Convergence of the endocytic and lysosomal pathways in soybean protoplasts

Ultrastructural analysis of endocytosis of cationized ferritin (CF) has been combined with ultrastructural localization of acid phosphatases (AcPase) in soybean (Glycine max (L.) Merr.) protoplasts. While CF is an electron-dense marker of organelles of the endocytic pathway, ultrastructural histochemistry of AcPase identifies the organelles involved in the synthesis, transport, and storage of lytic-compartment enzymes, i.e. the lysosomal pathway. Acid phosphatases have been localized using both lead- and cerium-precipitation techniques. Protoplasts have been exposed to CF for 5 min, 30 min, or 3 h and processed for AcPase localization. At 5 min, smooth vesicles contain both CF and AcPase. By 30 min, Golgi cisternae and multivesicular bodies contain both labels. By 3 h, vacuoles become labelled with both CF and AcPase. The large central vacuoles contain intraluminal membranes which are associated with both AcPase and CF. These observations extend the analogy between plant vacuoles and animal lysosomes and demonstrate the points at which the endocytic pathway of plants converges with the lysosomal pathway.Abbreviations AcPase acid phosphatase - CF cationized ferritin - ER endoplasmic reticulum - MVB multivesicular body - PCR partially coated reticulum - PM plasma membrane     

Break of symmetry in regenerating tobacco protoplasts is independent of nuclear positioning

Nuclear migration and positioning are crucial for the morphogenesis of plant cells. We addressed the potential role of nuclear positioning for polarity induction using an experimental system based on regenerating protoplasts, where the induction of a cell axis de novo can be followed by quantification of specific regeneration stages. Using overexpression of fluorescently tagged extranuclear (perinuclear actin basket, kinesins with a calponin homology domain (KCH)) as well as intranuclear (histone H2B) factors of nuclear positioning and time‐lapse series of the early stages of regeneration, we found that nuclear position is no prerequisite for polarity formation. However, polarity formation and nuclear migration were both modulated in the transgenic lines, indicating that both phenomena depend on factors affecting cytoskeletal tensegrity and chromatin structure. We integrated these findings into a model where retrograde signals are required for polarity induction. These signals travel via the cytoskeleton from the nucleus toward targets at the plasma membrane.     

Efficiency of transient transformation in tobacco protoplasts is independent of plasmid amount

We describe an optimized protocol for the transient transformation of tobacco protoplasts mediated by polyethylene-glycol (PEG). As expected, the quantitative beta-glucuronidase (Gus) activity driven by pCaMVGus was dependent on the amount of plasmid used. Nevertheless, we demonstrate by an immunodetection method that transformation efficiency did not depend on the amount of plasmid used but on the limitation imposed by cell competence. In fact, we obtained the same percentage of transformed cells (about 60%) using a wide range of plasmid concentrations (0.1-10 microg per test). Finally, we show that, when we used two plasmid types in a mixture at a concentration ranging from 0.1 to 10 microg for each, all transformed cells expressed proteins encoded by both plasmids. Transient expression and co-transformation experiments are routinely used methods and, probably, the major results from this work were assumed by many researchers in this field, but our data experimentally support this assumption.     

An improved method for electroporation in plant protoplasts: infection of tobacco protoplasts by tobacco mosaic virus particles

Conditions of electroporation were optimized for introduction of tobacco mosaic virus (TMV) particles into tobacco mesophyll protoplasts (Nicotiana tabacum L. cv. Petit Havana SR1). Compared with conditions for TMV-RNA uptake, a longer electric pulse was necessary at the same voltage to induce TMV particle entry. Up to 80–90% of the protoplasts were infected with TMV particles after exposure to a 10 msec pulse at 200 V (0.67 KV/cm) in a 0.5 M mannitol solution. Protoplast viability was slightly lower than for controls which did not undergo electroporation. The presence of buffer in the mannitol solution reduced the net voltage in the solution which resulted in a significant decrease of the level of infection. These results suggest that the membrane pores resulting from an electrical pulse were wide enough for TMV particles (300 × 18 nm) to enter protoplasts.     

Clathrin-dependent pathways and the cytoskeleton network are involved in ceramide endocytosis by a parasitic protozoan, Giardia lamblia

Although identified as an early-diverged protozoan, Giardia lamblia shares many similarities with higher eukaryotic cells, including an internal membrane system and cytoskeleton, as well as secretory pathways. However, unlike many other eukaryotes, Giardia does not synthesize lipids de novo, but rather depends on exogenous sources for both energy production and organelle or membrane biogenesis. It is not known how lipid molecules are taken up by this parasite and if endocytic pathways are involved in this process. In this investigation, we tested the hypothesis that highly regulated and selective lipid transport machinery is present in Giardia and necessary for the efficient internalization and intracellular targeting of ceramide molecules, the major sphingolipid precursor. Using metabolic and pathway inhibitors, we demonstrate that ceramide is internalized through endocytic pathways and is primarily targeted into perinuclear/endoplasmic reticulum membranes. Further investigations suggested that Giardia uses both clathrin-dependent pathways and the actin cytoskeleton for ceramide uptake, as well as microtubule filaments for intracellular localization and targeting. We speculate that this parasitic protozoan has evolved cytoskeletal and clathrin-dependent endocytic mechanisms for importing ceramide molecules from the cell exterior for the synthesis of membranes and vesicles during growth and differentiation.     

