Bochnia, M.; Sander, J.; Ziegler, J.; Terhardt, M.; Sander, S.; Janzen, N.; Cavalleri, J.-M. V.; Zuraw, A.; Wensch-Dorendorf, M.; Zeyner, A. Detection of MCPG metabolites in horses with atypical myopathy PLOS ONE 14, e0211698, (2019) DOI: 10.1371/journal.pone.0211698
Atypical myopathy (AM) in horses is caused by
ingestion of seeds of the Acer species (Sapindaceae family).
Methylenecyclopropylacetyl-CoA (MCPA-CoA), derived from hypoglycin A
(HGA), is currently the only active toxin in Acer pseudoplatanus or Acer
negundo seeds related to AM outbreaks. However, seeds or arils of
various Sapindaceae (e.g., ackee, lychee, mamoncillo, longan fruit) also
contain methylenecyclopropylglycine (MCPG), which is a structural
analogue of HGA that can cause hypoglycaemic encephalopathy in humans.
The active poison formed from MCPG is methylenecyclopropylformyl-CoA
(MCPF-CoA). MCPF-CoA and MCPA-CoA strongly inhibit enzymes that
participate in β-oxidation and energy production from fat. The aim of
our study was to investigate if MCPG is involved in Acer seed poisoning
in horses. MCPG, as well as glycine and carnitine conjugates
(MCPF-glycine, MCPF-carnitine), were quantified using high-performance
liquid chromatography-tandem mass spectrometry of serum and urine from
horses that had ingested Acer pseudoplatanus seeds and developed typical
AM symptoms. The results were compared to those of healthy control
horses. For comparison, HGA and its glycine and carnitine derivatives
were also measured. Additionally, to assess the degree of enzyme
inhibition of β-oxidation, several acyl glycines and acyl carnitines
were included in the analysis. In addition to HGA and the specific toxic
metabolites (MCPA-carnitine and MCPA-glycine), MCPG, MCPF-glycine and
MCPF-carnitine were detected in the serum and urine of affected horses.
Strong inhibition of β-oxidation was demonstrated by elevated
concentrations of all acyl glycines and carnitines, but the highest
correlations were observed between MCPF-carnitine and
isobutyryl-carnitine (r = 0.93) as well as between MCPA- (and MCPF-)
glycine and valeryl-glycine with r = 0.96 (and r = 0.87). As shown here,
for biochemical analysis of atypical myopathy of horses, it is
necessary to take MCPG and the corresponding metabolites into
Mitra, D.; Klemm, S.; Kumari, P.; Quegwer, J.; Möller, B.; Poeschl, Y.; Pflug, P.; Stamm, G.; Abel, S.; Bürstenbinder, K. Microtubule-associated protein IQ67 DOMAIN5 regulates morphogenesis of leaf pavement cells in Arabidopsis thaliana J Exp Bot 70, 529-543, (2019) DOI: 10.1093/jxb/ery395
Plant microtubules form a highly dynamic
intracellular network with important roles for regulating cell division,
cell proliferation and cell morphology. Its organization and dynamics
are coordinated by various microtubule-associated proteins (MAPs) that
integrate environmental and developmental stimuli to fine-tune and
adjust cytoskeletal arrays. IQ67 DOMAIN (IQD) proteins recently emerged
as a class of plant-specific MAPs with largely unknown functions. Here,
using a reverse genetics approach, we characterize Arabidopsis IQD5 in
terms of its expression domains, subcellular localization and biological
functions. We show that IQD5 is expressed mostly in vegetative tissues,
where it localizes to cortical microtubule arrays. Our phenotypic
analysis of iqd5 loss-of-function lines reveals functions of IQD5 in
pavement cell (PC) shape morphogenesis. Histochemical analysis of cell
wall composition further suggests reduced rates of cellulose deposition
in anticlinal cell walls, which correlate with reduced anisotropic
expansion. Lastly, we demonstrate IQD5-dependent recruitment of
calmodulin calcium sensors to cortical microtubule arrays and provide
first evidence for important roles of calcium in regulation of PC
morphogenesis. Our work thus identifies IQD5 as a novel player in PC
shape regulation, and, for the first time, links calcium signaling to
developmental processes that regulate anisotropic growth in PCs.
