Andreas Nebenführ

Brefeldin A: Deciphering an Enigmatic Inhibitor of Secretion

The fungal macrocyclic lactone brefeldin A (BFA) has proved to be of great value as an inhibitor of protein trafficking in the endomembrane system of mammalian cells ([Sciaky et al., 1997][1]). BFA has also often been used as an inhibitor of secretion and vacuolar protein transport in plant cells,

Reevaluation of the Effects of Brefeldin A on Plant Cells Using Tobacco Bright Yellow 2 Cells Expressing Golgi-Targeted Green Fluorescent Protein and COPI Antisera

Brefeldin A (BFA) causes a block in the secretory system of eukaryotic cells by inhibiting vesicle formation at the Golgi apparatus. Although this toxin has been used in many studies, its effects on plant cells are still shrouded in controversy. We have reinvestigated the early responses of plant cells to BFA with novel tools, namely, tobacco Bright Yellow 2 (BY-2) suspension-cultured cells expressing an in vivo green fluorescent protein–Golgi marker, electron microscopy of high-pressure frozen/freeze-substituted cells, and antisera against Atγ-COP, a component of COPI coats, and AtArf1, the GTPase necessary for COPI coat assembly. The first effect of 10 μg/mL BFA on BY-2 cells was to induce in <5 min the complete loss of vesicle-forming Atγ-COP from Golgi cisternae. During the subsequent 15 to 20 min, this block in Golgi-based vesicle formation led to a series of sequential changes in Golgi architecture, the loss of distinct Golgi stacks, and the formation of an endoplasmic reticulum (ER)–Golgi hybrid compartment with stacked domains. These secondary effects appear to depend in part on stabilizing intercisternal filaments and include the continued maturation of cis- and medial cisternae into trans-Golgi cisternae, as predicted by the cisternal progression model, the shedding of trans-Golgi network cisternae, the fusion of individual Golgi cisternae with the ER, and the formation of large ER-Golgi hybrid stacks. Prolonged exposure of the BY-2 cells to BFA led to the transformation of the ER-Golgi hybrid compartment into a sponge-like structure that does not resemble normal ER. Thus, although the initial effects of BFA on plant cells are the same as those described for mammalian cells, the secondary and tertiary effects have drastically different morphological manifestations. These results indicate that, despite a number of similarities in the trafficking machinery with other eukaryotes, there are fundamental differences in the functional architecture and properties of the plant Golgi apparatus that are the cause for the unique responses of the plant secretory pathway to BFA.

The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species

BackgroundPlant genome sequencing has resulted in the identification of a large number of uncharacterized genes. To investigate these unknown gene functions, several transient transformation systems have been developed as quick and convenient alternatives to the lengthy transgenic assay. These transient assays include biolistic bombardment, protoplast transfection and Agrobacterium-mediated transient transformation, each having advantages and disadvantages depending on the research purposes.ResultsWe present a novel transient assay based on cocultivation of young Arabidopsis (Arabidopsis thaliana) seedlings with Agrobacterium tumefaciens in the presence of a surfactant which does not require any dedicated equipment and can be carried out within one week from sowing seeds to protein analysis. This Fast Agro-mediated Seedling Transformation (FAST) was used successfully to express a wide variety of constructs driven by different promoters in Arabidopsis seedling cotyledons (but not roots) in diverse genetic backgrounds. Localizations of three previously uncharacterized proteins were identified by cotransformation with fluorescent organelle markers. The FAST procedure requires minimal handling of seedlings and was also adaptable for use in 96-well plates. The high transformation efficiency of the FAST procedure enabled protein detection from eight transformed seedlings by immunoblotting. Protein-protein interaction, in this case HY5 homodimerization, was readily detected in FAST-treated seedlings with Förster resonance energy transfer and bimolecular fluorescence complementation techniques. Initial tests demonstrated that the FAST procedure can also be applied to other dicot and monocot species, including tobacco, tomato, rice and switchgrass.ConclusionThe FAST system provides a rapid, efficient and economical assay of gene function in intact plants with minimal manual handling and without dedicated device. This method is potentially ideal for future automated high-throughput analysis.

