The characean internodal cell as a model system for studying wound healing.

This work describes the characean internodal cell as a model system for the study of wound healing and compares wounds induced by certain chemicals and UV irradiation with wounds occurring in the natural environment. We review the existing literature and define three types of wound response: (1) cortical window formation characterised by disassembly of microtubules, transient inhibition of actin-dependent cytoplasmic streaming and chloroplast detachment, (2) fibrillar wound walls characterised by exocytosis of vesicles carrying wall polysaccharides and membrane-bound cellulose synthase complexes coupled with endocytosis of surplus membrane and (3) amorphous, callose- and membrane-containing wound walls characterised by exocytosis of vesicles and endoplasmic reticulum cisternae in the absence of membrane recycling. We hypothesize that these three wound responses reflect the extent of damage, probably Ca(2+) influx, and that the secretion of Ca(2+) -loaded endoplasmic reticulum cisternae is an emergency reaction in case of severe Ca(2+) load. Microtubules are not required for wound healing but their disassembly could have a signalling function. Transient reorganisation of the actin cytoskeleton into a meshwork of randomly oriented filaments is required for the migration of wound wall forming organelles, just as occurs in tip-growing plant cells. New data presented in this study show that during the deposition of an amorphous wound wall numerous actin rings are present, which may indicate specific ion fluxes and/or a storage form for actin. In addition, we present new evidence for the exocytosis of FM1-43-stained organelles, putative endosomes, required for plasma membrane repair during wound healing. Finally, we show that quickly growing fibrillar wound walls, even when deposited in the absence of microtubules, have a highly ordered helical structure of consistent handedness comprised of cellulose microfibrils.

Actin-dependent deposition of putative endosomes and endoplasmic reticulum during early stages of wound healing in characean internodal cells.

We investigated the behaviour of organelles stained with FM1-43 (putative endosomes) and/or LysoTracker Red (LTred; acidic compartments) and of the endoplasmic reticulum (ER) during healing of puncture and UV-induced wounds in internodal cells of Nitella flexilis and Chara corallina. Immediately after puncture, wounds were passively sealed with a plug of solid vacuolar inclusions, onto which a bipartite wound wall was actively deposited. The outer, callose-containing amorphous layer consisted of remnants of FM1-43- and LTred-labelled organelles, ER cisternae and polysaccharide-containing secretory vesicles, which became deposited in the absence of membrane retrieval (compound exocytosis). During formation of the inner cellulosic layer, exocytosis of secretory vesicles with the newly formed plasma membrane is coupled to endocytosis via coated vesicles. Migration of FM1-43- and LTred-stained organelles, ER and secretory vesicles towards the cell cortex and deposition of a bipartite wound wall could also be induced by spot-like irradiation with ultraviolet light. Cytochalasin D reversibly inhibited the accumulation and deposition of organelles. Our study indicates that active actin-dependent deposition of putative recycling endosomes is required for wound healing (plasma membrane repair) and supports the hypothesis that deposition of ER cisternae helps to restore wounding-disturbed Ca(2+) metabolism.

Disturbance of endomembrane trafficking by brefeldin A and calyculin A reorganizes the actin cytoskeleton of Lilium longiflorum pollen tubes.

We investigated the effect of brefeldin A on membrane trafficking and the actin cytoskeleton of pollen tubes of Lilium longiflorum with fluorescent dyes, inhibitor experiments, and confocal laser scanning microscopy. The formation of a subapical brefeldin A-induced membrane aggregation (BIA) was associated with the formation of an actin basket from which filaments extended towards the tip. The orientation of these actin filaments correlated with the trajectories of membrane material stained by FM dyes, suggesting that the BIA-associated actin filaments are used as tracks for retrograde transport. Analysis of time series indicated that these tracks (actin filaments) were either stationary or glided along the plasma membrane towards the BIA together with the attached membranes or organelles. Disturbance of the actin cytoskeleton by cytochalasin D or latrunculin B caused immediate arrest of membrane trafficking, dissipation of the BIA and the BIA-associated actin basket, and reorganization into randomly oriented actin rods. Our observations suggest that brefeldin A causes ectopic activation of actin-nucleating proteins at the BIA, resulting in retrograde movement of membranes not only along but also together with actin filaments. We show further that subapical membrane aggregations and actin baskets supporting retrograde membrane flow can also be induced by calyculin A, indicating that dephosphorylation by type 2 protein phosphatases is required for proper formation of membrane coats and polar membrane trafficking.

