We assessed FM1-43 [ N -(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl)pyridinium dibromide] as a fluorescent endocytosis marker in intact, walled plant cells. At 4 ؇ C, FM1-43 stained the plasma membrane, and after 30 to 120 min of incubation at 26 ؇ C, FM1-43 labeled cytoplasmic vesicles and then the vacuole. Fluorimetric quantitation demonstrated dye uptake temperature sensitivity ( ‫ف‬ 65% reduction at 16 ؇ C, Ͼ 90% at 4 ؇ C). FM1-43 uptake in suspension cells was stimulated more than twofold by brefeldin A and inhibited ‫ف‬ 0.4-fold by wortmannin. FM1-43 delivery to the vacuole was largely inhibited by brefeldin A, although overall uptake was stimulated, and brefeldin A treatment caused the accumulation of large prevacuolar endosomal vesicles heavily labeled with FM1-43. Three-dimensional time lapse imaging revealed that FM1-43-labeled vacuoles and vesicles are highly dynamic. Thus, FM1-43 serves as a fluorescent marker for imaging and quantifying membrane endocytosis in intact plant cells. INTRODUCTIONEndocytosis in animal cells begins with the budding of invaginated coated pits from the plasma membrane into coated vesicles, which then uncoat and fuse with the endocytic organelles (Gruenberg et al., 1989;Gruenberg and Maxfield, 1995). By this action, both membrane proteins and fluid phase molecules are sequestered from the cell exterior into intracellular organelles, from where they can recycle to the plasma membrane or be transported to other destinations, such as the lysosomes. Thus, endocytosis serves to balance membrane secretion and to remove, transport, or degrade receptor ligand complexes from the cell surface (Goldstein et al., 1985). Although clathrin coatdependent endocytosis is involved in receptor internalization, clathrin-independent endocytosis pathways also contribute to internalization. These include uptake via nonclathrin-coated caveolae (Anderson, 1998), macropinocytosis (Swanson, 1989;Hewlett et al., 1994), and phagocytosis (Aderem and Underhill, 1999).Our understanding of animal cell endocytosis was advanced by the use of fluorescent and electron-opaque markers that can be internalized Maxfield, 1988, 1989;Griffiths et al., 1989). The use of fluid phase markers has permitted the development of assays that reconstitute endocytic vesicle fusion (Braell, 1987(Braell, , 1992Gruenberg et al., 1989;Emans et al., 1995;Emans and Verkman, 1996) and led to the identification of many proteins that catalyze and control fusion, such as the small GTPase rab5 (Gorvel et al., 1991) and its effector proteins (Christoforidis et al., 1999a(Christoforidis et al., , 1999bMcBride et al., 1999).In plants, there is evidence that endocytosis is likely to occur (Low and Chandra, 1994; for review, see Battey et al., 1999;Marcote et al., 2000). Endocytosis is postulated to counterbalance membrane secretion (Samuels and Bialputra, 1990) and permit cell volume to respond to changes in osmolality (Thiel et al., 1998;Kubitscheck et al., 2000). However, the study of plant cell endocytosis has been hampered by t...

Acyl homoserine lactones are synthesized by Pseudomonas aeruginosa as signaling molecules which control production of virulence factors and biofilm formation in a paracrine manner. We found that N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL), but not its 3-deoxo isomer or acyl-homoserine lactones with shorter fatty acids, induced the directed migration (chemotaxis) of human polymorphonuclear neutrophils (PMN) in vitro. By use of selective inhibitors a signaling pathway, comprising phosphotyrosine kinases, phospholipase C, protein kinase C, and mitogen-activated protein kinase C, could be delineated. In contrast to the well-studied chemokines complement C5a and interleukin 8, the chemotaxis did not depend on pertussis toxin-sensitive G proteins, indicating that 3OC12-HSL uses another signaling pathway. Strong evidence for the presence of a receptor for 3OC12-HSL on PMN was derived from uptake studies; by use of radiolabeled 3OC12-HSL, specific and saturable binding to PMN was seen. Taken together, our data provide evidence that PMN recognize and migrate toward a source of 3OC12-HSL (that is, to the site of a developing biofilm). We propose that this early attraction of PMN could contribute to prevention of biofilm formation.

