Four-dimensional imaging of transvacuolar strand dynamics in tobacco BY-2 cells.

The vacuole is a characteristic organelle of plant cells and fulfills several important functions related to metabolism and growth of the cell. To shed light on the details of vacuolar structural changes in plant cells, we explored the three-dimensional organization and dynamics of living Nicotiana tabacum L. cv. Bright Yellow 2 cell vacuoles by real-time confocal time-lapse imaging. For imaging, the cells were pulse-labeled with the amphipathic styryl dye FM1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide), which is delivered to the plant vacuole by endocytic uptake and then incubated overnight. Imaging of the membrane-labeled vacuole revealed a complex vacuole morphology underlaid by constant remodeling. The vacuole is traversed by multiple transvacuolar strands which move along each other and fuse in multiple manners. New strands were created by fission of large membrane sheets. Endocytic vesicle trafficking was followed within the dynamic transvacuolar strands. The movement occurred in a stop-and-go fashion with an average vesicle velocity of 0.46 microm/s and a peak velocity of 0.82 microm/s. Transvacuolar-strand reduction and creation is a characteristic event observed during mitosis. Here we propose a mechanistic model for the alteration of the number of transvacuolar strands, on the basis of their fusion and fission.

Expression pattern of fibroblast growth factors (FGFs), their receptors and antagonists in primary endothelial cells and vascular smooth muscle cells.

Fibroblast growth factors (FGFs) are important angiogenic growth factors. While basic FGF (FGF2) is well established as a potent inducer of angiogenesis much less is known about other FGFs possibly expressed by EC. We investigated the expression of all known FGFs, their main tyrosine kinase receptors and antagonists by RT-PCR analysis in human umbilical vascular endothelial cells (HUVECs) to obtain a complete expression profile of this important growth factor system in model endothelial cells (EC). In addition to FGFR1IIIc, which is considered as the major FGF receptor in EC, HUVECs express similar levels of FGFR3IIIc, detectable amounts of FGFR2IIIc and a new FGF receptor without an intracellular kinase domain (FGFR5). HUVECs express several secreted FGFs, including FGF5, 7, 8, 16 and 18 and two members of the fibroblast growth factor homologous factors (FHFs), not yet reported to be expressed in EC. The expression panel was compared with that obtained from human vascular smooth muscle cells (VSMCs) and human aortic tissue. Human umbilical artery smooth muscle cells (HUASMCs) and HUVECs express the identical FGF receptor and ligand panel implicating that both cell types act, according the FGF signals more as an entity than as individual cell types. Expression of Fgf1, 2, 7, 16 and 18 and the antagonists Sprouty 2,3 and 4 was demonstrated for all analysed cDNAs. The IIIc isoforms of FGFR1 and 2 and the novel FGFR5 were expressed in the aorta, but expression of the FGF receptor 3 was not detected in cDNAs derived from aortic tissue. In the VSMC of rat aortic tissue and in HUASM cultured cells we could demonstrate FGF18 immunoreactivity in the nucleus of the cells. The expression of several secreted FGFs by EC may focus the view more on their paracrine effects on neighbouring cells during tissue regeneration or tumor formation.

Syncytial fusion of human trophoblast depends on caspase 8.

Differentiation of human placental villous trophoblast includes syncytial fusion of cytotrophoblast forming syncytiotrophoblast. Early stages of the apoptosis cascade were described to be involved in this differentiation process. We investigated the role of the initiator caspase 8 in syncytial fusion in vitro, cultivating placental villous explants with or without caspase 8 antisense oligonucleotides or peptide inhibitors for up to 120 h. Trophoblast fusion and differentiation were assessed by confocal microscopy, immunohistochemistry and Western blot analysis. Culture with caspase 8 antisense oligonucleotides or peptide inhibitors reduced the fusion of cytotrophoblast with the syncytiotrophoblast, and resulted in multilayered cytotrophoblast. Caspase 8 expression was suppressed by antisense oligonucleotides and caspase 8 activities were reduced by peptide inhibitors. The organic anion-transporter hOAT-4 normally expressed in the cytotrophoblast and transferred into the syncytiotrophoblast by syncytial fusion was retained in the cytotrophoblast due to lack of fusion. We conclude that expression and activity of caspase 8 is a prerequisite for differentiation and syncytial fusion of cytotrophoblast cells.

Molecular farming of recombinant antibodies in plants.