Plant N-Glycan Processing Enzymes Employ Different Targeting Mechanisms for Their Spatial Arrangement along the Secretory Pathway

The processing of N-linked oligosaccharides in the secretory pathway requires the sequential action of a number of glycosidases and glycosyltransferases. We studied the spatial distribution of several type II membrane-bound enzymes from Glycine max, Arabidopsis thaliana, and Nicotiana tabacum. Glucosidase I (GCSI) localized to the endoplasmic reticulum (ER), α-1,2 mannosidase I (ManI) and N-acetylglucosaminyltransferase I (GNTI) both targeted to the ER and Golgi, and β-1,2 xylosyltransferase localized exclusively to Golgi stacks, corresponding to the order of expected function. ManI deletion constructs revealed that the ManI transmembrane domain (TMD) contains all necessary targeting information. Likewise, GNTI truncations showed that this could apply to other type II enzymes. A green fluorescent protein chimera with ManI TMD, lengthened by duplicating its last seven amino acids, localized exclusively to the Golgi and colocalized with a trans-Golgi marker (ST52-mRFP), suggesting roles for protein–lipid interactions in ManI targeting. However, the TMD lengths of other plant glycosylation enzymes indicate that this mechanism cannot apply to all enzymes in the pathway. In fact, removal of the first 11 amino acids of the GCSI cytoplasmic tail resulted in relocalization from the ER to the Golgi, suggesting a targeting mechanism relying on protein–protein interactions. We conclude that the localization of N-glycan processing enzymes corresponds to an assembly line in the early secretory pathway and depends on both TMD length and signals in the cytoplasmic tail.

Mobile factories: Golgi dynamics in plant cells

The plant Golgi apparatus plays a central role in the synthesis of cell wall material and the modification and sorting of proteins destined for the cell surface and vacuoles. Earlier perceptions of this organelle were shaped by static transmission electron micrographs and by its biosynthetic functions. However, it has become increasingly clear that many Golgi activities can only be understood in the context of its dynamic organization. Significant new insights have been gained recently into the molecules that mediate this dynamic behavior, and how this machinery differs between plants and animals or yeast. Most notable is the discovery that plant Golgi stacks can actively move through the cytoplasm along actin filaments, an observation that has major implications for trafficking to, through and from this organelle.

Organelle Targeting of Myosin XI Is Mediated by Two Globular Tail Subdomains with Separate Cargo Binding Sites

Myosin XI are actin-based molecular motors that are thought to drive organelle movements in plants, analogous to myosin V in animals and fungi. Similar domain structure of these myosins suggests that binding to organelles may occur via the globular tail domain in both types of motors, even though sequence similarity is low. To address this hypothesis, we developed a structure homology model for the globular tail of MYA1, a myosin XI from Arabidopsis, based on the known structure of yeast myosin V (Myo2p) globular tail. This model suggested an interaction between two subdomains of the globular tail which was verified by yeast two-hybrid assay and by in vivo bimolecular fluorescence complementation (BiFC). Interface mapping demonstrated that this subdomain interaction depends critically on the C terminus of helix H6 as well as three specific residues in helices H3 and H15, consistent with the structural prediction. The reconstituted globular tails of several Arabidopsis myosin XIs in BiFC assays targeted to peroxisomes in plant cells, identifying this domain as sufficient for cargo binding. Unlike myosin V, either subdomain of myosin XI alone was targeting-competent and responsible for association with different organelles. In addition, our data suggest that organelle binding is regulated by an allosteric interaction between two tail subdomains. We conclude that the globular tail of myosin XI shares a similar structure with that of myosin V, but has evolved plant-specific cargo binding mechanisms.

