Jozef Šamaj

Mrs2p is an essential component of the major electrophoretic Mg2+ influx system in mitochondria

Steady‐state concentrations of mitochondrial Mg2+ previously have been shown to vary with the expression of Mrs2p, a component of the inner mitochondrial membrane with two transmembrane domains. While its structural and functional similarity to the bacterial Mg2+ transport protein CorA suggested a role for Mrs2p in Mg2+ influx into the organelle, other functions in cation homeostasis could not be excluded. Making use of the fluorescent dye mag‐fura 2 to measure free Mg2+ concentrations continuously, we describe here a high capacity, rapid Mg2+ influx system in isolated yeast mitochondria, driven by the mitochondrial membrane potential Δψ and inhibited by cobalt(III)hexaammine. Overexpression of Mrs2p increases influx rates 5‐fold, while the deletion of the MRS2 gene abolishes this high capacity Mg2+ influx. Mg2+ efflux from isolated mitochondria, observed with low Δψ only, also requires the presence of Mrs2p. Cross‐linking experiments revealed the presence of Mrs2p‐containing complexes in the mitochondrial membrane, probably constituting Mrs2p homo‐ oligomers. Taken together, these findings characterize Mrs2p as the first molecularly identified metal ion channel protein in the inner mitochondrial membrane.

Involvement of the mitogen-activated protein kinase SIMK in regulation of root hair tip growth

Mitogen‐activated protein kinases (MAPKs) are involved in stress signaling to the actin cytoskeleton in yeast and animals. We have analyzed the function of the stress‐activated alfalfa MAP kinase SIMK in root hairs. In epidermal cells, SIMK is predominantly nuclear. During root hair formation, SIMK was activated and redistributed from the nucleus into growing tips of root hairs possessing dense F‐actin meshworks. Actin depolymerization by latrunculin B resulted in SIMK relocation to the nucleus. Conversely, upon actin stabilization with jasplakinolide, SIMK co‐localized with thick actin cables in the cytoplasm. Importantly, latrunculin B and jasplakinolide were both found to activate SIMK in a root‐derived cell culture. Loss of tip‐focused SIMK and actin was induced by the MAPK kinase inhibitor UO 126 and resulted in aberrant root hairs. UO 126 inhibited targeted vesicle trafficking and polarized growth of root hairs. In contrast, overexpression of gain‐of‐function SIMK induced rapid tip growth of root hairs and could bypass growth inhibition by UO 126. These data indicate that SIMK plays a crucial role in root hair tip growth.

Lipid microdomain polarization is required for NADPH oxidase-dependent ROS signaling in Picea meyeri pollen tube tip growth

The polarization of sterol-enriched lipid microdomains has been linked to morphogenesis and cell movement in diverse cell types. Recent biochemical evidence has confirmed the presence of lipid microdomains in plant cells; however, direct evidence for a functional link between these microdomains and plant cell growth is still lacking. Here, we reported the involvement of lipid microdomains in NADPH oxidase (NOX)-dependent reactive oxygen species (ROS) signaling in Picea meyeri pollen tube growth. Staining with di-4-ANEPPDHQ or filipin revealed that sterol-enriched microdomains were polarized to the growing tip of the pollen tube. Sterol sequestration with filipin disrupted membrane microdomain polarization, depressed tip-based ROS formation, dissipated tip-focused cytosolic Ca(2+) gradient and thereby arrested tip growth. NOX clustered at the growing tip, and corresponded with the ordered membrane domains. Immunoblot analysis and native gel assays demonstrated that NOX was partially associated with detergent-resistant membranes and, furthermore, that NOX in a sterol-dependent fashion depends on membrane microdomains for its enzymatic activity. In addition, in vivo time-lapse imaging revealed the coexistence of a steep tip-high apical ROS gradient and subapical ROS production, highlighting the reported signaling role for ROS in polar cell growth. Our results suggest that the polarization of lipid microdomains to the apical plasma membrane, and the inclusion of NOX into these domains, contribute, at least in part, to the ability to grow in a highly polarized manner to form pollen tubes.

