Anaesthetics stop diverse plant organ movements, affect endocytic vesicle recycling and ROS homeostasis, and block action potentials in Venus flytraps.

Background and Aims: Anaesthesia for medical purposes was introduced in the 19th century. However, the physiological mode of anaesthetic drug actions on the nervous system remains unclear. One of the remaining questions is how these different compounds, with no structural similarities and even chemically inert elements such as the noble gas xenon, act as anaesthetic agents inducing loss of consciousness. The main goal here was to determine if anaesthetics affect the same or similar processes in plants as in animals and humans. Methods: A single-lens reflex camera was used to follow organ movements in plants before, during and after recovery from exposure to diverse anaesthetics. Confocal microscopy was used to analyse endocytic vesicle trafficking. Electrical signals were recorded using a surface AgCl electrode. Key Results: Mimosa leaves, pea tendrils, Venus flytraps and sundew traps all lost both their autonomous and touch-induced movements after exposure to anaesthetics. In Venus flytrap, this was shown to be due to the loss of action potentials under diethyl ether anaesthesia. The same concentration of diethyl ether immobilized pea tendrils. Anaesthetics also impeded seed germination and chlorophyll accumulation in cress seedlings. Endocytic vesicle recycling and reactive oxygen species (ROS) balance, as observed in intact Arabidopsis root apex cells, were also affected by all anaesthetics tested. Conclusions: Plants are sensitive to several anaesthetics that have no structural similarities. As in animals and humans, anaesthetics used at appropriate concentrations block action potentials and immobilize organs via effects on action potentials, endocytic vesicle recycling and ROS homeostasis. Plants emerge as ideal model objects to study general questions related to anaesthesia, as well as to serve as a suitable test system for human anaesthesia.

Immunogold-EM analysis reveal brefeldin a-sensitive clusters of auxin in Arabidopsis root apex cells.

Immunogold electron microscopy (EM) study of Arabidopsis root apices analyzed using specific IAA antibody and high-pressure freeze fixation technique allowed, for the first time, vizualization of subcellular localization of IAA in cells assembled intactly within plant tissues. Our quantitative analysis reveals that there is considerable portion of IAA gold particles that clusters within vesicles and membraneous compartments in all root apex cells. There are clear tissue-specific and developmental differences of clustered IAA in root apices. These findings have significant consequences for our understanding of this small molecule which is controlling plant growth, development and behavior.

A new hypothesis of pathogenesis based on the divorce between mitochondria and their host cells: possible relevance for Alzheimer's disease.

On the basis of not only the endosymbiotic theory of eukaryotic cell organization and evolution but also of observations of transcellular communication via Tunneling NanoTubes (TNTs), the hypothesis is put forward that when mitochondria, which were once independently living prokaryote-like organisms, are subjected to detrimental genetic, toxic, or environmental conditions, including age-related endogenous factors, they can regress towards their original independent state. At that point, they can become potentially pathogenic intruders within their eukaryotic host cell. Because of the protoplasmic disequilibrium caused by an altered, or mutated, mitochondral population, certain host cells with a minimal capacity for self-renewal, such as dopaminergic neurons, risk a loss of function and degenerate. It is also proposed that altered mitochondria, as well as their mutated mtDNA, can migrate, via TNTs, into adjacent cells. In this way, neurodegenerative states are propagated between cells (glia and/or neurons) of the Central Nervous System (CNS) and that this leads to conditions such as Alzheimer's and Parkinson's disease. This proposal finds indirect support from observations on rotenone-poisoned glioblastoma cells which have been co-cultured with non-poisoned cells. Immunocytochemical techniques revealed that mitochondria, moving along the TNTs, migrated from the poisoned cells towards the healthy cells. It has also been demonstrated by means of immunocytochemistry that, in glioblastoma cell cultures, Amyloid Precursor Protein (APP) is present in TNTs, hence it may migrate from one cell to neighbouring cells. This datum may be of high relevance for a better understanding of Alzheimer's Disease (AD) since molecular, cellular, and animal model studies have revealed that the formation of amyloid beta (Abeta) and other derivatives of the APP are key pathogenic factors in AD, causing mitochondrial dysfunction, free radical generation, oxidative damage, and inflammation. Furthermore, the present data demonstrate the presence of alpha-synuclein (alpha-syn) within TNTs, hence a similar pathogenic mechanism to the one surmised for AD, but centred on alpha-syn rather than on Abeta, may play a role in Parkinson's Disease (PD). As a matter of fact, alpha-syn can enter mitochondria and interact with complex I causing respiratory deficiency and increased oxygen free radical production. In agreement with this view, it has been demonstrated that, in comparison with normal subjects, PD patients show a significant accumulation of alpha-syn at Substantia Nigra and Striatal level, predominantly associated with the inner mitochondrial membrane,. These observations suggest that potentially neuropathogenic proteins, such as Abeta and alpha-syn, can not only diffuse via the extracellular space but also move from cell to cell also via TNTs where they can cause mitochondrial damage and cell degeneration. A mathematical model (see Appendix) is proposed to simulate the pathogenic consequences of the migration of altered mitochondria and/or of their mtDNA via TNTs. The results of the present simulation is compatible with the proposal that mutated mitochondrial agents behave as though they were infectious particles migrating through a continuum of interconnected cells.