Microfilaments and microtubules control the shape, motility, and subcellular distribution of cortical mitochondria in characean internodal cells.

The shape, motility, and subcellular distribution of mitochondria in characean internodal cells were studied by visualizing fluorescent dyes with confocal laser scanning microscopy and conducting drug-inhibitor experiments. Shape, size, number, and distribution of mitochondria varied according to the growth status and the metabolic activity within the cell. Vermiform (sausage-shaped), disc-, or amoeba-like mitochondria were present in elongating internodes, whereas very young cells and older cells that had completed growth contained short, rodlike organelles only. Mitochondria were evenly distributed and passively transported in the streaming endoplasm. In the cortex, mitochondria were sandwiched between the plasma membrane and the stationary chloroplast files and distributed in relation to the pattern of pH banding. Highest mitochondrial densities were found at the acid, photosynthetically more active regions, whereas the alkaline sites contained fewer and smaller mitochondria. In the cortex of elongating cells, small mitochondria moved slowly along microtubules or actin filaments. The shape and motility of giant mitochondria depended on the simultaneous interaction with both cytoskeletal systems. There was no microtubule-dependent motility in the cortex of nonelongating mature cells and mitochondria only occasionally travelled along actin filaments. These observations suggest that mitochondria of characean internodes possess motor proteins for microtubules and actin filaments, both of which can be used either as tracks for migration or for immobilization. The cortical cytoskeleton probably controls the spatiotemporal distribution of mitochondria within the cell and promotes their association with chloroplasts, which is necessary for exchange of metabolites during photosynthesis and detoxification.

In vivo imaging of an elicitor-induced nitric oxide burst in tobacco.

A growing body of evidence suggests that nitric oxide (NO), an important signalling and defence molecule in mammals, plays a key role in activating disease resistance in plants, acting as signalling molecule and possibly as direct anti-microbial agent. Recently, a novel fluorophore (diaminofluorescein diacetate, DAF-2 DA) has been developed which allows bio-imaging of NO in vivo. Here we use the cell-permeable DAF-2 DA, in conjunction with confocal laser scanning microscopy, for real-time imaging of NO in living plant cells. Epidermal tobacco cells treated with cryptogein, a fungal elicitor from Phytophthora cryptogea, respond to the elicitor with a strong increase of intracellular NO. NO-induced fluorescence was found in several cellular compartments, and could be inhibited by a NO scavenger and an inhibitor of nitric oxide synthase. The NO burst was triggered within minutes, reminiscent of the oxidative burst during hypersensitive response reactions. These results reveal additional similarities between plant and animal host responses to infection.

Actin-based vesicle dynamics and exocytosis during wound wall formation in characean internodal cells.

Characean internodal cells readily form wound walls upon local membrane damage. In the present study we documented the dynamics of vesicles involved in wound wall secretion and compared them with actin organization in equivalent cells using immunofluorescence. Single exocytotic events (spreading of vesicle contents) could be visualized using image enhancement by video microscopy. In control unwounded cells vesicles moved unidirectionally along parallel actin bundles and rarely contacted the plasma membrane. The wound response started with (1) local inhibition of active cytoplasmic streaming (unidirectional movements) due to inactivation, depolymerization, or mechanical displacement of the subcortical actin bundles. Accordingly, vesicles performed only oscillating motions and moved slowly with the same velocity and direction as passive endoplasmic flow. (2) Several minutes after wounding, vesicles started to perform random saltatory movements with frequently changing velocities, punctuated by oscillating motion and periods of immobility (docking) at the plasma membrane. Vesicle trajectories correlated with a fine-meshed actin network at the wound site. (3) Several hours after wounding, vesicles moved again unidirectionally along regenerated subcortical actin bundles. Spreading of vesicles (vesicle contents) was observed during wound wall formation, i.e., during the period of saltatory movements when vesicles had access to the plasma membrane. Dependent on the type of wound wall being secreted, three variants could be distinguished: (1) slow and continuous spreading over a time period of several seconds up to 30 min near the plasma membrane, (2) fast spreading within 80 ms inside an already formed wound wall, and/or (3) fast spreading at the plasma membrane. We conclude from our study that wounding-induced changes in vesicle dynamics are due to transient reorganization of the actin cytoskeleton from parallel bundles to a fine-meshed network. Furthermore, our results indicate that spreading of vesicle contents varies considerably with time and may be delayed by vesicle docking and/or discharge.

