Papers by Riet M De Rycke
Nature Cell Biology, Jan 23, 2023
Analytica Chimica Acta, Apr 1, 2020
Radiation based X-ray Fluorescence (SR nano-XRF) techniques were compared for a specific single c... more Radiation based X-ray Fluorescence (SR nano-XRF) techniques were compared for a specific single cell imaging case study: high pressure frozen (HPF) and cryo-substituted polymorphonuclear neutrophils (PMNs). Nano-SIMS enabled nanoscale mapping of isotopes of C, N, O, P and S, while SR based nano-XRF enabled trace level imaging of metals like Ca, Mn, Fe, Ni, Cu and Zn at a resolution of approx. 50 nm. The obtained elemental distributions were compared with those of whole, cryofrozen PMNs measured at the newly developed ID16A nano-imaging beamline at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Similarities were observed for elements more tightly bound to the cell structure such as phosphorus and sulphur, while differences for mobile ions such as chlorine and potassium were more pronounced. Due to the observed elemental redistribution of mobile ions such as potassium and chlorine, elemental analysis of high pressure frozen (HPF), cryo-substituted samples should therefore be interpreted critically. Although decreasing analytical sensitivity occurs due to the presence of ice, analysis of cryofrozen cells -close to their native state -remains the reference method par excellence. In general, we found nanoscale secondary ion mass spectrometry (nano-SIMS) and synchrotron radiation based nanoscopic X-ray fluorescence (SR nano-XRF) to be two supplementary alternatives, each with their own pros and cons, to investigate single cells at the nanoscale.
Analytical and Bioanalytical Chemistry, Feb 21, 2019
This paper describes a workflow towards the reconstruction of the three-dimensional elemental dis... more This paper describes a workflow towards the reconstruction of the three-dimensional elemental distribution profile within human cervical carcinoma cells (HeLa), at a spatial resolution down to 1 µm, employing state-of-the-art laser ablationinductively coupled plasma-mass spectrometry (LA-ICP-MS) instrumentation. The suspended cells underwent a series of fixation/embedding protocols, and were stained with uranyl acetate and an Ir-based DNA-intercalator. A priori, laboratorybased absorption micro-computed tomography (µ-CT) was applied to acquire a reference frame of the morphology of the cells and their distribution before sectioning. After CT-analysis, a trimmed 300 × 300 × 300 𝜇𝑚 3 block was sectioned into a sequential series of 132 sections with a thickness of 2 𝜇𝑚 and subjected to LA-ICP-MS imaging. A pixel acquisition rate of 250 pixels s -1 was achieved, in combination with a bidirectional scanning strategy. After acquisition, the twodimensional elemental images were reconstructed using the timestamps in the laser log file. The synchronization of the data required an improved optimization algorithm, which forces the pixels of scans in different ablation directions to be spatially coherent in the direction orthogonal to the scan direction. The volume was reconstructed using multiple registration approaches. Registration using the section outline itself as a fiducial marker resulted into a volume, which was in good agreement with the morphology visualized in the µ-CT volume. The 3D µ-CT volume could be registered to the LA-ICP-MS volume, consisting of 2.9 x 10 7 voxels, and the nucleus dimensions in 3D space could be derived.
Plant Physiology, Apr 1, 1990
2S albumin seed storage proteins undergo a complex series of posttranslational proteolytic cleava... more 2S albumin seed storage proteins undergo a complex series of posttranslational proteolytic cleavages. In order to determine if this process is correctly carried out in transgenic plants, the gene AT2S1 encoding an Arabidopsis thaliana 2S albumin isoform has been expressed in transgenic tobacco. Initial experiments using a reporter gene demonstrated that the AT2S1 promoter directs seed specific expression in both transgenic tobacco and Brassica napus plants. The entire AT2S1 gene was then transferred into tobacco plants, where it showed a tissue specific and developmentally regulated expression. Arabidopsis 2S albumin accumulates up to 0.1% of the total high-salt extractable seed protein. Protein sequencing demonstrated that the amino termini of the two Arabidopsis 2S albumin subunits were correctly processed, suggesting that the protease(s) necessary for posttranslational processing of 2S albumin precursors may display com- mon specificities among different dicot plant species. Immunocytochemical studies showed that the Arabidopsis 2S albumin is localized in the protein body matrix of tobacco endosperm and embryo. Correct processing and targeting of the 2S albumin in transgenic plants suggests that modified versions could be ex- pressed, allowing the study of 2S albumin processing and in particular the possible roles of the processed fragments in protein stability and/or targeting. 'A. D. C. is indebted to the "Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw" for a predoctoral fellowship, and J. V. was a Research Associate of the National Fund for Scientific Research (Belgium).