Antibodies represent a large proportion of therapeutic drugs currently in development. In most cases, they are produced in mammalian cell lines or transgenic animals because these have been shown to fold and assemble the proteins correctly and generate authentic glycosylation patterns. However, such expression systems are expensive, difficult to scale up and there are safety concerns due to potential contamination with pathogenic organisms or oncogenic DNA sequences. Plants represent an inexpensive, efficient and safe alternative for the production of recombinant antibodies. Research over the last 10 years has shown that plants can produce a variety of functional antibodies and there is now intense interest in scaling up production to commercial levels. In this review, we discuss the advantages of plants over traditional expression systems, describe how antibody expression in plants is achieved and optimized and then consider the practical issues concerning large-scale molecular farming in plants. The first plant-produced therapeutic antibodies are already in clinical trials, and, given the economic benefits of this production system, we are likely to see many more recombinant antibodies produced in this manner in the future.

Molecular farming of pharmaceutical proteins.

Molecular farming is the production of pharmaceutically important and commercially valuable proteins in plants. Its purpose is to provide a safe and inexpensive means for the mass production of recombinant pharmaceutical proteins. Complex mammalian proteins can be produced in transformed plants or transformed plant suspension cells. Plants are suitable for the production of pharmaceutical proteins on a field scale because the expressed proteins are functional and almost indistinguishable from their mammalian counterparts. The breadth of therapeutic proteins produced by plants range from interleukins to recombinant antibodies. Molecular farming in plants has the potential to provide virtually unlimited quantities of recombinant proteins for use as diagnostic and therapeutic tools in health care and the life sciences. Plants produce a large amount of biomass and protein production can be increased using plant suspension cell culture in fermenters, or by the propagation of stably transformed plant lines in the field. Transgenic plants can also produce organs rich in a recombinant protein for its long-term storage. This demonstrates the promise of using transgenic plants as bioreactors for the molecular farming of recombinant therapeutics, including vaccines, diagnostics, such as recombinant antibodies, plasma proteins, cytokines and growth factors.

Antibody production by molecular farming in plants.

"Molecular farming" is the production of pharmaceutical proteins in transgenic plants and has great potential for the production of therapeutic anti-cancer antibodies and recombinant therapeutic proteins. Plants make fully functional recombinant human or animal antibodies. Cultivating transgenic plants on an agricultural scale will produce almost unlimited supplies of recombinant proteins for uses in medicine. Combinatorial library technology is a key tool for the generation and optimisation of therapeutic antibodies ahead of their expression in plants. Optimised antibody expression can be rapidly verified using transient expression assays in plants before creation of transgenic suspension cells or plant lines. Subcellular targeting signals that increase expression levels and optimise protein stability can be identified and exploited using transient expression to create high expresser plant lines. When high expresser lines have been selected, the final step is the development of efficient purification methods to retrieve functional antibody. Antibody production on an industrial scale is then possible using plant suspension cell culture in fermenters, or by the propagation of stably transformed plant lines in the field. Recombinant proteins can be produced either in whole plants or in seeds and tubers, which can be used for the long-term storage of both the protein and its production system. The review will discuss these developments and how we are moving toward the molecular farming of therapeutic antibodies becoming an economic and clinical reality.

Towards molecular farming in the future: moving from diagnostic protein and antibody production in microbes to plants.

Molecular farming of pharmaceuticals in plants has the potential to provide almost unlimited amounts of recombinant proteins for use in disease diagnosis and therapy. Transgenic plants are attracting interest as bioreactors for the inexpensive production of large amounts of safe, functional, recombinant macromolecules, such as blood substitutes, vaccines and antibodies. In some cases, the function of expressed recombinant proteins can be rapidly analysed by expression in microbes or by transient expression in intact or virally infected plants. Protein production can be increased by upscaling production in fermenters, using yeast- or plant-suspension cells or by using transient-expression systems. Stable transgenic plants can be used to produce leaves or seeds rich in the recombinant protein for long-term storage or direct processing. This demonstrates the promise for using plants as bioreactors for the molecular farming of recombinant therapeutics, diagnostics, blood substitutes and antibodies. We anticipate that this technology has the potential to greatly benefit human health by making safe recombinant pharmaceuticals widely available.

Towards molecular farming in the future: using plant-cell-suspension cultures as bioreactors.

Plant-suspension cells are an in vitro system that can be used for recombinant protein production under carefully controlled certified conditions. Plant-suspension cells can be grown in shake flasks or fermenters to produce secondary metabolites, like vincristine and vinblastine, and to produce recombinant proteins after transformation. This review article focuses on discussing the generation of transformed suspension-cell lines expressing recombinant proteins, like antibodies, and recombinant-protein downstream processing and purification.

Towards molecular farming in the future: pichia pastoris-based production of single-chain antibody fragments.

