel competitive solid-phase radioimmunoassay was developed for BrE-Ags that consists of the NP5 fusion protein, produced in Escherichia coli, that is composed of beta-galactosidase and polypeptide sequence obtained from a breast carcinoma cell line cDNA library, and anti-human milk fat globule monoclonal antibody Mc5. The fusion protein carries an altered epitope sequence (mimotope) that is similar, but not identical, to that found in the native antigen. This new competitive assay configuration has two essential features, a solid-phase affinity step that purifies the fusion protein carrying the mimotope for Mc5 and a competitive step that provides quantitation. Serum values for this assay show high specificity and sensitivity for breast cancer patients when compared to normal subjects and post-surgical breast cancer patients during their disease-free period.     

Massive Ca-induced membrane fusion and phospholipid changes triggered by reverse Na/Ca exchange in BHK fibroblasts

Baby hamster kidney (BHK) fibroblasts increase their cell capacitance by 25-100% within 5 s upon activating maximal Ca influx via constitutively expressed cardiac Na/Ca exchangers (NCX1). Free Ca, measured with fluo-5N, transiently exceeds 0.2 mM with total Ca influx amounting to approximately 5 mmol/liter cell volume. Capacitance responses are half-maximal when NCX1 promotes a free cytoplasmic Ca of 0.12 mM (Hill coefficient approximately 2). Capacitance can return to baseline in 1-3 min, and responses can be repeated several times. The membrane tracer, FM 4-64, is taken up during recovery and can be released at a subsequent Ca influx episode. Given recent interest in signaling lipids in membrane fusion, we used green fluorescent protein (GFP) fusions with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and diacylglycerol (DAG) binding domains to analyze phospholipid changes in relation to these responses. PI(4,5)P(2) is rapidly cleaved upon activating Ca influx and recovers within 2 min. However, PI(4,5)P(2) depletion by activation of overexpressed hM1 muscarinic receptors causes only little membrane fusion, and subsequent fusion in response to Ca influx remains massive. Two results suggest that DAG may be generated from sources other than PI(4,5)P in these protocols. First, acylglycerols are generated in response to elevated Ca, even when PI(4,5)P(2) is metabolically depleted. Second, DAG-binding C1A-GFP domains, which are brought to the cell surface by exogenous ligands, translocate rapidly back to the cytoplasm in response to Ca influx. Nevertheless, inhibitors of PLCs and cPLA2, PI(4,5)P(2)-binding peptides, and PLD modification by butanol do not block membrane fusion. The cationic agents, FM 4-64 and heptalysine, bind profusely to the extracellular cell surface during membrane fusion. While this binding might reflect phosphatidylserine (PS) "scrambling" between monolayers, it is unaffected by a PS-binding protein, lactadherin, and by polylysine from the cytoplasmic side. Furthermore, the PS indicator, annexin-V, binds only slowly after fusion. Therefore, we suggest that the luminal surfaces of membrane vesicles that fuse to the plasmalemma may be rather anionic. In summary, our results provide no support for any regulatory or modulatory role of phospholipids in Ca-induced membrane fusion in fibroblasts.     

Mitotic phosphorylation of rab4 prevents binding to a specific receptor on endosome membranes.

Phosphorylation of the monomeric GTPase rab4 in mitotic cells leads to its relocalization from endosome membranes to the cytosol. To determine the mechanism underlying this change in distribution, we established an in vitro assay that reconstituted specific binding of rab4 when endosome-containing membranes were incubated with rab4 complexed with its cytosolic chaperone, GDP dissociation inhibitor (GDI). rab4 was found to bind to a saturable receptor associated with highly purified endosomes. Membrane binding and nucleotide exchange were physically distinct, since an active soluble fragment of the rab4 receptor, but not rab4 nucleotide exchange activity, could be released from membranes by elastase cleavage. Interestingly, the soluble fragment could be used to fully reconstitute rab4 membrane binding. In vitro phosphorylation of rab4 by cdc2/cyclin B kinase did not affect formation of rab4-GDI complexes, but did completely inhibit rab4 binding to its receptor. In contrast, in vitro phosphorylation of membranes did not result in the dissociation of bound rab4, nor were mitotic membranes deficient with respect to binding non-phosphorylated rab4. Thus, mitotic cells appear to accumulate rab4 in the cytosol by phosphorylating rab4 during the soluble phase of its normal activity cycle, thereby preventing membrane attachment.     

