Publications by authors named "Arie J Verkleij"

59 Publications

Optimizing immuno-labeling for correlative fluorescence and electron microscopy on a single specimen.

J Struct Biol 2012 Nov 13;180(2):382-6. Epub 2012 Sep 13.

Molecular Biophysics, Department of Physics and Astronomy, Utrecht University, Princetonplein 1, Utrecht, The Netherlands.

Correlative fluorescence and electron microscopy has become an indispensible tool for research in cell biology. The integrated Laser and Electron Microscope (iLEM) combines a Fluorescence Microscope (FM) and a Transmission Electron Microscope (TEM) within one set-up. This unique imaging tool allows for rapid identification of a region of interest with the FM, and subsequent high resolution TEM imaging of this area. Sample preparation is one of the major challenges in correlative microscopy of a single specimen; it needs to be apt for both FM and TEM imaging. For iLEM, the performance of the fluorescent probe should not be impaired by the vacuum of the TEM. In this technical note, we have compared the fluorescence intensity of six fluorescent probes in a dry, oxygen free environment relative to their performance in water. We demonstrate that the intensity of some fluorophores is strongly influenced by its surroundings, which should be taken into account in the design of the experiment. Furthermore, a freeze-substitution and Lowicryl resin embedding protocol is described that yields excellent membrane contrast in the TEM but prevents quenching of the fluorescent immuno-labeling. The embedding protocol results in a single specimen preparation procedure that performs well in both FM and TEM. Such procedures are not only essential for the iLEM, but also of great value to other correlative microscopy approaches.
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http://dx.doi.org/10.1016/j.jsb.2012.09.002DOI Listing
November 2012

Arabidopsis PLETHORA transcription factors control phyllotaxis.

Curr Biol 2011 Jul 23;21(13):1123-8. Epub 2011 Jun 23.

Molecular Genetics, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

The pattern of plant organ initiation at the shoot apical meristem (SAM), termed phyllotaxis, displays regularities that have long intrigued botanists and mathematicians alike. In the SAM, the central zone (CZ) contains a population of stem cells that replenish the surrounding peripheral zone (PZ), where organs are generated in regular patterns. These patterns differ between species and may change in response to developmental or environmental cues [1]. Expression analysis of auxin efflux facilitators of the PIN-FORMED (PIN) family combined with modeling of auxin transport has indicated that organ initiation is associated with intracellular polarization of PIN proteins and auxin accumulation [2-10]. However, regulators that modulate PIN activity to determine phyllotactic patterns have hitherto been unknown. Here we reveal that three redundantly acting PLETHORA (PLT)-like AP2 domain transcription factors control shoot organ positioning in the model plant Arabidopsis thaliana. Loss of PLT3, PLT5, and PLT7 function leads to nonrandom, metastable changes in phyllotaxis. Phyllotactic changes in plt3plt5plt7 mutants are largely attributable to misregulation of PIN1 and can be recapitulated by reducing PIN1 dosage, revealing that PLT proteins are key regulators of PIN1 activity in control of phyllotaxis.
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http://dx.doi.org/10.1016/j.cub.2011.05.009DOI Listing
July 2011

A biparatopic anti-EGFR nanobody efficiently inhibits solid tumour growth.

Int J Cancer 2011 Oct 8;129(8):2013-24. Epub 2011 Aug 8.

Cell Biology, Department of Biology, Science Faculty, Utrecht University, Utrecht, The Netherlands.

The epidermal growth factor receptor (EGFR) has been shown to be a valid cancer target for antibody-based therapy. At present, several anti-EGFR monoclonal antibodies have been successfully used, such as cetuximab and matuzumab. X-ray crystallography data show that these antibodies bind to different epitopes on the ecto-domain of EGFR, providing a rationale for the combined use of these two antibody specificities. We have previously reported on the successful isolation of antagonistic anti-EGFR nanobodies. In our study, we aimed to improve the efficacy of these molecules by combining nanobodies with specificities similar to both cetuximab and matuzumab into a single biparatopic molecule. Carefully designed phage nanobody selections resulted in two sets of nanobodies that specifically blocked the binding of either matuzumab or cetuximab to EGFR and that did not compete for each others' binding. A combination of nanobodies from both epitope groups into the biparatopic nanobody CONAN-1 was shown to block EGFR activation more efficiently than monovalent or bivalent (monospecific) nanobodies. In addition, this biparatopic nanobody potently inhibited EGF-dependent cell proliferation. Importantly, in an in vivo model of athymic mice bearing A431 xenografts, CONAN-1 inhibited tumour outgrowth with an almost similar potency as the whole mAb cetuximab, despite the fact that CONAN-1 is devoid of an Fc portion that could mediate immune effector functions. Compared to therapy using bivalent, monospecific nanobodies, CONAN-1 was clearly more potent in tumour growth inhibition. These results show that the rational design of biparatopic nanobody-based anticancer therapeutics may yield potent lead molecules for further development.
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http://dx.doi.org/10.1002/ijc.26145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4197845PMC
October 2011

VIS2FIX: a high-speed fixation method for immuno-electron microscopy.

Traffic 2011 Jul 28;12(7):806-14. Epub 2011 Apr 28.

Molecular Biophysics, Department of Physics and Astronomy, Utrecht University, Princetonplein 1, NL-3508 TA Utrecht, The Netherlands.

