Simultaneous multiple immunolabeling in field emission scanning electron microscope (FESEM) is li... more Simultaneous multiple immunolabeling in field emission scanning electron microscope (FESEM) is limited by the number of suitable markers which is possible to distinguish in the microscope. At present two sizes of gold nanoparticles, e.g. 5 and 15 nm, are used for simultaneous labeling of two antigens in the observed cell surface structures. The detection of backscattered electrons in FESEM offers to use colloidal metal nanoparticles (NPs) made from other elements, because the BSE signal is very sensitive to the atomic number Z of the observed specimen area. The aim of this study was to select colloidal metal NPs that might be suitable markers, and test their stability in FESEM, their ability to conjugate them to antibodies and possibility to distinguish them from the gold nanoparticles using BSE signal. Based on our measurements, laboratory synthesized Pd nanoparticles in sizes between 8-15 nm and double shell Ag Au nanoparticles with the diameter around 15 nm were selected as the m...
Proceedings of the National Academy of Sciences of the United States of America, Jan 13, 2020
The adaptation of eukaryotic cells to anaerobic conditions is reflected by substantial changes to... more The adaptation of eukaryotic cells to anaerobic conditions is reflected by substantial changes to mitochondrial metabolism and functional reduction. Hydrogenosomes belong among the most modified mitochondrial derivative and generate molecular hydrogen concomitant with ATP synthesis. The reduction of mitochondria is frequently associated with loss of peroxisomes, which compartmentalize pathways that generate reactive oxygen species (ROS) and thus protect against cellular damage. The biogenesis and function of peroxisomes are tightly coupled with mitochondria. These organelles share fission machinery components, oxidative metabolism pathways, ROS scavenging activities, and some metabolites. The loss of peroxisomes in eukaryotes with reduced mitochondria is thus not unexpected. Surprisingly, we identified peroxisomes in the anaerobic, hydrogenosome-bearing protist Mastigamoeba balamuthi . We found a conserved set of peroxin (Pex) proteins that are required for protein import, peroxisomal growth, and division. Key membrane-associated Pexs ( Mb Pex3, Mb Pex11, and Mb Pex14) were visualized in numerous vesicles distinct from hydrogenosomes, the endoplasmic reticulum (ER), and Golgi complex. Proteomic analysis of cellular fractions and prediction of peroxisomal targeting signals (PTS1/PTS2) identified 51 putative peroxisomal matrix proteins. Expression of selected proteins in Saccharomyces cerevisiae revealed specific targeting to peroxisomes. The matrix proteins identified included components of acyl-CoA and carbohydrate metabolism and pyrimidine and CoA biosynthesis, whereas no components related to either β-oxidation or catalase were present. In conclusion, we identified a subclass of peroxisomes, named “anaerobic” peroxisomes that shift the current paradigm and turn attention to the reductive evolution of peroxisomes in anaerobic organisms.
Extended abstract of a paper presented at MC 2007, 33rd DGE Conference in Saarbrücken, Germany, S... more Extended abstract of a paper presented at MC 2007, 33rd DGE Conference in Saarbrücken, Germany, September 2 – September 7, 2007
Lyme disease (LD) spirochetes are well known to be able to disseminate into the tissues of infect... more Lyme disease (LD) spirochetes are well known to be able to disseminate into the tissues of infected hosts, including humans. The diverse strategies used by spirochetes to avoid the host immune system and persist in the host include active immune suppression, induction of immune tolerance, phase and antigenic variation, intracellular seclusion, and, importantly, incursion into immune privileged sites such as the brain. Invasion of immune privileged sites, like the brain allows the spirochetes not only escape from the host immune system but also can reduce the efficacy of antibiotic therapy. Here we present a case of the detection of DNA of spirochetes from Borrelia burgdorferi sensu lato complex from multiple loci of LD patient’s post-mortem brain. The presence of co-infection with Borrelia burgdorferi sensu stricto and Borrelia garinii in LD patient’s brain was confirmed by PCR. The presence of atypical spirochete morphology was confirmed by immunohistochemistry of the brain samples...