Arabidopsis Homologs of Nucleus- and Phragmoplast-Localized Kinase 2 and 3 and Mitogen-Activated Protein Kinase 4 Are Essential for Microtubule Organization

Arabidopsis mutants defective in MAPK signaling were found to have aberrant microtubule organization and cell growth. This study shows that two mitogen-activated protein kinase kinase kinase isoforms, mitogen-activated protein kinase 4 and microtubule-associated protein 65, play a role in the organization of cortical microtubules. A double homozygous recessive mutant in the Arabidopsis thaliana homologs of nucleus- and phragmoplast-localized kinase 2 (ANP2) and 3 (ANP3) genes and a homozygous recessive mutant in the mitogen-activated protein kinase 4 (MPK4) gene of Arabidopsis exhibit deficiencies in the overall microtubule (MT) organization, which result in abnormal cell growth patterns, such as branching of root hairs and swelling of diffusely growing epidermal cells. Genetic, pharmacological, molecular, cytological, and biochemical analyses show that the major underlying mechanism for these phenotypes is excessive MT stabilization manifested in both mutants as heavy MT bundling, disorientation, and drug stability. The above defects in MAPK signaling result in the adverse regulation of members of the microtubule-associated protein (MAP65) protein family, including strongly diminished phosphorylation of MAP65-1. These data suggest that ANP2/ANP3, MPK4, and the microtubule-associated protein MAP65-1, a putative target of MPK4 signaling, are all essential for the proper organization of cortical microtubules in Arabidopsis epidermal cells.

Extracellular matrix surface network of embryogenic units of friable maize callus contains arabinogalactan-proteins recognized by monoclonal antibody JIM4

Abstract Embryogenic units of friable maize callus are formed as globular or oblong packets of tightly associated meristematic cells. These units are surrounded by conspicuous cell walls visible in light microscopy after staining with basic fuchsin. Transmission electron microscopy revealed that embryogenic cells are rich in endoplasmic reticulum, polysomes and small protein bodies, and that the outermost layer of their cell walls is composed of fibrillar material. Electron microscopy has also shown that this material covers the surface of embryogenic cells as a distinct layer which we denote as extracellular matrix surface network (ECMSN). Employing histochemical staining with β-glucosyl Yariv phenylglycoside, we localized arabinogalactan-proteins (AGPs) to the outer cell walls of embryogenic units including ECMSN. The most prominent staining was found in cell-cell junction domains. Large non-embryogenic callus cells were not stained with this AGP-specific dye. Immunofluorescence and silver-enhanced immunogold labelling using monoclonal antibody JIM4 has shown that the ECMSN of embryogenic cells is equipped with JIM4 epitope, while non-embryogenic callus cells are devoid of this epitope. We propose that some specific AGPs of the ECMSN might be relevant for cell-cell adhesion and recognition of embryogenic cells during early embryogenic stages, and that the JIM4 antibody can serve as an early marker of embryogenic competence in maize callus culture.

Nitric oxide modulates the influx of extracellular Ca2+ and actin filament organization during cell wall construction in Pinus bungeana pollen tubes

Nitric oxide (NO) plays a key role in many physiological processes in plants, including pollen tube growth. Here, effects of NO on extracellular Ca(2+) flux and microfilaments during cell wall construction in Pinus bungeana pollen tubes were investigated. Extracellular Ca(2+) influx, the intracellular Ca(2+) gradient, patterns of actin organization, vesicle trafficking and cell wall deposition upon treatment with the NO donor S-nitroso-N-acetylpenicillamine (SNAP), the NO synthase (NOS) inhibitor N(omega)-nitro-L-arginine (L-NNA) or the NO scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) were analyzed. SNAP enhanced pollen tube growth in a dose-dependent manner, while L-NNA and cPTIO inhibited NO production and arrested pollen tube growth. Noninvasive detection and microinjection of a Ca(2+) indicator revealed that SNAP promoted extracellular Ca(2+) influx and increased the steepness of the tip-focused Ca(2+) gradient, while cPTIO and L-NNA had the opposite effect. Fluorescence labeling indicated that SNAP, cPTIO and L-NNA altered actin organization, which subsequently affected vesicle trafficking. Finally, the configuration and/or distribution of cell wall components such as pectins and callose were significantly altered in response to L-NNA. Fourier transform infrared (FTIR) microspectroscopy confirmed the changes in the chemical composition of walls. Our results indicate that NO affects the configuration and distribution of cell wall components in pollen tubes by altering extracellular Ca(2+) influx and F-actin organization.

