Papers by Vincent Schoonderwoert
Microscopy Today, 2018
Light sheet fluorescence microscopy (LSFM) allows for highresolution three-dimensional imaging wi... more Light sheet fluorescence microscopy (LSFM) allows for highresolution three-dimensional imaging with minimal photo-damage. By viewing the sample from different directions, different regions of large specimens can be imaged optimally. Moreover, owing to their good spatial resolution and high signal-to-noise ratio, LSFM data are well suited for image deconvolution. Here we present the Huygens Fusion and Deconvolution Wizard, a unique integrated solution for restoring LSFM images, and show that improvements in signal and resolution of 1.5 times and higher are feasible.
European Journal of Biochemistry, 2000
Vacuolar H+‐ATPases (V‐ATPases) are multisubunit enzymes that acidify various intracellular organ... more Vacuolar H+‐ATPases (V‐ATPases) are multisubunit enzymes that acidify various intracellular organelles, including secretory pathway compartments. We have examined the effects of the specific V‐ATPase inhibitor bafilomycin A1 (Baf) on the intracellular transport, sorting, processing and release of a number of neuroendocrine secretory proteins in primary Xenopus intermediate pituitary cells. Ultrastructural examination of Baf‐treated intermediate pituitary cells revealed a reduction in the amount of small dense‐core secretory granules and the appearance of vacuolar structures in the trans‐Golgi area. Pulse–chase incubations in combination with immunoprecipitation analysis showed that in treated cells, the proteolytic processing of the newly synthesized prohormone proopiomelanocortin, prohormone convertase PC2 and secretogranin III (SgIII) was inhibited, and an intracellular accumulation of intact precursor forms and intermediate cleavage products became apparent. Moreover, we found th...
European Journal of Biochemistry, 1999
Vacuolar H+-ATPases (V-ATPases) mediate the acidification of multiple intracellular compartments,... more Vacuolar H+-ATPases (V-ATPases) mediate the acidification of multiple intracellular compartments, including secretory granules in which an acidic milieu is necessary for prohormone processing. A search for genes coordinately expressed with the prohormone proopiomelanocortin (POMC) in the melanotrope cells of Xenopus intermediate pituitary led to the isolation of a cDNA encoding the complete amino-acid sequence of the type I transmembrane V-ATPase accessory subunit Ac45 (predicted size 48 kDa). Comparison of Xenopus and mammalian Ac45 sequences revealed conserved regions in the protein that may be of functional importance. Western blot analysis showed that immunoreactive Ac45 represents a approximately 40-kDa product that is expressed predominantly in neuroendocrine tissues; deglycosylation resulted in a approximately 27-kDa immunoreactive Ac45 product which is smaller than predicted for the intact protein. Biosynthetic studies revealed that newly synthesized Xenopus Ac45 is an N-glycosylated protein of approximately 60 kDa; the nonglycosylated, newly synthesized form is approximately 46 kDa which is similar to the predicted size. Immunocytochemical analysis showed that in Xenopus pituitary, Ac45 is highly expressed in the biosynthetically active melanotrope cells. We conclude that the regionally conserved Xenopus Ac45 protein is synthesized as an N-glycosylated approximately 60-kDa precursor that is intracellularly cleaved to an approximately 40-kDa product and speculate that it may assist in the V-ATPase-mediated acidification of neuroendocrine secretory granules.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1994
Sea urchin fascin and the Drosophila singed gene product form a unique class of actin cross-linki... more Sea urchin fascin and the Drosophila singed gene product form a unique class of actin cross-linking proteins involved in the bundling of filamentous actin by an as yet unknown mechanism. From a Xenopus laevis intermediate pituitary cDNA library we have isolated a cDNA encoding a 53-kDa protein that shares approximately 36% amino acid sequence identity with both fascin and the singed gene product, and thus likely represents a vertebrate homolog of these actin-bundling proteins. RNase-protection experiments revealed that in Xenopus the gene is expressed in a wide variety of tissues but with the highest levels of expression in oocytes and testis. This raises the possibility that fascin has a role in microfilament dynamics associated with the formation and/or fertilization of vertebrate germ cells.
