Galactose induces formation of cell wall stubs and cell death in Arabidopsis roots.

MAIN CONCLUSION: Arabidopsis seedlings growing on low concentration of galactose stop regular root growth. Incomplete cell division with cell wall stubs, binuclear and giant cells and lignified root tips are observed. Galactose is a sugar abundant in root cell walls of Arabidopsis. Nevertheless, we found that the germination of Arabidopsis seedlings on galactose containing media causes a strong modification of the root development, as shown by analysing the root with microscopy methods ranging from the bright field over confocal to transmission electron microscopy. At concentrations of about 1 mM, the growth of the primary root stops after a few days though stem cell markers like WOX5 are still expressed. The root tip swells and forms a slightly opaque, partially lignified structure in parts of the cortex and the central cylinder. The formation of the cell plate after mitosis is impaired, often leading to cell wall stubs and binuclear cells. Some cells in the cortex and the central cylinder degenerate, while some rhizodermal and cortical cells increase massively in size. The galactose toxicity phenotype in Arabidopsis depends on the activity of galactokinase and is completely diminished in galactokinase knock-out lines. From the comparison of the galactose toxicity phenotype with those of cytokinesis mutants and plants treated with appropriate inhibitors we speculate that the toxicity syndrome of galactose is caused by interference with intracellular vesicle transport or cell wall biogenesis.

The molecular identity of the characean OH- transporter: a candidate related to the SLC4 family of animal pH regulators.

Characeae are closely related to the ancient algal ancestors of all land plants. The long characean cells display a pH banding pattern to facilitate inorganic carbon import in the acid zones for photosynthetic efficiency. The excess OH-, generated in the cytoplasm after CO2 is taken into the chloroplasts, is disposed of in the alkaline band. To identify the transporter responsible, we searched the Chara australis transcriptome for homologues of mouse Slc4a11, which functions as OH-/H+ transporter. We found a single Slc4-like sequence CL5060.2 (named CaSLOT). When CaSLOT was expressed in Xenopus oocytes, an increase in membrane conductance and hyperpolarization of resting potential difference (PD) was observed with external pH increase to 9.5. These features recall the behavior of Slc4a11 in oocytes and are consistent with the action of a pH-dependent OH-/H+ conductance. The large scatter in the data might reflect intrinsic variability of CaSLOT transporter activation, inefficient expression in the oocyte due to evolutionary distance between ancient algae and frogs, or absence of putative activating factor present in Chara cytoplasm. CaSLOT homologues were found in chlorophyte and charophyte algae, but surprisingly not in related charophytes Zygnematophyceae or Coleochaetophyceae.

Fluid-phase and membrane markers reveal spatio-temporal dynamics of membrane traffic and repair in the green alga Chara australis.

We investigated the mechanisms and the spatio-temporal dynamics of fluid-phase and membrane internalization in the green alga Chara australis using fluorescent hydrazides markers alone, or in conjunction with styryl dyes. Using live-cell imaging, immunofluorescence and inhibitor studies we revealed that both fluid-phase and membrane dyes were actively taken up into the cytoplasm by clathrin-mediated endocytosis and stained various classes of endosomes including brefeldin A- and wortmannin-sensitive organelles (trans-Golgi network and multivesicular bodies). Uptake of fluorescent hydrazides was poorly sensitive to cytochalasin D, suggesting that actin plays a minor role in constitutive endocytosis in Chara internodal cells. Sequential pulse-labelling experiments revealed novel aspects of the temporal progression of endosomes in Chara internodal cells. The internalized fluid-phase marker distributed to early compartments within 10 min from dye exposure and after about 30 min, it was found almost exclusively in late endocytic compartments. Notably, fluid cargo consecutively internalized at time intervals of more than 1h, was not targeted to the same vesicular structures, but was sorted into distinct late compartments. We further found that fluorescent hydrazide dyes distributed not only to rapidly recycling endosomes but also to long-lived compartments that participated in plasma membrane repair after local laser injury. Our approach highlights the benefits of combining different fluid-phase markers in conjunction with membrane dyes in simultaneous and sequential application modus for investigating vesicle traffic, especially in organisms, which are still refractory to genetic transformation like characean algae.