Implications of the 'Energide' concept for communication and information handling in the central nervous system.

Recently a revision of the cell theory has been proposed, which has several implications both for physiology and pathology. This revision is founded on adapting the old Julius von Sach's proposal (1892) of the Energide as the fundamental universal unit of eukaryotic life. This view maintains that, in most instances, the living unit is the symbiotic assemblage of the cell periphery complex organized around the plasma membrane, some peripheral semi-autonomous cytosol organelles (as mitochondria and plastids, which may be or not be present), and of the Energide (formed by the nucleus, microtubules, and other satellite structures). A fundamental aspect is the proposal that the Energide plays a pivotal and organizing role of the entire symbiotic assemblage (see Appendix 1). The present paper discusses how the Energide paradigm implies a revision of the concept of the internal milieu. As a matter of fact, the Energide interacts with the cytoplasm that, in turn, interacts with the interstitial fluid, and hence with the medium that has been, classically, known as the internal milieu. Some implications of this aspect have been also presented with the help of a computational model in a mathematical Appendix 2 to the paper. Finally, relevances of the Energide concept for the information handling in the central nervous system are discussed especially in relation to the inter-Energide exchange of information.

Spatiotemporal dynamics of the electrical network activity in the root apex.

The study of electrical network systems, integrated with chemical signaling networks, is becoming a common trend in contemporary biology. Classical techniques are limited to the assessment of signals from doublets or triplets of cells at a fixed temporal bin width. At present, full characteristics of the electrical network distribution and dynamics in plant cells and tissues has not been established. Here, a 60-channels multielectrode array (MEA) is applied to study spatiotemporal characteristics of the electrical network activity of the root apex. Both intense spontaneous electrical activities and stimulation-elicited bursts of locally propagating electrical signals have been observed. Propagation of the spikes indicates the existence of excitable traveling waves in plants, similar to those observed in non-nerve electrogenic tissues of animals. Obtained data reveal synchronous electric activities of root cells emerging in a specific root apex region. The dynamic electrochemical activity of root apex cells is proposed to continuously integrate internal and external signaling for developmental adaptations in a changing environment.

Gravity: one of the driving forces for evolution.

Mechanical load is 10(3) larger for land-living than for water-living organisms. As a consequence, antigravitational material in form of compound materials like lignified cell walls in plants and mineralised bones in animals occurs in land-living organisms preferentially. Besides cellulose, pectic substances of plant cell walls seem to function as antigravitational material in early phases of plant evolution and development. A testable hypothesis including vesicular recycling processes into the tensegrity concept is proposed for both sensing of gravitational force and responding by production of antigravitational material at the cellular level.

Fine Structural Analysis of Brefeldin A-Induced Compartment Formation After High-Pressure Freeze Fixation of Maize Root Epidermis: Compound Exocytosis Resembling Cell Plate Formation during Cytokinesis.