Microscopy Today, 2020
Single-molecule localization microscopy (SMLM) is a family of super-resolution microscopy techniq... more Single-molecule localization microscopy (SMLM) is a family of super-resolution microscopy techniques based on localizing clusters of detected photons that are emitted by single molecules. The localization procedure is based on careful statistical analysis of long image sequences to derive the nanometer positions of the molecules. By introducing additional optics, such as cylindrical lenses in the optical system, SMLM techniques have been extended to 3D super-resolution imaging. This adds a calibration step, thereby further complicating the data analysis. Here we present Huygens Localizer, a well-supported user-friendly package that carries out these tasks quickly by offloading carefully designed 2D and 3D analysis and visualization procedures to massively parallel graphical processors (GPUs).
Molecular biology of the cell, 2017
Homophilic binding of immunoglobulin superfamily molecules such as the Aplysia cell adhesion mole... more Homophilic binding of immunoglobulin superfamily molecules such as the Aplysia cell adhesion molecule (apCAM) leads to actin filament assembly near nascent adhesion sites. Such actin assembly can generate significant localized forces that have not been characterized in the larger context of axon growth and guidance. We used apCAM-coated bead substrates applied to the surface of neuronal growth cones to characterize the development of forces evoked by varying stiffness of mechanical restraint. Unrestrained bead propulsion matched or exceeded rates of retrograde network flow and was dependent on Arp2/3 complex activity. Analysis of growth cone forces applied to beads at low stiffness of restraint revealed switching between two states: frictional coupling to retrograde flow and Arp2/3-dependent propulsion. Stiff mechanical restraint led to formation of an extensive actin cup matching the geometric profile of the bead target and forward growth cone translocation; pharmacological inhibit...
European Journal of Biochemistry, 2002
The vacuolar H + -ATPase (V-ATPase) is a multimeric enzyme complex that acidifies organelles of t... more The vacuolar H + -ATPase (V-ATPase) is a multimeric enzyme complex that acidifies organelles of the vacuolar system in eukaryotic cells. Proteins that interact with the V-ATPase may play an important role in controlling the intracellular localization and activity of the proton pump. The neuroendocrine-enriched V-ATPase accessory subunit Ac45 may represent such a protein as it has been shown to interact with the membrane sector of the V-ATPase in only a subset of organelles. Here, we examined the fate of newly synthesized Ac45 in the secretory pathway of a neuroendocrine cell. A major portion of intact 46-kDa Ac45 was found to be N-linked glycosylated to 62 kDa and a minor fraction to 64 kDa. Trimming of the N-linked glycans gave rise to glycosylated Ac45-forms of 61 and 63 kDa that are cleaved to a C-terminal fragment of 42-44 kDa (the deglycosylated form is 23 kDa), and a previously not detected 22-kDa N-terminal cleavage fragment (the deglycosylated form is 20 kDa). Degradation of the N-terminal fragment is rapid, does not occur in lysosomes and is inhibited by brefeldin A. Both the N-and C-terminal fragment pass the medial Golgi, as they become partially endoglycosidase H resistant. The Ac45 cleavage event is a relatively slow process (half-life of intact Ac45 is 4-6 h) and takes place in the early secretory pathway, as it is not affected by brefeldin A and monensin. Tunicamycin inhibited N-linked glycosylation of Ac45 and interfered with the cleavage process, suggesting that Ac45 needs proper folding for the cleavage to occur. Together, our results indicate that Ac45 folding and cleavage occur slowly and early in the secretory pathway, and that the cleavage event may be linked to V-ATPase activation.