A non-coat protein synthesized in tobacco mesophyll protoplasts infected by tobacco mosaic virus

Summary A high molecular weight protein unrelated to the viral coat was detected in tobacco mesophyll protoplasts infected by tobacco mosaic virus.     

Membrane domains in the secretory and endocytic pathways

Progress in identifying, characterizing, and localizing the constituents of distinct membrane bound compartments has revealed a new level of intracellular subcompartmentation. Proteins and lipids are not uniformly distributed in a given organelle, and subdomains are formed by a combination of hierarchical assembly processes and protein exclusion. Thus, functionally distinct specializations of a given organelle are physically segregated to a greater extent than previously believed.     

Axonal and dendritic endocytic pathways in cultured neurons

The endocytic pathways from the axonal and dendritic surfaces of cultured polarized hippocampal neurons were examined. The dendrites and cell body contained extensive networks of tubular early endosomes which received endocytosed markers from the somatodendritic domain. In axons early endosomes were confined to presynaptic terminals and to varicosities. The somatodendritic but not the presynaptic early endosomes were labeled by internalized transferrin. In contrast to early endosomes, late endosomes and lysosomes were shown to be predominantly located in the cell body. Video microscopy was used to follow the transport of internalized markers from the periphery of axons and dendrites back to the cell body. Labeled structures in both domains moved unidirectionally by retrograde fast transport. Axonally transported organelles were sectioned for EM after video microscopic observation and shown to be large multivesicular body-like structures. Similar structures accumulated at the distal side of an axonal lesion. Multivesicular bodies therefore appear to be the major structures mediating transport of endocytosed markers between the nerve terminals and the cell body. Late endocytic structures were also shown to be highly mobile and were observed moving within the cell body and proximal dendritic segments. The results show that the organization of the endosomes differs in the axons and dendrites of cultured rat hippocampal neurons and that the different compartments or stages of the endocytic pathways can be resolved spatially.     

Surface labelling of plant protoplasts with fluorochromes for discrimination of heterokaryons by microscopy and flow cytometry

Surface labelling of plant protoplasts was tested for use in mass fusion systems and heterokaryon detection. Parameters have been established for biotinylation and subsequent incubation with avidin-coupled fluorochromes. The procedure is rapid (less than 3 hours) and does not affect viability. Fusion responses were the same as with unlabelled protoplasts. From a range of fluorochromes tested, fluorescein and phycoerythrin proved best suited for detection experiments with protoplasts of both suspension and leaf origin. With this standard combination of labels, as applied in experiments with animal cells, heterokaryons from fused plant protoplasts could clearly be discriminated from other protoplasts by means of fluorescence microscopy or flow cytometry with a single combination of filters and wavelengths.     

Phenylalanine ammonia-lyase levels in protoplasts isolated from hypersensitive tobacco pre-infected with tobacco mosaic virus

Leaves of tobacco varieties carrying the N gene for hypersensitiviy react to tobacco mosaic virus (TMV) infection by forming necrotic lesions and by localizing the virus in the vicinity of these lesions. These changes are accompanied in the host by an increased metabolic activity, in particular by an increased production of phenolic compounds derived from phenylalanine. Necrogenesis apparently destroys cells which have become heavily infected despite this strong defense reaction. However, it has been demonstrated previously (Otsuki et al., 1972) that protoplasts derived from leaves which normally respond in vivo to virus inoculation by forming necrotic local lesions, show no such response when inoculated in vitro. In the present study we have investigated the effect of pre-infecting hypersensitive leaves with TMV on the production or the non-production of the factor(s) of necrosis at the level of either protoplasts or mesophyll cells isolated from these preinfected leaves. Phenylalanine ammonia-lyase (PAL), whose rate of synthesis has been shown (Duchesne et al., 1977) to increase in stimulated cells of infected leaves, was used as a biochemical marker in the search for the stimulus preceding necrogenesis. We found that this stimulus concerning PAL activity was never elicited in either protoplasts or mesophyll cells which were prepared just before the appearance of necrotic local lesions. This result did not depend on the conditions of pre-infection or on the methods used to isolate the protoplasts or mesophyll cells. We also assayed samples derived from pre-infected leaves that were already carrying local lesions, i.e., in which the stimulus and necrogenesis were already operating: not only did the isolated protoplasts and mesophyll cells not sustain the stimulus concerning PAL activity, but the stimulated enzyme activity decreased abruptly and, in most of the experiments, had disappeared within the time necessary for maceration. Evidence is presented showing that the non-elicitation or the abrupt decrease of stimulated PAL activity could not result from a selection of unstimulated cells or from a preferential destruction of stimulated cells during maceration of the leaves.Our results support the view that hypertonic osmotic pressure is responsible for the non-occurence of the hypersensitive response by acting according to one or both of the following processes: it suppresses the contacts through plasmodesmata between neighboring cells and, hence, it also suppresses the cell-to-cell diffusion of the factor(s) eliciting the stimulus; and/or since hypertonic osmotic pressure causes striking differences between leaf cells and protoplasts in total RNA and protein synthesis, these differences might include the suppression of synthesis of the elicitor of hypersensitivity.Abbreviations OMT O-methyltransferase - PAL phenylalanine ammonia-lyase - TMV Tobacco mosaic virus     

