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Daniel F. Bogenhagen - One of the best experts on this subject based on the ideXlab platform.
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initial steps in rna processing and ribosome assembly occur at mitochondrial dna Nucleoids
Cell Metabolism, 2014Co-Authors: Daniel F. Bogenhagen, Dwight W Martin, Antonius KollerAbstract:Summary Mammalian mitochondrial DNA (mtDNA) resides in compact Nucleoids, where it is replicated and transcribed into long primary transcripts processed to generate rRNAs, tRNAs, and mRNAs encoding 13 proteins. This situation differs from bacteria and eukaryotic nucleoli, which have dedicated rRNA transcription units. The assembly of rRNAs into mitoribosomes has received little study. We show that mitochondrial RNA processing enzymes involved in tRNA excision, ribonuclease P (RNase P) and ELAC2, as well as a subset of nascent mitochondrial ribosomal proteins (MRPs) associate with Nucleoids to initiate RNA processing and ribosome assembly. SILAC pulse-chase labeling experiments show that nascent MRPs recruited to the Nucleoid fraction were highly labeled after the pulse in a transcription-dependent manner and decreased in labeling intensity during the chase. These results provide insight into the landscape of binding events required for mitochondrial ribosome assembly and firmly establish the mtDNA Nucleoid as a control center for mitochondrial biogenesis.
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superresolution fluorescence imaging of mitochondrial Nucleoids reveals their spatial range limits and membrane interaction
Molecular and Cellular Biology, 2011Co-Authors: Timothy A Brown, Daniel F. Bogenhagen, Ariana N Tkachuk, Gleb Shtengel, Benjamin G Kopek, Harald F Hess, David A ClaytonAbstract:A fundamental objective in molecular biology is to understand how DNA is organized in concert with various proteins, RNA, and biological membranes. Mitochondria maintain and express their own DNA (mtDNA), which is arranged within structures called Nucleoids. Their functions, dimensions, composition, and precise locations relative to other mitochondrial structures are poorly defined. Superresolution fluorescence microscopy techniques that exceed the previous limits of imaging within the small and highly compartmentalized mitochondria have been recently developed. We have improved and employed both two- and three-dimensional applications of photoactivated localization microscopy (PALM and iPALM, respectively) to visualize the core dimensions and relative locations of mitochondrial Nucleoids at an unprecedented resolution. PALM reveals that Nucleoids differ greatly in size and shape. Three-dimensional volumetric analysis indicates that, on average, the mtDNA within ellipsoidal Nucleoids is extraordinarily condensed. Two-color PALM shows that the freely diffusible mitochondrial matrix protein is largely excluded from the Nucleoid. In contrast, Nucleoids are closely associated with the inner membrane and often appear to be wrapped around cristae or crista-like inner membrane invaginations. Determinations revealing high packing density, separation from the matrix, and tight association with the inner membrane underscore the role of mechanisms that regulate access to mtDNA and that remain largely unknown.
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the layered structure of human mitochondrial dna Nucleoids
Journal of Biological Chemistry, 2008Co-Authors: Daniel F. Bogenhagen, Denis L. Rousseau, Stephanie BurkeAbstract:Abstract Mitochondrial DNA (mtDNA) occurs in cells in Nucleoids containing several copies of the genome. Previous studies have identified proteins associated with these large DNA structures when they are biochemically purified by sedimentation and immunoaffinity chromatography. In this study, formaldehyde cross-linking was performed to determine which Nucleoid proteins are in close contact with the mtDNA. A set of core Nucleoid proteins is found in both native and cross-linked Nucleoids, including 13 proteins with known roles in mtDNA transactions. Several other metabolic proteins and chaperones identified in native Nucleoids, including ATAD3, were not observed to cross-link to mtDNA. Additional immunofluorescence and protease susceptibility studies showed that an N-terminal domain of ATAD3 previously proposed to bind to the mtDNA D-loop is directed away from the mitochondrial matrix, so it is unlikely to interact with mtDNA in vivo. These results are discussed in relation to a model for a layered structure of mtDNA Nucleoids in which replication and transcription occur in the central core, whereas translation and complex assembly may occur in the peripheral region.