European Journal of Biochemistry, 1999
Vacuolar H + -ATPases (V-ATPases) mediate the acidification of multiple intracellular compartment... more Vacuolar H + -ATPases (V-ATPases) mediate the acidification of multiple intracellular compartments, including secretory granules in which an acidic milieu is necessary for prohormone processing. A search for genes coordinately expressed with the prohormone proopiomelanocortin (POMC) in the melanotrope cells of Xenopus intermediate pituitary led to the isolation of a cDNA encoding the complete amino-acid sequence of the type I transmembrane V-ATPase accessory subunit Ac45 (predicted size 48 kDa). Comparison of Xenopus and mammalian Ac45 sequences revealed conserved regions in the protein that may be of functional importance. Western blot analysis showed that immunoreactive Ac45 represents a <40-kDa product that is expressed predominantly in neuroendocrine tissues; deglycosylation resulted in a <27-kDa immunoreactive Ac45 product which is smaller than predicted for the intact protein. Biosynthetic studies revealed that newly synthesized Xenopus Ac45 is an N-glycosylated protein of <60 kDa; the nonglycosylated, newly synthesized form is <46 kDa which is similar to the predicted size. Immunocytochemical analysis showed that in Xenopus pituitary, Ac45 is highly expressed in the biosynthetically active melanotrope cells. We conclude that the regionally conserved Xenopus Ac45 protein is synthesized as an N-glycosylated <60-kDa precursor that is intracellularly cleaved to an <40-kDa product and speculate that it may assist in the V-ATPase-mediated acidification of neuroendocrine secretory granules.
Neuron, 2003
grade actin flow ( ). The second structure, actin arcs, appear in the T zone and move into the C ... more grade actin flow ( ). The second structure, actin arcs, appear in the T zone and move into the C domain, where they contribute to central actin bundle structure. Interestingly, MTs associated with actin arcs are less dynamic than those in the P domain, exhibiting prolonged periods of slow growth due to dramatically reduced catastrophe frequencies. Our initial report identi-New Haven, Connecticut 06520 fied arcs as a novel motile actin structure in growth cones and raised questions about their physiological significance.
Molecular Membrane Biology, 2002
Acidification of organelles of the eukaryotic vacuolar system is important for multiple intracell... more Acidification of organelles of the eukaryotic vacuolar system is important for multiple intracellular processes including receptor-mediated endocytosis, proteolytic activity in lysosomes, and prohormone sorting and processing in secretory granules. Responsible for the generation of a proton gradient across a membrane is vacuolar H(+)-ATPase (V-ATPase). How the activity of this multisubunit enzyme is regulated remains to be established. Accessory subunits of the V-ATPase may be involved in the organelle-specific regulation, one candidate being the chromaffin granular V-ATPase-associated protein Ac45. To assess the function of Ac45, we disrupted its gene by gene targeting in male mouse embryonic stem cells. We have successfully generated Ac45 null mutant (-IY) embryonic stem cells and injected them into C57BL/6 recipient blastocysts. The blastocysts were replaced into pseudopregnant foster mothers, giving rise to 16 littermates. One of these appeared to be a low-chimeric female mouse that died 6 weeks after birth. No signs of late abortion were detected in the foster mothers. The results suggest that the injected Ac45 null mutant embryonic stem cells affect the normal development of the blastocyst and are in line with knockout studies on other V-ATPase subunits that point to an essential role for the V-ATPase in early embryonic development.