Brefeldin A inhibits clathrin-dependent endocytosis and ion transport in Chara internodal cells.

BACKGROUND: The Characeae are multicellular green algae, which are closely related to higher plants. Their internodal cells are a convenient model to study membrane transport and organelle interactions. RESULTS: In this study, we report on the effect of brefeldin A (BFA), an inhibitor of vesicle trafficking, on internodal cells of Chara australis. BFA induced the commonly observed agglomeration of Golgi bodies and trans Golgi network into 'brefeldin compartments' at concentrations between 6 and 500 µM and within 30-120 min treatment. In contrast to most other cells, however, BFA inhibited endocytosis and significantly decreased the number of clathrin-coated pits and clathrin-coated vesicles at the plasma membrane. BFA did not inhibit secretion of organelles at wounds induced by puncturing or local light damage but prevented the formation of cellulosic wound walls probably because of insufficient membrane recycling. We also found that BFA inhibited the formation of alkaline and acid regions along the cell surface ('pH banding pattern') which facilitates carbon uptake required for photosynthesis; we hypothesise that this is due to insufficient recycling of ion transporters. During long-term treatments over several days, BFA delayed the formation of complex 3D plasma membranes (charasomes). Interestingly, BFA had no detectable effect on clathrin-dependent charasome degradation. Protein sequence analysis suggests that the peculiar effects of BFA in Chara internodal cells are due to a mutation in the guanine-nucleotide exchange factor GNOM required for recruitment of membrane coats via activation of ADP-ribosylation factor proteins. CONCLUSIONS AND SIGNIFICANCE: This work provides an overview on the effects of BFA on different processes in C. australis. It revealed similarities but also distinct differences in vesicle trafficking between higher plant and algal cells. It shows that characean internodal cells are a promising model to study interactions between seemingly distant metabolic pathways.

Inhibition of endosomal trafficking by brefeldin A interferes with long-distance interaction between chloroplasts and plasma membrane transporters.

The huge internodal cells of the characean green algae are a convenient model to study long-range interactions between organelles via cytoplasmic streaming. It has been shown previously that photometabolites and reactive oxygen species released by illuminated chloroplasts are transmitted to remote shaded regions where they interfere with photosynthetic electron transport and the differential activity of plasma membrane transporters, and recent findings indicated the involvement of organelle trafficking pathways. In the present study, we applied pulse amplitude-modulated microscopy and pH-sensitive electrodes to study the effect of brefeldin A (BFA), an inhibitor of vesicle trafficking, on long-distance interactions in Chara australis internodal cells. These data were compared with BFA-induced changes in organelle number, size and distribution using fluorescent dyes and confocal laser scanning microscopy. We found that BFA completely and immediately inhibited endocytosis in internodal cells and induced the aggregation of organelles into BFA compartments within 30-120 min of treatment. The comparison with the physiological data suggests that the early response, the arrest of endocytosis, is related to the attenuation of differences in surface pH, whereas the longer lasting formation of BFA compartments is probably responsible for the acceleration of the cyclosis-mediated interaction between chloroplasts. These data indicate that intracellular turnover of membrane material might be important for the circulation of electric currents between functionally distinct regions in illuminated characean internodes and that translational movement of metabolites is delayed by transient binding of the transported substances to organelles.

PH-dependent cell-cell interactions in the green alga Chara.