We report for the first time the use of two live-cell imaging agents from the group of luminescen... more We report for the first time the use of two live-cell imaging agents from the group of luminescent transition metal complexes (IRAZOLVE-MITO and REZOLVE-ER) as cathodoluminescent probes. This first experimental demonstration shows the application of both probes for the identification of cellular structures at the nanoscale and near the native state directly in the cryo-scanning electron microscope. This approach can potentially be applied to correlative and multimodal approaches and used to target specific regions within vitrified samples at low electron beam energies.
SUMMARYBarium (Ba) and strontium (Sr) are often used as proxies for the reconstruction of past ma... more SUMMARYBarium (Ba) and strontium (Sr) are often used as proxies for the reconstruction of past marine productivity and global climate. The ability to accumulate Ba2+ and Sr2+ in the form of crystals is rare among eukaryotes. Here we report that unicellular heterotrophs called diplonemids (Euglenozoa), one of the most abundant groups of marine planktonic protists, accumulate conspicuous amounts of these trace elements in the form of intracellular barite (BaSO4) and celestite (SrSO4) crystals, in concentrations greater than in other known Ba/Sr-accumulating organisms. Moreover, these flagellates can uptake Sr2+ exclusively or together with Ba2+ and form (Ba,Sr)SO4. One species, Namystinia karyoxenos, is naturally capable of intracellular accumulation of Ba2+ and Sr2+ 42,000 and 10,000 times relative to the surrounding medium. Altering the amounts of both elements in the medium resulted in corresponding changes in the quantity and composition of crystals. Planktonic copepods fed with d...
Kinesins are motor proteins found in all eukaryotic lineages that move along microtubules to medi... more Kinesins are motor proteins found in all eukaryotic lineages that move along microtubules to mediate cellular processes such as mitosis and intracellular transport. In trypanosomatids, the kinesin superfamily has undergone a prominent expansion, resulting in one of the most diverse kinesin repertoires that includes the two kinetoplastid‐restricted families X1 and X2. Here, we characterize in Trypanosoma brucei TbKifX2A, an orphaned X2 kinesin. TbKifX2A tightly interacts with TbPH1, a kinesin‐like protein with a likely inactive motor domain, a rarely reported occurrence. Both TbKifX2A and TbPH1 localize to the microtubule quartet (MtQ), a characteristic but poorly understood cytoskeletal structure that wraps around the flagellar pocket as it extends to the cell body anterior. The proximal proteome of TbPH1 revealed two other interacting proteins, the flagellar pocket protein FP45 and intriguingly another X2 kinesin, TbKifX2C. Simultaneous ablation of TbKifX2A/TbPH1 results in the dep...
Extended abstract of a paper presented at MC 2007, 33rd DGE Conference in Saarbrücken, Germany, S... more Extended abstract of a paper presented at MC 2007, 33rd DGE Conference in Saarbrücken, Germany, September 2 – September 7, 2007
ABSTRACTTriphenylphosphonium (TPP) derivatives are commonly used to target chemical into mitochon... more ABSTRACTTriphenylphosphonium (TPP) derivatives are commonly used to target chemical into mitochondria. We show that alkyl-TPP cause reversible, dose- and hydrophobicity-dependent alterations of mitochondrial morphology and function and a selective decrease of mitochondrial inner membrane proteins including subunits of the respiratory chain complexes, as well as components of the mitochondrial calcium uniporter complex. The treatment with alkyl-TPP resulted in the cleavage of the pro-fusion and cristae organisation regulator Optic atrophy-1. The structural and functional effects of alkyl-TPP were found to be reversible and not merely due to loss of membrane potential. A similar effect was observed with the mitochondria-targeted antioxidant MitoQ.
