Three-dimensional analysis of syncytial-type cell plates during endosperm cellularization visualized by high resolution electron tomography.

The three-dimensional architecture of syncytial-type cell plates in the endosperm of Arabidopsis has been analyzed at approximately 6-nm resolution by means of dual-axis high-voltage electron tomography of high-pressure frozen/freeze-substituted samples. Mini-phragmoplasts consisting of microtubule clusters assemble between sister and nonsister nuclei. Most Golgi-derived vesicles appear connected to these microtubules by two molecules that resemble kinesin-like motor proteins. These vesicles fuse with each other to form hourglass-shaped intermediates, which become wide (approximately 45 nm in diameter) tubules, the building blocks of wide tubular networks. New mini-phragmoplasts also are generated de novo around the margins of expanding wide tubular networks, giving rise to new foci of cell plate growth, which later become integrated into the main cell plate. Spiral-shaped rings of the dynamin-like protein ADL1A constrict but do not fission the wide tubules at irregular intervals. These rings appear to maintain the tubular geometry of the network. The wide tubular network matures into a convoluted fenestrated sheet in a process that involves increases of 45 and 130% in relative membrane surface area and volume, respectively. The proportionally larger increase in volume appears to reflect callose synthesis. Upon fusion with the parental plasma membrane, the convoluted fenestrated sheet is transformed into a planar fenestrated sheet. This transformation involves clathrin-coated vesicles that reduce the relative membrane surface area and volume by approximately 70%. A ribosome-excluding matrix encompasses the cell plate membranes from the fusion of the first vesicles until the onset of the planar fenestrated sheet formation. We postulate that this matrix contains the molecules that mediate cell plate assembly.

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.

Amyloplast sedimentation dynamics in maize columella cells support a new model for the gravity-sensing apparatus of roots.

Quantitative analysis of statolith sedimentation behavior was accomplished using videomicroscopy of living columella cells of corn (Zea mays) roots, which displayed no systematic cytoplasmic streaming. Following 90 degrees rotation of the root, the statoliths moved downward along the distal wall and then spread out along the bottom with an average velocity of 1.7 microm min(-1). When statolith trajectories traversed the complete width or length of the cell, they initially moved horizontally toward channel-initiation sites and then moved vertically through the channels to the lower side of the reoriented cell where they again dispersed. These statoliths exhibited a significantly lower average velocity than those sedimenting on distal-to-side trajectories. In addition, although statoliths undergoing distal-to-side sedimentation began at their highest velocity and slowed monotonically as they approached the lower cell membrane, statoliths crossing the cell's central region remained slow initially and accelerated to maximum speed once they reached a channel. The statoliths accelerated sooner, and the channeling effect was less pronounced in roots treated with cytochalasin D. Parallel ultrastructural studies of high-pressure frozen-freeze-substituted columella cells suggest that the low-resistance statolith pathway in the cell periphery corresponds to the sharp interface between the endoplasmic reticulum (ER)-rich cortical and the ER-devoid central region of these cells. The central region is also shown to contain an actin-based cytoskeletal network in which the individual, straight, actin-like filaments are randomly distributed. To explain these findings as well as the results of physical simulation experiments, we have formulated a new, tensegrity-based model of gravity sensing in columella cells. This model envisages the cytoplasm as pervaded by an actin-based cytoskeletal network that is denser in the ER-devoid central region than in the ER-rich cell cortex and is linked to stretch receptors in the plasma membrane. Sedimenting statoliths are postulated to produce a directional signal by locally disrupting the network and thereby altering the balance of forces acting on the receptors in different plasma membrane regions.

Nodal endoplasmic reticulum, a specialized form of endoplasmic reticulum found in gravity-sensing root tip columella cells.