Characean internodal cells develop alternating patterns of acid and alkaline zones along their surface in order to facilitate uptake of carbon required for photosynthesis. In this study, we used a pH-indicating membrane dye, 4-heptadecylumbiliferone, to study the kinetics of alkaline band formation and decomposition. The differences in growth/decay kinetics suggested that growth occurred as an active, autocatalytic process, whereas decomposition was due to diffusion. We further investigated mutual interactions between internodal cells and found that their alignment parallel to each other induced matching of the pH banding patterns, which was mirrored by chloroplast activity. In non-aligned cells, the lowered photosynthetic activity was noted upon a rise of the external pH, suggesting that the matching of pH bands was due to a local elevation of membrane conductance by the high pH of the alkaline zones of neighboured cells. Finally, we show that the altered pH banding pattern caused the reorganization of the cortical cytoplasm. Complex plasma membrane elaborations (charasomes) were degraded via endocytosis, and mitochondria were moved away from the cortex when a previously acid region became alkaline and vice versa. Our data show that characean internodal cells react flexibly to environmental cues, including those originating from neighboured cells.

Chemical Fixation, Immunofluorescence, and Immunogold Labeling of Electron Microscopical Sections.

Knowledge about the spatiotemporal distribution patterns of proteins and other molecules of the cell is essential for understanding their function. A widely used technique is immunolabeling which uses specific antibodies to reveal the distribution of molecular components at various structural levels. Immunofluorescence gives an overview about the distribution of molecules at the level of the fluorescence or confocal laser scanning microscope. Electron microscopy offers the highest resolution of morphological techniques and is thus an indispensable tool for the analysis of molecule distribution patterns at the subcellular level. In this chapter we describe selected routine methods for immunofluorescence and for labeling ultrathin sections of resin-embedded material with antibodies conjugated to colloidal gold, including protocols for chemical fixation, embedding, and sectioning.

Dissecting the subcellular membrane proteome reveals enrichment of H+ (co-)transporters and vesicle trafficking proteins in acidic zones of Chara internodal cells.

The Characeae are multicellular green algae with very close relationship to land plants. Their internodal cells have been the subject of numerous (electro-)physiological studies. When exposed to light, internodal cells display alternating bands of low and high pH along their surface in order to facilitate carbon uptake required for photosynthesis. Here we investigated for the first time the subcellular membrane protein composition of acidic and alkaline regions in internodal cells of Chara australis R. Br. using MS-proteomics. The identified peptides were annotated to Chara unigenes using a custom-made Chara database generated from a transcriptome analysis and to orthologous Arabidopsis genes using TAIR (The Arabidopsis Information Resource) database. Apart from providing the first public-available, functionally-annotated sequence database for Chara australis, the proteome study, which is supported by immunodetection, identified several membrane proteins associated with acidic regions that contain a high density of specific plasma membrane (PM) invaginations, the charasomes, which locally increase the membrane area to overcome diffusion limitation in membrane transport. An increased abundance of PM H+ ATPases at charasomes is consistent with their role in the acidification of the environment, but the characean PM H+ ATPase sequence suggests a different regulation compared to higher plant PM H+ ATPases. A higher abundance of H+ co-transporters in the charasome-rich, acidic regions possibly reflects enhanced uptake of ions and nutrients. The increase in mitochondrial proteins confirms earlier findings about the accumulation of cortical mitochondria in the acidic zones. The significant enrichment of clathrin heavy chains and clathrin adaptor proteins as well as other proteins involved in trafficking indicate a higher activity of membrane transport in the charasome-rich than in charasome-poor areas. New and unexpected data, for instance the upregulation and abundance of vacuolar transporters correlating with the charasome-rich, acidic cell regions account for new perspectives in the formation of charasomes.

Surface pH changes suggest a role for H+/OH- channels in salinity response of Chara australis.

