127 research outputs found
UNC-108/RAB-2 and its effector RIC-19 are involved in dense core vesicle maturation in C. elegans
No full textSmall GTPases of the Rab family regulate intracellular vesicular trafficking. In C. elegans, two dominant alleles of unc-108/rab-2 have been isolated based on their movement defects. Using a combination of pharmacological assays and electron microscopy, we show that the locomotion defects of rab-2 mutant animals are cause by a reduced facilitation of synaptic vesicle (SV) release from pre-synaptic sites due to lower levels of diacylglycerol (DAG). DAG levels at release sites can be regulated by neuro-peptides secreted from dense core vesicles (DCV). We show that while their number and distribution is not affected, DCVs in dominant rab-2 mutants are enlarged and contain less neuropeptides. This implicates RAB-2 in the biogenesis of DCVs at the Golgi complex. In rab-2 mutants DCV cargo inappropriately enters late endosomal compartments. Finally we show that RIC-19, the C. elegans ortholog of the human diabetes autoantigen ICA69, is recruited to Golgi membranes by RAB-2 for proper DCV biogenesis.sponsorship: We thank Francis Barr for the hRab2 cDNA, Shweta Aggarwal, Reinhard Jahn, and Gottfried Mieskes for support, Marc Pilon for RIC-19 antibodies, Ken Miller and Josh Kaplan for strains, and Ken Miller for sharing unpublished results and discussions. We specially thank Virginie Lecaudey, Nikhil Sasidharan, and Reinhard Jahn for their comments on the manuscript. Some strains were provided by the Caenorhabditis Genetics Center, which is supported by the National Institutes of Health National Center for Research Resources. We gratefully acknowledge funding by the European Union (EU) Sixth Framework Programme coordination action program European Network of Neuroscience Institutes, Neuroscience Early Stage Research Training fellowships of the EU to L. Luo and M. Sumakovic, and a postdoctoral fellowship from the Research Foundation Flanders (FWO) to S.J. Husson. L. Schoofs is supported by the FWO, and S. Eimer is supported by the German Research Foundation Research Center for Molecular Physiology of the Brain. (National Institutes of Health National Center for Research Resources, European Union (EU), Research Foundation Flanders (FWO), German Research Foundation Research Center for Molecular Physiology of the Brain)status: Publishe
TBC-8, a putative RAB-2 GAP, regulates dense core vesicle maturation in Caenorhabditis elegans.
No full textDense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2-specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation
In vivo synaptic recovery following optogenetic hyperstimulation
No full textLocal recycling of synaptic vesicles (SVs) allows neurons to sustain transmitter release. Extreme activity (e.g., during seizure) may exhaust synaptic transmission and, in vitro, induces bulk endocytosis to recover SV membrane and proteins; how this occurs in animals is unknown. Following optogenetic hyperstimulation of Caenorhabditis elegans motoneurons, we analyzed synaptic recovery by time-resolved behavioral, electrophysiological, and ultrastructural assays. Recovery of docked SVs and of evoked-release amplitudes (indicating readily-releasable pool refilling) occurred within similar to 8-20 s (tau = 9.2 s and tau = 11.9 s), whereas locomotion recovered only after similar to 60 s (tau = 20 s). During similar to 11-s stimulation, 50- to 200-nm noncoated vesicles ("100nm vesicles") formed, which disappeared similar to 8 s poststimulation, likely representing endocytic intermediates from which SVs may regenerate. In endophilin, synaptojanin, and dynamin mutants, affecting endocytosis and vesicle scission, resolving 100nm vesicles was delayed (>20 s). In dynamin mutants, 100nm vesicles were abundant and persistent, sometimes continuous with the plasma membrane; incomplete budding of smaller vesicles from 100nm vesicles further implicates dynamin in regenerating SVs from bulk-endocytosed vesicles. Synaptic recovery after exhaustive activity is slow, and different time scales of recovery at ultrastructural, physiological, and behavioral levels indicate multiple contributing processes. Similar processes may jointly account for slow recovery from acute seizures also in higher animals
Human CFTR deficient iPSC-macrophages reveal impaired functional and transcriptomic response upon Pseudomonas aeruginosa infection