Immuno-transmission electron microscopy (TEM) is the technique of choice for high-resolution localization of proteins in fixed specimen. Here we introduce 2 novel methods for the fixation of sections from cryo-immobilized samples that result in excellent ultrastructural preservation. These high-speed fixation techniques, both called VIS2FIX, allow for a reduction in sample preparation time from at least 1 week to only 8 h. The methods were validated in immuno-TEM experiments on THP-1 monocytes, human umbilical vein endothelial cells (HUVECs) and Madin-Darby canine kidney (MDCK-II) cells. The fixation and retention of neutral lipids is demonstrated, offering unique prospects for the application of immuno-TEM in the lipidomics field. Furthermore, the VIS2FIX methods were successfully employed in correlative fluorescence and electron microscopy.
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http://dx.doi.org/10.1111/j.1600-0854.2011.01199.xDOI Listing
July 2011

Ligand-induced EGF receptor oligomerization is kinase-dependent and enhances internalization.

J Biol Chem 2010 Dec 12;285(50):39481-9. Epub 2010 Oct 12.

Department of Cellular Dynamics, Science Faculty, Utrecht University, 3584 CH Utrecht, The Netherlands.

The current activation model of the EGF receptor (EGFR) predicts that binding of EGF results in dimerization and oligomerization of the EGFR, leading to the allosteric activation of the intracellular tyrosine kinase. Little is known about the regulatory mechanism of receptor oligomerization. In this study, we have employed FRET between identical fluorophores (homo-FRET) to monitor the dimerization and oligomerization state of the EGFR before and after receptor activation. Our data show that, in the absence of ligand, ∼40% of the EGFR molecules were present as inactive dimers or predimers. The monomer/predimer ratio was not affected by deletion of the intracellular domain. Ligand binding induced the formation of receptor oligomers, which were found in both the plasma membrane and intracellular structures. Ligand-induced oligomerization required tyrosine kinase activity and nine different tyrosine kinase substrate residues. This indicates that the binding of signaling molecules to activated EGFRs results in EGFR oligomerization. Induction of EGFR predimers or pre-oligomers using the EGFR fused to the FK506-binding protein did not affect signaling but was found to enhance EGF-induced receptor internalization. Our data show that EGFR oligomerization is the result of EGFR signaling and enhances EGFR internalization.
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http://dx.doi.org/10.1074/jbc.M110.164731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2998144PMC
December 2010

The making of frozen-hydrated, vitreous lamellas from cells for cryo-electron microscopy.

J Struct Biol 2010 Nov 16;172(2):180-90. Epub 2010 Jul 16.

FEI Company, Achtseweg Noord 5, 5600 KA Eindhoven, The Netherlands.

There has been a long standing desire to produce thick (up to 500 nm) cryo-sections of fully hydrated cells and tissue for high-resolution analysis in their natural state by cryo-transmission electron microscopy. Here, we present a method that can successfully produce sections (lamellas in FIB-SEM terminology) of fully hydrated, unstained cells from high-pressure frozen samples by focused ion beam (FIB) milling. The samples are therefore placed in thin copper tubes and vitrified by high-pressure freezing. For transfer, handling and subsequent milling, the tubes are placed in a novel connective device (ferrule) that protects the sample from devitrification and contamination and passes through all operation steps. A piezo driven sample positioning stage (cryo-nano-bench, CNB) with three degrees of freedom was additionally developed to enable accurate milling of frozen-hydrated lamellas. With the CNB, high-pressure frozen samples can be milled to produce either thin lamellas (<100 nm), for direct imaging by high-resolution cryo-TEM or thicker lamellas (300-500 nm) for cryo-electron tomography. The sample remains vitreous throughout the process by using the presented tools and methods. The results are an important step towards investigating larger cells and even tissue in there natural state which in the end will enable us to gain better insights into cellular processes.
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http://dx.doi.org/10.1016/j.jsb.2010.07.004DOI Listing
November 2010

Spiral coating of the endothelial caveolar membranes as revealed by electron tomography and template matching.

Traffic 2010 Jan;11(1):138-50

Department of Biomolecular Imaging, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Caveolae are invaginations of the plasma membrane involved in multiple cellular processes, including transcytosis. In this paper we present an extensive 3-D electron tomographic study of the endothelial caveolar system in situ. Analysis of large cellular volumes of (high-pressure frozen, freeze-substituted and epon-embedded) human umbilical vein endothelial cells (HUVECs) provided a notable view on the architecture of the caveolar system that comprises--as confirmed by 3-D immunolabeling for caveolin of 'intact' cells--bona fide caveolae, free plasmalemmal vesicles, racemose invaginations and free multi-caveolar bodies. Application of template matching to tomograms allowed the 3-D localization of caveolar membrane coatings in a robust manner. In this way we observed that bona fide endothelial caveolae, cryofixed and embedded in their cellular context, show a spiral organization of the coating as shown in the past for chemically fixed and freeze-etched caveolae from fibroblasts. Meticulous 3-D analysis further revealed that the coatings are distributed in triads of spirals over the caveolar bulb and neck. Remarkably, this coating distribution is consistently present over the membranes of the other members of the caveolar system in HUVECs. The novel observations that we present clarify the ultrastructural complexity of the 'intact' caveolar system, setting a detailed morphological basis for its functional diversity.
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http://dx.doi.org/10.1111/j.1600-0854.2009.01008.xDOI Listing
January 2010

AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis.