The endoplasmic reticulum (ER) of columella root cap cells has been postulated to play a role in gravity sensing. We have re-examined the ultrastructure of columella cells in tobacco (Nicotiana tabacum) root tips preserved by high-pressure freezing/freeze-substitution techniques to gain more precise information about the organization of the ER in such cells. The most notable findings are: the identification of a specialized form of ER, termed "nodal ER," which is found exclusively in columella cells; the demonstration that the bulk of the ER is organized in the form of a tubular network that is confined to a peripheral layer under the plasma membrane; and the discovery that this ER-rich peripheral region excludes Golgi stacks, vacuoles, and amyloplasts but not mitochondria. Nodal ER domains consist of an approximately 100-nm-diameter central rod composed of oblong subunits to which usually seven sheets of rough ER are attached along their margins. These domains form patches at the interface between the peripheral ER network and the ER-free central region of the cells, and they occupy defined positions within central and flanking columella cells. Over one-half of the nodal ER domains are located along the outer tangential walls of the flanking cells. Cytochalasin D and latrunculin A cause an increase in size and a decrease in numbers of nodal ER domains. We postulate that the nodal ER membranes locally modulate the gravisensing signals produced by the sedimenting amyloplasts, and that the confinement of all ER membranes to the cell periphery serves to enhance the sedimentability of the amyloplasts in the central region of columella cells.

Correlation between persistent forms of zeaxanthin-dependent energy dissipation and thylakoid protein phosphorylation.

High light stress induced not only a sustained form of xanthophyll cycle-dependent energy dissipation but also sustained thylakoid protein phosphorylation. The effect of protein phosphatase inhibitors (fluoride and molybdate ions) on recovery from a 1-h exposure to a high PFD was examined in leaf discs of Parthenocissus quinquefolia (Virginia creeper). Inhibition of protein dephosphorylation induced zeaxanthin retention and sustained energy dissipation (NPQ) upon return to low PFD for recovery, but had no significant effects on pigment and Chl fluorescence characteristics under high light exposure. In addition, whole plants of Monstera deliciosa and spinach grown at low to moderate PFDs were transferred to high PFDs, and thylakoid protein phosphorylation pattern (assessed with anti-phosphothreonine antibody) as well as pigment and Chl fluorescence characteristics were examined over several days. A correlation was obtained between dark-sustained D1/D2 phosphorylation and dark-sustained zeaxanthin retention and maintenance of PS II in a state primed for energy dissipation in both species. The degree of these dark-sustained phenomena was more pronounced in M. deliciosa compared with spinach. Moreover, M. deliciosa but not spinach plants showed unusual phosphorylation patterns of Lhcb proteins with pronounced dark-sustained Lhcb phosphorylation even under low PFD growth conditions. Subsequent to the transfer to a high PFD, dark-sustained Lhcb protein phosphorylation was further enhanced. Thus, phosphorylation patterns of D1/D2 and Lhcb proteins differed from each other as well as among plant species. The results presented here suggest an association between dark-sustained D1/D2 phosphorylation and sustained retention of zeaxanthin and energy dissipation (NPQ) in light-stressed, and particularly 'photoinhibited', leaves. Functional implications of these observations are discussed.

Cytokinesis in flowering plants: more than one way to divide a cell.

Several different cytokinetic mechanisms operate in flowering plants. During 'conventional' somatic cytokinesis, the mitotic spindle remnants give rise to a phragmoplast that serves as a framework for the assembly of the cell plate. Cell plates fuse with the parental plasma membrane at specific cortical sites previously defined by the preprophase band of microtubules. In nuclear endosperms, meiocytes, and gametophytic cells, cytokinesis occurs without preprophase bands. The position of the new cell walls is determined instead by interacting arrays of microtubules that radiate from the nuclear envelope surfaces. The nuclear cytoplasmic domains defined by these microtubule arrays demarcate the boundaries of the future cells. Recent studies have provided new insights into the ultrastructural similarities and dissimilarities between conventional and non-conventional cytokinesis. Numerous proteins have also been localized to cytokinesis-related cytoskeletal arrays and cell plates but the functions of most of them have yet to be elucidated.

Redistribution of Golgi stacks and other organelles during mitosis and cytokinesis in plant cells.