Morphogenesis and ultrastructure of the soil ciliate Engelmanniella mobilis (Ciliophora, Hypotrichida).

The morphogenetic pattern, freeze-fracture, transmission, and scanning electron-microscopy of interphasic cells were used to elucidate the enigmatic systematic position of Engelmanniella mobilis. Frontal, buccal, parabuccal, and marginal cirri are distinguishable during cortical development; transverse, frontoterminal (migratory), and caudal cirri are absent. Ontogenetic peculiarities include the conservation of parental and grandparental marginal cirri and the apokinetal origin of a dorsal kinety in the opisthe. Thus, interphasic cells of E. mobilis possess three generations of cirri. The pellicle is multilamellated. Prominent, undischargeable subpellicular granules consisting of a homogeneous osmiophilic mass develop from bundles of long fibers. Rhomboid crystal-like structures projecting from the lithosomes and regularly patterned mitoribosomes are additional remarkable ultrastructural characteristics. The somatic and frontal cirri comprise 2-10 kinetosomes. Very likely, all cirri are composed of closely adjacent "dikinetids" because their kinetosomes form pairs each possessing a transverse and a postciliary microtubular ribbon. The fourth row of kinetosomes of the adorai membranelies shows the usual transverse microtubular ribbons, while one or two transverse microtubules are closely adjacent to the third and the second row of its basal bodies. Our data suggest that Engelmanniella has descended from a kahliellid lineage. However, its placement in a well-founded (!) stichotrichid family is impossible at present.

The hemimastigophora (Hemimastix amphikineta nov. gen., nov. spec.), a new protistan phylum from gondwanian soils.

The morphology, morphogenesis and ultrastructure of Hemimastix amphikineta nov. gen., nov. spec, are described. This species occurred in some Australian and in 1 Chilean soil, but was absent from more than 1000 soil samples from Laurasian localities. Thus, it has probably a restricted Gondwanian distribution. Hemimastix amphikineta is a small (14-20 × 7-10 µn), colourless organism that looks distinctly Ciliophora-like because of its posteriorly located contractile vacuole and its 2 longitudinal somatic kineties each composed of about 12 cilia-like flagella. These 2 kineties are interposed between 2 large plicated and microtubule-bearing pellicular plates which are arranged inversely mirror-image like ("diagonal symmetry"). Hemimastix amphikineta has saccular to tubular mitochondrial cristae and complex extrusomes. It has 2 microtubular systems and a membranous sac associated with each kinetid. The nucleolus persists throughout nuclear division. A permanent cytostome-cytopharyngeal complex, pharyngeal rods, striated fibres, mastigonemes, and a paraflagellar rod are absent. This unique combination of characters dictates a very separate position for H. amphikineta within the known protists. Thus, the phylum Hemimastigophora nov. phylum (Hemimastigea nov. cl. and Hemimastigida nov. ord.), is established to include H. amphikineta and possibly Spironema multiciliatum Klebs, 1892. The structure of the pellicle and the nuclear apparatus of H. amphikineta indicate some relationship with the Euglenophyta. However, clear evidence for a certain affinity is lacking. Thus, the Hemimastigophora are placed in an incertae sedis position within the kingdom Protista Haeckel, 1866.

A rotation model for microtubule and filament sliding.

Simple model experiments show that the cyclic motion of myosin cross-bridges in muscle which is assumed to be active ("sliding model" by "power-stroke" or "rowing-stroke" of the crossbridges) can be interpreted equally well as a passive process during which the myosin heads simply lock mechanically into the grooves of the thin filaments. In order to explain the sliding process a filament or microtubule rotation is assumed to be combined with the winding and unwinding of associated helical protein filaments ("MAPs", "dynein"). As shown in further model experiments the direction of helix winding or unwinding along a rod (microtubule) determines the direction of rod displacement ("parallel" or "antiparallel sliding"). The "sidearms" and "bridges" visible in the electron microscope along the cytoskeletal elements might correspond to the winding or unwinding filaments. On the basis of this conception simple models for the behavior of spindle microtubules and the anaphase movement of chromosomes are presented. The latter is assumed to occur via the unwinding of helical filaments accompanying the kinetochore microtubules, which causes their simultaneous depolymerization.