J Cell Biol 2009 Oct;187(2):247-64

Structure and Membrane Compartments, Centre Nationale de la Recherche Scientifique, UMR 144 Institut Curie, Centre de Recherche, Paris F-75248, France.

Specialized cell types exploit endosomal trafficking to deliver protein cargoes to cell type-specific lysosome-related organelles (LROs), but how endosomes are specified for this function is not known. In this study, we show that the clathrin adaptor AP-1 and the kinesin motor KIF13A together create peripheral recycling endosomal subdomains in melanocytes required for cargo delivery to maturing melanosomes. In cells depleted of AP-1 or KIF13A, a subpopulation of recycling endosomes redistributes to pericentriolar clusters, resulting in sequestration of melanosomal enzymes like Tyrp1 in vacuolar endosomes and consequent inhibition of melanin synthesis and melanosome maturation. Immunocytochemistry, live cell imaging, and electron tomography reveal AP-1- and KIF13A-dependent dynamic close appositions and continuities between peripheral endosomal tubules and melanosomes. Our results reveal that LRO protein sorting is coupled to cell type-specific positioning of endosomes that facilitate endosome-LRO contacts and are required for organelle maturation.
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http://dx.doi.org/10.1083/jcb.200907122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2768840PMC
October 2009

Cell division ring, a new cell division protein and vertical inheritance of a bacterial organelle in anammox planctomycetes.

Mol Microbiol 2009 Sep 24;73(6):1009-19. Epub 2009 Aug 24.

Department of Microbiology, Institute for Water and Wetland Research, Faculty of Science, Radboud University Nijmegen, Nijmegen, The Netherlands.

Anammox bacteria are members of the phylum Planctomycetes that oxidize ammonium anaerobically and produce a significant part of the atmosphere's dinitrogen gas. They contain a unique bacterial organelle, the anammoxosome, which is the locus of anammox catabolism. While studying anammox cell and anammoxosome division with transmission electron microscopy including electron tomography, we observed a cell division ring in the outermost compartment of dividing anammox cells. In most Bacteria, GTP hydrolysis drives the tubulin-analogue FtsZ to assemble into a ring-like structure at the cell division site where it functions as a scaffold for the molecular machinery that performs cell division. However, the genome of the anammox bacterium 'Candidatus Kuenenia stuttgartiensis' does not encode ftsZ. Genomic analysis of open reading frames with potential GTPase activity indicated a possible novel cell division ring gene: kustd1438, which was unrelated to ftsZ. Immunogold localization specifically localized kustd1438 to the cell division ring. Genomic analyses of other members of the phyla Planctomycetes and Chlamydiae revealed no putative functional homologues of kustd1438, suggesting that it is specific to anammox bacteria. Electron tomography also revealed that the bacterial organelle was elongated along with the rest of the cell and divided equally among daughter cells during the cell division process.
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http://dx.doi.org/10.1111/j.1365-2958.2009.06841.xDOI Listing
September 2009

Golgi-associated cPLA2alpha regulates endothelial cell-cell junction integrity by controlling the trafficking of transmembrane junction proteins.

Mol Biol Cell 2009 Oct 12;20(19):4225-34. Epub 2009 Aug 12.

Cellular Architecture and Dynamics, Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands.

In endothelial cells specifically, cPLA2alpha translocates from the cytoplasm to the Golgi complex in response to cell confluence. Considering the link between confluence and cell-cell junction formation, and the emerging role of cPLA2alpha in intracellular trafficking, we tested whether Golgi-associated cPLA2alpha is involved in the trafficking of junction proteins. Here, we show that the redistribution of cPLA2alpha from the cytoplasm to the Golgi correlates with adherens junction maturation and occurs before tight junction formation. Disruption of adherens junctions using a blocking anti-VE-cadherin antibody reverses the association of cPLA2alpha with the Golgi. Silencing of cPLA2alpha and inhibition of cPLA2alpha enzymatic activity using various inhibitors result in the diminished presence of the transmembrane junction proteins VE-cadherin, occludin, and claudin-5 at cell-cell contacts, and in their accumulation at the Golgi. Altogether, our data support the idea that VE-cadherin triggers the relocation of cPLA2alpha to the Golgi and that in turn, Golgi-associated cPLA2alpha regulates the transport of transmembrane junction proteins through or from the Golgi, thereby controlling the integrity of endothelial cell-cell junctions.
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http://dx.doi.org/10.1091/mbc.e08-02-0210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2754936PMC
October 2009

Sol-gel transitions and liquid crystal phase transitions in concentrated aqueous suspensions of colloidal gibbsite platelets.

J Phys Chem B 2009 Aug;113(34):11604-13

van 't Hoff Laboratory for Physical and Colloid Chemistry, Faculty of Sciences, Utrecht University, P.O. Box 80.056, 3508 TB Utrecht, The Netherlands.