This review article focuses on the use of the methylotrophic yeast Pichia pastoris as a recombinant protein-expression system. P. pastoris is a useful system for the expression of milligram-to-gram quantities of a protein, which can be scaled up to fermentation to meet greater demands. Compared with mammalian cells, Pichia do not require a complex growth medium or culture conditions, they are as easy to manipulate genetically as Escherichia coli and have a eukaryotic protein-synthesis pathway. They seem suited to laboratory-scale production of recombinant proteins for in-house use or, in some cases, molecular farming of recombinant products. This review article focuses on the use of P. pastoris, describes a fermentation production run of a single-chain antibody fragment and includes a discussion of fermentation as a production strategy.

Towards molecular farming in the future: transient protein expression in plants.

Molecular farming in plants can be achieved by stable or transient expression of a recombinant protein. Transient expression of recombinant proteins in plants can rapidly provide large amounts of the proteins for detailed characterization. It is fast, flexible and can be carried out at field scale using viral vectors, but it lacks the increases in production volume that can be achieved easily with stable transgenic crops. This review article focuses on discussing the applications of transient expression using viral vectors, biolistic methods or agroinfiltration.

Molecular farming of recombinant antibodies in plants.

'Molecular farming' is the production of recombinant proteins in plants. It is intended to harness the power of agriculture to cultivate and harvest transgenic plants producing recombinant therapeutics. Molecular farming has the potential to provide virtually unlimited quantities of recombinant antibodies for use as diagnostic and therapeutic tools in both health care and the life sciences. Importantly, recombinant antibody expression can be used to modify the inherent properties of plants, for example by using expressed antipathogen antibodies to increase disease resistance. Plant transformation is technically straightforward for model plant species and some cereals, and the functional expression of recombinant proteins can be rapidly analyzed using transient expression systems in intact or virally infected plants. Protein production can then be increased using plant suspension cell production in fermenters, or by the propagation of stably transformed plant lines in the field. Transgenic plants can be exploited to produce organs rich in a recombinant protein for its long-term storage. This demonstrates the promise of using transgenic plants as bioreactors for the 'molecular farming' of recombinant therapeutics, blood substitutes and diagnostics, such as recombinant antibodies.

Protein phosphorylation during phagosome maturation.

Phagolysosome biogenesis is driven by a series of interactions between phagosomes and organelles of the biosynthetic and endocytic pathways. The presence of endocytic markers on phagosomes suggests that phagosomes and endosomes share common structural and functional characteristics. In that line of thought, protein phosphorylation has been shown to be involved in regulatory aspects of the fusion properties of endosomes and other vacuolar organelles. To study further the mechanisms involved in phagolysosome biogenesis, we have investigated the presence of phagosome proteins that can be phosphorylated in vitro by endogenous phagosome-associated kinases. The results obtained show that proteins phosphorylated on tyrosine residues are present on phagosomes. Moreover, complex phosphorylation/dephosphorylation cycles appear to occur during phagolysosome biogenesis. The addition of endosome fractions to phagosomes inhibit the phosphorylation of phagosome proteins. These results suggest that phosphorylation and dephosphorylation events could play roles in the biogenesis of phagolysosomes and regulate, in part, the complex in vivo interactions between phagosomes and endosomes.

Cystic fibrosis transmembrane conductance regulator activation stimulates endosome fusion in vivo.

Previous studies have suggested a role for cystic fibrosis transmembrane conductance regulator (CFTR) in the regulation of intracellular vesicular trafficking. A quantitative fluorescence method was used to test the hypothesis that CFTR expression and activation affects endosome-endosome fusion in intact cells. Endosomes from CFTR-expressing and control (vector-transfected) Swiss 3T3 fibroblasts were labeled by internalization with 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene (Bodipy)-avidin, a fluid-phase marker whose fluorescence increases approximately 8-fold upon biotin binding. Cells were washed, chased, and then labeled with biotin-albumin or biotin-transferrin. The fraction of Bodipy-avidin-labeled endosomes that fused with biotin-containing endosomes (f(fusion)) was quantified by ratio imaging microfluorimetry. Endosome fusion in unstimulated CFTR-expressing cells was similar to that in control cells. However, in CFTR-expressing cells activated by forskolin, ffusion was increased by 1.30 +/- 0.18- and 2.65 +/- 0.17-fold for a 0 and 10 min chase time between avidin and biotin-albumin pulses; f(fusion) also increased (1.32 +/- 0.11-fold) when biotin-transferrin replaced biotin-albumin. The stimulation of endosome fusion was not due to differences in rates of endocytosis or endosomal acidification. Endosome fusion was not stimulated by forskolin in Cl--depleted CFTR-expressing cells, suggesting that the increase in endosome fusion is due to the CFTR chloride channel activity. These results provide evidence that CFTR is involved in the regulation of endosome fusion and, thus, a possible basis for the cellular defects associated with cystic fibrosis.

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.

Imaging of endosome fusion in BHK fibroblasts based on a novel fluorimetric avidin-biotin binding assay.