No full textIntroduction Cystic fibrosis (CF) is a hereditary autosomal recessive disease driven by deleterious variants of the CFTR gene, leading, among other symptoms, to increased lung infection susceptibility. Mucus accumulation in the CF lung is, as of yet, considered as one important factor contributing to its colonization by opportunistic pathogens such as Pseudomonas aeruginosa . However, in recent years evidence was provided that alveolar macrophages, which form the first line of defense against airborne pathogens, seem to be intrinsically defective with regard to bactericidal functionality in the CF lung. To assess the impact of CFTR deficiency in human macrophages only insufficient systems are available. Methods To address this problem and to evaluate the role of CFTR in human macrophages, we successfully differentiated human induced pluripotent stem cells (iPSC) from a CF p.Phe508del homozygous individual and a healthy donor into primitive macrophages (iMac ΔF508 and iMac WT ), respectively, and compared the bactericidal functionality in the relevant cell type. Results iMac ΔF508 showed impaired P. aeruginosa clearance and intracellular killing capacity in comparison to iMac WT . Furthermore, iMac ΔF508 exhibited a less acidic lysosomal pH, and upon P. aeruginosa infection, there were signs of mitochondrial fragmentation and autophagosome formation together with a hyperinflammatory phenotype and deficient type I interferon response. Conclusion In summary, we present a defective phenotype in iMac ΔF508 upon P. aeruginosa infection, which will constitute an ideal platform to further study the role of macrophages in the context of CF
RAB-5 and RAB-10 cooperate to regulate neuropeptide release in Caenorhabditis elegans
No full textNeurons secrete neuropeptides from dense core vesicles (DCVs) to modulate neuronal activity. Little is known about how neurons manage to differentially regulate the release of synaptic vesicles (SVs) and DCVs. To analyze this, we screened all Caenorhabditis elegans Rab GTPases and Tre2/Bub2/Cdc16 (TBC) domain containing GTPase-activating proteins (GAPs) for defects in DCV release from C. elegans motoneurons. rab-5 and rab-10 mutants show severe defects in DCV secretion, whereas SV exocytosis is unaffected. We identified TBC-2 and TBC-4 as putative GAPs for RAB-5 and RAB-10, respectively. Multiple Rabs and RabGAPs are typically organized in cascades that confer directionality to membrane-trafficking processes. We show here that the formation of release-competent DCVs requires a reciprocal exclusion cascade coupling RAB-5 and RAB-10, in which each of the two Rabs recruits the other's GAP molecule. This contributes to a separation of RAB-5 and RAB-10 domains at the Golgi-endosomal interface, which is lost when either of the two GAPs is inactivated. Taken together, our data suggest that RAB-5 and RAB-10 cooperate to locally exclude each other at an essential stage during DCV sorting
Investigations of cytoskeletal components and motility in Mycoplasma pneumoniae
No full textDie Zellstruktur des Bakteriums Mycoplasma pneumoniae wurde durch elektronenmikroskopische und lichtmikroskopische Methoden untersucht. Ultrastrukturelle Details des Cytoskeletts von M. pneumoniae wurden mittels verschiedener Präparationstechniken abgebildet. Ein im Tip der Zelle lokalisiertes Cytoskelettelement, der Rod", weist am proximalen Ende eine rad-ähnliche Struktur auf, lateral ist er über Linkermoleküle mit der inneren Seite der Zellhülle verbunden. Eine Längsspaltung des Rod konnte nachgewiesen werden, die wahrscheinlich ein frühes Stadium der Zellteilung darstellt.Der Rod ist abgeflacht, seine Länge beträgt ca. 280 nm, die Breite misst, abhängig vom Betrachtungswinkel, bis zu 40 nm. Hier wird eine Methode zur partiellen Lyse von M. pneumoniae-Zellen im Detergens Triton X-100 vorgestellt, welche es ermöglicht, die Cytoplasmamembran von den Zellen abzulösen, ohne dass die restliche Zelle lysiert. Die Ablösung der Cytoplasmamembran wurde hierbei elektronenmikroskopisch nachgewiesen. Die bei M. pneumoniae unter der Cytoplasmamembran gelegene Schicht konnte so sichtbar gemacht werden und weist im elektronenmikroskopischen Bild ein hexagonales Muster mit einer Periodizität von ca. 10 nm auf. Auf Grundlage dessen wurde ein Modell für ein peripheres, funktional am Erhalt der Zellform beteiligtes Cytoskelett entworfen.Durch Immunelektronenmikroskopie und Immunfluoreszenzmikroskopie konnten einige Proteine von M. pneumoniae am Tip nachgewiesen werden. Das Protein P01_orf1033 konnte dabei der Cytoplasmamembran zugeordnet werden. Für weitere Untersuchungen wurde das Gen für P01_orf1033 kloniert und als Fusion mit dem monomeren rotfluoreszierenden Protein in E. coli exprimiert.The cell structure of the bacterium