In this paper, we present a comprehensive study of the sol-gel transitions and liquid crystal phase transitions in aqueous suspensions of positively charged colloidal gibbsite platelets at pH 4-5 over a wide range of particle concentrations (50-600 g/L) and salt concentrations (10(-4)-10(-1) M NaCl). A detailed sol-gel diagram was established by oscillatory rheological experiments. These demonstrate the presence of kinetically arrested states both at high and at low salt concentrations, enclosing a sol region. Birefringence and iridescence show that in the sol state nematic and hexagonal columnar liquid crystal phases are formed. The gel and liquid crystal structures are studied in further detail using small-angle X-ray scattering (SAXS) and cryo-focused ion beam/scanning electron microscopy (cryo-FIB-SEM). The gel formed at high salt concentration shows signatures of a sponge-like structure and does not display birefringence. In the sol region, by lowering the salt concentration and/or increasing the gibbsite concentration, the nematic phase gradually transforms from the discotic nematic (ND) into the columnar nematic (NC) with much stronger side-to-side interparticle correlations. Subsequently, this NC structure can be either transformed into the hexagonal columnar phase or arrested into a birefringent repulsive gel state with NC structure.
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http://dx.doi.org/10.1021/jp903783bDOI Listing
August 2009

Septal pore complex morphology in the Agaricomycotina (Basidiomycota) with emphasis on the Cantharellales and Hymenochaetales.

Mycol Res 2009 May 27;113(5):559-76. Epub 2009 Jan 27.

CBS Fungal Biodiversity Centre, Royal Netherlands Academy of Arts and Sciences (KNAW), Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.

The ultrastructure of septa and septum-associated septal pore caps are important taxonomic markers in the Agaricomycotina (Basidiomycota, Fungi). The septal pore caps covering the typical basidiomycetous dolipore septum are divided into three main phenotypically recognized morphotypes: vesicular-tubular (including the vesicular, sacculate, tubular, ampulliform, and globular morphotypes), imperforate, and perforate. Until recently, the septal pore cap-type reflected the higher-order relationships within the Agaricomycotina. However, the new classification of Fungi resulted in many changes including revision of existing and addition of new orders. Therefore, the septal pore cap ultrastructure of more than 325 species as reported in literature was related to this new classification. In addition, the septal pore cap ultrastructures of Rickenella fibula and Cantharellus formosus were examined by transmission electron microscopy. Both fungi have dolipore septa associated with perforate septal pore caps. These results combined with data from the literature show that the septal pore cap-type within orders of the Agaricomycotina is generally monomorphic, except for the Cantharellales and Hymenochaetales. It appears from the fungal phylogeny combined with the septal pore cap ultrastructure that the vesicular-tubular and the imperforate type both may have arisen from endoplasmic reticulum. Thereafter, the imperforate type eventually gave rise to the perforate septal pore cap-type.
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http://dx.doi.org/10.1016/j.mycres.2008.12.007DOI Listing
May 2009

Membrane contact sites between apicoplast and ER in Toxoplasma gondii revealed by electron tomography.

Traffic 2009 Oct 9;10(10):1471-80. Epub 2009 Jun 9.

Electron Microscopy and Structural Analysis, Department of Biology, Faculty of Sciences, Utrecht University, 3584 CH Utrecht, The Netherlands.

Toxoplasma gondii is an obligate intracellular parasite from the phylum Apicomplexa. A hallmark of these protozoans is the presence of a unique apical complex of organelles that includes the apicoplast, a plastid acquired by secondary endosymbiosis. The apicoplast is indispensible for parasite viability. It harbours a fatty acid biosynthesis type II (FAS II) pathway and plays a key role in the parasite lipid metabolism. Possibly, the apicoplast provides components for the establishment and the maturation of the parasitophorous vacuole, ensuring the successful infection of the host cell. This implies the presence of a transport mechanism for fast and accurate allocation of lipids between the apicoplast and other membrane-bound compartments in the parasite cell. Using a combination of high-pressure freezing, freeze-substitution and electron tomography, we analysed the ultrastructural organization of the apicoplast of T. gondii in relation with the endoplasmic reticulum (ER). This allowed us to clearly show the presence of four continuous membranes surrounding the apicoplast. We present, for the first time, the existence of membrane contact sites between the apicoplast outermost membrane and the ER. We describe the morphological characteristics of these structures and discuss their potential significance for the subcellular distribution of lipids in the parasite.
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http://dx.doi.org/10.1111/j.1600-0854.2009.00954.xDOI Listing
October 2009

Quantitative structural analysis of binary nanocrystal superlattices by electron tomography.

Nano Lett 2009 Jul;9(7):2719-24

Inorganic Chemistry and Catalysis, Debye Institute for Nanomaterials Science, Utrecht University, Sorbonnelaan 16, Utrecht 3584 CA, The Netherlands.

Binary nanocrystal superlattices, that is, ordered structures of two sorts of nanocolloids, hold promise for a series of functional materials with novel collective properties. Here we show that based on electron tomography a comprehensive, quantitative, three-dimensional characterization of these systems down to the single nanocrystal level can be achieved, which is key in understanding the emerging materials properties. On four binary lattices composed of PbSe, CdSe, and Au nanocrystals, we illustrate that ambiguous interpretations based on two-dimensional transmission electron microscopy can be prevented, nanocrystal sizes and superlattice parameters accurately determined, individual crystallographic point and plane defects studied, and the order/disorder at the top and bottom surfaces imaged. Furthermore, our results suggest that superlattice nucleation and growth occurred at the suspension/air interface and that the unit cells of some lattices are anisotropically deformed upon drying.
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http://dx.doi.org/10.1021/nl901212mDOI Listing
July 2009

Induced membrane domains as visualized by electron tomography and template matching.