A fluorescence assay of in vivo endosome fusion was developed and applied to define the kinetics of endosome fusion in baby hamster kidney (BHK) fibroblasts. The assay is based on an approximately 10-fold enhancement of the green fluorescence of BODIPY-avidin upon biotin binding. The BODIPY-avidin fluorescence enhancement occurred in < 25 ms, was pH-independent, and involved a BODIPY-tryptophan interaction. For endocytosis in vivo, BHK fibroblasts were pulse-labeled with BODIPY-avidin together with a red (rhodamine) fluorescent fusion-independent chromophore (TMR). After specified chase times in a nonfluorescent medium, a second cohort of endosomes was pulse-labeled with biotin-conjugated albumin, dextran, or transferrin. Fusion of biotin-containing endosomes with avidin-containing endosomes was quantified by ratio imaging of BODIPY-to-TMR fluorescence in individual endosomes, using imaging methods developed for endosome pH studies. Analysis of BODIPY-to-TMR ratio distributions in avidin-labeled endosomes exposed to zero and maximum biotin indicated > 90% sensitivity for detection of endosome fusion. In avidin pulse (10 min) -chase-biotin albumin pulse (10 min) studies, both fused and unfused endosomes were identified; the fractions of avidin-labeled endosomes that fused with biotin-labeled endosomes were 0.48, 0.21, 0.16, and 0.07 for 0-, 5-, 10-, and 20-min chase times. Fitting of fusion data to a mathematical model of in vivo endosome fusion required the existence of an intermediate fusion compartment. Pulse-chase studies performed with biotin-transferrin to label the early/recycling endosomes indicated that after a 10-min chase, avidin-labeled endosomes reached a compartment that was inaccessible to biotin-transferrin. The assay was also applied to determine whether endosome fusion was influenced by temperature, pH (bafilomycin A1), second messengers (cAMP agonists, phorbol 12-myristate 13-acetate, staurosporine), and growth-related factors (platelet-derived growth factor, genistein). The results establish a sensitive fluorescence assay to quantify the fusion of vesicular compartments in living cells.

Cytoplasmic dynein-dependent vesicular transport from early to late endosomes.

We have used an in vitro fusion assay to study the mechanisms of transport from early to late endosomes. Our data show that the late endosomes share with the early endosomes a high capacity to undergo homotypic fusion in vitro. However, direct fusion of early with late endosomes does not occur. We have purified vesicles which are intermediates during transport from early to late endosomes in vivo, and analyzed their protein composition in two-dimensional gels. In contrast to either early or late endosomes, these vesicles do not appear to contain unique proteins. Moreover, these vesicles undergo fusion with late endosomes in vitro, but not with each other or back with early endosomes. In vitro, fusion of these endosomal vesicles with late endosomes is stimulated by polymerized microtubules, consistent with the known role of microtubules during early to late endosome transport in vivo. In contrast, homotypic fusion of early or late endosomes is microtubule-independent. Finally, this stimulation by microtubules depends on microtubule-associated proteins and requires the presence of the minus-end directed motor cytoplasmic dynein, but not the plus-end directed motor kinesin, in agreement with the microtubule organization in vivo. Our data strongly suggest that early and late endosomes are separate, highly dynamic organelles, which are connected by a microtubule-dependent vesicular transport step.

Annexins in membrane traffic.

Annexins have long been though to be involved in exocytosis, possibly by helping to create close interactions between membranes destined to undergo fusion. In this article, we examine recent observations that implicate annexins in three different steps of the endocytic pathway, suggesting that annexins may be universal modulators of membrane trafficking.

Annexin II is a major component of fusogenic endosomal vesicles.

We have used an in vitro assay to follow the proteins transferred from a donor to an acceptor upon fusion of early endosomes. The acceptor was a purified early endosomal fraction immunoisolated on beads and the donor was a metabolically-labeled early endosomal fraction in suspension. In the assay, both fractions were mixed in the presence of unlabeled cytosol, and then the beads were retrieved and washed. The donor proteins transferred to the acceptor were identified by two-dimensional gel electrophoresis and autoradiography. Approximately 50 major proteins were transferred and this transfer fulfilled all criteria established for endosome fusion in vitro. However, only a small subset of proteins was efficiently transferred, if donor endosomes were briefly sonicated to generate small (0.1 micron diam) vesicles before the assay. These include two acidic membrane proteins, and three alkaline peripheral proteins exposed on the cytoplasmic face of the membrane. Partial sequencing and Western blotting indicated that one of the latter components is annexin II, a protein known to mediate membrane-membrane interactions. Immunogold labeling of cryosections confirmed that annexin II is present on early endosomes in vivo. These data demonstrate that annexin II, together with the other four proteins we have identified, is a major component of fusogenic endosomal vesicles, suggesting that these proteins are involved in the binding and/or fusion process.