GTP gamma S stimulation of endosome fusion suggests a role for a GTP-binding protein in the priming of vesicles before fusion.

Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), a non-hydrolyzable analogue of GTP, inhibits in vitro fusion among early endocytic vesicles in the presence of high concentrations of cytosol. In this report we show that fusion is remarkably stimulated by GTP gamma S under conditions where cytosolic components are the limiting factors for the process. The amount of cytosolic factors required for maximal fusion activity is several-fold decreased by the presence of GTP gamma S. Moreover, preincubation of vesicles in the presence of cytosol and GTP gamma S allows fusion to proceed even in the absence of cytosol. Our results indicate that a GTP-binding protein facilitates the binding of cytosolic factor(s) required for endosome fusion to the endosomal membrane and stabilizes a dilution-resistant intermediate of the fusion process.     

A fluorimetric assay for real-time monitoring of adenylyl cyclase activity based on terbium norfloxacin

Adenylyl cyclases catalyze the production of the second messenger cyclic AMP from ATP. Until now, there has been no fluorescent adenylyl cyclase assay known that is applicable to high-throughput screening and kinetic determinations that can directly monitor the turnover of the unmodified substrate ATP. In this study, a fluorescence-based assay is described using the Ca(II)- and calmodulin-dependent adenylyl cyclase edema factor (EF) from Bacillus anthracis and Tb(III)-norfloxacin as probe for the enzyme activity. This assay can be used to study enzyme regulators, allows real-time monitoring of adenylyl cyclase activity, and does not substitute ATP by fluorescent derivatives. These derivatives must be judged critically due to their interference on the activity of enzymes. Furthermore, the new assay makes redundant the application of radioactively labeled substrates such as [α-³²P]ATP or fluorescently labeled antibodies such as anti-cyclic AMP. We determined the Michaelis-Menten constant (K_M), the v₀^max value of ATP turnover, and the IC₅₀ values for three inhibitors of EF by this newly developed fluorescent method.     

Interaction of peroxisomes with microtubules. In vitro studies using a novel peroxisome-microtubule binding assay.

The association of membrane-bounded cell organelles to microtubules is crucial for determination of their shape, intracellular localization and translocation. We have shown previously the high affinity binding of peroxisomes to microtubules which appears to be of static nature as in vivo studies indicate that only a few peroxisomes move along the microtubular tracks. In order to characterize the interactions of peroxisomes with microtubules, we have developed a semiquantitative in vitro binding assay, which is based on the association of highly purified rat liver peroxisomes to microtubules coated onto microtiterplates. The binding was visualized by differential interference contrast and immunofluorescence using a confocal laser scanning microscope. The binding was concentration dependent and saturable, being affected by time, temperature, and pH. Addition of ATP or the motor proteins kinesin and dynein increased the binding capacity, while ATP-depletion or microtubule associated proteins (MAPs) decreased it. KCl treatment of peroxisomes reduced the binding, which was restored by dialyzed KCl-stripping eluate as well as by rat liver cytosol. The reconstituting effect of cytosol was abolished by its pretreatment with proteases or N-ethylmaleimide. Moreover, the treatment of peroxisomes with proteases or N-ethylmaleimide reduced their binding, which was not reversed by cytosol. These results suggest the involvement of a peroxisomal membrane protein and cytosolic factor(s) in the binding of peroxisomes to microtubules. This notion is supported by the observation that distinct subfractions of dialyzed KCl-stripping eluate obtained by gel chromatography augmented the binding. Those subfractions, as well as purified peroxisome fractions, exhibited strong immunoreactivity with an antibody to cytoplasmic linker protein (CLIP)-115, revealing a 70-kDa polypeptide. Moreover, immunodepletion of KCl-stripping eluate and its subfractions with an antibody to the conserved microtubule binding domain of CLIPs, abolished their promoting effect on the binding, thus suggesting the involvement of a CLIP-related protein in the binding of peroxisomes to microtubules.     