J Struct Biol 2009 May;166(2):156-61

Cellular Architecture & Dynamics, Utrecht University, P.O. Box 80.056, 3508 TB Utrecht, The Netherlands.

Membranes play a crucial role in many cellular processes, and it is therefore not surprising that many electron tomographic studies in life sciences concern membranous structures. While these tomographic studies provide many new insights into membrane connections and continuities in three dimensions, they are mostly limited to a macro-morphological level. In this paper, we demonstrate that by combining electron tomography and three-dimensional template matching we are able to investigate membrane morphology at a new level: membrane domains in three dimensions. To test this, temperature induced lipid phase separation in the biological model system of the Escherichia coli bacteria was used. We compared the inner (containing phospholipids) and outer (containing lipopolysaccharides) leaflet of the E. coli outer membrane at both 37 and -20 degrees C, and could visualize how these leaflets react differently to temperature shifts. These findings can be explained by the physico-chemical nature of the building blocks and are in line with earlier published data. This study shows that the combination of electron tomography and template matching is robust enough to visualize membrane domains that are beyond the perception of manual annotation.
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http://dx.doi.org/10.1016/j.jsb.2009.01.006DOI Listing
May 2009

Electron tomography for heterogeneous catalysts and related nanostructured materials.

Chem Rev 2009 May;109(5):1613-29

Inorganic Chemistry and Catalysis, Debye Institute for Nanomaterials Science, Utrecht University, Sorbonnelaan 16, 3584 CA, Utrecht, The Netherlands.

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http://dx.doi.org/10.1021/cr800434tDOI Listing
May 2009

Tannic acid-mediated osmium impregnation after freeze-substitution: a strategy to enhance membrane contrast for electron tomography.

J Struct Biol 2009 Apr 30;166(1):103-6. Epub 2008 Dec 30.

Department of Cellular Architecture and Dynamics, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

In this technical note we report a tannic acid-mediated osmium impregnation method that, applied after freeze-substitution, increases membrane contrast in cells for transmission electron microscopy and tomography studies. The general staining that is achieved allows visualization of organelles, plasma membrane and associated specializations (e.g. caveolae) in non-post-stained plastic sections by conventional transmission electron microscopy. In combination with electron tomography it results in membranes with a proper contrast and equal staining pattern through the depth of the tomograms. The protocol that we contribute can serve as starting point for those willing to improve the membrane contrast of their specimens or to make 3D studies on the architecture of membranous compartments by electron tomography.
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http://dx.doi.org/10.1016/j.jsb.2008.12.009DOI Listing
April 2009

Electron tomography of early melanosomes: implications for melanogenesis and the generation of fibrillar amyloid sheets.

Proc Natl Acad Sci U S A 2008 Dec 25;105(50):19726-31. Epub 2008 Nov 25.

Institut Curie, Centre de Recherche, and Unité Mixte de Recherche 144, Centre National de la Recherche Scientifique, F-75248 Paris, France.

Melanosomes are lysosome-related organelles (LROs) in which melanins are synthesized and stored. Early stage melanosomes are characterized morphologically by intralumenal fibrils upon which melanins are deposited in later stages. The integral membrane protein Pmel17 is a component of the fibrils, can nucleate fibril formation in the absence of other pigment cell-specific proteins, and forms amyloid-like fibrils in vitro. Before fibril formation Pmel17 traffics through multivesicular endosomal compartments, but how these compartments participate in downstream events leading to fibril formation is not fully known. By using high-pressure freezing of MNT-1 melanoma cells and freeze substitution to optimize ultrastructural preservation followed by double tilt 3D electron tomography, we show that the amyloid-like fibrils begin to form in multivesicular compartments, where they radiate from the luminal side of intralumenal membrane vesicles. The fibrils in fully formed stage II premelanosomes organize into sheet-like arrays and exclude the remaining intralumenal vesicles, which are smaller and often in continuity with the limiting membrane. These observations indicate that premelanosome fibrils form in association with intralumenal endosomal membranes. We suggest that similar processes regulate amyloid formation in pathological models.
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http://dx.doi.org/10.1073/pnas.0803488105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2604932PMC
December 2008

Effective lead selection for improved protein production in Aspergillus niger based on integrated genomics.

Fungal Genet Biol 2009 Mar 12;46 Suppl 1:S141-52. Epub 2008 Sep 12.

DSM Food Specialties, PO Box 1, NL-2600 MA Delft, The Netherlands.

The filamentous fungus Aspergillus niger is widely exploited for industrial production of enzymes and organic acids. An integrated genomics approach was developed to determine cellular responses of A. niger to protein production in well-controlled fermentations. Different protein extraction methods in combination with automated sample processing and protein identification allowed quantitative analysis of 898 proteins. Three different enzyme overproducing strains were compared to their isogenic fungal host strains. Clear differences in response to the amount and nature of the overproduced enzymes were observed. The corresponding genes of the differentially expressed proteins were studied using transcriptomics. Genes that were up-regulated both at the proteome and transcriptome level were selected as leads for generic strain improvement. Up-regulated proteins included proteins involved in carbon and nitrogen metabolism as well as (oxidative) stress response, and proteins involved in protein folding and endoplasmic reticulum-associated degradation (ERAD). Reduction of protein degradation through the removal of the ERAD factor doaA combined with overexpression of the oligosaccharyl transferase sttC in A. niger overproducing beta-glucuronidase (GUS) strains indeed resulted in a small increase in GUS expression.
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http://dx.doi.org/10.1016/j.fgb.2008.08.012DOI Listing
March 2009

Discovery of a new RNA-containing nuclear structure in UVC-induced apoptotic cells by integrated laser electron microscopy.

