Investigating Cellulose Binding of Peptides Derived from Carbohydrate Binding Module 1.

Carbohydrate-binding modules (CBM) have emerged as useful tools for a wide range of tasks, including the use as purification tags or for cellulose fiber modification. For this purpose, the CBM needs to be attached to a target protein leading to large constructs. We investigated if short peptides from the carbohydrate binding site of CBMs can bind in a similar way as native, full-length CBMs to nanocrystalline cellulose (NCC) or cotton linter paper. We designed our cellulose-binding peptides to be less hydrophobic and shorter than those previously reported. Starting from the binding site of Cel7A-CBM1, we incorporated the essential amino acids involved in cellulose binding into our peptides. These peptides, as well as control peptides with scrambled sequences or a lack of essential amino acids, bound to cellulose with similar affinity as CBM regardless of their secondary structure, sequence, or hydrophobicity. This unspecific mode of cellulose binding displayed by the presented peptides may be exploited to functionalize cellulose-based biomaterials by means of peptide-conjugates.

Combination of hydrophobicity and codon usage bias determines sorting of model K+ channel protein to either mitochondria or endoplasmic reticulum.

When the K+ channel-like protein Kesv from Ectocarpus siliculosus virus 1 is heterologously expressed in mammalian cells, it is sorted to the mitochondria. This targeting can be redirected to the endoplasmic reticulum (ER) by altering the codon usage in distinct regions of the gene or by inserting a triplet of hydrophobic amino acids (AAs) into the protein's C-terminal transmembrane domain (ct-TMD). Systematic variations in the flavor of the inserted AAs and/or its codon usage show that a positive charge in the inserted AA triplet alone serves as strong signal for mitochondria sorting. In cases of neutral AA triplets, mitochondria sorting are favored by a combination of hydrophilic AAs and rarely used codons; sorting to the ER exhibits the inverse dependency. This propensity for ER sorting is particularly high when a common codon follows a rarer one in the AA triplet; mitochondria sorting in contrast is supported by codon uniformity. Since parameters like positive charge, hydrophobic AAs, and common codons are known to facilitate elongation of nascent proteins in the ribosome the data suggest a mechanism in which local changes in elongation velocity and co-translational folding in the ct-TMD influence intracellular protein sorting.

Modulation of Differentiation and Bone Resorbing Activity of Human (Pre-) Osteoclasts After X-Ray Exposure.

Low-dose radiotherapy (LD-RT) is a local treatment option for patients with chronic degenerative and inflammatory diseases, in particular musculoskeletal diseases. Despite reported analgesic and anti-inflammatory effects, cellular and molecular mechanisms related to osteoimmunological effects are still elusive. Here we test the hypothesis that X-irradiation inhibits the differentiation of precursor osteoclasts into mature osteoclasts (mOC) and their bone resorbing activity. Circulating monocytes from healthy donors were isolated and irradiated after attachment with single or fractionated X-ray doses, comparable to an LD-RT treatment scheme. Then monocytes underwent ex vivo differentiation into OC during cultivation up to 21 days, under conditions mimicking the physiological microenvironment of OC on bone. After irradiation, apoptotic frequencies were low, but the total number of OC precursors and mOC decreased up to the end of the cultivation period. On top, we observed an impairment of terminal differentiation, i.e. a smaller fraction of mOC, reduced resorbing activity on bone, and release of collagen fragments. We further analyzed the effect of X-irradiation on multinucleation, resulting from the fusion of precursor OC, which occurs late during OC differentiation. At 21 days after exposure, the observation of smaller cellular areas and a reduced number of nuclei per mOC suggest an impaired fusion of OC precursors to form mOC. Before, at 14 days, the nuclear translocation of Nuclear Factor Of Activated T Cells 1 (NFATc1), a master regulator of osteoclast differentiation and fusion, was decreased. In first results, obtained in the frame of a longitudinal LD-RT study, we previously reported a pain-relieving effect in patients. However, in a subgroup of patients suffering from Calcaneodynia or Achillodynia, we did not observe a consistent decrease of established blood markers for resorption and formation of bone, or modified T cell subtypes involved in regulating these processes. To assess the relevance of changes in bone metabolism for other diseases treated with LD-RT will be subject of further studies. Taken together, we observed that in vitro X-irradiation of monocytes results in an inhibition of the differentiation into bone-resorbing OC and a concomitant reduction of resorbing activity. The detected reduced NFATc1 signaling could be one underlying mechanism.