To understand salt stress, the full impact of salinity on plant cell physiology has to be resolved. Electrical measurements suggest that salinity inhibits the proton pump and opens putative H+/OH- channels all over the cell surface of salt sensitive Chara australis (Beilby and Al Khazaaly 2009; Al Khazaaly and Beilby 2012). The channels open transiently at first, causing a characteristic noise in membrane potential difference (PD), and after longer exposure remain open with a typical current-voltage (I/V) profile, both abolished by the addition of 1 mM ZnCl2, the main known blocker of animal H+ channels. The cells were imaged with confocal microscopy, using fluorescein isothiocyanate (FITC) coupled to dextran 70 to illuminate the pH changes outside the cell wall in artificial fresh water (AFW) and in saline medium. In the early saline exposure, we observed alkaline patches (bright fluorescent spots) appearing transiently in random spatial distribution. After longer exposure, some of the spots became fixed in space. Saline also abolished or diminished the pH banding pattern observed in the untreated control cells. ZnCl2 suppressed the alkaline spot formation in saline and the pH banding pattern in AFW. The osmotic component of the saline stress did not produce transient bright spots or affect banding. The displacement of H+ from the cell wall charges, the H+/OH- channel conductance/density, and self-organization are discussed. No homologies to animal H+ channels were found. Salinity activation of the H+/OH- channels might contribute to saline response in roots of land plants and leaves of aquatic angiosperms.

Pathways for external alkalinization in intact and in microwounded Chara cells are differentially sensitive to wortmannin.

Proton flows across the plant cell membranes play a major role in electrogenesis and regulation of photosynthesis and ion balance. The profiles of external pH along the illuminated internodal cells of characean algae consist of alternating high- and low-pH zones that are spatially coordinated with the distribution of photosynthetic activity of chloroplasts underlying these zones. The results based on confocal laser scanning fluorescence microscopy, pH microsensors, and pulse-amplitude-modulated chlorophyll microfluorometry revealed that the coordination of H+ transport and photosynthesis is disrupted by the 2 different environmental cues (low light and wounding) and by a chemical, wortmannin interfering with the inositol phospholipid metabolism. On the one hand, the transition from moderate to low irradiance diminished the peaks in the profiles of photosystem II (PSII) quantum efficiency but did not remove the pH bands. On the other hand, the microwounding of the internode with a glass micropipette, impacting primarily the cell wall, resulted in a rapid local alkalinization of the external medium (by 2-2.5 pH units) near the cell surface, thus mimicking the appearance of natural pH bands. Despite their seeming similarity, the alkaline bands of intact cells were eliminated by wortmannin, whereas the wound-induced alkalinization was insensitive to this drug. Furthermore, the attenuation of natural pH bands in wortmannin-treated cells was accompanied by the enhancement in spatial heterogeneity of PSII efficiency and electron transport rates, which indicates the complexity of chloroplast-plasma membrane interactions. The results suggest that the light- and wound-induced alkaline areas on the cell surface are associated with different ion-transport systems.

Poly-lactic acid nanoparticles (PLA-NP) promote physiological modifications in lung epithelial cells and are internalized by clathrin-coated pits and lipid rafts.

BACKGROUND: Poly-lactic acid nanoparticles (PLA-NP) are a type of polymeric NP, frequently used as nanomedicines, which have advantages over metallic NP such as the ability to maintain therapeutic drug levels for sustained periods of time. Despite PLA-NP being considered biocompatible, data concerning alterations in cellular physiology are scarce. METHODS: We conducted an extensive evaluation of PLA-NP biocompatibility in human lung epithelial A549 cells using high throughput screening and more complex methodologies. These included measurements of cytotoxicity, cell viability, immunomodulatory potential, and effects upon the cells' proteome. We used non- and green-fluorescent PLA-NP with 63 and 66 nm diameters, respectively. Cells were exposed with concentrations of 2, 20, 100 and 200 µg/mL, for 24, 48 and 72 h, in most experiments. Moreover, possible endocytic mechanisms of internalization of PLA-NP were investigated, such as those involving caveolae, lipid rafts, macropinocytosis and clathrin-coated pits. RESULTS: Cell viability and proliferation were not altered in response to PLA-NP. Multiplex analysis of secreted mediators revealed a low-level reduction of IL-12p70 and vascular epidermal growth factor (VEGF) in response to PLA-NP, while all other mediators assessed were unaffected. However, changes to the cells' proteome were observed in response to PLA-NP, and, additionally, the cellular stress marker miR155 was found to reduce. In dual exposures of staurosporine (STS) with PLA-NP, PLA-NP enhanced susceptibility to STS-induced cell death. Finally, PLA-NP were rapidly internalized in association with clathrin-coated pits, and, to a lesser extent, with lipid rafts. CONCLUSIONS: These data demonstrate that PLA-NP are internalized and, in general, tolerated by A549 cells, with no cytotoxicity and no secretion of pro-inflammatory mediators. However, PLA-NP exposure may induce modification of biological functions of A549 cells, which should be considered when designing drug delivery systems. Moreover, the pathways of PLA-NP internalization we detected could contribute to the improvement of selective uptake strategies.