Biol Cell 2009 May;101(5):287-99

Electron Microscopy and Structure Analysis, Cellular Architecture and Dynamics, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands.

Background Information: Treatment of cells with UVC radiation leads to the formation of DNA cross-links which, if not repaired, can lead to apoptosis. gamma-H2AX and cleaved caspase 3 are proteins formed during UVC-induced DNA damage and apoptosis respectively. The present study sets out to identify early morphological markers of apoptosis using a new method of correlative microscopy, ILEM (integrated laser electron microscopy). Cleaved caspase 3 and gamma-H2AX were immunofluorescently labelled to mark the cells of interest. These cells were subsequently searched in the fluorescence mode of the ILEM and further analysed at high resolution with TEM (transmission electron microscopy).

Results: Following the treatment of HUVECs (human umbilical vein endothelial cells) with UVC radiation, in the majority of the cells gamma-H2AX was formed, whereas only in a subset of cells caspase 3 was activated. In severely damaged cells with high levels of gamma-H2AX a round, electron-dense nuclear structure was found, which was hitherto not identified in UV-stressed cells. This structure exists only in nuclei of cells containing cleaved caspase 3 and is present during all stages of the apoptotic process. Energy-loss imaging showed that the nuclear structure accumulates phosphorus, indicating that it is rich in nucleic acids. Because the nuclear structure did not label for DNA and was not affected by regressive EDTA treatment, it is suggested that the UV-induced nuclear structure contains a high amount of RNA.

Conclusions: Because the UV-induced nuclear structure was only found in cells labelled for cleaved caspase 3 it is proposed as an electron microscopic marker for all stages of apoptosis. Such a marker will especially facilitate the screening for early apoptotic cells, which lack the well-known hallmarks of apoptosis within a cell population. It also raises new questions on the mechanisms involved in the UV-induced apoptotic pathway.
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http://dx.doi.org/10.1042/BC20080076DOI Listing
May 2009

Septal pore cap protein SPC18, isolated from the basidiomycetous fungus Rhizoctonia solani, also resides in pore plugs.

Eukaryot Cell 2008 Oct 29;7(10):1865-73. Epub 2008 Aug 29.

CBS Fungal Biodiversity Centre, Institute of the Royal Netherlands Academy of Arts and Sciences, KNAW, Utrecht, The Netherlands.

The hyphae of filamentous fungi are compartmentalized by septa that have a central pore. The fungal septa and septum-associated structures play an important role in maintaining cellular and intrahyphal homeostasis. The dolipore septa in the higher Basidiomycota (i.e., Agaricomycotina) are associated with septal pore caps. Although the ultrastructure of the septal pore caps has been studied extensively, neither the biochemical composition nor the function of these organelles is known. Here, we report the identification of the glycoprotein SPC18 that was found in the septal pore cap-enriched fraction of the basidiomycetous fungus Rhizoctonia solani. Based on its N-terminal sequence, the SPC18 gene was isolated. SPC18 encodes a protein of 158 amino acid residues, which contains a hydrophobic signal peptide for targeting to the endoplasmic reticulum and has an N-glycosylation motif. Immunolocalization showed that SPC18 is present in the septal pore caps. Surprisingly, we also observed SPC18 being localized in some plugs. The data reported here strongly support the hypothesis that septal pore caps are derived from endoplasmic reticulum and are involved in dolipore plugging and, thus, contribute to hyphal homeostasis in basidiomycetous fungi.
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http://dx.doi.org/10.1128/EC.00125-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2568075PMC
October 2008

Integrated fluorescence and transmission electron microscopy.

J Struct Biol 2008 Nov 11;164(2):183-9. Epub 2008 Jul 11.

Molecular Biophysics, Department of Physics, Faculty of Science, Utrecht University, Princetonplein 1, 3584 CC Utrecht, The Netherlands.

Correlative microscopy is a powerful technique that combines the strengths of fluorescence microscopy and electron microscopy. The first enables rapid searching for regions of interest in large fields of view while the latter exhibits superior resolution over a narrow field of view. Routine use of correlative microscopy is seriously hampered by the cumbersome and elaborate experimental procedures. This is partly due to the use of two separate microscopes for fluorescence and electron microscopy. Here, an integrated approach to correlative microscopy is presented based on a laser scanning fluorescence microscope integrated in a transmission electron microscope. Using this approach the search for features in the specimen is greatly simplified and the time to carry out the experiment is strongly reduced. The potential of the integrated approach is demonstrated at room temperature on specimens of rat intestine cells labeled with AlexaFluor488 conjugated to wheat germ agglutinin and on rat liver peroxisomes immunolabeled with anti-catalase antibodies and secondary AlexaFluor488 antibodies and 10nm protein A-gold.
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http://dx.doi.org/10.1016/j.jsb.2008.07.003DOI Listing
November 2008

EGF induces coalescence of different lipid rafts.