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human mitochondrial dna Nucleoids are linked to protein folding machinery and metabolic enzymes at the mitochondrial inner membrane
Journal of Biological Chemistry, 2006Co-Authors: Yousong Wang, Daniel F. BogenhagenAbstract:Mitochondrial DNA (mtDNA) is packaged into bacterial Nucleoid-like structures, each containing several mtDNA molecules. The distribution of Nucleoids during mitochondrial fission and fusion events and during cytokinesis is important to the segregation of mitochondrial genomes in heteroplasmic cells bearing a mixture of wild-type and mutant mtDNA molecules. We report fractionation of HeLa cell mtDNA Nucleoids into two subsets of complexes that differ in their sedimentation velocity and their association with cytoskeletal proteins. Pulse labeling studies indicated that newly replicated mtDNA molecules are evenly represented in the rapidly and slowly sedimenting fractions. Slowly sedimenting Nucleoids were immunoaffinity purified using antibodies to either of two abundant mtDNA-binding proteins, TFAM or mtSSB. These two different immunoaffinity procedures yielded very similar sets of proteins, with 21 proteins in common, including most of the proteins previously shown to play roles in mtDNA replication and transcription. In addition to previously identified mitochondrial proteins, multiple peptides were observed for one novel DNA metabolic protein, the DEAH-box helicase DHX30. Antibodies raised against a recombinant fragment of this protein confirmed the mitochondrial localization of a specific isoform of DHX30.
Kunio Takeyasu - One of the best experts on this subject based on the ideXlab platform.
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Dynamic state of DNA topology is essential for genome condensation in bacteria.
The EMBO journal, 2006Co-Authors: Ryosuke L. Ohniwa, Kazuya Morikawa, Joongbaek Kim, Toshiko Ohta, Akira Ishihama, Chieko Wada, Kunio TakeyasuAbstract:In bacteria, Dps is one of the critical proteins to build up a condensed Nucleoid in response to the environmental stresses. In this study, we found that the expression of Dps and the Nucleoid condensation was not simply correlated in Escherichia coli, and that Fis, which is an E. coli (gamma-Proteobacteria)-specific Nucleoid protein, interfered with the Dps-dependent Nucleoid condensation. Atomic force microscopy and Northern blot analyses indicated that the inhibitory effect of Fis was due to the repression of the expression of Topoismerase I (Topo I) and DNA gyrase. In the Δfis strain, both topA and gyrA/B genes were found to be upregulated. Overexpression of Topo I and DNA gyrase enhanced the nulceoid condensation in the presence of Dps. DNA-topology assays using the cell extract showed that the extracts from the Δfis and Topo I-/DNA gyrase-overexpressing strains, but not the wild-type extract, shifted the population toward relaxed forms. These results indicate that the topology of DNA is dynamically transmutable and that the topology control is important for Dps-induced Nucleoid condensation.
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bacterial Nucleoid dynamics oxidative stress response in staphylococcus aureus
Genes to Cells, 2006Co-Authors: Kazuya Morikawa, Ryosuke L. Ohniwa, Toshiko Ohta, Atsushi Maruyama, Kunio TakeyasuAbstract:A single-molecule-imaging technique, atomic force microscopy (AFM) was applied to the analyses of the genome architecture of Staphylococcus aureus. The staphylococcal cells on a cover glass were subjected to a mild lysis procedure that had maintained the fundamental structural units in Escherichia coli. The Nucleoids were found to consist of fibrous structures with diameters of 80 and 40 nm. This feature was shared with the E. coli Nucleoid. However, whereas the E. coli Nucleoid dynamically changed its structure to a highly compacted one towards the stationary phase, the S. aureus Nucleoid never underwent such a tight compaction under a normal growth condition. Bioinformatic analysis suggested that this was attributable to the lack of IHF that regulate the expression of a Nucleoid protein, Dps, required for Nucleoid compaction in E. coli. On the other hand, under oxidative conditions, MrgA (a staphylococcal Dps homolog) was over-expressed and a drastic compaction of the Nucleoid was detected. A knock-out mutant of the gene encoding the transcription factor (perR) constitutively expressed mrgA, and its Nucleoid was compacted without the oxidative stresses. The regulatory mechanisms of Dps/MrgA expression and their biological significance were postulated in relation to the Nucleoid compaction.
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fundamental structural units of the escherichia coli Nucleoid revealed by atomic force microscopy
Nucleic Acids Research, 2004Co-Authors: Joongbaek Kim, Ryosuke L. Ohniwa, Akira Ishihama, Shige H Yoshimura, Kohji Hizume, Kunio TakeyasuAbstract:A small container of several to a few hundred µm3 (i.e. bacterial cells and eukaryotic nuclei) contains extremely long genomic DNA (i.e. mm and m long, respectively) in a highly organized fashion. To understand how such genomic architecture could be achieved, Escherichia coli Nucleoids were subjected to structural analyses under atomic force microscopy, and found to change their structure dynamically during cell growth, i.e. the Nucleoid structure in the stationary phase was more tightly compacted than in the log phase. However, in both log and stationary phases, a fundamental fibrous structure with a diameter of ∼80 nm was found. In addition to this ‘80 nm fiber’, a thinner ‘40 nm fiber’ and a higher order ‘loop’ structure were identified in the log phase Nucleoid. In the later growth phases, the Nucleoid turned into a ‘coral reef structure’ that also possessed the 80 nm fiber units, and, finally, into a ‘tightly compacted Nucleoid’ that was stable in a mild lysis buffer. Mutant analysis demonstrated that these tight compactions of the Nucleoid required a protein, Dps. From these results and previously available information, we propose a structural model of the E.coli Nucleoid.