Journal of Membrane Biology, 2001
Developmental Cell, 2008
Although much evidence suggests that axon growth and guidance depend on well-coordinated cytoskel... more Although much evidence suggests that axon growth and guidance depend on well-coordinated cytoskeletal dynamics, direct characterization of the corresponding molecular events has remained a challenge. Here, we address this outstanding problem by examining neurite outgrowth stimulated by local application of cell adhesion substrates. During acute outgrowth, the advance of organelles and underlying microtubules into the central domain was correlated with regions of attenuated retrograde actin network flow in the periphery. Interestingly, as adhesion sites matured, contractile actin arc structures, known to be regulated by the Rho/Rho Kinase/myosin II signaling cascade, became more robust and coordinated microtubule movements in the growth cone neck. When Rho Kinase was inhibited, although growth responses occurred with less of a delay, microtubules failed to consolidate into a single axis of growth. These results reveal a new role for Rho Kinase and myosin II contractility in regulation of microtubule behavior during neuronal growth.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1994
Sea urchin fascin and the Drosophila singed gene product form a unique class of actin cross-linki... more Sea urchin fascin and the Drosophila singed gene product form a unique class of actin cross-linking proteins involved in the bundling of filamentous actin by an as yet unknown mechanism. From a Xenopus laevis intermediate pituitary cDNA library we have isolated a cDNA encoding a 53-kDa protein that shares approximately 36% amino acid sequence identity with both fascin and the singed gene product, and thus likely represents a vertebrate homolog of these actin-bundling proteins. RNase-protection experiments revealed that in Xenopus the gene is expressed in a wide variety of tissues but with the highest levels of expression in oocytes and testis. This raises the possibility that fascin has a role in microfilament dynamics associated with the formation and/or fertilization of vertebrate germ cells.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 2002
The vacuolar H+-ATPase (V-ATPase) is a multisubunit enzyme that couples ATP hydrolysis to proton ... more The vacuolar H+-ATPase (V-ATPase) is a multisubunit enzyme that couples ATP hydrolysis to proton pumping across membranes. The intracellular targeting and activity of the V-ATPase may be regulated via proteins that interact with the pump such as the accessory subunit Ac45. Here we report the isolation and characterization of the gene encoding Ac45. This single-copy gene is located in a gene-dense region of chromosome Xq and consists of 10 exons spanning approximately 8 kb in the mouse and human genomes. The gene structure is poorly conserved in that its invertebrate orthologs of Caenorhabditis elegans and Drosophila melanogaster encompass only six and four exons extending over 4.1 and 2.1 kb, respectively. Furthermore, the overall degree of amino acid sequence identity between the mammalian and invertebrate Ac45 proteins is very low (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;18%), except for a surprisingly highly conserved putative targeting motif in the carboxy-terminal region. Primer extension analysis revealed that the mouse Ac45 gene contains two major transcription initiation sites. The start sites are not preceded by a clear CAAT-box and are located in a CpG island. The most downstream start site contains a TATA-box and transcriptional regulatory elements such as PEA-3, F2F, Maz and Sp1. The limited number of regulatory DNA elements common in the genes encoding Ac45 and V-ATPase subunits suggests a differential regulation of these genes. Together with the finding that Ac45 appears to occur only in multicellular organisms, these results indicate that this accessory subunit directs the V-ATPase to specialized and complex vacuolar systems.
European Journal of Biochemistry, 2000
Vacuolar H 1 -ATPases (V-ATPases) are multisubunit enzymes that acidify various intracellular org... more Vacuolar H 1 -ATPases (V-ATPases) are multisubunit enzymes that acidify various intracellular organelles, including secretory pathway compartments. We have examined the effects of the specific V-ATPase inhibitor bafilomycin A1 (Baf ) on the intracellular transport, sorting, processing and release of a number of neuroendocrine secretory proteins in primary Xenopus intermediate pituitary cells. Ultrastructural examination of Baf-treated intermediate pituitary cells revealed a reduction in the amount of small dense-core secretory granules and the appearance of vacuolar structures in the trans-Golgi area. Pulse±chase incubations in combination with immunoprecipitation analysis showed that in treated cells, the proteolytic processing of the newly synthesized prohormone proopiomelanocortin, prohormone convertase PC2 and secretogranin III (SgIII) was inhibited, and an intracellular accumulation of intact precursor forms and intermediate cleavage products became apparent. Moreover, we found that treated cells secreted considerable amounts of a PC2 processing intermediate and unprocessed SgIII in a constitutive fashion. Collectively, these data indicate that in the secretory pathway, V-ATPases play an important role in creating the microenvironment that is essential for proper transport, sorting, processing and release of regulated secretory proteins. q FEBS 2000 V-ATPase activity in the secretory pathway (Eur. J. Biochem. 267) 5647 q FEBS 2000 V-ATPase activity in the secretory pathway (Eur. J. Biochem. 267) 5649 q FEBS 2000 V-ATPase activity in the secretory pathway (Eur. J. Biochem. 267) 5651 q FEBS 2000 V-ATPase activity in the secretory pathway (Eur. J. Biochem. 267) 5653
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Papers by Vincent Schoonderwoert