Reducing Unspecific Protein Adsorption in Microfluidic Papers Using Fiber-Attached Polymer Hydrogels.

Microfluidic paper combines pump-free water transport at low cost with a high degree of sustainability, as well as good availability of the paper-forming cellulosic material, thus making it an attractive candidate for point-of-care (POC) analytics and diagnostics. Although a number of interesting demonstrators for such paper devices have been reported to date, a number of challenges still exist, which limit a successful transfer into marketable applications. A strong limitation in this respect is the (unspecific) adsorption of protein analytes to the paper fibers during the lateral flow assay. This interaction may significantly reduce the amount of analyte that reaches the detection zone of the microfluidic paper-based analytical device (µPAD), thereby reducing its overall sensitivity. Here, we introduce a novel approach on reducing the nonspecific adsorption of proteins to lab-made paper sheets for the use in µPADs. To this, cotton linter fibers in lab-formed additive-free paper sheets are modified with a surrounding thin hydrogel layer generated from photo-crosslinked, benzophenone functionalized copolymers based on poly-(oligo-ethylene glycol methacrylate) (POEGMA) and poly-dimethyl acrylamide (PDMAA). This, as we show in tests similar to lateral flow assays, significantly reduces unspecific binding of model proteins. Furthermore, by evaporating the transport fluid during the microfluidic run at the end of the paper strip through local heating, model proteins can almost quantitatively be accumulated in that zone. The possibility of complete, almost quantitative protein transport in a µPAD opens up new opportunities to significantly improve the signal-to-noise (S/N) ratio of paper-based lateral flow assays.

Toward Fabrication of Bioactive Papers: Covalent Immobilization of Peptides and Proteins.

Herein, we report a novel two-step method for the covalent, site-directed, and efficient immobilization of proteins on lab-made paper sheets. First, paper fibers were modified with a peptidic anchor comprising enzyme recognition motifs. Four different conjugation strategies for peptide immobilization were evaluated with respect to reproducibility and fiber loading efficiency. After manufacturing of the peptide-preconditioned paper, oriented conjugation of the model protein tGFP containing a C-terminal recognition sequence for either sortase A or microbial transglutaminase was assessed semiquantitatively by fluorescence measurement and inspected by confocal laser scanning microscopy (CLSM). The two enzymes utilized for protein conjugation used the same oligoglycine peptide anchor, and both proved to be suitable for controlled oriented linkage of substrate proteins at physiological conditions.

Codon Bias Can Determine Sorting of a Potassium Channel Protein.

Due to the redundancy of the genetic code most amino acids are encoded by multiple synonymous codons. It has been proposed that a biased frequency of synonymous codons can affect the function of proteins by modulating distinct steps in transcription, translation and folding. Here, we use two similar prototype K+ channels as model systems to examine whether codon choice has an impact on protein sorting. By monitoring transient expression of GFP-tagged channels in mammalian cells, we find that one of the two channels is sorted in a codon and cell cycle-dependent manner either to mitochondria or the secretory pathway. The data establish that a gene with either rare or frequent codons serves, together with a cell-state-dependent decoding mechanism, as a secondary code for sorting intracellular membrane proteins.

Carbohydrate binding module-fused antibodies improve the performance of cellulose-based lateral flow immunoassays.

Since the pandemic outbreak of Covid-19 in December 2019, several lateral flow assay (LFA) devices were developed to enable the constant monitoring of regional and global infection processes. Additionally, innumerable lateral flow test devices are frequently used for determination of different clinical parameters, food safety, and environmental factors. Since common LFAs rely on non-biodegradable nitrocellulose membranes, we focused on their replacement by cellulose-composed, biodegradable papers. We report the development of cellulose paper-based lateral flow immunoassays using a carbohydrate-binding module-fused to detection antibodies. Studies regarding the protein binding capacity and potential protein wash-off effects on cellulose paper demonstrated a 2.7-fold protein binding capacity of CBM-fused antibody fragments compared to the sole antibody fragment. Furthermore, this strategy improved the spatial retention of CBM-fused detection antibodies to the test area, which resulted in an enhanced sensitivity and improved overall LFA-performance compared to the naked detection antibody. CBM-assisted antibodies were validated by implementation into two model lateral flow test devices (pregnancy detection and the detection of SARS-CoV-2 specific antibodies). The CBM-assisted pregnancy LFA demonstrated sensitive detection of human gonadotropin (hCG) in synthetic urine and the CBM-assisted Covid-19 antibody LFA was able to detect SARS-CoV-2 specific antibodies present in serum. Our findings pave the way to the more frequent use of cellulose-based papers instead of nitrocellulose in LFA devices and thus potentially improve the sustainability in the field of POC diagnostics.