Endosperm Cell Death Promoted by NAC Transcription Factors Facilitates Embryo Invasion in Arabidopsis
In this work, the three-dimensional elemental distribution profile within the freshwater crustace... more In this work, the three-dimensional elemental distribution profile within the freshwater crustacean Ceriodaphnia dubia was constructed at a spatial resolution down to 5 µm via a data fusion approach employing state-of-theart laser ablation-inductively coupled plasma-time-of-flight mass spectrometry (LA-ICP-TOF-MS) and laboratory-based absorption micro-computed tomography (µ-CT). C. dubia was exposed to elevated Cu, Ni and Zn concentrations, chemically fixed, dehydrated, stained, and embedded, prior to µ-CT analysis. Subsequently, the sample was cut into 5 µm thin sections and subjected to LA-ICP-TOF-MS imaging. Multimodal image registration was performed to spatially align the 2D LA-ICP-TOF-MS images relative to the corresponding slices of the 3D µ-CT reconstruction. Mass channels corresponding to the isotopes of a single element were merged to improve the signal-to-noise ratios within the elemental images. In order to aid the visual interpretation of the data, LA-ICP-TOF-MS data were projected onto the µ-CT voxels representing tissue. Additionally, the image resolution and elemental sensitivity were compared to those obtained with synchrotron radiation based 3D confocal µ-X-ray fluorescence imaging upon a chemically fixed and air-dried C. dubia specimen.
Immuno-fluorescence microscopic analysis of post-transcriptional silencing of neomycin phosphotransferase II transgenes in Nicotiana tabacum
Archives of Physiology and Biochemistry, 1998
Water-mediated transport of plant made antibodies in Arabidopsis thaliana
MEDEDELINGEN VAN DE FACULTEIT LANDBOUWKUNDIGE EN TOEGEPASTE BIOLOGISCHE WETENSCHAPPEN, UNIVERSITEIT GENT, 1997
Molecular-physiological aspects of ethylene in Arabidopsis: hormonal control of an Arabidopsis thaliana ACC synthase gene
MEDEDELINGEN VAN DE FACULTEIT LANDBOUWKUNDIGE EN TOEGEPASTE BIOLOGISCHE WETENSCHAPPEN, UNIVERSITEIT GENT, 1995
Knockout of <scp> <i>RSN1</i> </scp> , <scp> <i>TVP18</i> </scp> or <scp> <i>CSC1</i> </scp> <i>‐2</i> causes perturbation of Golgi cisternae in <scp> <i>Pichia pastoris</i> </scp>
Traffic, Dec 12, 2020
The structural organization of the Golgi stacks in mammalian cells is intrinsically linked to fun... more The structural organization of the Golgi stacks in mammalian cells is intrinsically linked to function, including glycosylation, but the role of morphology is less clear in lower eukaryotes. Here we investigated the link between the structural organization of the Golgi and secretory pathway function using Pichia pastoris as a model system. To unstack the Golgi cisternae, we disrupted 18 genes encoding proteins in the secretory pathway without loss of viability. Using biosensors, confocal microscopy and transmission electron microscopy we identified three strains with irreversible perturbations in the stacking of the Golgi cisternae, all of which had disruption in genes that encode proteins with annotated function as or homology to calcium/calcium permeable ion channels. Despite this, no variation in the secretory pathway for ER size, whole cell glycomics or recombinant protein glycans was observed. Our investigations showed the robust nature of the secretory pathway in P. pastoris and suggest that Ca2+ concentration, homeostasis or signalling may play a significant role for Golgi stacking in this organism and should be investigated in other organisms.
Plant Physiology, Mar 1, 2016
Structural characterization of extracellular polysaccharides of Azorhizobium caulinodans and importance for nodule initiation on Sesbania rostrata
Molecular Microbiology, Mar 19, 2004
SummaryDuring lateral root base nodulation, the microsymbiont Azorhizobium caulinodans enters its... more SummaryDuring lateral root base nodulation, the microsymbiont Azorhizobium caulinodans enters its host plant, Sesbania rostrata, via the formation of outer cortical infection pockets, a process that is characterized by a massive production of H2O2. Infection threads guide bacteria from infection pockets towards nodule primordia. Previously, two mutants were constructed that produce lipopolysaccharides (LPSs) similar to one another but different from the wild‐type LPS, and that are affected in extracellular polysaccharide (EPS) production. Mutant ORS571‐X15 was blocked at the infection pocket stage and unable to produce EPS. The other mutant, ORS571‐oac2, was impaired in the release from infection threads and was surrounded by a thin layer of EPS in comparison to the wild‐type strain that produced massive amounts of EPS. Structural characterization revealed that EPS purified from cultured and nodule bacteria was a linear homopolysaccharide of α‐1,3‐linked 4,6‐O‐(1‐carboxyethylidene)‐d‐galactosyl residues. In situ H2O2 localization demonstrated that increased EPS production during early stages of invasion prevented the incorporation of H2O2 inside the bacteria, suggesting a role for EPS in protecting the microsymbiont against H2O2. In addition, ex planta assays confirmed a positive correlation between increased EPS production and enhanced protection against H2O2.