Fabai Wu - One of the best experts on this subject based on the ideXlab platform.
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cell boundary confinement sets the size and position of the e coli chromosome
Current Biology, 2019Co-Authors: Fabai Wu, Pinaki Swain, Xuan Zheng, Kevin M Felter, Margot Guurink, Jacopo Solari, Louis Kuijpers, Thomas S. Shimizu, Debasish ChaudhuriAbstract:While the spatiotemporal structure of the genome is crucial to its biological function, many basic questions remain unanswered on the morphology and segregation of chromosomes. Here, we experimentally show in Escherichia coli that spatial confinement plays a dominant role in determining both the chromosome size and position. In non-dividing cells with lengths up to 10 times normal, single chromosomes are observed to expand more than 4 fold in size, an effect only modestly influenced by deletions of various Nucleoid-associated proteins. Chromosomes show pronounced internal dynamics but exhibit a robust positioning where single Nucleoids reside strictly at mid-cell, while two Nucleoids self-organize at 1/4 and 1/4 cell positions. Molecular dynamics simulations of model chromosomes recapitulate these phenomena and indicate that these observations can be attributed to depletion effects induced by cytosolic crowders. These findings highlight boundary confinement as a key causal factor that needs to be considered for understanding chromosome organization.
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cell boundary confinement sets the size and position of the e coli chromosome
Current Biology, 2019Co-Authors: Fabai Wu, Pinaki Swain, Xuan Zheng, Kevin M Felter, Margot Guurink, Jacopo Solari, Louis Kuijpers, Thomas S. Shimizu, Debasish ChaudhuriAbstract:While the spatiotemporal structure of the genome is crucial to its biological function, many basic questions remain unanswered on the morphology and segregation of chromosomes. Here, we experimentally show in Escherichia coli that spatial confinement plays a dominant role in determining both the chromosome size and position. In non-dividing cells with lengths up to 10 times normal, single chromosomes are observed to expand more than 4 fold in size, an effect only modestly influenced by deletions of various Nucleoid-associated proteins. Chromosomes show pronounced internal dynamics but exhibit a robust positioning where single Nucleoids reside strictly at mid-cell, while two Nucleoids self-organize at 1/4 and 1/4 cell positions. Molecular dynamics simulations of model chromosomes recapitulate these phenomena and indicate that these observations can be attributed to depletion effects induced by cytosolic crowders. These findings highlight boundary confinement as a key causal factor that needs to be considered for understanding chromosome organization.
Debasish Chaudhuri - One of the best experts on this subject based on the ideXlab platform.
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cell boundary confinement sets the size and position of the e coli chromosome
Current Biology, 2019Co-Authors: Fabai Wu, Pinaki Swain, Xuan Zheng, Kevin M Felter, Margot Guurink, Jacopo Solari, Louis Kuijpers, Thomas S. Shimizu, Debasish ChaudhuriAbstract:While the spatiotemporal structure of the genome is crucial to its biological function, many basic questions remain unanswered on the morphology and segregation of chromosomes. Here, we experimentally show in Escherichia coli that spatial confinement plays a dominant role in determining both the chromosome size and position. In non-dividing cells with lengths up to 10 times normal, single chromosomes are observed to expand more than 4 fold in size, an effect only modestly influenced by deletions of various Nucleoid-associated proteins. Chromosomes show pronounced internal dynamics but exhibit a robust positioning where single Nucleoids reside strictly at mid-cell, while two Nucleoids self-organize at 1/4 and 1/4 cell positions. Molecular dynamics simulations of model chromosomes recapitulate these phenomena and indicate that these observations can be attributed to depletion effects induced by cytosolic crowders. These findings highlight boundary confinement as a key causal factor that needs to be considered for understanding chromosome organization.