Simultaneous multiple immunolabeling in field emission scanning electron microscope (FESEM) is li... more Simultaneous multiple immunolabeling in field emission scanning electron microscope (FESEM) is limited by the number of suitable markers which is possible to distinguish in the microscope. At present two sizes of gold nanoparticles, e.g. 5 and 15 nm, are used for simultaneous labeling of two antigens in the observed cell surface structures. The detection of backscattered electrons in FESEM offers to use colloidal metal nanoparticles (NPs) made from other elements, because the BSE signal is very sensitive to the atomic number Z of the observed specimen area. The aim of this study was to select colloidal metal NPs that might be suitable markers, and test their stability in FESEM, their ability to conjugate them to antibodies and possibility to distinguish them from the gold nanoparticles using BSE signal. Based on our measurements, laboratory synthesized Pd nanoparticles in sizes between 8-15 nm and double shell Ag Au nanoparticles with the diameter around 15 nm were selected as the m...
Proceedings of the National Academy of Sciences of the United States of America, Jan 13, 2020
The adaptation of eukaryotic cells to anaerobic conditions is reflected by substantial changes to... more The adaptation of eukaryotic cells to anaerobic conditions is reflected by substantial changes to mitochondrial metabolism and functional reduction. Hydrogenosomes belong among the most modified mitochondrial derivative and generate molecular hydrogen concomitant with ATP synthesis. The reduction of mitochondria is frequently associated with loss of peroxisomes, which compartmentalize pathways that generate reactive oxygen species (ROS) and thus protect against cellular damage. The biogenesis and function of peroxisomes are tightly coupled with mitochondria. These organelles share fission machinery components, oxidative metabolism pathways, ROS scavenging activities, and some metabolites. The loss of peroxisomes in eukaryotes with reduced mitochondria is thus not unexpected. Surprisingly, we identified peroxisomes in the anaerobic, hydrogenosome-bearing protist Mastigamoeba balamuthi . We found a conserved set of peroxin (Pex) proteins that are required for protein import, peroxisomal growth, and division. Key membrane-associated Pexs ( Mb Pex3, Mb Pex11, and Mb Pex14) were visualized in numerous vesicles distinct from hydrogenosomes, the endoplasmic reticulum (ER), and Golgi complex. Proteomic analysis of cellular fractions and prediction of peroxisomal targeting signals (PTS1/PTS2) identified 51 putative peroxisomal matrix proteins. Expression of selected proteins in Saccharomyces cerevisiae revealed specific targeting to peroxisomes. The matrix proteins identified included components of acyl-CoA and carbohydrate metabolism and pyrimidine and CoA biosynthesis, whereas no components related to either β-oxidation or catalase were present. In conclusion, we identified a subclass of peroxisomes, named “anaerobic” peroxisomes that shift the current paradigm and turn attention to the reductive evolution of peroxisomes in anaerobic organisms.
Extended abstract of a paper presented at MC 2007, 33rd DGE Conference in Saarbrücken, Germany, S... more Extended abstract of a paper presented at MC 2007, 33rd DGE Conference in Saarbrücken, Germany, September 2 – September 7, 2007
Lyme disease (LD) spirochetes are well known to be able to disseminate into the tissues of infect... more Lyme disease (LD) spirochetes are well known to be able to disseminate into the tissues of infected hosts, including humans. The diverse strategies used by spirochetes to avoid the host immune system and persist in the host include active immune suppression, induction of immune tolerance, phase and antigenic variation, intracellular seclusion, and, importantly, incursion into immune privileged sites such as the brain. Invasion of immune privileged sites, like the brain allows the spirochetes not only escape from the host immune system but also can reduce the efficacy of antibiotic therapy. Here we present a case of the detection of DNA of spirochetes from Borrelia burgdorferi sensu lato complex from multiple loci of LD patient’s post-mortem brain. The presence of co-infection with Borrelia burgdorferi sensu stricto and Borrelia garinii in LD patient’s brain was confirmed by PCR. The presence of atypical spirochete morphology was confirmed by immunohistochemistry of the brain samples...