Bochnia, M.; Scheidemann, W.; Ziegler, J.; Sander, J.; Vollstedt, S.; Glatter, M.; Janzen, N.; Terhardt, M.; Zeyner, A. Predictive value of hypoglycin A and methylencyclopropylacetic acid conjugates in a horse with atypical myopathy in comparison to its cograzing partners Equine Vet Educ 30, 24-28, (2018) DOI: 10.1111/eve.12596
Hypoglycin A (HGA) was detected in blood and urine of a horse suffering from atypical myopathy (AM; Day 2, serum, 8290 μg/l; urine: Day 1, 574, Day 2, 742 μg/l) and in its cograzing partners with a high variability (46–1570 μg/l serum). Over the period of disease, the level of the toxic metabolites (methylencyclopropylacetic acid [MCPA]-conjugates) increased in body fluids of the AM horse (MCPA-carnitine: Day 2, 0.246, Day 3, 0.581 μmol/l serum; MCPA-carnitine: Day 2, 0.621, Day 3, 0.884 μmol/mmol creatinine in urine) and HGA decreased rapidly (Day 3, 2430 μg/l serum). In cograzing horses MCPA-conjugates were not detected. HGA in seeds ranged from 268 to 367 μg/g. Although HGA was present in body fluids of healthy cograzing horses, MCPA-conjugates were not detectable, in contrast to the AM horse. Therefore, increasing concentrations of MCPA-conjugates are supposed to be linked with the onset of AM and both parameters seem to indicate the clinical stage of disease. However, detection of HGA in body fluids of cograzing horses might be a promising step in preventing the disease.
Mitra, D.; Kumari, P.; Quegwer, J.; Klemm, S.; Moeller, B.; Poeschl, Y.; Pflug, P.; Stamm, G.; Abel, S.; Bürstenbinder, K. Microtubule-associated protein IQ67 DOMAIN5 regulates interdigitation of leaf pavement cells in Arabidopsis thaliana bioRxiv (2018) DOI: 10.1101/268466
Plant microtubules form a highly dynamic intracellular network with important roles for regulating cell division, cell proliferation and cell morphology. Its organization and dynamics are coordinated by various microtubule-associated proteins (MAPs) that integrate environmental and developmental stimuli to fine-tune and adjust cytoskeletal arrays. IQ67 DOMAIN (IQD) proteins recently emerged as a class of plant-specific MAPs with largely unknown functions. Here, using a reverse genetics approach, we characterize Arabidopsis IQD5 in terms of its expression domains, subcellular localization and biological functions. We show that IQD5 is expressed mostly in vegetative tissues, where it localizes to cortical microtubule arrays. Our phenotypic analysis of iqd5 loss-of-function lines reveals functions of IQD5 in pavement cell (PC) shape morphogenesis, as indicated by reduced interdigitation of neighboring cells in the leaf epidermis of iqd5 mutants. Histochemical analysis of cell wall composition further suggests reduced rates of cellulose deposition in anticlinal cell walls, which correlate with reduced asymmetric expansion. Lastly, we provide evidence for IQD5-dependent recruitment of calmodulin calcium sensors to cortical microtubule arrays. Our work thus identifies IQD5 as a novel player in PC shape regulation, and, for the first time, links calcium signaling to developmental processes that regulate multi-polar growth in PCs.
Bürstenbinder, K.; Mitra, D.; Quegwer, J. Functions of IQD proteins as hubs in cellular calcium and auxin signaling: a toolbox for shape formation and tissue-specification in plants? Plant Signal Behav 12 , e1331198, (2017) DOI: 10.1080/15592324.2017.1331198
Ca2+ ions play pivotal roles as second messengers in intracellular signal transduction, and coordinate many biological processes. Changes in intracellular Ca2+ levels are perceived by Ca2+ sensors such as CaM/CML proteins, which transduce Ca2+ signals into cellular responses by regulation of diverse target proteins. Insights into molecular functions of CaM targets are thus essential to understand the molecular and cellular basis of Ca2+ signaling. During the last decade, IQD proteins emerged as the largest class of CaM targets in plants with mostly unknown functions. In the March issue of Plant Physiology, we presented the first comprehensive characterization of the 33-membered IQD family in Arabidopsis thaliana. We showed, by analysis of the subcellular localization of translational GFP fusion proteins, that most IQD members label MTs, and additionally often localize to the cell nucleus or to membranes, where the recruit CaM Ca2+ sensors. Important functions at MTs are supported by altered MT organization and plant growth in IQD gain-of-function lines. Because IQD proteins share structural hallmarks of scaffold proteins, we propose roles of IQDs in the assembly of macromolecular complexes to orchestrate Ca2+ CaM signaling from membranes to the nucleus.