The EMBO Journal, Jun 1, 1992
We studied protein sorting signals which are responsible for the retention of reticuloplasmins in... more We studied protein sorting signals which are responsible for the retention of reticuloplasmins in the lumen of the plant endoplasmic reticulum (ER). A non-specific passenger protein, previously shown to be secreted by default, was used as a carrier for such signals. Tagging with C-terminal tetrapeptide sequences of mammalian (KDEL) and yeast (HDEL) reticuloplasmins led to effective accumulation of the protein chineras in the lumen of the plant ER. Some single amino acid substitutions within the tetrapeptide tag (-SDEL, -KDDL, -KDEI and -KDEV) can cause a complete loss of its function as a retention signal, demonstrating the high specificity of the retention machinery. However, other modifications confer efficient (-RDEL) or partial (-KEEL) retention. It is also shown that the efficiency of protein retention is not significantly impaired by an increased ligand concentration in plants. The efficiently retained chimeras (-KDEL, -HDEL and -RDEL) were shown to be recognized by a monoclonal antibody directed against the C-terminus of the mammaLian reticuloplasmin protein disulfide isomerase (PDI). The recognized epitope is also present in several putative reticuloplasmins in microsomal fractions of plant and mammalian cells, suggesting that the antibodies recognize an important structural determinant of the retention signal. In addition, data are discussed which support the view that upstream sequences beyond the C-terminal tetrapeptide can influence or may be part of the structure of reticuloplasmin retention signals.
The Plant Cell, Apr 18, 2017
In addition to the nucleus, mitochondria and chloroplasts in plant cells also contain genomes. Ef... more In addition to the nucleus, mitochondria and chloroplasts in plant cells also contain genomes. Efficient DNA repair pathways are crucial in these organelles to fix damage resulting from endogenous and exogenous factors. Plant organellar genomes are complex compared with their animal counterparts, and although several plant-specific mediators of organelle DNA repair have been reported, many regulators remain to be identified. Here, we show that a mitochondrial SWI/SNF (nucleosome remodeling) complex B protein, SWIB5, is capable of associating with mitochondrial DNA (mtDNA) in Arabidopsis thaliana. Gain-and loss-of-function mutants provided evidence for a role of SWIB5 in influencing mtDNA architecture and homologous recombination at specific intermediate-sized repeats both under normal and genotoxic conditions. SWIB5 interacts with other mitochondrial SWIB proteins. Gene expression and mutant phenotypic analysis of SWIB5 and SWIB family members suggests a link between organellar genome maintenance and cell proliferation. Taken together, our work presents a protein family that influences mtDNA architecture and homologous recombination in plants and suggests a link between organelle functioning and plant development.
Journal of Investigative Dermatology, Sep 1, 2016
RIPK4 is a member of the Receptor Interacting Protein Kinases, which are mediators of cellular st... more RIPK4 is a member of the Receptor Interacting Protein Kinases, which are mediators of cellular stress that trigger cell survival, cell death and inflammatory responses. Humans with autosomal recessive mutations in RIPK4 suffer from the Bartsocas-Papas syndrome, a lethal form of popliteal pterygium syndromes. Bartsocas-Papas patients are characterized by pterygia, syndactyly, clefting and hypoplastic genitalia. RIPK4-deficient mice largely phenocopy the defects observed in humans suffering from Bartsocas-Papas syndrome. Previously, we reported that RIPK4 is required for epidermal barrier formation. To further characterize the role of RIPK4 in epidermal development we generated keratinocyte-specific RIPK4 deficient mice (RIPK4 EKO ). RIPK4 EKO mice died shortly after birth due to dehydration caused by a defective inside-out barrier suggesting the involvement of tight junctions. In contrast, RIP-K4 EKO mice were largely rescued from the defect in epidermal outside-in barrier observed in full RIPK4 deficient mice. In vitro experiments showed that RIPK4 knockdown in the HaCaT cell line results in a down regulation of several tight junction proteins. Similarly, inducible overexpression of a kinase-dead dominant negative RIPK4 mutant decreased the protein levels of tight junction proteins. This suggests that RIPK4 kinase activity is necessary to maintain tight junction integrity and barrier formation. We also found that PKCh is required for constitutive RIPK4 activation in the HaCaT keratinocyte cell line and in primary keratinocytes. In addition, the levels of active RIPK4 are tightly regulated by proteasomal degradation mediated by the SCF b-TrCP1/2 complex. Our data identified a novel PKCh-RIPK4 signaling axis in keratinocytes.