We have followed the redistribution of Golgi stacks during mitosis and cytokinesis in living tobacco BY-2 suspension culture cells by means of a green fluorescent protein-tagged soybean alpha-1,2 mannosidase, and correlated the findings to cytoskeletal rearrangements and to the redistribution of endoplasmic reticulum, mitochondria, and plastids. In preparation for cell division, when the general streaming of Golgi stacks stops, about one-third of the peripheral Golgi stacks redistributes to the perinuclear cytoplasm, the phragmosome, thereby reversing the ratio of interior to cortical Golgi from 2:3 to 3:2. During metaphase, approximately 20% of all Golgi stacks aggregate in the immediate vicinity of the mitotic spindle and a similar number becomes concentrated in an equatorial region under the plasma membrane. This latter localization, the "Golgi belt," accurately predicts the future site of cell division, and thus forms a novel marker for this region after the disassembly of the preprophase band. During telophase and cytokinesis, many Golgi stacks redistribute around the phragmoplast where the cell plate is formed. At the end of cytokinesis, the daughter cells have very similar Golgi stack densities. The sites of preferential Golgi stack localization are specific for this organelle and largely exclude mitochondria and plastids, although some mitochondria can approach the phragmoplast. This segregation of organelles is first observed in metaphase and persists until completion of cytokinesis. Maintenance of the distinct localizations does not depend on intact actin filaments or microtubules, although the mitotic spindle appears to play a major role in organizing the organelle distribution patterns. The redistribution of Golgi stacks during mitosis and cytokinesis is consistent with the hypothesis that Golgi stacks are repositioned to ensure equal partitioning between daughter cells as well as rapid cell plate assembly.

Syncytial-type cell plates: a novel kind of cell plate involved in endosperm cellularization of Arabidopsis.

Cell wall formation in the syncytial endosperm of Arabidopsis was studied by using high-pressure-frozen/freeze-substituted developing seeds and immunocytochemical techniques. The endosperm cellularization process begins at the late globular embryo stage with the synchronous organization of small clusters of oppositely oriented microtubules ( approximately 10 microtubules in each set) into phragmoplast-like structures termed mini-phragmoplasts between both sister and nonsister nuclei. These mini-phragmoplasts produce a novel kind of cell plate, the syncytial-type cell plate, from Golgi-derived vesicles approximately 63 nm in diameter, which fuse by way of hourglass-shaped intermediates into wide ( approximately 45 nm in diameter) tubules. These wide tubules quickly become coated and surrounded by a ribosome-excluding matrix; as they grow, they branch and fuse with each other to form wide tubular networks. The mini-phragmoplasts formed between a given pair of nuclei produce aligned tubular networks that grow centrifugally until they merge into a coherent wide tubular network with the mini-phragmoplasts positioned along the network margins. The individual wide tubular networks expand laterally until they meet and eventually fuse with each other at the sites of the future cell corners. Transformation of the wide tubular networks into noncoated, thin ( approximately 27 nm in diameter) tubular networks begins at multiple sites and coincides with the appearance of clathrin-coated budding structures. After fusion with the syncytial cell wall, the thin tubular networks are converted into fenestrated sheets and cell walls. Immunolabeling experiments show that the cell plates and cell walls of the endosperm differ from those of the embryo and maternal tissue in two features: their xyloglucans lack terminal fucose residues on the side chain, and callose persists in the cell walls after the cell plates fuse with the parental plasma membrane. The lack of terminal fucose residues on xyloglucans suggests that these cell wall matrix molecules serve both structural and storage functions.

Columella cells revisited: novel structures, novel properties, and a novel gravisensing model.

A hundred years of research has not produced a clear understanding of the mechanism that transduces the energy associated with the sedimentation of starch-filled amyloplast statoliths in root cap columella cells into a growth response. Most models postulate that the statoliths interact with microfilaments (MF) to transmit signals to the plasma membrane (or ER), or that sedimentation onto these organelles produces the signals. However, no direct evidence for statolith-MF links has been reported, and no asymmetric structures of columella cells have been identified that might explain how a root turned by 90 degrees knows which side is up. To address these and other questions, we have (1) quantitatively examined the effects of microgravity on the size, number, and spatial distribution of statoliths; (2) re-evaluated the ultrastructure of columella cells in high-pressure frozen/freeze-substituted roots; and (3) followed the sedimentation dynamics of statolith movements in reoriented root tips. The findings have led to the formulation of a new model for the gravity-sensing apparatus of roots, which envisages the cytoplasm pervaded by an actin-based cytoskeletal network. This network is denser in the ER-devoid central region of the cell than in the ER-rich cell cortex and is coupled to receptors in the plasma membrane. Statolith sedimentation is postulated to disrupt the network and its links to receptors in some regions of the cell cortex, while allowing them to reform in other regions and thereby produce a directional signal.