Kinetic timing: a novel mechanism that improves the accuracy of GTPase timers in endosome fusion and other biological processes

The GTPase superfamily contains a large number of proteins that function as molecular switches by binding and hydrolyzing GTP molecules. They are localized at various intracellular organelles and control diverse cellular processes. For many GTPases, the lifetime of the activated, GTP-bound state is believed to serve as a timer in determining the activation time of a biological event such as membrane fusion and signal transduction. However, such a timer is intrinsically stochastic due to thermal noise at the level of single GTPase molecules. Here, we describe a mathematical model that shows how a directional GTPase cycle, in a nonequilibrium steady-state driven by GTP hydrolysis, can significantly reduce the variance in the lifetime of an activated GTPase molecule and thereby increase the accuracy and efficiency of the timer. This mechanism, termed kinetic timing, articulates a clear function for the energy consumption in GTPase-controlled biological processes. It provides a rationale for why biological timers utilize a GTP hydrolysis cycle rather than a simple GTP binding–dissociation equilibrium, and why the GTP-bound state is a better timer than the GDP-bound state. It also explains the necessity for the existence of multiple GTP-bound intermediates identified by fluorescence spectroscopy and nuclear magnetic resonance studies.     

Schizosaccharomyces pombe ypt5: a homologue of the rab5 endosome fusion regulator.

The ypt/rab proteins are a family of small GTP-binding proteins thought to be required for different stages of membrane traffic. From the fission yeast Schizosaccharomyces pombe we have isolated and characterized ypt5, a gene encoding a homologue of rab5, a mammalian protein apparently involved in regulating fusion of early endosomes. Recombinant ypt5 protein bound GTP. The ypt5 gene was found to be essential for viability on minimal media, but ypt5-disrupted cells grew slowly on some rich media and accumulated a population of small vesicles not observed in wild-type cells. Canine rab5 cDNA could replace the ypt5 gene in S. pombe and restore normal growth and viability. Ypt5 protein expressed in mammalian cells colocalized with the transferrin receptor to early endosomes. Thus, molecular aspects of the early endocytic pathway may be conserved between mammalian cells and S. pombe and hence may be amenable to genetic analysis.     

Quantitative measurement of mast cell degranulation using a novel flow cytometric annexin-V binding assay.

BACKGROUND: Mast cells are primary mediators of allergic inflammation. Antigen-mediated crosslinking of their cell surface immunoglobulin E (IgE) receptors results in degranulation and the release of proinflammatory mediators including histamine, tumor necrosis factor-alpha, and leukotrienes. METHODS: Mast cells were stimulated to degranulate by using either IgE crosslinking or ionophore treatment. Exogenously added annexin-V was used to stain exocytosing granules, and the extent of binding was measured flow cytometrically. Release of the enzyme beta-hexosaminidase was used for population-based measurements of degranulation. Two known inhibitors of degranulation, the phosphatidylinositol 3 kinase inhibitor wortmannin and overexpression of a mutant rab3d protein, were used as controls to validate the annexin-V binding assay. RESULTS: Annexin-V specifically bound to mast cell granules exposed after stimulation in proportion to the extent of degranulation. Annexin-V binding was calcium dependent and was blocked by phosphatidylserine containing liposomes, consistent with specific binding to this membrane lipid. Visualization of annexin-V staining showed granular cell surface patches that colocalized with the exocytic granule marker VAMP-green fluorescent protein (GFP). Wortmannin inhibited both annexin-V binding and beta-hexosaminidase release in RBL-2H3 cells, as did the expression of a dominant negative rab3d mutant protein. CONCLUSIONS: The annexin-V binding assay represents a powerful new flow cytometric method to monitor mast cell degranulation for functional analysis.     

The distribution of spontaneous chromosome aberrations in BHK21/C13 fibroblasts.

Previous investigations of spontaneous aberrations in mammalian cells have been carried out on large heterogeneous samples of individuals, each of whom had had a different exposure to exogenous clastogens. In the present analysis using Syrian Hamster cells, a large number of metaphases were analysed from one sample of control cells. In this way all cells were exposed to the same doses of any unknown clastogens. The overall distribution of spontaneous breaks was found to be nonrandom. Breaks involved in different types of aberration had a nonrandom distribution, which was specific for each type. (e.g. terminal deletion and rearrangement).     