We report for the first time the use of two live-cell imaging agents from the group of luminescen... more We report for the first time the use of two live-cell imaging agents from the group of luminescent transition metal complexes (IRAZOLVE-MITO and REZOLVE-ER) as cathodoluminescent probes. This first experimental demonstration shows the application of both probes for the identification of cellular structures at the nanoscale and near the native state directly in the cryo-scanning electron microscope. This approach can potentially be applied to correlative and multimodal approaches and used to target specific regions within vitrified samples at low electron beam energies.
SUMMARYBarium (Ba) and strontium (Sr) are often used as proxies for the reconstruction of past ma... more SUMMARYBarium (Ba) and strontium (Sr) are often used as proxies for the reconstruction of past marine productivity and global climate. The ability to accumulate Ba2+ and Sr2+ in the form of crystals is rare among eukaryotes. Here we report that unicellular heterotrophs called diplonemids (Euglenozoa), one of the most abundant groups of marine planktonic protists, accumulate conspicuous amounts of these trace elements in the form of intracellular barite (BaSO4) and celestite (SrSO4) crystals, in concentrations greater than in other known Ba/Sr-accumulating organisms. Moreover, these flagellates can uptake Sr2+ exclusively or together with Ba2+ and form (Ba,Sr)SO4. One species, Namystinia karyoxenos, is naturally capable of intracellular accumulation of Ba2+ and Sr2+ 42,000 and 10,000 times relative to the surrounding medium. Altering the amounts of both elements in the medium resulted in corresponding changes in the quantity and composition of crystals. Planktonic copepods fed with d...
Kinesins are motor proteins found in all eukaryotic lineages that move along microtubules to medi... more Kinesins are motor proteins found in all eukaryotic lineages that move along microtubules to mediate cellular processes such as mitosis and intracellular transport. In trypanosomatids, the kinesin superfamily has undergone a prominent expansion, resulting in one of the most diverse kinesin repertoires that includes the two kinetoplastid‐restricted families X1 and X2. Here, we characterize in Trypanosoma brucei TbKifX2A, an orphaned X2 kinesin. TbKifX2A tightly interacts with TbPH1, a kinesin‐like protein with a likely inactive motor domain, a rarely reported occurrence. Both TbKifX2A and TbPH1 localize to the microtubule quartet (MtQ), a characteristic but poorly understood cytoskeletal structure that wraps around the flagellar pocket as it extends to the cell body anterior. The proximal proteome of TbPH1 revealed two other interacting proteins, the flagellar pocket protein FP45 and intriguingly another X2 kinesin, TbKifX2C. Simultaneous ablation of TbKifX2A/TbPH1 results in the dep...
Extended abstract of a paper presented at MC 2007, 33rd DGE Conference in Saarbrücken, Germany, S... more Extended abstract of a paper presented at MC 2007, 33rd DGE Conference in Saarbrücken, Germany, September 2 – September 7, 2007
ABSTRACTTriphenylphosphonium (TPP) derivatives are commonly used to target chemical into mitochon... more ABSTRACTTriphenylphosphonium (TPP) derivatives are commonly used to target chemical into mitochondria. We show that alkyl-TPP cause reversible, dose- and hydrophobicity-dependent alterations of mitochondrial morphology and function and a selective decrease of mitochondrial inner membrane proteins including subunits of the respiratory chain complexes, as well as components of the mitochondrial calcium uniporter complex. The treatment with alkyl-TPP resulted in the cleavage of the pro-fusion and cristae organisation regulator Optic atrophy-1. The structural and functional effects of alkyl-TPP were found to be reversible and not merely due to loss of membrane potential. A similar effect was observed with the mitochondria-targeted antioxidant MitoQ.
Uploads
Papers by Marie Vancová