Formation of large perinuclear brefeldin A (BFA)-induced compartments is a characteristic feature of root apex cells, but it does not occur in shoot apex cells. BFA-induced compartments have been studied mostly using low resolution fluorescence microscopy techniques. Here, we have employed a high-resolution ultrastructural method based on ultra rapid freeze fixation of samples in order to study the formation of BFA-induced compartments in intact maize root epidermis cells in detail. This approach reveals five novel findings. Firstly, plant TGN/PGN elements are not tubular networks, as generally assumed, but rather vesicular compartments. Secondly, TGN/PGN vesicles interact with one another extensively via stalk-like connections and even fuse together via bridge-like structures. Thirdly, BFA-induced compartments are formed via extensive homotypic fusions of the TGN/PGN vesicles. Fourthly, multivesicular bodies (MVBs) are present within the BFA-induced compartments. Fifthly, mitochondria and small vacuoles accummulate abundantly around the large perinuclear BFA-induced compartments.

Endocytosis and vesicle trafficking during tip growth of root hairs.

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.

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.

Actin-dependent fluid-phase endocytosis in inner cortex cells of maize root apices.

The fluorescent dye Lucifer Yellow (LY) is a well-known and widely-used marker for fluid-phase endocytosis. In this paper, both light and electron microscopy revealed that LY was internalized into transition zone cells of the inner cortex of intact maize root apices. The internalized LY was localized within tubulo-vesicular compartments invaginating from the plasma membrane at actomyosin-enriched pit-fields and individual plasmodesmata, as well as within adjacent small peripheral vacuoles. The internalization of LY was blocked by pretreating the roots with the F-actin depolymerizing drug latrunculin B, but not with the F-actin stabilizer jasplakinolide. F-actin enriched plasmodesmata and pit-fields of the inner cortex also contain abundant plant-specific unconventional class VIII myosin(s). In addition, 2,3 butanedione monoxime, a general inhibitor of myosin ATPases, partially inhibited the uptake of LY into cells of the inner cortex. Conversely, loss of microtubules did not inhibit fluid-phase endocytosis of LY into these cells. In conclusion, specialized actin- and myosin VIII-enriched membrane domains perform a tissue-specific form of fluid-phase endocytosis in maize root apices. The possible physiological relevance of this process is discussed.

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.

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.

Auxin deprivation induces a developmental switch in maize somatic embryogenesis involving redistribution of microtubules and actin filaments from endoplasmic to cortical cytoskeletal arrays.

A developmental switch from non-polar pre-embryogenic units to polarized transition units in maize embryogenic callus is caused by auxin deprivation from the culture medium. This switch is accompanied by cytoskeletal rearrangements in embryogenic cells. An immunofluorescence study revealed prominent endoplasmic microtubules and actin filament meshworks radiating from the nuclear surfaces in pre-embryogenic cells growing on medium supplemented with auxin. On the other hand, parallel-organized cortical microtubules and cortical actin filament networks are inherently associated with polarized embryogenic cells of transition units growing on medium without auxin. These results indicate that fine-tuning of the dynamic equilibrium between endoplasmic and cortical cytoskeletal arrays is important for progress in somatic embryogenesis.

Mastoparan alters subcellular distribution of profilin and remodels F-actin cytoskeleton in cells of maize root apices.

Indirect immunofluorescence localization of profilin in cells of maize root apices revealed that this abundant protein was present both in the cytoplasm and within nuclei. Nucleo-cytoplasmic partitioning of profilin exhibits tissue-specific and developmental features. Mastoparan-mediated activation of heterotrimeric G-proteins, presumably through triggering a phosphoinositide-signaling pathway based on phosphatidylinositol-4,5-bisphosphate (PIP(2)), induced relocalization of profilin from nuclei into the cytoplasm of root apex cells. In contrast, PIP(2) accumulated within nuclei of mastoparan-treated root cells. Intriguingly, cytoplasmic accumulation of profilin was associated with remodeling of F-actin arrays in root apex cells. Specifically, dense F-actin networks were dismantled and distinct actin patches became associated with the periphery of small vacuoles. On the other hand, disruption of F-actin with the G-actin sequestering agent latrunculin B does not affect the subcellular distribution of profilin or PIP(2). These data suggest that nuclear profilin can mediate a stimulus-response action on the actin cytoskeleton which is somehow linked to a phosphoinositide-signaling cascade.

Lilliputian mutant of maize lacks cell elongation and shows defects in organization of actin cytoskeleton.