Analytical and Bioanalytical Chemistry, Mar 28, 2019
A lipid nanoparticle platform for mRNA delivery through repurposing of cationic amphiphilic drugs
Journal of Controlled Release, Oct 1, 2022
Clinical Genetics, Dec 12, 2019
Biallelic MFSD8 variants are an established cause of severe late-infantile subtype of neuronal ce... more Biallelic MFSD8 variants are an established cause of severe late-infantile subtype of neuronal ceroid lipofuscinosis (v-LINCL), a severe lysosomal storage disorder, but have also been associated with nonsyndromic adult-onset maculopathy. Here, we functionally characterized two novel MFSD8 variants found in a child with juvenile isolated maculopathy, in order to establish a refined prognosis. ABCA4 locus resequencing was followed by the analysis of other inherited retinal disease genes by whole exome sequencing (WES). Minigene assays and cDNA sequencing were used to assess the effect of a novel MFSD8 splice variant. MFSD8 expression was quantified with qPCR and overexpression studies were analyzed by immunoblotting. Transmission electron microscopy (TEM) was performed on a skin biopsy and ophthalmological and neurological re-examinations were conducted. WES revealed two novel MFSD8 variants: c.[590del];[439+3A>C] p.[Gly197Valfs*2];[Ile67Glufs*3]. Characterization of the c.439+3A>C variant via splice assays showed exon-skipping (p.Ile67Glufs*3), while overexpression studies of the corresponding protein indicated expression of a truncated polypeptide. In addition, a significantly reduced MFSD8 RNA expression was noted in patient's lymphocytes. TEM of a skin biopsy revealed typical v-LINCL
Analytical Chemistry, Mar 16, 2017
In this work, the three-dimensional elemental distribution profile within the freshwater crustace... more In this work, the three-dimensional elemental distribution profile within the freshwater crustacean Ceriodaphnia dubia was constructed at a spatial resolution down to 5 µm via a data fusion approach employing state-of-theart laser ablation-inductively coupled plasma-time-of-flight mass spectrometry (LA-ICP-TOF-MS) and laboratory-based absorption micro-computed tomography (µ-CT). C. dubia was exposed to elevated Cu, Ni and Zn concentrations, chemically fixed, dehydrated, stained, and embedded, prior to µ-CT analysis. Subsequently, the sample was cut into 5 µm thin sections and subjected to LA-ICP-TOF-MS imaging. Multimodal image registration was performed to spatially align the 2D LA-ICP-TOF-MS images relative to the corresponding slices of the 3D µ-CT reconstruction. Mass channels corresponding to the isotopes of a single element were merged to improve the signal-to-noise ratios within the elemental images. In order to aid the visual interpretation of the data, LA-ICP-TOF-MS data were projected onto the µ-CT voxels representing tissue. Additionally, the image resolution and elemental sensitivity were compared to those obtained with synchrotron radiation based 3D confocal µ-X-ray fluorescence imaging upon a chemically fixed and air-dried C. dubia specimen.
The Plant Cell, Jul 1, 2014
Plant plasma membrane intrinsic proteins (PIPs) are aquaporins that facilitate the passive moveme... more Plant plasma membrane intrinsic proteins (PIPs) are aquaporins that facilitate the passive movement of water and small neutral solutes through biological membranes. Here, we report that post-Golgi trafficking of PIP2;7 in Arabidopsis thaliana involves specific interactions with two syntaxin proteins, namely, the Qc-SNARE SYP61 and the Qa-SNARE SYP121, that the proper delivery of PIP2;7 to the plasma membrane depends on the activity of the two SNAREs, and that the SNAREs colocalize and physically interact. These findings are indicative of an important role for SYP61 and SYP121, possibly forming a SNARE complex. Our data support a model in which direct interactions between specific SNARE proteins and PIP aquaporins modulate their post-Golgi trafficking and thus contribute to the fine-tuning of the water permeability of the plasma membrane.
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Papers by Riet M De Rycke