Stop-and-go movements of plant Golgi stacks are mediated by the acto-myosin system.

The Golgi apparatus in plant cells consists of a large number of independent Golgi stack/trans-Golgi network/Golgi matrix units that appear to be randomly distributed throughout the cytoplasm. To study the dynamic behavior of these Golgi units in living plant cells, we have cloned a cDNA from soybean (Glycine max), GmMan1, encoding the resident Golgi protein alpha-1,2 mannosidase I. The predicted protein of approximately 65 kD shows similarity of general structure and sequence (45% identity) to class I animal and fungal alpha-1,2 mannosidases. Expression of a GmMan1::green fluorescent protein fusion construct in tobacco (Nicotiana tabacum) Bright Yellow 2 suspension-cultured cells revealed the presence of several hundred to thousands of fluorescent spots. Immuno-electron microscopy demonstrates that these spots correspond to individual Golgi stacks and that the fusion protein is largely confined to the cis-side of the stacks. In living cells, the stacks carry out stop-and-go movements, oscillating rapidly between directed movement and random "wiggling." Directed movement (maximal velocity 4.2 microm/s) is related to cytoplasmic streaming, occurs along straight trajectories, and is dependent upon intact actin microfilaments and myosin motors, since treatment with cytochalasin D or butanedione monoxime blocks the streaming motion. In contrast, microtubule-disrupting drugs appear to have a small but reproducible stimulatory effect on streaming behavior. We present a model that postulates that the stop-and-go motion of Golgi-trans-Golgi network units is regulated by "stop signals" produced by endoplasmic reticulum export sites and locally expanding cell wall domains to optimize endoplasmic reticulum to Golgi and Golgi to cell wall trafficking.

Golgi structure in three dimensions: functional insights from the normal rat kidney cell.

Three-dimensional reconstructions of portions of the Golgi complex from cryofixed, freeze-substituted normal rat kidney cells have been made by dual-axis, high-voltage EM tomography at approximately 7-nm resolution. The reconstruction shown here ( approximately 1 x 1 x 4 microm3) contains two stacks of seven cisternae separated by a noncompact region across which bridges connect some cisternae at equivalent levels, but none at nonequivalent levels. The rest of the noncompact region is filled with both vesicles and polymorphic membranous elements. All cisternae are fenestrated and display coated buds. They all have about the same surface area, but they differ in volume by as much as 50%. The trans-most cisterna produces exclusively clathrin-coated buds, whereas the others display only nonclathrin coated buds. This finding challenges traditional views of where sorting occurs within the Golgi complex. Tubules with budding profiles extend from the margins of both cis and trans cisternae. They pass beyond neighboring cisternae, suggesting that these tubules contribute to traffic to and/or from the Golgi. Vesicle-filled "wells" open to both the cis and lateral sides of the stacks. The stacks of cisternae are positioned between two types of ER, cis and trans. The cis ER lies adjacent to the ER-Golgi intermediate compartment, which consists of discrete polymorphic membranous elements layered in front of the cis-most Golgi cisterna. The extensive trans ER forms close contacts with the two trans-most cisternae; this apposition may permit direct transfer of lipids between ER and Golgi membranes. Within 0.2 microm of the cisternae studied, there are 394 vesicles (8 clathrin coated, 190 nonclathrin coated, and 196 noncoated), indicating considerable vesicular traffic in this Golgi region. Our data place structural constraints on models of trafficking to, through, and from the Golgi complex.

Early root cap development and graviresponse in white clover (Trifolium repens) grown in space and on a two-axis clinostat.