ROS- and Radiation Source-Dependent Modulation of Leukocyte Adhesion to Primary Microvascular Endothelial Cells.

Anti-inflammatory effects of low-dose irradiation often follow a non-linear dose-effect relationship. These characteristics were also described for the modulation of leukocyte adhesion to endothelial cells. Previous results further revealed a contribution of reactive oxygen species (ROS) and anti-oxidative factors to a reduced leukocyte adhesion. Here, we evaluated the expression of anti-oxidative enzymes and the transcription factor Nrf2 (Nuclear factor-erythroid-2-related factor 2), intracellular ROS content, and leukocyte adhesion in primary human microvascular endothelial cells (HMVEC) upon low-dose irradiation under physiological laminar shear stress or static conditions after irradiation with X-ray or Carbon (C)-ions (0-2 Gy). Laminar conditions contributed to increased mRNA expression of anti-oxidative factors and reduced ROS in HMVEC following a 0.1 Gy X-ray and 0.5 Gy C-ion exposure, corresponding to reduced leukocyte adhesion and expression of adhesion molecules. By contrast, mRNA expression of anti-oxidative markers and adhesion molecules, ROS, and leukocyte adhesion were not altered by irradiation under static conditions. In conclusion, irradiation of endothelial cells with low doses under physiological laminar conditions modulates the mRNA expression of key factors of the anti-oxidative system, the intracellular ROS contents of which contribute at least in part to leucocyte adhesion, dependent on the radiation source.

Light-Regulated Transcription of a Mitochondrial-Targeted K+ Channel.

The inner membranes of mitochondria contain several types of K+ channels, which modulate the membrane potential of the organelle and contribute in this way to cytoprotection and the regulation of cell death. To better study the causal relationship between K+ channel activity and physiological changes, we developed an optogenetic platform for a light-triggered modulation of K+ conductance in mitochondria. By using the light-sensitive interaction between cryptochrome 2 and the regulatory protein CIB1, we can trigger the transcription of a small and highly selective K+ channel, which is in mammalian cells targeted into the inner membrane of mitochondria. After exposing cells to very low intensities (≤0.16 mW/mm2) of blue light, the channel protein is detectable as an accumulation of its green fluorescent protein (GFP) tag in the mitochondria less than 1 h after stimulation. This system allows for an in vivo monitoring of crucial physiological parameters of mitochondria, showing that the presence of an active K+ channel causes a substantial depolarization compatible with the effect of an uncoupler. Elevated K+ conductance also results in a decrease in the Ca2+ concentration in the mitochondria but has no impact on apoptosis.

Lipid-rafts remain stable even after ionizing radiation induced disintegration of ß1 integrin containing focal adhesions.

OBJECTIVE: Adhesion of cells to the extracellular matrix is facilitated by integrin receptors. We recently found that a nanoscale organization of plasma membrane located integrins containing the ß1 subunit is responsible for an enhanced radio-resistance in 3D cultured cells over cells grown in 2D. While ionizing radiation is known to have broad effects on the lipid composition of the plasma membrane and their organization in lipid-rafts, it is not clear whether the effects of ionizing radiation on the nanoscale clustering of integrins is lipid-raft dependent. RESULTS: Using single molecule microscopy we can show that ß1 integrins colocalize with cholesterol in lipid-rafts. Ionizing radiation, as an extrinsic stressor, causes the separation of ß1 integrins from cholesterol lipid raft suggesting that the effects of ionizing radiation on the clustering of ß1 integrins are lipid-raft independent.

Direct evidence for cell adhesion-mediated radioresistance (CAM-RR) on the level of individual integrin ß1 clusters.