Bürstenbinder, K.; Möller, B.; Plötner; R.; Stamm, G.; Hause, G.; Mitra, D.; Abel, S. The IQD family of calmodulin-binding proteins links calcium signaling to microtubules, membrane subdomains, and the nucleus. Plant Physiol 173, 1692-1708, (2017) DOI: 10.1104/pp.16.01743
Calcium (Ca2+) signaling and dynamic reorganization of the cytoskeleton are essential processes for the coordination and control of plant cell shape and cell growth. Calmodulin (CaM) and closely related CaM-like polypeptides (CML) are principal sensors of Ca2+ signals. CaM/CMLs decode and relay information encrypted by the second messenger via differential interactions with a wide spectrum of targets to modulate their diverse biochemical activities. The plant-specific IQ67-DOMAIN (IQD) family emerged as the possibly largest class of CaM interacting proteins with undefined molecular functions and biological roles. Here, we show that the 33 members of the IQD family in Arabidopsis thaliana differentially localize, using GFP-tagged proteins, to multiple and distinct subcellular sites, including microtubule (MT) arrays, plasma membrane microdomains, and nuclear compartments. Intriguingly, the various IQD-specific localization patterns coincide with the subcellular patterns of IQD-dependent recruitment of CaM, suggesting that the diverse IQD members sequester Ca2+-CaM signaling modules to specific subcellular sites for precise regulation of Ca2+-dependent processes. Because MT localization is a hallmark of most IQD family members, we quantitatively analyzed GFP-labeled MT arrays in tobacco cells transiently expressing GFP-IQD fusions and observed IQD-specific MT patterns, which point to a role of IQDs in MT organization and dynamics. Indeed, stable overexpression of select IQD proteins in Arabidopsis altered cellular MT orientation, cell shape, and organ morphology. Because IQDs share biochemical properties with scaffold proteins, we propose that IQD families provide an assortment of platform proteins for integrating CaM-dependent Ca2+ signaling at multiple cellular sites to regulate cell function, shape, and growth.
Bochnia, M.; Ziegler, J.; Sander, J.; Uhlig, A.; Schaefer, S.; Vollstedt, S.; Glatter, M.; Abel, S.; Recknagel, S.; Schusser, G. F.; Wensch-Dorendorf, M.; Zeyner, A. Hypoglycin A Content in Blood and Urine Discriminates Horses with Atypical Myopathy from Clinically Normal Horses Grazing on the Same Pasture PLoS ONE 10, e0136785, (2015) DOI: 10.1371/journal.pone.0136785
Hypoglycin A (HGA) in seeds of Acer spp. is suspected to cause seasonal pasture myopathy in North America and equine atypical myopathy (AM) in Europe, fatal diseases in horses on pasture. In previous studies, this suspicion was substantiated by the correlation of seed HGA content with the concentrations of toxic metabolites in urine and serum (MCPA-conjugates) of affected horses. However, seed sampling was conducted after rather than during an outbreak of the disease. The aim of this study was to further confirm the causality between HGA occurrence and disease outbreak by seed sampling during an outbreak and the determination of i) HGA in seeds and of ii) HGA and MCPA-conjugates in urine and serum of diseased horses. Furthermore, cograzing healthy horses, which were present on AM affected pastures, were also investigated. AM-pastures in Germany were visited to identify seeds of Acer pseudoplatanus and serum (n = 8) as well as urine (n = 6) from a total of 16 diseased horses were analyzed for amino acid composition by LC-ESI-MS/MS, with a special focus on the content of HGA. Additionally, the content of its toxic metabolite was measured in its conjugated form in body fluids (UPLC-MS/MS). The seeds contained 1.7–319.8 μg HGA/g seed. The content of HGA in serum of affected horses ranged from 387.8–8493.8 μg/L (controls < 10 μg/L), and in urine from 143.8–926.4 μg/L (controls < 10 μg/L), respectively. Healthy cograzing horses on AM-pastures showed higher serum (108.8 ± 83.76 μg/L) and urine concentrations (26.9 ± 7.39 μg/L) compared to control horses, but lower concentrations compared to diseased horses. The range of MCPA-carnitine and creatinine concentrations found in diseased horses in serum and urine were 0.17–0.65 mmol/L (controls < 0.01), and 0.34–2.05 μmol/mmoL (controls < 0.001), respectively. MCPA-glycine levels in urine of cograzing horses were higher compared to controls. Thus, the causal link between HGA intoxication and disease outbreak could be further substantiated, and the early detection of HGA in cograzing horses, which are clinically normal, might be a promising step in prophylaxis.