J Cell Sci 2008 Aug 15;121(Pt 15):2519-28. Epub 2008 Jul 15.

Department of Cellular Architecture and Dynamics, Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands.

The suggestion that microdomains may function as signaling platforms arose from the presence of growth factor receptors, such as the EGFR, in biochemically isolated lipid raft fractions. To investigate the role of EGFR activation in the organization of lipid rafts we have performed FLIM analyses using putative lipid raft markers such as ganglioside GM1 and glycosylphosphatidylinositol (GPI)-anchored GFP (GPI-GFP). The EGFR was labeled using single domain antibodies from Llama glama that specifically bind the EGFR without stimulating its kinase activity. Our FLIM analyses demonstrate a cholesterol-independent colocalization of GM1 with EGFR, which was not observed for the transferrin receptor. By contrast, a cholesterol-dependent colocalization was observed for GM1 with GPI-GFP. In the resting state no colocalization was observed between EGFR and GPI-GFP, but stimulation of the cell with EGF resulted in the colocalization at the nanoscale level of EGFR and GPI-GFP. Moreover, EGF induced the enrichment of GPI-GFP in a detergent-free lipid raft fraction. Our results suggest that EGF induces the coalescence of the two types of GM1-containing microdomains that might lead to the formation of signaling platforms.
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http://dx.doi.org/10.1242/jcs.028753DOI Listing
August 2008

Membrane damage by human islet amyloid polypeptide through fibril growth at the membrane.

Proc Natl Acad Sci U S A 2008 Apr 11;105(16):6033-8. Epub 2008 Apr 11.

Department of Metabolic and Endocrine Diseases, Division of Biomedical Genetics, University Medical Center Utrecht, P. O. Box 85090, 3508 AB Utrecht, The Netherlands.

Fibrillar protein deposits (amyloid) in the pancreatic islets of Langerhans are thought to be involved in death of the insulin-producing islet beta cells in type 2 diabetes mellitus. It has been suggested that the mechanism of this beta cell death involves membrane disruption by human islet amyloid polypeptide (hIAPP), the major constituent of islet amyloid. However, the molecular mechanism of hIAPP-induced membrane disruption is not known. Here, we propose a hypothesis that growth of hIAPP fibrils at the membrane causes membrane damage. We studied the kinetics of hIAPP-induced membrane damage in relation to hIAPP fibril growth and found that the kinetic profile of hIAPP-induced membrane damage is characterized by a lag phase and a sigmoidal transition, which matches the kinetic profile of hIAPP fibril growth. The observation that seeding accelerates membrane damage supports the hypothesis. In addition, variables that are well known to affect hIAPP fibril formation, i.e., the presence of a fibril formation inhibitor, hIAPP concentration, and lipid composition, were found to have the same effect on hIAPP-induced membrane damage. Furthermore, electron microscopy analysis showed that hIAPP fibrils line the surface of distorted phospholipid vesicles, in agreement with the notion that hIAPP fibril growth at the membrane and membrane damage are physically connected. Together, these observations point toward a mechanism in which growth of hIAPP fibrils, rather than a particular hIAPP species, is responsible for the observed membrane damage. This hypothesis provides an additional mechanism next to the previously proposed role of oligomers as the main cytotoxic species of amyloidogenic proteins.
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http://dx.doi.org/10.1073/pnas.0708354105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2329711PMC
April 2008

Linking ultrastructure and function in four genera of anaerobic ammonium-oxidizing bacteria: cell plan, glycogen storage, and localization of cytochrome C proteins.

J Bacteriol 2008 Jan 9;190(2):708-17. Epub 2007 Nov 9.

Department of Microbiology, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands.

Anaerobic ammonium oxidation (anammox) is an ecologically and industrially important process and is performed by a clade of deeply branching Planctomycetes. Anammox bacteria possess an intracytoplasmic membrane-bounded organelle, the anammoxosome. In the present study, the ultrastructures of four different genera of anammox bacteria were compared with transmission electron microscopy and electron tomography. The four anammox genera shared a common cell plan and contained glycogen granules. Differences between the four genera included cell size (from 800 to 1,100 nm in diameter), presence or absence of cytoplasmic particles, and presence or absence of pilus-like appendages. Furthermore, cytochrome c proteins were detected exclusively inside the anammoxosome. This detection provides further support for the hypothesis that this organelle is the locus of anammox catabolism.
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http://dx.doi.org/10.1128/JB.01449-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2223682PMC
January 2008

Enrichment of perforate septal pore caps from the basidiomycetous fungus Rhizoctonia solani by combined use of French press, isopycnic centrifugation, and Triton X-100.

J Microbiol Methods 2007 Dec 18;71(3):298-304. Epub 2007 Oct 18.

Centraalbureau voor Schimmelcultures (CBS), Royal Netherlands Academy of Arts and Sciences (KNAW), Utrecht, The Netherlands.