White clover (Trifolium repens) was germinated and grown in microgravity aboard the Space Shuttle (STS-60, 1994; STS-63, 1995), on Earth in stationary racks and in a slow-rotating two-axis clinostat. The objective of this study was to determine if normal root cap development and early plant gravity responses were dependent on gravitational cues. Seedlings were germinated in space and chemically fixed in orbit after 21, 40, and 72 h. Seedlings 96 h old were returned viable to earth. Germination and total seedling length were not dependent on gravity treatment. In space-flown seedlings, the number of cell stories in the root cap and the geometry of central columella cells did not differ from those of the Earth-grown seedlings. The root cap structure of clinorotated plants appeared similar to that of seedlings from microgravity, with the exception of three-day rotated plants, which displayed significant cellular damage in the columella region. Nuclear polarity did not depend on gravity; however, the positions of amyloplasts in the central columella cells were dependent on both the gravity treatment and the age of the seedlings. Seedlings from space, returned viable to earth, responded to horizontal stimulation as did 1 g controls, but seedlings rotated on the clinostat for the same duration had a reduced curvature response. This study demonstrates that initial root cap development is insensitive to either chronic clinorotation or microgravity. Soon after differentiation, however, clinorotation leads to loss of normal root cap structure and plant graviresponse while microgravity does not.

Auxin deprivation induces synchronous Golgi differentiation in suspension-cultured tobacco BY-2 cells.

To date, the lack of a method for inducing plant cells and their Golgi stacks to differentiate in a synchronous manner has made it difficult to characterize the nature and extent of Golgi retailoring in biochemical terms. Here we report that auxin deprivation can be used to induce a uniform population of suspension-cultured tobacco (Nicotiana tabacum cv BY-2) cells to differentiate synchronously during a 4-d period. Upon removal of auxin, the cells stop dividing, undergo elongation, and differentiate in a manner that mimics the formation of slime-secreting epidermal and peripheral root-cap cells. The morphological changes to the Golgi apparatus include a proportional increase in the number of trans-Golgi cisternae, a switch to larger-sized secretory vesicles that bud from the trans-Golgi cisternae, and an increase in osmium staining of the secretory products. Biochemical alterations include an increase in large, fucosylated, mucin-type glycoproteins, changes in the types of secreted arabinogalactan proteins, and an increase in the amounts and types of molecules containing the peripheral root-cap-cell-specific epitope JIM 13. Taken together, these findings support the hypothesis that auxin deprivation can be used to induce tobacco BY-2 cells to differentiate synchronously into mucilage-secreting cells.

Cellular responses to gravity: extracellular, intracellular and in-between.

Our understanding of gravitational effects (inertial effects in the vicinity of 1 x g) on cells has matured to a stage at which it is possible to define, on the basis of experimental evidence, extracellular effects on small cells and intracellular effects on eukaryotic gravisensing cells. Yet undetermined is the nature of response, if any, of those classes of cells that are not governed solely by extracellular physical events (as are prokaryotes) and are devoid of obvious mechanical devices for sensing inertial forces (such as those possessed by certain plant cells and sensory cells of animals). This "in-between" class of cells needs to be understood on the basis of the combination of intracellular and extracellular gravity-dependent processes that govern experimentally-measurable variables that are relevant to the cell's responses to modified inertial forces. The forces that certain cell types generate or respond to are therefore compared to those imposed by approximately 1 x g in the context of cytoskeletal action and symmetry-breaking pathways.

The plant ER: a dynamic organelle composed of a large number of discrete functional domains.

The endoplasmic reticulum (ER) of plants is comprised of a three-dimensional network of continuous tubules and sheets that underlies the plasma membrane, courses through the cytoplasm, and links up with the nuclear envelope. Aside from discussing the dynamic properties of this versatile and adaptable organelle, the review highlights the structure and the functional properties of 16 types of morphologically defined ER membrane domains. Owing to their labile or transient nature, several of these domains can only be visualized reliably through the use of ultrarapid freezing techniques. The ER domains discussed are: the lamin receptor domain; the nuclear pores; the nuclear envelope-ER gates, the microtubule nucleation domains; the protein and oil body-forming domains; the vacuole-forming ER; the actin-binding, the plasma membrane-anchoring and the vacuole and mitochondrion-attachment domains; the lipid recycling ER cisternae and the plasmodesmata. Preliminary evidence suggests that this list will have to be expanded in the near future. Understanding the assembly, the functional roles, and the developmental regulation of these domains has implications both for understanding cell structure and function, and for exploiting plants for agricultural and biotechnological purposes.

Xyloglucan sidechains modulate binding to cellulose during in vitro binding assays as predicted by conformational dynamics simulations.