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We describe the expression of the bispecific antibody biscFv2429 in transgenic suspension culture cells and tobacco plants. biscFv2429 consists of two single-chain antibodies, scFv24 and scFv29, connected by the Trichoderma reesi cellobiohydrolase I linker. biscFv2429 binds two epitopes of tobacco mosaic virus (TMV): the scFv24 domain recognizes neotopes of intact virions, and the scFv29 domain recognizes a cryptotope of the TMV coat protein monomer. biscFv2429 was functionally expressed either in the cytosol (biscFv2429-cyt) or targeted to the apoplast using a murine leader peptide sequence (biscFv2429-apoplast). A third construct contained the C-terminal KDEL sequence for retention in the ER (biscFv2429-KDEL). Levels of cytoplasmic biscFv2429 expression levels were low. The highest levels of antibody expression were for apoplast-targeted biscFv2429-apoplast and ER-retained biscFv2429-KDEL that reached a maximum expression level of 1.65% total soluble protein in transgenic plants. Plant-expressed biscFv2429 retained both epitope specificities, and bispecificity and bivalency were confirmed by ELISA and surface plasmon resonance analysis. This study establishes plant cells as an expression system for bispecific single-chain antibodies for use in medical and biological applications.Keywords: bispecific single-chain antibody; recombinant antibodies; protein targeting; tobacco mosaic virus; transgenic plants.Antibodies are essential tools in medicine, biology and biochemistry. Their high affinity and specificity make them invaluable for diagnostic and therapeutic applications in medicine and human healthcare. To broaden the potential use of antibodies in medical and biological applications, bispecific antibody molecules, which have two independent binding sites for two epitopes are often desirable. Bispecific antibody molecules are unique therapeutic agents with their ability to crosslink two different antigens, which can be exploited in cancer therapy for the recruitment of cytotoxic T cells to a tumor cell [1,2].Various strategies have been used to generate bispecific antibodies. The first bivalent bispecific full-size antibodies or F(ab H ) 2 fragments were produced by in-vitro chemical crosslinking of two different antibodies [3±5]. In addition, bispecific F(ab H ) 2 fragments have been created by heterodimerization of Escherichia coli-expressed Fab fragments through cysteine residues [6] or leucine zippers [7]. Bispecific antibodies have been produced in vivo using the hybrid hybridoma (quadroma) technology [8], but a limitation of this procedure is the low yield of bispecific antibody of the desired dual specificity. Protein engineering has permitted the design of even smaller bispecific fragments based on single-chain Fv fragments (scFv) fragments, such as the scFv heterodimer diabody, which is formed in vivo by noncovalent association of two single-chain fusion products [9]. Alternatively, the two different binding specificities can be combined in a single polypeptide using a flexible linker peptide to form...

The P. aeruginosa quorum-sensing molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL) interacts not only with bacteria, but also with mammalian cells, among others with those of the immune defence system. We focussed on the possible interaction of 3OC12-HSL with human polymorphonuclear neutrophils (PMN), because these cells are the first to enter an infected site. We found that 3OC12-HSL attracts PMN, and up-regulates expression of receptors known to be involved in host defence, including the adhesion proteins CD11b/CD18 and the immunoglobulin receptors CD16 and CD64. Furthermore, the uptake of bacteria (phagocytosis), which is crucial for an efficient defence against infection, was enhanced. Thus, recognising and responding to 3OC12-HSL not only attracts the PMN to the site of a developing biofilm, but also reinforces their defence mechanisms, and hence could be a means to control the infection in an early stage and to prevent biofilm formation.