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.

Immunolocalization of LM2 arabinogalactan protein epitope associated with endomembranes of plant cells

SummaryArabinogalactan proteins (AGPs) are proteoglycans detected in high amounts at plant cell surfaces; however, details of their subcellular localization are largely unknown. Immunolocalization studies with the anti-AGP monoclonal antibody LM2 have indicated that this AGP epitope is associated with secretory compartments such as endoplasmic reticulum and Golgi apparatus within plant cells actively producing and secreting AGPs. The LM2 epitope contains a β-linked glucuronic acid residue and occurs in the polysaccharide moiety of AGPs. We have localized this AGP epitope also to the tonoplast and to cytoplasmic strands. Endomembrane association of AGPs was confirmed with two other monoclonal antibodies, JIM13 and MAC207, both reacting with carbohydrate AGP epitopes containing GlcpA-β(1→3)-D-GalpA-α(1→2)-L-Rha residues. Immunocytochemistry is supported by biochemical analysis which shows that LM2 reacts with the microsomal fraction and also with low-molecular-weight material of the detergent phase after Triton X-114 phase separation prepared from maize roots. Our results indicate that some AGP epitopes are closely associated with endomembranes.

Actin Turnover Is Required for Myosin-Dependent Mitochondrial Movements in Arabidopsis Root Hairs

Background Previous studies have shown that plant mitochondrial movements are myosin-based along actin filaments, which undergo continuous turnover by the exchange of actin subunits from existing filaments. Although earlier studies revealed that actin filament dynamics are essential for many functions of the actin cytoskeleton, there are little data connecting actin dynamics and mitochondrial movements. Methodology/Principal Findings We addressed the role of actin filament dynamics in the control of mitochondrial movements by treating cells with various pharmaceuticals that affect actin filament assembly and disassembly. Confocal microscopy of Arabidopsis thaliana root hairs expressing GFP-FABD2 as an actin filament reporter showed that mitochondrial distribution was in agreement with the arrangement of actin filaments in root hairs at different developmental stages. Analyses of mitochondrial trajectories and instantaneous velocities immediately following pharmacological perturbation of the cytoskeleton using variable-angle evanescent wave microscopy and/or spinning disk confocal microscopy revealed that mitochondrial velocities were regulated by myosin activity and actin filament dynamics. Furthermore, simultaneous visualization of mitochondria and actin filaments suggested that mitochondrial positioning might involve depolymerization of actin filaments on the surface of mitochondria. Conclusions/Significance Base on these results we propose a mechanism for the regulation of mitochondrial speed of movements, positioning, and direction of movements that combines the coordinated activity of myosin and the rate of actin turnover, together with microtubule dynamics, which directs the positioning of actin polymerization events.

Comparison of Cryofixation and Aldehyde Fixation for Plant Actin Immunocytochemistry: Aldehydes do not Destroy F-actin

For walled plant cells, the immunolocalization of actin microfilaments, also known as F-actin, has proved to be much trickier than that of microtubules. These difficulties are commonly attributed to the high sensitivity of F-actin to aldehyde fixatives. Therefore, most plant studies have been accomplished using fluorescent phallotoxins in fresh tissues. Nevertheless, concerns regarding the questionable ability of phallotoxins to bind the whole complement of F-actin necessitate further optimization of actin immunofluorescence methods. We have compared two procedures: (1) formaldehyde fixation and (2) rapid freezing and freeze substitution (cryofixation), both followed by embedding in low-melting polyester wax. Actin immunofluorescence in sections of garden cress (Lepidium sativum L.) root gave similar results with both methods. The compatibility of aldehydes with actin immunodetection was further confirmed by the freeze-shattering technique that does not require embedding after aldehyde fixation. It appears that rather than aldehyde fixation, some further steps in the procedures used for actin visualization are critical for preserving F-actin. Wax embedding, combined with formaldehyde fixation, has proved to be also suitable for the detection of a wide range of other antigens.