Cross-links between cellulose microfibrils and xyloglucan (XG) molecules play a major role in defining the structural properties of plant cell walls and the regulation of growth and development of dicotyledonous plants. How these cross-links are established and how they are regulated has yet to be determined. In a previous study, preliminary data were presented which suggested that the different sidechains of XG may play a role in controlling cellulose microfibril-XG interactions. In this study, this question is addressed directly by analyzing to what extent the different sidechains of pea cell wall XG and nasturtium seed storage XG affect their binding to cellulose microfibrils. Of particular importance to this study are the chemical data indicating that pea XG possesses a trisaccharide sidechain, which is not found in nasturtium XG. To this end, conformational dynamic simulations have been used to predict whether oligosaccharides representative of pea and nasturtium XG can adopt a hypothesized cellulose-binding conformation and which of these XGs exhibits a preferential ability to bind cellulose. Extensive analysis of the conformational forms populated during 300 K and high-temperature Monte Carlo simulations established that a planar, sterically accessible, glucan backbone is essential for optimal cellulose-binding. For the trisaccharide sidechain-containing oligosaccharide as found in pea XG, sidechain orientation appeared to regulate the gradual acquisition of this hypothesized cellulose binding conformation. Thus, conformational forms were identified that included the twisted backbone (non-planar) putative solution form of XG, forms in which the trisaccharide sidechain orientation enables increased backbone planarity and steric accessibility, and finally a planar, sterically accessible, backbone. By applying these conformational requirements for cellulose binding, it has been determined that pea XG possesses a two- to threefold occurrence of the cellulose binding conformation than nasturtium XG. Based on this finding, it was predicted that pea XG would bind to cellulose at a higher rate than nasturtium XG. In vitro binding assays showed that pea XG-avicel binding does indeed occur at a twofold higher rate than nasturtium XG-avicel binding. The enhanced ability of pea cell wall XG over nasturtium seed storage XG to associate with cellulose is consistent with a structural role of the former during epicotyl growth where efficient association with cellulose is a requirement. In contrast, the relatively low ability of nasturtium XG to bind cellulose is consistent with the need to enhance the accessibility of this polymer to glycanases during germination. These findings suggest potential roles for XG sidechain substitution, enabling XG to function in a variety of different biological contexts.

7-Dehydrobrefeldin A, a naturally occurring brefeldin A derivative, inhibits secretion and causes a cis-to-trans breakdown of Golgi stacks in plant cells.

7-Dehydrobrefeldin A (7-oxo-BFA) is a brefeldin A (BFA) analog that, like BFA, is a potent phytotoxin of Alternaria carthami, a fungal pathogen of safflower (Carthamus tinctorius L.) plants. Both BFA and 7-oxo-BFA have been shown to be causal agents of the leaf spot disease of these plants. We have investigated the effects of 7-oxo-BFA on the secretion and the structure of the Golgi stacks of sycamore maple (Acer pseudoplatanus) suspension-cultured cells to determine whether 7-oxo-BFA affects these cells in the same manner as BFA. When applied at 10 micrograms/mL for 1 h, 7-oxo-BFA inhibits secretion of proteins by approximately 80%, the same value obtained for BFA. However, electron micrographs of high-pressure frozen/freeze-substituted cells demonstrated that 7-oxo-BFA is a more potent disrupter of the Golgi stacks of sycamore maple cells than BFA. In cells treated for 1 h with 10 micrograms/mL 7-oxo-BFA, very few Golgi stacks can be discerned. Most of those that are left consist of fewer than three cisternae, all of which stain like trans-Golgi cisternae. They are surrounded by clusters of large (150-300 nm in diameter), darkly staining vesicles that are embedded in a fine-filamentous, ribosome-excluding matrix. Similarly sized and stained vesicles are seen budding from the rims of the residual trans-Golgi cisternae. Both the large vesicles and the residual Golgi stack buds stain with anti-xyloglucan polysaccharide antibodies. Recovery of Golgi stacks after removal of 7-oxo-BFA from 1-h-treated cells takes 2 to 6 h, compared with 1 to 2 h for cells treated with BFA. In contrast to 7-oxo-BFA, the BFA breakdown product BFA acid had no effect either on secretion or on the secretory apparatus. This is the first report, to our knowledge of a BFA analog inhibiting secretion in a eukaryotic cell system.