Immunological evidence for the presence of plant homologues of the actin- related protein Arp3 in tobacco and maize: subcellular localization to actin-enriched pit fields and emerging root hairs

Summary. The actin-nucleating and -organizing Arp2/3 protein complex is well known to be conserved throughout the eukaryotic kingdom. For higher plants, however, only limited evidence is available for the presence of the Arp2/3 complex so far. Using heterologous antibodies against the Dictyostelium discoideum and Schizosaccharomyces pombe proteins and a bovine peptide, we found immunological evidence for the presence of Arp3 homologues in plants. First, proteins with a molecular mass of about 47–50 kDa were clearly recognized in extracts of both a dicotyledonous plant (tobacco) and a monocotyledonous plant (maize) in immunoblots with the anti-Arp3 antibodies. Second, immunolocalization with these Arp3 antibodies was performed on different plant cells, selected for their diverse actin organizations and functions. On isolated plasma membrane ghosts derived from tobacco leaf protoplasts, a putative Arp3 was localized along cortical actin filaments. In the inner cortex of maize roots, Arp3 was localized to actin-rich plasmodesmata and pit fields and to multivesicular bodies in the cytoplasm. During root hair formation, distinct site-specific localization was found at the protruding apical plasma membrane portions of these tip-growing cells.Correspondence and reprints: Department of Biology, Universitaire Instelling Antwerpen, Universiteitsplein 1, 2610 Wilrijk, Belgium.Received January 3, 2003; accepted February 7, 2003; published online August 26, 2003     

Plasmid mediated silver resistance in Acinetobacter baumannii

Acinetobacter baumannii BL88, an environmental isolate, was resistant to 13 metals and 10 antibiotics. Plumbagin cured resistance to silver, cadmium, antimony, streptomycin and ampicillin at varying frequencies. However, only silver resistance transferred (1 × 10^–6 recepient^–1) to Escherichia coli K12 during conjugation. Correspondingly there was transfer of a 54 kb plasmid (pUPI199) from A. baumannii BL88. The plasmid transformed E. coli DH5 cells at a frequency of 1 × 10^–8 recepient^–1. The growth rate of E. coli DH5; (pUPI199) was slower as compared with E. coli DH5. Plasmid pUPI199 was 76 and 9.6% stable in the host A. baumannii BL88 in the presence and absence of selection pressure, respectively. A. baumannii BL88 was found to accumulate and retain silver whereas E. coli DH5 (pUPI199) effluxed 63% of the accumulated silver ions.     

Endocytosis and vesicle trafficking during tip growth of root hairs

Summary. The directional elongation of root hairs, “tip growth”, depends on the coordinated and highly regulated trafficking of vesicles which fill the tip cytoplasm and are active in secretion of cell wall material. So far, little is known about the dynamics of endocytosis in living root hairs. We analyzed the motile behaviour of vesicles in the apical region of living root hairs of Arabidopsis thaliana and of Triticum aestivum by live cell microscopy. For direct observation of endocytosis and of the fate of endocytic vesicles, we used the fluorescent endocytosis marker dyes FM 1-43 and FM 4-64. Rapid endocytosis was detected mainly in the tip, where it caused a bright fluorescence of the apical cytoplasm. The internalized membranes proceeded through highly dynamic putative early endosomes in the clear zone to larger endosomal compartments in the subapical region that are excluded from the clear zone. The internalized cargo ended up in the dynamic vacuole by fusion of large endosomal compartments with the tonoplast. Before export to these lytic compartments, putative early endosomes remained in the apical zone, where they most probably recycled to the plasma membrane and back into the cytoplasm for more than 30 min. Endoplasmic reticulum was not involved in trafficking pathways of endosomes. Actin cytoskeleton was needed for the endocytosis itself, as well as for further membrane trafficking. The actin-depolymerizing drug latrunculin B modified the dynamic properties of vesicles and endosomes; they became immobilized and aggregated in the tip. Treatment with brefeldin A inhibited membrane trafficking and caused the disappearance of FM-containing vesicles and putative early endosomes from the clear zone; labelled structures accumulated in motile brefeldin A-induced compartments. These large endocytic compartments redispersed upon removal of the drug. Our results hence prove that endocytosis occurs in growing root hairs. We show the localization of endocytosis in the tip and indicate specific endomembrane compartments and their recycling. Correspondence and reprints: Institute of Botany, Slovak Academy of Sciences, Dubravska cesta 14, 845 23 Bratislava, Slovak Republic.     