Vesicle traffic in the endomembrane system: a tale of COPs, Rabs and SNAREs

Recent years have seen remarkable progress in our understanding of the endomembrane system of plants. A large number of genes and proteins that are involved in membrane exchange between the different compartments of this system have been identified on the basis of their similarity to animal and yeast homologs. These proteins indicate that the endomembrane system in plants functions in essentially the same way as those in other eukaryotes. However, a growing number of examples demonstrate that the dynamic interplay between membrane-exchange proteins can be regulated differently in plant cells. Novel tools and a better understanding of the molecular effects of the inhibitor brefeldin A are helping to unravel these plant-specific adaptations.

The diageotropica mutation alters auxin induction of a subset of the Aux/IAA gene family in tomato.

The diageotropica (dgt) mutation has been proposed to affect either auxin perception or responsiveness in tomato plants. It has previously been demonstrated that the expression of one member of the Aux/IAA family of auxin-regulated genes is reduced in dgt plants. Here, we report the cloning of ten new members of the tomato Aux/IAA family by PCR amplification based on conserved protein domains. All of the gene family members except one (LeIAA7) are expressed in etiolated tomato seedlings, although they demonstrate tissue specificity (e.g. increased expression in hypocotyls vs. roots) within the seedling. The wild-type auxin-response characteristics of the expression of these tomato LeIAA genes are similar to those previously described for Aux/IAA family members in Arabidopsis. In dgt seedlings, auxin stimulation of gene expression was reduced in only a subset of LeIAA genes (LeIAA5, 8, 10, and 11), with the greatest reduction associated with those genes with the strongest wild-type response to auxin. The remaining LeIAA genes tested exhibited essentially the same induction levels in response to the hormone in both dgt and wild-type hypocotyls. These results confirm that dgt plants can perceive auxin and suggest that a specific step in early auxin signal transduction is disrupted by the dgt mutation.

Dynamic rearrangements of transvacuolar strands in BY-2 cells imply a role of myosin in remodeling the plant actin cytoskeleton

Summary.Plant cells typically contain a large central vacuole that confines the cytoplasm and organelles to the periphery of the cell and the vicinity of the nucleus. These two domains are often connected by transvacuolar strands (TVS), thin tubular structures that traverse the vacuole. The TVS are thought to act as important transport routes for the distribution of organelles and metabolites, and also to play a role in the positioning of the nucleus. Most TVS depend on internal actin filaments for their existence, and rearrangements of TVS can therefore indicate modifications in the actin cytoskeleton. In this study we describe time-lapse observations of tobacco BY-2 suspension-cultured cells that document the dynamic behavior of TVS. The TVS formed, branched, and collapsed, and their attachment points in the nuclear or cortical cytoplasm, as well as on other TVS, moved around. These dynamic rearrangements were inhibited within 5 min by the myosin inhibitor 2,3-butanedione monoxime (BDM). In particular, the movements of TVS attachment points and the variations in TVS length were significantly reduced in the presence of the drug. Similarly, movements of the nucleus were reduced by two thirds in BDM-treated cells. The number of TVS, together with the number of attachment and branch points, also dropped during BDM treatment. All effects of BDM on TVS dynamics were reversible upon removal of the drug. These results suggest a role for myosin motors in the rearrangement of TVS, which is likely to occur through their interaction with actin filaments.

The Tail that Wags the Dog: The Globular Tail Domain Defines the Function of Myosin V/XI

Actin‐based organelle movements are driven by the related multifunctional myosin motors of class V in animals and fungi and class XI in plants. The versatility of these motors depends critically on their C‐terminal globular tail domain that allows them to bind to a broad variety of cargo molecules. Regulation of this motor–cargo attachment is frequently employed to modulate organelle movement. While the overall structure of the cargo‐binding globular tail appears to be conserved between myosin V and XI, it has become apparent that the motor–cargo interactions differ widely even within a single organism and involve protein complexes with different architecture and completely unrelated protein domains. At the same time, indirect evidence suggests that adaptor or receptor dimerization could facilitate efficient myosin capture. Comparison of myosin V and XI across the large evolutionary distance between animals and plants will likely reveal more fundamental insights into these important motors.