TY - JOUR A1 - Capetian, Philipp A1 - Müller, Lorenz A1 - Volkmann, Jens A1 - Heckmann, Manfred A1 - Ergün, Süleyman A1 - Wagner, Nicole T1 - Visualizing the synaptic and cellular ultrastructure in neurons differentiated from human induced neural stem cells - an optimized protocol JF - International Journal of Molecular Sciences N2 - The size of the synaptic subcomponents falls below the limits of visible light microscopy. Despite new developments in advanced microscopy techniques, the resolution of transmission electron microscopy (TEM) remains unsurpassed. The requirements of tissue preservation are very high, and human post mortem material often does not offer adequate quality. However, new reprogramming techniques that generate human neurons in vitro provide samples that can easily fulfill these requirements. The objective of this study was to identify the culture technique with the best ultrastructural preservation in combination with the best embedding and contrasting technique for visualizing neuronal elements. Two induced neural stem cell lines derived from healthy control subjects underwent differentiation either adherent on glass coverslips, embedded in a droplet of highly concentrated Matrigel, or as a compact neurosphere. Afterward, they were fixed using a combination of glutaraldehyde (GA) and paraformaldehyde (PFA) followed by three approaches (standard stain, Ruthenium red stain, high contrast en-bloc stain) using different combinations of membrane enhancing and contrasting steps before ultrathin sectioning and imaging by TEM. The compact free-floating neurospheres exhibited the best ultrastructural preservation. High-contrast en-bloc stain offered particularly sharp staining of membrane structures and the highest quality visualization of neuronal structures. In conclusion, compact neurospheres growing under free-floating conditions in combination with a high contrast en-bloc staining protocol, offer the optimal preservation and contrast with a particular focus on visualizing membrane structures as required for analyzing synaptic structures. KW - transmission electron microscopy KW - human neurons KW - induced neural stem cells KW - synapse KW - synaptic vesicles KW - high contrast Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-236053 SN - 1422-0067 VL - 21 IS - 5 ER - TY - JOUR A1 - Cull, Benjamin A1 - Lima Prado Godinho, Joseane A1 - Fernandes Rodrigues, Juliany Cola A1 - Frank, Benjamin A1 - Schurigt, Uta A1 - Williams, Roderick AM A1 - Coombs, Graham H A1 - Mottram, Jeremy C T1 - Glycosome turnover in Leishmania major is mediated by autophagy JF - Autophagy N2 - Autophagy is a central process behind the cellular remodeling that occurs during differentiation of Leishmania, yet the cargo of the protozoan parasite's autophagosome is unknown. We have identified glycosomes, peroxisome-like organelles that uniquely compartmentalize glycolytic and other metabolic enzymes in Leishmania and other kinetoplastid parasitic protozoa, as autophagosome cargo. It has been proposed that the number of glycosomes and their content change during the Leishmania life cycle as a key adaptation to the different environments encountered. Quantification of RFP-SQL-labeled glycosomes showed that promastigotes of L. major possess ~20 glycosomes per cell, whereas amastigotes contain ~10. Glycosome numbers were significantly greater in promastigotes and amastigotes of autophagy-defective L. major Δatg5 mutants, implicating autophagy in glycosome homeostasis and providing a partial explanation for the previously observed growth and virulence defects of these mutants. Use of GFP-ATG8 to label autophagosomes showed glycosomes to be cargo in ~15% of them; glycosome-containing autophagosomes were trafficked to the lysosome for degradation. The number of autophagosomes increased 10-fold during differentiation, yet the percentage of glycosome-containing autophagosomes remained constant. This indicates that increased turnover of glycosomes was due to an overall increase in autophagy, rather than an upregulation of autophagosomes containing this cargo. Mitophagy of the single mitochondrion was not observed in L. major during normal growth or differentiation; however, mitochondrial remnants resulting from stress-induced fragmentation colocalized with autophagosomes and lysosomes, indicating that autophagy is used to recycle these damaged organelles. These data show that autophagy in Leishmania has a central role not only in maintaining cellular homeostasis and recycling damaged organelles but crucially in the adaptation to environmental change through the turnover of glycosomes. KW - ATG8 KW - Leishmania KW - TEM KW - glycosome KW - protozoan parasite KW - ATG KW - autophagy-related KW - GFP KW - green fluorescent protein KW - MVT KW - multivesicular tubule KW - RFP KW - red fluorescent protein KW - transmission electron microscopy KW - adaptation KW - autophagy KW - mC KW - mCherry KW - fluorescent protein Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-150277 VL - 10 IS - 12 ER - TY - JOUR A1 - Frank, Benjamin A1 - Marcu, Ana A1 - de Oliveira Almeida Petersen, Antonio Luis A1 - Weber, Heike A1 - Stigloher, Christian A1 - Mottram, Jeremy C. A1 - Scholz, Claus Jürgen A1 - Schurigt, Uta T1 - Autophagic digestion of Leishmania major by host macrophages is associated with differential expression of BNIP3, CTSE, and the miRNAs miR-101c, miR-129, and miR-210 JF - Parasites & Vectors N2 - Background Autophagy participates in innate immunity by eliminating intracellular pathogens. Consequently, numerous microorganisms have developed strategies to impair the autophagic machinery in phagocytes. In the current study, interactions between Leishmania major (L. m.) and the autophagic machinery of bone marrow-derived macrophages (BMDM) were analyzed. Methods BMDM were generated from BALB/c mice, and the cells were infected with L. m. promastigotes. Transmission electron microscopy (TEM) and electron tomography were used to investigate the ultrastructure of BMDM and the intracellular parasites. Affymetrix® chip analyses were conducted to identify autophagy-related messenger RNAs (mRNAs) and microRNAs (miRNAs). The protein expression levels of autophagy related 5 (ATG5), BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), cathepsin E (CTSE), mechanistic target of rapamycin (MTOR), microtubule-associated proteins 1A/1B light chain 3B (LC3B), and ubiquitin (UB) were investigated through western blot analyses. BMDM were transfected with specific small interfering RNAs (siRNAs) against autophagy-related genes and with mimics or inhibitors of autophagy-associated miRNAs. The infection rates of BMDM were determined by light microscopy after a parasite-specific staining. Results The experiments demonstrated autophagy induction in BMDM after in vitro infection with L. m.. The results suggested a putative MTOR phosphorylation-dependent counteracting mechanism in the early infection phase and indicated that intracellular amastigotes were cleared by autophagy in BMDM in the late infection phase. Transcriptomic analyses and specific downregulation of protein expression with siRNAs suggested there is an association between the infection-specific over expression of BNIP3, as well as CTSE, and the autophagic activity of BMDM. Transfection with mimics of mmu-miR-101c and mmu-miR-129-5p, as well as with an inhibitor of mmu-miR-210-5p, demonstrated direct effects of the respective miRNAs on parasite clearance in L. m.-infected BMDM. Furthermore, Affymetrix® chip analyses revealed a complex autophagy-related RNA network consisting of differentially expressed mRNAs and miRNAs in BMDM, which indicates high glycolytic and inflammatory activity in the host macrophages. Conclusions Autophagy in L. m.-infected host macrophages is a highly regulated cellular process at both the RNA level and the protein level. Autophagy has the potential to clear parasites from the host. The results obtained from experiments with murine host macrophages could be translated in the future to develop innovative and therapeutic antileishmanial strategies for human patients. KW - autophagy KW - BNIP3 KW - CTSE KW - electron tomography KW - leishmania major KW - macrophages KW - miRNAs KW - MTOR KW - siRNAs KW - transmission electron microscopy Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-124997 VL - 8 IS - 404 ER -