The cellular interaction with the extracellular matrix (ECM) modulates many key processes such as proliferation, migration, differentiation and survival. In addition, cells cultured under 3D conditions in presence of an ECM display a marked radioresistance towards ionizing radiation (IR) in comparison to conventionally 2D cultured cells. This process, also known as "cell-adhesion-mediated-radio-resistance" (CAM-RR), has been linked to the chromatin structure that differs between cells cultured on stiff surfaces versus cell grown on soft planar supports or in 3D environments. As integrins are the key mediators of cell adhesion and mechanosensing, they originate the molecular signalling towards chromatin remodelling in response to a cell's microenvironment. We aimed to investigate this molecular origin that leads to CAM-RR by investigating the distribution of integrins at the single molecule level and show that cells cultured in 2D keep a lower fraction of integrin ß1 in clusters and maintain a less defined cluster status than 3D cultured cells. Upon X-irradiation this nanoscale distribution of integrin ß1 is disturbed at much lower dosages in 2D versus 3D cultured cells. Radioresistance is thus linked to the ability to maintain a well defined organization of integrins in clusters, making integrin distribution a potential drug target for radiosensitization.

The right motifs for plant cell adhesion: what makes an adhesive site?

Cells of multicellular organisms are surrounded by and attached to a matrix of fibrous polysaccharides and proteins known as the extracellular matrix. This fibrous network not only serves as a structural support to cells and tissues but also plays an integral part in the process as important as proliferation, differentiation, or defense. While at first sight, the extracellular matrices of plant and animals do not have much in common, a closer look reveals remarkable similarities. In particular, the proteins involved in the adhesion of the cell to the extracellular matrix share many functional properties. At the sequence level, however, a surprising lack of homology is found between adhesion-related proteins of plants and animals. Both protein machineries only reveal similarities between small subdomains and motifs, which further underlines their functional relationship. In this review, we provide an overview on the similarities between motifs in proteins known to be located at the plant cell wall-plasma membrane-cytoskeleton interface to proteins of the animal adhesome. We also show that by comparing the proteome of both adhesion machineries at the level of motifs, we are also able to identify potentially new candidate proteins that functionally contribute to the adhesion of the plant plasma membrane to the cell wall.

Single-molecule detection and tracking in plants.

Combining optical properties with a limited choice of fluorophores turns single-molecule imaging in plants into a challenging task. This explains why the technique, despite its success in the field of animal cell biology, is far from being routinely applied in plant cell research. The same challenges, however, also apply to the application of single-molecule microscopy to any intact tissue or multicellular 3D cell culture. As recent and upcoming progress in fluorescence microscopy will permit single-molecule detection in the context of multicellular systems, plant tissue imaging will experience a huge benefit from this progress. In this review, we address every step of a single-molecule experiment, highlight the critical aspects of each and elaborate on optimizations and developments required for improvements. We relate each step to recent achievements, which have so far been conducted exclusively on the root epidermis of Arabidopsis thaliana seedlings with inclined illumination and show examples of single-molecule measurements using different cells or illumination schemes.

Putative p24 complexes in Arabidopsis contain members of the delta and beta subfamilies and cycle in the early secretory pathway.

p24 proteins are a family of type I membrane proteins localized to compartments of the early secretory pathway and to coat protein I (COPI)- and COPII-coated vesicles. They can be classified, by sequence homology, into four subfamilies, named p24α, p24ß, p24γ, and p24δ. In contrast to animals and fungi, plants contain only members of the p24ß and p24δ subfamilies, the latter probably including two different subclasses. It has previously been shown that transiently expressed red fluorescent protein (RFP)-p24δ5 (p24δ1 subclass) localizes to the endoplasmic reticulum (ER) at steady state as a consequence of highly efficient COPI-based recycling from the Golgi apparatus. It is now shown that transiently expressed RFP-p24δ9 (p24δ2 subclass) also localizes to the ER. In contrast, transiently expressed green fluorescent protein (GFP)-p24ß3 mainly localizes to the Golgi apparatus (as p24ß2) and exits the ER in a COPII-dependent manner. Immunogold electron microscopy in Arabidopsis root tip cells using specific antibodies shows that endogenous p24δ9 localizes mainly to the ER but also partially to the cis-Golgi. In contrast, endogenous p24ß3 mainly localizes to the Golgi apparatus. By a combination of experiments using transient expression, knock-out mutants, and co-immunoprecipitation, it is proposed that Arabidopsis p24 proteins form different heteromeric complexes (including members of the ß and δ subfamilies) which are important for their stability and their coupled trafficking at the ER-Golgi interface. Evidence is also provided for a role for p24δ5 in retrograde Golgi-ER transport of the KDEL-receptor ERD2.