Mycoplasma pneumoniae was investigated by electron- and light-microscopic methods. Ultrastructural details of the cytoskeleton of M. pneumoniae were displayed by different preparation techniques. A cytoskeletal element localized to the tip of the cell, the "rod", exhibits a wheel-like complex at its proximal end and is connected lateral to the inner face of the cell envelope by linkers. A lengthwise splitting of the rod could be demonstrated, which is probably an early state in cell division.The rod is flat, it is about 280 nm long and appears, depending on the viewing angle, up to 40 nm wide. A method for a partial lysis of M. pneumoniae-cells in the detergent Triton X-100 is demonstrated by which the cytoplasmic membrane can be removed from the cells without lysis of the remaining cell. The removal of the cytoplasmic membrane was monitored by electron microscopy. The layer beneath the cytoplasmic membrane could be vizualized and exhibits a hexagonal pattern with a periodicity of about 10 nm. Based on this, a model for a peripheral cytoskeleton, functionally involved in stabilizing cell shape, is presented.Different proteins of M. pneumoniae could be localized to the tip via immuno-electron and immuno-fluorescence-microscopy. The protein P01_orf1033 was shown to be associated with the cytoplasmic membrane. For further investigations the gene for P01_orf1033 was cloned and expressed as a fusion protein with the monomeric red fluorescent protein in E. coli
The endoplasmic reticulum localized PIN8 is a pollen-specific auxin carrier involved in intracellular auxin homeostasis
No full textThe plant hormone auxin is a mobile signal which affects nuclear transcription by regulating the stability of auxin/indole-3-acetic acid (IAA) repressor proteins. Auxin is transported polarly from cell to cell by auxin efflux proteins of the PIN family, but it is not as yet clear how auxin levels are regulated within cells and how access of auxin to the nucleus may be controlled. The Arabidopsis genome contains eight PINs, encoding proteins with a similar membrane topology. While five of the PINs are typically targeted polarly to the plasma membranes, the smallest members of the family, PIN5 and PIN8, seem to be located not at the plasma membrane but in endomembranes. Here we demonstrate by electron microscopy analysis that PIN8, which is specifically expressed in pollen, resides in the endoplasmic reticulum and that it remains internally localized during pollen tube growth. Transgenic Arabidopsis and tobacco plants were generated overexpressing or ectopically expressing functional PIN8, and its role in control of auxin homeostasis was studied. PIN8 ectopic expression resulted in strong auxin-related phenotypes. The severity of phenotypes depended on PIN8 protein levels, suggesting a rate-limiting activity for PIN8. The observed phenotypes correlated with elevated levels of free IAA and ester-conjugated IAA. Activation of the auxin-regulated synthetic DR5 promoter and of auxin response genes was strongly repressed in seedlings overexpressing PIN8 when exposed to 1-naphthalene acetic acid. Thus, our data show a functional role for endoplasmic reticulum-localized PIN8 and suggest a mechanism whereby PIN8 controls auxin thresholds and access of auxin to the nucleus, thereby regulating auxin-dependent transcriptional activity
Volume electron microscopy: analyzing the lung
No full textSince its entry into biomedical research in the first half of the twentieth century, electron microscopy has been a valuable tool for lung researchers to explore the lung's delicate ultrastructure. Among others, it proved the existence of a continuous alveolar epithelium and demonstrated the surfactant lining layer. With the establishment of serial sectioning transmission electron microscopy, as the first 'volume electron microscopic' technique, electron microscopy entered the third dimension and investigations of the lung's three-dimensional ultrastructure became possible. Over the years, further techniques, ranging from electron tomography over serial block-face and focused ion beam scanning electron microscopy to array tomography became available. All techniques cover different volumes and resolutions, and, thus, different scientific questions. This review gives an overview of these techniques and their application in lung research, focusing on their fields of application and practical implementation. Furthermore, an introduction is given how the output raw data are processed and the final three-dimensional models can be generated
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