Septal pore caps occur in many filamentous basidiomycetes located at both sides of the dolipore septum and are at their base connected to the endoplasmic reticulum. The septal pore cap ultrastructure has been described extensively by the use of electron microscopy, but its composition and function are not yet known. To enable biochemical and functional analyses in the future, we here describe an enrichment method for perforate septal pore caps from Rhizoctonia solani. Our method is based on the combined use of French press and isopycnic centrifugation, using a discontinuous sucrose gradient followed by a treatment with Triton X-100. Enrichment was monitored by the use of scanning electron microscopy and transmission electron microscopy. Using the same isolation method, smaller septal pore caps were isolated from two other basidiomycetes as well. Furthermore, we showed pore-occluding material co-purified with the septal pore caps. This observation supports the hypothesis that septal pore caps play a key role in the plugging process of the septal pores in filamentous basidiomycetes.
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http://dx.doi.org/10.1016/j.mimet.2007.09.013DOI Listing
December 2007

Human embryonic stem cell-derived cardiomyocytes survive and mature in the mouse heart and transiently improve function after myocardial infarction.

Stem Cell Res 2007 Oct 17;1(1):9-24. Epub 2007 Aug 17.

Heart Lung Center, University Medical Center, GA Utrecht, The Netherlands.

Regeneration of the myocardium by transplantation of cardiomyocytes is an emerging therapeutic strategy. Human embryonic stem cells (HESC) form cardiomyocytes readily but until recently at low efficiency, so that preclinical studies on transplantation in animals are only just beginning. Here, we show the results of the first long-term (12 weeks) analysis of the fate of HESC-derived cardiomyocytes transplanted intramyocardially into healthy, immunocompromised (NOD-SCID) mice and in NOD-SCID mice that had undergone myocardial infarction (MI). Transplantation of mixed populations of differentiated HESC containing 20-25% cardiomyocytes in control mice resulted in rapid formation of grafts in which the cardiomyocytes became organized and matured over time and the noncardiomyocyte population was lost. Grafts also formed in mice that had undergone MI. Four weeks after transplantation and MI, this resulted in significant improvement in cardiac function measured by magnetic resonance imaging. However, at 12 weeks, this was not sustained despite graft survival. This suggested that graft size was still limiting despite maturation and organization of the transplanted cells. More generally, the results argued for requiring a minimum of 3 months follow-up in studies claiming to observe improved cardiac function, independent of whether HESC or other (adult) cell types are used for transplantation.
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http://dx.doi.org/10.1016/j.scr.2007.06.001DOI Listing
October 2007

Measuring location, size, distribution, and loading of NiO crystallites in individual SBA-15 pores by electron tomography.

J Am Chem Soc 2007 Aug 27;129(33):10249-54. Epub 2007 Jul 27.

Inorganic Chemistry and Catalysis, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.

By the combination of electron tomography with image segmentation, the properties of 299 NiO crystallites contained in 6 SBA-15 pores were studied. A statistical analysis of the particle size showed that crystallites between 2 and 6 nm were present with a distribution maximum at 3 and 4 nm, for the number-weighted and volume-weighted curves, respectively. Interparticle distances between nearest neighbors were 1-3 nm with very few isolated crystallites. In the examined pores, a local loading twice the applied average of 24 wt % NiO was found. This suggests that a very high local loading combined with a high dispersion is achievable.
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http://dx.doi.org/10.1021/ja0728876DOI Listing
August 2007

Combined structural and chemical analysis of the anammoxosome: a membrane-bounded intracytoplasmic compartment in anammox bacteria.

J Struct Biol 2008 Mar 2;161(3):401-10. Epub 2007 Jun 2.

Department of Microbiology, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands.

Anammox bacteria have unique intracellular membranes that divide their cytoplasm into three separate compartments. The largest and innermost cytoplasmic compartment, the anammoxosome, is hypothesized to be the locus of all catabolic reactions in the anammox metabolism. Electron tomography showed that the anammoxosome and its membrane were highly folded. This finding was confirmed by a transmission electron microscopy study using different sample preparation methods. Further, in this study electron-dense particles were observed and electron tomography showed that they were confined to the anammoxosome compartment. Energy dispersive X-ray analysis revealed that these particles contained iron. The functional significance of a highly folded anammoxosome membrane and intracellular iron storage particles are discussed in relation to their possible function in energy generation.
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http://dx.doi.org/10.1016/j.jsb.2007.05.005DOI Listing
March 2008

Template matching as a tool for annotation of tomograms of stained biological structures.

J Struct Biol 2007 Jun 22;158(3):327-35. Epub 2006 Dec 22.

Cellular Architecture and Dynamics, Utrecht University, The Netherlands.

In recent years, electron tomography has improved our three-dimensional (3D) insight in the structural architecture of cells and organelles. For studies that involve the 3D imaging of stained sections, manual annotation of tomographic data has been an important method to help understand the overall 3D morphology of cellular compartments. Here, we postulate that template matching can provide a tool for more objective annotation and contouring of cellular structures. Also, this technique can extract information hitherto unharvested in tomographic studies. To evaluate the performance of template matching on tomograms of stained sections, we generated several templates representing a piece of microtubule or patches of membranes of different staining-thicknesses. These templates were matched to tomograms of stained electron microscopy sections. Both microtubules and ER-Golgi membranes could be detected using this method. By matching cuboids of different thicknesses, we were able to distinguish between coated and non-coated endosomal membrane-domains. Finally, heterogeneity in staining-thickness of endosomes could be observed. Template matching can be a useful addition to existing annotation-methods, and provide additional insights in cellular architecture.
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http://dx.doi.org/10.1016/j.jsb.2006.12.001DOI Listing
June 2007
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