Comparison of membrane targeting strategies for the accumulation of the human immunodeficiency virus p24 protein in transgenic tobacco.

Membrane anchorage was tested as a strategy to accumulate recombinant proteins in transgenic plants. Transmembrane domains of different lengths and topology were fused to the cytosolic HIV antigen p24, to promote endoplasmic reticulum (ER) residence or traffic to distal compartments of the secretory pathway in transgenic tobacco. Fusions to a domain of the maize seed storage protein γ-zein were also expressed, as a reference strategy that leads to very high stability via the formation of large polymers in the ER lumen. Although all the membrane anchored constructs were less stable compared to the zein fusions, residence at the ER membrane either as a type I fusion (where the p24 sequence is luminal) or a tail-anchored fusion (where the p24 sequence is cytosolic) resulted in much higher stability than delivery to the plasma membrane or intermediate traffic compartments. Delivery to the tonoplast was never observed. The inclusion of a thrombin cleavage site allowed for the quantitative in vitro recovery of p24 from all constructs. These results point to the ER as suitable compartment for the accumulation of membrane-anchored recombinant proteins in plants.

Synthesis and biological properties of novel brefeldin A analogues.

New brefeldin A (1) analogues were obtained by introducing a variety of substituents at C15. Most of the analogues exhibited significant biological activity. (15R)-Trifluoromethyl-nor-brefeldin A (3), (15R)-vinyl-nor-brefeldin A (5), their epimers 4 and 6 as well as (15S)-ethyl-nor-brefeldin A (2) were prepared from the key building blocks 12 or 24 by Julia-Kocienski olefination with tetrazolyl sulfones and subsequent macrolactonization. The vinyl derivative 5 allowed analogues to be synthesized by hydroboration and Suzuki-Miyaura coupling. The following biological properties were assessed: (a) inhibition of cell growth of human cancer cells (NCI), (b) induction of morphological changes of the Golgi apparatus of plant and mammalian cells, and (c) influence on the replication of the enterovirus CVB3. Furthermore, conformational aspects were studied by X-ray crystal structure analysis and molecular mechanics calculations, including docking of the analogues into the brefeldin A binding site of an Arf1/Sec7-complex.

PDMP induces rapid changes in vacuole morphology in Arabidopsis root cells.

PDMP (D-L-threo-1-phenyl-2-decanoyl amino-3-morpholino-1-propanol) is a well-known inhibitor of glucosylceramide synthase (GCS), a key enzyme in sphingolipid biosynthesis. Through the resultant increase in ceramides which interact with mTOR and Beclin1 (Atg6), this drug is also known to induce macroautophagy in mammalian cells. This study investigated the response of Arabidopsis root cells to PDMP, and what are probably numerous tightly packed small vacuoles in the control cells appear to fuse to form a single globular-shaped vacuole. However, during this fusion process, cytoplasm channels between the individual vacuoles become trapped in deep invaginations of the tonoplast. In both optical sections in the confocal laser scanning microscope and in ultrathin sections in the electron microscope, these invaginations have the appearance of cytoplasmic inclusions in the vacuole lumen. These changes in vacuole morphology are rapid (occurring within minutes after application of PDMP) and are independent of ongoing protein synthesis. The tonoplast invaginations remain visible for hours, but after 24h almost all disappear. Experiments designed to examine whether ceramide levels might be the cause of the PDMP effect have not proved conclusive. On the other hand, this study has been able to rule out the release of Ca(2+) ions from intracellular stores as a contributing factor.

ER Import Sites and Their Relationship to ER Exit Sites: A New Model for Bidirectional ER-Golgi Transport in Higher Plants.