The maize mutant lilliputian is characterized by miniature seedling stature, reduced cell elongation, and aberrant root anatomy. Here, we document that root cells of this mutant show several defects in the organization of actin filaments (AFs). Specifically, cells within the meristem lack dense perinuclear AF baskets and fail to redistribute AFs during mitosis. In contrast, mitotic cells of wild-type roots accumulate AFs at plasma membrane-associated domains that face the mitotic spindle poles. Both mitotic and early postmitotic mutant cells fail to assemble transverse arrays of cortical AFs, which are characteristic for wild-type root cells. In addition, early postmitotic cells show aberrant distribution of endoplasmic AF bundles that are normally organized through anchorage sites at cross-walls and nuclear surfaces. In wild-type root apices, these latter AF bundles are organized in the form of symmetrically arranged conical arrays and appear to be essential for the onset of rapid cell elongation. Exposure of wild-type and cv. Alarik maize root apices to the F-actin drugs cytochalasin D and latrunculin B mimics the phenotype of lilliputian root apices. In contrast to AFs, microtubules are more or less normally organized in root cells of lilliputian mutant. Collectively, these data suggest that the LILLIPUTIAN protein, the nature of which is still unknown, impinges on plant development via its action on the actin cytoskeleton.

Motile plant cell body: a "bug" within a "cage".

Analysis of the cytoskeleton in morphogenetically active plant cells allows us to propose a unified concept for the structural organization of eukaryotic cells. Their cytoarchitecture is determined by two principal structural complexes: nucleus-microtubule-based cell bodies ("bugs") and plasma-membrane-F-actin-based cell periphery complexes ("cages"). There are dynamic interactions between each of these entities in response to extracellular and intracellular signals. In the case of the cell body, these signals determine its polarization, rotation and migration. Interactions between cell body and cell periphery complexes determine cell growth polarity and morphogenesis throughout the eukaryotic kingdom.

Latrunculin B-induced plant dwarfism: Plant cell elongation is F-actin-dependent.

Marine macrolides latrunculins are highly specific toxins which effectively depolymerize actin filaments (generally F-actin) in all eukaryotic cells. We show that latrunculin B is effective on diverse cell types in higher plants and describe the use of this drug in probing F-actin-dependent growth and in plant development-related processes. In contrast to other eukaryotic organisms, cell divisions occurs in plant cells devoid of all actin filaments. However, the alignment of the division planes is often distorted. In addition to cell division, postembryonic development and morphogenesis also continue in the absence of F-actin. These experimental data suggest that F-actin is of little importance in the morphogenesis of higher plants, and that plants can develop more or less normally without F-actin. In contrast, F-actin turns out to be essential for cell elongation. When latrunculin B was added during germination, morphologically normal Arabidopsis and rye seedlings developed but, as a result of the absence of cell elongation, these were stunted, resembling either genetic dwarfs or environmental bonsai plants. In conclusion, F-actin is essential for the plant cell elongation, while this F-actin-dependent cell elongation is not an essential feature of plant-specific developmental programs.

Root hair formation: F-actin-dependent tip growth is initiated by local assembly of profilin-supported F-actin meshworks accumulated within expansin-enriched bulges.

Plant root hair formation is initiated when specialized elongating root epidermis cells (trichoblasts) assemble distinct domains at the plasma membrane/cell wall cell periphery complexes facing the root surface. These localities show accumulation of expansin and progressively transform into tip-growing root hair apices. Experimentation showed that trichoblasts made devoid of microtubules (MTs) were unaffected in root hair formation, whereas those depleted of F-actin by the G-actin sequestering agent latrunculin B had their root hair formation blocked after the bulge formation stage. In accordance with this, MTs are naturally depleted from early outgrowing bulges in which dense F-actin meshworks accumulate. These F-actin caps remain associated with tips of emerging and growing root hairs. Constitutive expression of the GFP-mouse talin fusion protein in transgenic Arabidopsis, which visualizes all classes of F-actin in a noninvasive mode, allowed in vivo confirmation of the presence of distinct F-actin meshworks within outgrowing bulges and at tips of young root hairs. Profilin accumulates, at both the protein and the mRNA levels, within F-actin-enriched bulges and at tips of emerging hairs. ER-based calreticulin and HDEL proteins also accumulate within outgrowing bulges and remain enriched at tips of emerging hairs. All this suggests that installation of the actin-based tip growth machinery takes place only after expansin-associated bulge formation and requires assembly of profilin-supported dynamic F-actin meshworks.