A novel cell-free mitochondrial fusion assay amenable for high-throughput screenings of fusion modulators

Background  

Mitochondria are highly dynamic organelles whose morphology and position within the cell is tightly coupled to metabolic function. There is a limited list of essential proteins that regulate mitochondrial morphology and the mechanisms that govern mitochondrial dynamics are poorly understood. However, recent evidence indicates that the core machinery that governs mitochondrial dynamics is linked within complex intracellular signalling cascades, including apoptotic pathways, cell cycle transitions and nuclear factor kappa B activation. Given the emerging importance of mitochondrial plasticity in cell signalling pathways and metabolism, it is essential that we develop tools to quantitatively analyse the processes of fission and fusion. In terms of mitochondrial fusion, the field currently relies upon on semi-quantitative assays which, even under optimal conditions, are labour-intensive, low-throughput and require complex imaging techniques.     

Identification of selective class II histone deacetylase inhibitors using a novel dual-parameter binding assay based on fluorescence anisotropy and lifetime

Histone deacetylases (HDACs) are important epigenetic factors regulating a variety of vital cellular functions such as cell cycle progression, differentiation, cell migration, and apoptosis. Consequently, HDACs have emerged as promising targets for cancer therapy. The drugability of HDACs has been shown by the discovery of several structural classes of inhibitors (HDACis), particularly by the recent approval of two HDACis, vorinostat (ZOLINZA) and romidepsin (Istodax), for the treatment of cutaneous T-cell lymphoma by the US Food and Drug Administration. The outstanding potential of HDACis, with a defined isoform selectivity profile as drugs against a plurality of diseases, vindicates increased effort in developing high-throughput capable assays for screening campaigns. In this study, a dual-competition assay exploiting changes in fluorescence anisotropy and lifetime was used to screen the LOPAC (Sigma-Aldrich, St Louis, MO) library against the bacterial histone deacetylase homologue HDAH from Bordetella, which shares 35% identity with the second deacetylase domain of HDAC6. The binding assay proved to be highly suitable for high-throughput screening campaigns. Several LOPAC compounds have been identified to inhibit HDAH in the lower micromolar range. Most interestingly, some of the hit compounds turned out to be weak but selective inhibitors of human class IIa and IIb HDACs.     

Real-time fluorescence measurement of cell-free endosome fusion: regulation by second messengers.

A quantitative real-time assay of cell-free endosomal vesicle fusion was developed and applied to study fusion mechanisms in endosomes from baby hamster kidney (BHK-21) cells. The assay is based on an irreversible approximately 10-fold increase in BODIPY-avidin fluorescence on binding of biotinylated conjugates. BODIPY-avidin and biotin-dextran were internalized for 10 min at 37 degrees C into separate populations of BHK-21 cells, and endosome fractions were prepared. Postnuclear supernatant fractions underwent ATP- and temperature-dependent fusion, as measured in a sensitive custom-built microfluorimeter by the continuous increase in BODIPY-avidin fluorescence. Fusion processes of efficiency > 2.5% could be detected with 200-ms time resolution in sample volumes of 50 microL containing endosomes derived from approximately 4 x 10(4) cells. The fusion time course consisted of a distinct lag phase (up to 10 min) in which little fusion occurred, followed by an approximately exponential rise (t 1/2 10-30 min; fusion efficiency approximately 15%). The lag phase was reduced by preincubation of separate endosome fractions with ATP at 37 degrees C and by coincubation of endosomes at 22 degrees C before the assay, suggesting a rate-limiting step involving binding of a soluble protein to the endosome membrane. Endosome fusion was strongly inhibited by GTP gamma S, N-ethylmaleimide, and AIF4-. Endosome fusion was not affected by phorbol myristate acetate but was significantly inhibited by cAMP and bovine brain calmodulin. The results establish a sensitive real-time fluorescence assay to quantify the kinetics and extent of endosome fusion in a cell-free system and demonstrate regulation of early endosome fusion by cytosolic second messengers.