Per definition, ER exit sites are COPII vesiculation events at the surface of the ER and in higher plants are only visualizable in the electron microscope through cryofixation techniques. Fluorescent COPII labeling moves with Golgi stacks and locates to the interface between the ER and the Golgi. In contrast, the domain of the ER where retrograde COPI vesicles fuse, i.e., ER import sites (ERIS), has remained unclear. To identify ERIS we have employed ER-located SNAREs and tethering factors. We screened several SNAREs (SYP81, the SYP7 family, and USE1) to find a SNARE whose overexpression did not disrupt ER-Golgi traffic and which gave rise to discrete fluorescent punctae when expressed with an XFP tag. Only the Qc-SNARE SYP72 fulfilled these criteria. When coexpressed with SYP72-YFP, both the type I-membrane protein RFP-p24δ5 and the luminal marker CFP-HDEL whose ER localization are due to an efficient COPI-mediated recycling, form nodules along the tubular ER network. SYP72-YFP colocalizes with these nodules which are not seen when RFP-p24δ5 or CFP-HDEL is expressed alone or when SYP72-YFP is coexpressed with a mutant form of RFP-p24δ5 that cannot exit the ER. SYP72-YFP does not colocalize with Golgi markers, except when the Golgi stacks are immobilized through actin depolymerization. Endogenous SYP7 SNAREs, also colocalize with immobilized COPII/Golgi. In contrast, XFP-tagged versions of plant homologs to TIP20 of the Dsl1 COPI-tethering factor complex, and the COPII-tethering factor p115 colocalize perfectly with Golgi stacks irrespective of the motile status. These data suggest that COPI vesicle fusion with the ER is restricted to periods when Golgi stacks are stationary, but that when moving both COPII and COPI vesicles are tethered and collect in the ER-Golgi interface. Thus, the Golgi stack and an associated domain of the ER thereby constitute a mobile secretory and recycling unit: a unique feature in eukaryotic cells.

Coupled transport of Arabidopsis p24 proteins at the ER-Golgi interface.

p24 proteins are a family of type I membrane proteins localized to compartments of the early secretory pathway and to coat protein I (COPI)- and COPII-coated vesicles. They can be classified, by sequence homology, into four subfamilies, named p24α, p24ß, p24γ, and p24δ. In contrast to animals and fungi, plants contain only members of the p24ß and p24δ subfamilies. It has previously been shown that transiently expressed red fluorescent protein (RFP)-p24δ5 localizes to the endoplasmic reticulum (ER) as a consequence of highly efficient COPI-based recycling from the Golgi apparatus. Using specific antibodies, endogenous p24δ5 has now been localized to the ER and p24ß2 to the Golgi apparatus in Arabidopsis root tip cells by immunogold electron microscopy. The relative contributions of the cytosolic tail and the luminal domains to p24δ5 trafficking have also been characterized. It is demonstrated that whereas the dilysine motif in the cytoplasmic tail determines the location of p24δ5 in the early secretory pathway, the luminal domain may contribute to its distribution downstream of the Golgi apparatus. By using knock-out mutants and co-immunoprecipitation experiments, it is shown that p24δ5 and p24ß2 interact with each other. Finally, it is shown that p24δ5 and p24ß2 exhibit coupled trafficking at the ER-Golgi interface. It is proposed that p24δ5 and p24ß2 interact with each other at ER export sites for ER exit and coupled transport to the Golgi apparatus. Once in the Golgi, p24δ5 interacts very efficiently with the COPI machinery for retrograde transport back to the ER.

An Exo2 derivative affects ER and Golgi morphology and vacuolar sorting in a tissue-specific manner in arabidopsis.

We screened a panel of compounds derived from Exo2 - a drug that perturbs post-Golgi compartments and trafficking in mammalian cells - for their effect on the secretory pathway in Arabidopsis root epidermal cells. While Exo2 and most related compounds had no significant effect, one Exo2 derivative, named LG8, induced severe morphological alterations in both the Golgi (at high concentrations) and the endoplasmic reticulum (ER). LG8 causes the ER to form foci of interconnecting tubules, which at the ultrastructural level appear similar to those previously reported in Arabidopsis roots after treatment with the herbicide oryzalin. In cotyledonary leaves, LG8 causes redistribution of a trans Golgi network (TGN) marker to the vacuole. LG8 affects the anterograde secretory pathway by inducing secretion of vacuolar cargo and preventing the brassinosteroid receptor BRI1 from reaching the plasma membrane. Uptake and arrival at the TGN of the endocytic marker FM4-64 is not affected. Unlike the ADP ribosylation factor-GTP exchange factor (ARF-GEF) inhibitor brefeldin A (BFA), LG8 affects these post-Golgi events without causing the formation of BFA bodies. Up to concentrations of 50 µm, the effects of LG8 are reversible.