Transformation of tobacco protoplasts with DNA entrapped in pH-sensitive liposomes

A system for the transformation of tobacco mesophyll protoplasts using pH-sensitive liposomes was developed. Plasmid DNA (plGVneo23) encoding the NPT-II gene for kanamycin resistance was entrapped in pH-sensitive liposomes composed of dioleolphosphatidylethanolamine, cholesterol and oleic acid. These liposomes release their contents at low pH and are capable of delivering their contents into the cytoplasm of protoplasts. Kanamycin-resistant colonies were reproducibly recovered from transformed protoplasts at an average frequency of 1.62×10^-4 at pH 7.5. Plants regenerated from transformed cell lines were normal in appearance and were fertile. NPT-II activity was detected in leaf extracts of transformed, kanamycin-resistant plants and the presence of NPT-II DNA in the tobacco genome was shown by Southern blots. Analysis of self-pollinations and reciprocal crosses to non-transformed plants indicated that kanamycin resistance segregated as a dominant nuclear marker. Co-transformation of protoplasts with liposomes containing two selectable markers indicated that co-transformation occurred with a frequency of approximately 23%.Abbreviations DOPE dioleoylphosphatidylethanolamine - DOPC dioleoylphosphatidylcholine - Chol cholesterol - OA oleic acid - PEG polyethylene glycol 6000 - NAA -napthaleneacetic acid - BAP 6-benzylaminopurine     

Fusion accessibility of endocytic compartments along the recycling and lysosomal endocytic pathways in intact cells

A fluorescence assay developed for the quantitation of intracellular fusion of sequentially formed endocytic compartments (Salzman, N. H., and F. R. Maxfield. 1988 J. Cell Biol. 106:1083-1091) has been used to measure the time course of endosome fusion accessibility along the recycling and degradative endocytic pathways. Transferrin (Tf) was used to label the recycling pathway, and alpha2-macroglobulin (alpha 2 M) was used to label the lysosomal degradative pathway. Along the degradative pathway, accessibility of vesicles containing alpha 2M to fusion with subsequently formed endocytic vesicles decreased with apparent first order kinetics. The t12 for the loss of fusion accessibility was approximately 8 min. The behavior of Tf is more complex. Initially the fusion accessibility of Tf decayed rapidly (t1/2 less than 3 min), but a constant level of fusion accessibility was then observed for 10 min. This suggests that Tf moves through one fusion accessible endosome rapidly and then enters a second fusion accessible compartment on the recycling pathway. At 18 degrees C, fusion of antifluorescein antibodies (AFA) containing vesicles with F-alpha 2M was observed when the interval between additions was 10 min. However, if the interval was increased to 1 h, no fusion with incoming vesicles was observed. These results identify the site of F-alpha 2M accumulation at 18 degrees C as a prelysosomal late endosome that no longer fuses with newly formed endosomes since no delivery to lysosomes is observed at this temperature.     

Aminoglycoside binding sites in the cochlea as revealed by neomycin-gold labelling

Summary Neomycin/bovine serum albumin/gold was used as a probe to detect the binding sites of aminoglycosides on the thin sections of the cochlea embedded in Spurr. The binding sites were mainly located on the stereocilia, the cuticular plate of hair cells, the head plates of Deiters' cells, fibrous structures in pillar cells, in the spiral limbus and tectorial membrane and basilar membrane, plasma membranes, mitochondria and the chromatin of various kinds of cells. Triphosphoinositide, acidic glycosaminoglycans, and RNA were considered to be responsible for the binding activity.     

Aminoglycoside binding sites in the cochlea as revealed by neomycin-gold labelling

Neomycin/bovine serum albumin/gold was used as a probe to detect the binding sites of aminoglycosides on the thin sections of the cochlea embedded in Spurr. The binding sites were mainly located on the stereocilia, the cuticular plate of hair cells, the head plates of Deiters' cells, fibrous structures in pillar cells, in the spiral limbus and tectorial membrane and basilar membrane, plasma membranes, mitochondria and the chromatin of various kinds of cells. Triphosphoinositide, acidic glycosaminoglycans, and RNA were considered to be responsible for the binding activity.