Cell wall pectins and xyloglucans are internalized into dividing root cells and accumulate within cell plates during cytokinesis

Recently, we have reported that cell wall pectins are internalized into apical meristem root cells. In cells exposed to the fungal metabolite brefeldin A, all secretory pathways were inhibited, while endocytic pathways remained intact, resulting in accumulation of internalized cell wall pectins within brefeldin A-induced compartments. Here we report that, in addition to the already published cell wall epitopes, rhamnogalacturonan I and xyloglucans also undergo large-scale internalization into dividing root cells. Interestingly, multilamellar endosomes were identified as compartments internalizing arabinan cell wall pectins reactive to the 6D7 antibody, while large vacuole-like endosomes internalized homogalacturonans reactive to the 2F4 antibody. As all endosomes belong topographically to the exocellular space, cell wall pectins deposited in these "cell wall islands", enclosed by the plasma-membrane-derived membrane, are ideally suited to act as temporary stores for rapid formation of cell wall and generation of new plasma membrane. In accordance with this notion, we report that all cell wall pectins and xyloglucans that internalize into endosomes are highly enriched within cytokinetic cell plates and accumulate within brefeldin A compartments. On the other hand, only small amounts of the pectins reactive to the JIM7 antibody, which are produced in the Golgi apparatus, localize to cell plates and they do not accumulate within brefeldin A compartments. In conclusion, meristematic root cells have developed pathways for internalization and recycling of cell wall molecules which are relevant for plant-specific cytokinesis.     

Anaesthetics and plants: from sensory systems to cognition-based adaptive behaviour

Protoplasma - Plants are not only sensitive to exogenous anaesthetics, but they also produce multitudes of endogenous substances, especially when stressed, that often have anaesthetic and anelgesic...     

A Phenazine based colorimetric and fluorescent chemosensor for sequential detection of Ag+ and I− in aqueous media

A new colorimetric and fluorescent probe MNTPZ based on 1H‐imidazo[4,5‐b]phenazine derivative has been designed and synthesized for successive detection of Ag⁺ and I^?. The probe MNTPZ shows selective colorimetric response by a change in color from yellow to orange and “turn‐off” fluorometric response upon binding with Ag⁺ in DMSO: Water (pH = 7, 1:1, v/v) over other cations. The binding mode of probe MNTPZ to Ag⁺ was studied by Job's plot, ¹H NMR studies, FT‐IR spectroscopy and DFT calculations. Moreover, the situ generated probe MNTPZ + Ag⁺ complex acted as an efficient fluorometric “turn‐on” probe for I^? via Ag⁺ displacement approach. The detection limit of probe MNTPZ for Ag⁺ and the resultant complex probe MNTPZ + Ag⁺ for I^? were determined to be 1.36 μmol/L and 1.03 μmol/L respectively. Notably, the developed probe was successfully used for quantitative determination of I^? in real samples with satisfactory results.     

Calcium-sensing receptor antagonist (calcilytic) NPS 2143 specifically blocks the increased secretion of endogenous Aβ42 prompted by exogenous fibrillary or soluble Aβ25–35 in human cortical astrocytes and neurons—Therapeutic relevance to Alzheimer's disease

The “amyloid-β (Aβ) hypothesis” posits that accumulating Aβ peptides (Aβs) produced by neurons cause Alzheimer's disease (AD). However, the Aβs contribution by the more numerous astrocytes remains undetermined. Previously we showed that fibrillar (f)Aβ_25–35, an Aβ₄₂ proxy, evokes a surplus endogenous Aβ₄₂ production/accumulation in cortical adult human astrocytes. Here, by using immunocytochemistry, immunoblotting, enzymatic assays, and highly sensitive sandwich ELISA kits, we investigated the effects of fAβ_25–35 and soluble (s)Aβ_25–35 on Aβ₄₂ and Aβ₄₀ accumulation/secretion by human cortical astrocytes and HCN-1A neurons and, since the calcium-sensing receptor (CaSR) binds Aβs, their modulation by NPS 2143, a CaSR allosteric antagonist (calcilytic). The fAβ_25–35-exposed astrocytes and surviving neurons produced, accumulated, and secreted increased amounts of Aβ₄₂, while Aβ₄₀ also accrued but its secretion was unchanged. Accordingly, secreted Aβ₄₂/Aβ₄₀ ratio values rose for astrocytes and neurons. While slightly enhancing Aβ₄₀ secretion by fAβ_25–35-treated astrocytes, NPS 2143 specifically suppressed the fAβ_25–35-elicited surges of endogenous Aβ₄₂ secretion by astrocytes and neurons. Therefore, NPS 2143 addition always kept Aβ₄₂/Aβ₄₀ values to baseline or lower levels. Mechanistically, NPS 2143 decreased total CaSR protein complement, transiently raised proteasomal chymotrypsin activity, and blocked excess NO production without affecting the ongoing increases in BACE1/β-secretase and γ-secretase activity in fAβ_25–35-treated astrocytes. Compared to fAβ_25–35, sAβ_25–35 also stimulated Aβ₄₂ secretion by astrocytes and neurons and NPS 2143 specifically and wholly suppressed this effect. Therefore, since NPS 2143 thwarts any Aβ/CaSR-induced surplus secretion of endogenous Aβ₄₂ and hence further vicious cycles of Aβ self-induction/secretion/spreading, calcilytics might effectively prevent/stop the progression to full-blown AD.