Clathrin in Chara australis: Molecular Analysis and Involvement in Charasome Degradation and Constitutive Endocytosis.

Charasomes are convoluted plasma membrane domains in characean green algae. They are known to form in response to light via secretion of trans-Golgi network (TGN) vesicles and local inhibition of endocytosis. Charasomes are involved in the acidification of their aqueous environment, thereby facilitating photosynthesis-dependent carbon uptake. Charasome formation is reversible to allow cells to adapt to different light conditions. Here, we show that darkness-induced degradation of charasomes involves the formation of coated pits and coated vesicles. The darkness-induced degradation of charasomes can be inhibited by 1-2 µM ikarugamycin (IKA), which is considered to be a specific inhibitor of clathrin-dependent endocytosis. At a much higher concentration (100 µM), IKA also significantly reduces the internalization of styryl dyes, indicating uptake via clathrin-coated vesicles (CV). We are the first to present evidence, based on fine structure investigation, that IKA does not interfere with the formation of clathrin coat, but inhibits the detachment and/or further processing of coated vesicles. Both charasome degradation and constitutive endocytosis are also significantly inhibited by sterol complexing agents (methyl-ß-cyclodextrin and filipin). The absence of an additive effect, when applied together with IKA, suggests that charasome degradation and constitutive endocytosis (measured via styryl dye uptake) is not inhibited due to membrane retrieval via lipid rafts, but due to clathrin coat formation requirement of a specific set of sterols. Analysis of Chara australis clathrin proteins revealed two heavy chains and several light chains with sequence peculiarities, suggesting functional and/or species specific differences. The data obtained indicate that clathrin plays a central role not only in constitutive endocytosis but also in the degradation of charasomes, thereby representing a valuable system for studying targeted exo- and endocytosis.

Is Wortmannin-Induced Reorganization of the trans-Golgi Network the Key to Explain Charasome Formation?

Wortmannin, a fungal metabolite and an inhibitor of phosphatidylinositol-3 (PI3) and phosphatidylinositol-4 (PI4) kinases, is widely used for the investigation and dissection of vacuolar trafficking routes and for the identification of proteins located at multivesicular bodies (MVBs). In this study, we applied wortmannin on internodal cells of the characean green alga Chara australis. Wortmannin was used at concentrations of 25 and 50 µM which, unlike in other cells, arrested neither constitutive, nor wounding-induced endocytosis via coated vesicles. Wortmannin caused the formation of "mixed compartments" consisting of MVBs and membranous tubules which were probably derived from the trans-Golgi network (TGN) and within these compartments MVBs fused into larger organelles. Most interestingly, wortmannin also caused pronounced changes in the morphology of the TGNs. After transient hypertrophy, the TGNs lost their coat and formed compact, three-dimensional meshworks of anastomosing tubules containing a central core. These meshworks had a size of up to 4 µm and a striking resemblance to charasomes, which are convoluted plasma membrane domains, and which serve to increase the area available for transporters. Our findings indicate that similar mechanisms are responsible for the formation of charasomes and the wortmannin-induced reorganization of the TGN. We hypothesize that both organelles grow because of a disturbance of clathrin-dependent membrane retrieval due to inhibition of PI3 and/or PI4 kinases. This leads to local inhibition of clathrin-mediated endocytosis during charasome formation in untreated cells and to inhibition of vesicle release from the TGN in wortmannin-treated cells, respectively. The morphological resemblance between charasomes and wortmannin-modified TGN compartments suggests that homologous proteins are involved in membrane curvature and organelle architecture.

Convoluted Plasma Membrane Domains in the Green Alga Chara are Depleted of Microtubules and Actin Filaments.

Charasomes are convoluted plasma membrane domains in the green alga Chara australis. They harbor H(+)-ATPases involved in acidification of the medium, which facilitates carbon uptake required for photosynthesis. In this study we investigated the distribution of cortical microtubules and cortical actin filaments in relation to the distribution of charasomes. We found that microtubules and actin filaments were largely lacking beneath the charasomes, suggesting the absence of nucleating and/or anchoring complexes or an inhibitory effect on polymerization. We also investigated the influence of cytoskeleton inhibitors on the light-dependent growth and the darkness-induced degradation of charasomes. Inhibition of cytoplasmic streaming by cytochalasin D significantly inhibited charasome growth and delayed charasome degradation, whereas depolymerization of microtubules by oryzalin or stabilization of microtubules by paclitaxel had no effect. Our data indicate that the membrane at the cytoplasmic surface of charasomes has different properties in comparison with the smooth plasma membrane. We show further that the actin cytoskeleton is necessary for charasome growth and facilitates charasome degradation presumably via trafficking of secretory and endocytic vesicles, respectively. However, microtubules are required neither for charasome growth nor for charasome degradation.

Molecular Analysis and Localization of CaARA7 a Conventional RAB5 GTPase from Characean Algae.

RAB5 GTPases are important regulators of endosomal membrane traffic. Among them Arabidopsis thaliana ARA7/RABF2b is highly conserved and homologues are present in fungal, animal and plant kingdoms. In land plants ARA7 and its homologues are involved in endocytosis and transport towards the vacuole. Here we report on the isolation of an ARA7 homologue (CaARA7/CaRABF2) in the highly evolved characean green alga Chara australis. It encodes a polypeptide of 202 amino acids with a calculated molecular mass of 22.2 kDa and intrinsic GTPase activity. Immunolabelling of internodal cells with a specific antibody reveals CaARA7 epitopes at multivesicular endosomes (MVEs) and at MVE-containing wortmannin (WM) compartments. When transiently expressed in epidermal cells of Nicotiana benthamiana leaves, fluorescently tagged CaARA7 localizes to small organelles (putative MVEs) and WM compartments, and partially colocalizes with AtARA7 and CaARA6, a plant specific RABF1 GTPase. Mutations in membrane anchoring and GTP binding sites alter localization of CaARA7 and affect GTPase activity, respectively. This first detailed study of a conventional RAB5 GTPase in green algae demonstrates that CaARA7 is similar to RAB5 GTPases from land plants and other organisms and shows conserved structure and localization.

Photosynthesis-dependent formation of convoluted plasma membrane domains in Chara internodal cells is independent of chloroplast position.

The characean green alga Chara australis forms complex plasma membrane convolutions called charasomes when exposed to light. Charasomes are involved in local acidification of the surrounding medium which facilitates carbon uptake required for photosynthesis. They have hitherto been only described in the internodal cells and in close contact with the stationary chloroplasts. Here, we show that charasomes are not only present in the internodal cells of the main axis, side branches, and branchlets but that the plasma membranes of chloroplast-containing nodal cells, protonemata, and rhizoids are also able to invaginate into complex domains. Removal of chloroplasts by local irradiation with intense light revealed that charasomes can develop at chloroplast-free "windows" and that the resulting pH banding pattern is independent of chloroplast or window position. Charasomes were not detected along cell walls containing functional plasmodesmata. However, charasomes formed next to a smooth wound wall which was deposited onto the plasmodesmata-containing wall when the neighboring cell was damaged. In contrast, charasomes were rarely found at uneven, bulged wound walls which protrude into the streaming endoplasm and which were induced by ligation or puncturing. The results of this study show that charasome formation, although dependent on photosynthesis, does not require intimate contact with chloroplasts. Our data suggest further that the presence of plasmodesmata inhibits charasome formation and/or that exposure to the outer medium is a prerequisite for charasome formation. Finally, we hypothesize that the absence of charasomes at bulged wound walls is due to the disturbance of uniform laminar mass streaming.

Characean internodal cells as a model system for the study of cell organization.

Giant internodal cells of characean green algae have been widely used for studying cellular physiology. This review emphasizes their significance for understanding cytoarchitecture and cytoplasmic reorganization. The cytoarchitecture of internodal cells undergoes pronounced, cytoskeleton-dependent changes during development and in response to environmental cues. Under bright light, internodes develop alternating bands of acid and alkaline pH at their surface that correlate with the differential size and abundance of cortical organelles and, in the genus Chara, with the size and distribution of convoluted plasma membrane domains known as charasomes. Wounding induces responses ranging from chloroplast detachment to deposition of wound walls. These properties and the possibility for mechanical manipulation make the internodal cell ideal for exploring plasma membrane domains, organelle interactions, vesicle trafficking, and local cell wall deposition. The significance of this model system will further increase with the application of molecular biological methods in combination with metabolomics and proteomics.

Vesicular trafficking in characean green algae and the possible involvement of a VAMP72-family protein.

The RAB5 GTPase ARA6 (AtARA6) of Arabidopsis thaliana is known to be involved in endosomal trafficking by targeting vesicles to the plasma membrane. During this process AtARA6 is working in close relationship with the SNARE protein VAMP727 (vesicle associated membrane protein 727). Recently, ARA6 of the characean green algae Chara australis (CaARA6) was shown to have properties similar to AtARA6, pointing to similar trafficking pathways. In order to gain further insight into the vesicle trafficking machinery of characeae, C. australis was analyzed for homologous proteins of the VAMP72-family. A CaVAMP72 protein was detected and classified by protein sequence alignment and phylogenetic analyses.

Trafficking of the myrosinase-associated protein GLL23 requires NUC/MVP1/GOLD36/ERMO3 and the p24 protein CYB.

Proteins detrimental to endoplasmic reticulum (ER) morphology need to be efficiently exported. Here, we identify two mechanisms that control trafficking of Arabidopsis thalianaGLL23, a 43 kDa GDSL-like lipase implicated in glucosinolate metabolism through its association with the ß-glucosidase myrosinase. Using immunofluorescence, we identified two mutants that showed aberrant accumulation of GLL23: large perinuclear ER aggregates in the nuclear cage (nuc) mutant; and small compartments contiguous with the peripheral ER in the cytoplasmic bodies (cyb) mutant. Live imaging of fluorescently tagged GLL23 confirmed its presence in the nuc and cyb compartments, but lack of fluorescent signals in the wild-type plants suggested that GLL23 is normally post-translationally modified for ER export. NUC encodes the MVP1/GOLD36/ERMO3 myrosinase-associated protein, previously shown to have vacuolar distribution. CYB is an ER and Golgi-localized p24 type I membrane protein component of coat protein complex (COP) vesicles, animal and yeast homologues of which are known to be involved in selective cargo sorting for ER-Golgi export. Without NUC, GLL23 accumulates in the ER this situation suggests that NUC is in fact active in the ER. Without CYB, both GLL23 and NUC were found to accumulate in cyb compartments, consistent with a role for NUC in GLL23 processing and indicated that GLL23 is the likely sorting target of the CYB p24 protein.

Immunogold labeling of resin-embedded electron microscopical sections.

Knowledge about the spatio-temporal distribution patterns of proteins and other molecules of the cell is essential for understanding their function. A widely used technique is immunolabeling which uses specific antibodies to reveal the distribution of molecular components at various structural levels. Electron microscopy offers the highest resolution of morphological techniques and is thus an indispensable tool for the analysis of molecule distribution patterns at the subcellular level. In this chapter we describe a routine method for labeling ultrathin sections of resin-embedded material with antibodies conjugated to colloidal gold.