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cell boundary confinement sets the size and position of the e coli chromosome
Current Biology, 2019Co-Authors: Pinaki Swain, Xuan Zheng, Kevin M Felter, Margot Guurink, Jacopo Solari, Debasish Chaudhuri, Louis Kuijpers, Thomas S. Shimizu, Suckjoon Jun, Bela M MulderAbstract:Although the spatiotemporal structure of the genome is crucial to its biological function, many basic questions remain unanswered on the morphology and segregation of chromosomes. Here, we experimentally show in Escherichia coli that spatial confinement plays a dominant role in determining both the chromosome size and position. In non-dividing cells with lengths increased to 10 times normal, single chromosomes are observed to expand > 4-fold in size. Chromosomes show pronounced internal dynamics but exhibit a robust positioning where single Nucleoids reside robustly at mid-cell, whereas two Nucleoids self-organize at 1/4 and 3/4 positions. The cell-size-dependent expansion of the Nucleoid is only modestly influenced by deletions of Nucleoid-associated proteins, whereas osmotic manipulation experiments reveal a prominent role of molecular crowding. Molecular dynamics simulations with model chromosomes and crowders recapitulate the observed phenomena and highlight the role of entropic effects caused by confinement and molecular crowding in the spatial organization of the chromosome.
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cell boundary confinement sets the size and position of the e coli chromosome
Current Biology, 2019Co-Authors: Fabai Wu, Pinaki Swain, Xuan Zheng, Kevin M Felter, Margot Guurink, Jacopo Solari, Louis Kuijpers, Thomas S. Shimizu, Debasish ChaudhuriAbstract:While the spatiotemporal structure of the genome is crucial to its biological function, many basic questions remain unanswered on the morphology and segregation of chromosomes. Here, we experimentally show in Escherichia coli that spatial confinement plays a dominant role in determining both the chromosome size and position. In non-dividing cells with lengths up to 10 times normal, single chromosomes are observed to expand more than 4 fold in size, an effect only modestly influenced by deletions of various Nucleoid-associated proteins. Chromosomes show pronounced internal dynamics but exhibit a robust positioning where single Nucleoids reside strictly at mid-cell, while two Nucleoids self-organize at 1/4 and 1/4 cell positions. Molecular dynamics simulations of model chromosomes recapitulate these phenomena and indicate that these observations can be attributed to depletion effects induced by cytosolic crowders. These findings highlight boundary confinement as a key causal factor that needs to be considered for understanding chromosome organization.
John A. Fuerst - One of the best experts on this subject based on the ideXlab platform.
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the cell cycle of the planctomycete gemmata obscuriglobus with respect to cell compartmentalization
BMC Cell Biology, 2009Co-Authors: Richard I Webb, John A. FuerstAbstract:Gemmata obscuriglobus is a distinctive member of the divergent phylum Planctomycetes, all known members of which are peptidoglycan-less bacteria with a shared compartmentalized cell structure and divide by a budding process. G. obscuriglobus in addition shares the unique feature that its Nucleoid DNA is surrounded by an envelope consisting of two membranes forming an analogous structure to the membrane-bounded Nucleoid of eukaryotes and therefore G. obscuriglobus forms a special model for cell biology. Draft genome data for G. obscuriglobus as well as complete genome sequences available so far for other planctomycetes indicate that the key bacterial cell division protein FtsZ is not present in these planctomycetes, so the cell division process in planctomycetes is of special comparative interest. The membrane-bounded nature of the Nucleoid in G. obscuriglobus also suggests that special mechanisms for the distribution of this nuclear body to the bud and for distribution of chromosomal DNA might exist during division. It was therefore of interest to examine the cell division cycle in G. obscuriglobus and the process of Nucleoid distribution and nuclear body formation during division in this planctomycete bacterium via light and electron microscopy. Using phase contrast and fluorescence light microscopy, and transmission electron microscopy, the cell division cycle of G. obscuriglobus was determined. During the budding process, the bud was formed and developed in size from one point of the mother cell perimeter until separation. The matured daughter cell acted as a new mother cell and started its own budding cycle while the mother cell can itself initiate budding repeatedly. Fluorescence microscopy of DAPI-stained cells of G. obscuriglobus suggested that translocation of the Nucleoid and formation of the bud did not occur at the same time. Confocal laser scanning light microscopy applied to cells stained for membranes as well as DNA confirmed the behaviour of the Nucleoid and Nucleoid envelope during cell division. Electron microscopy of cryosubstituted cells confirmed deductions from light microscopy concerning Nucleoid presence in relation to the stage of budding, and showed that the Nucleoid was observed to occur in both mother and bud cells only at later budding stages. It further suggested that Nucleoid envelope formed only after the Nucleoid was translocated into the bud, since envelopes only appeared in more mature buds, while naked Nucleoids occurred in smaller buds. Nucleoid envelope appeared to originate from the intracytoplasmic membranes (ICM) of both mother cell and bud. There was always a connecting passage between mother cell and bud during the budding process until separation of the two cells. The division cycle of the nucleated planctomycete G. obscuriglobus appears to be a complex process in which chromosomal DNA is transported to the daughter cell bud after initial formation of the bud, and this can be performed repeatedly by a single mother cell. The division cycle of the nucleated planctomycete G. obscuriglobus is a complex process in which chromosomal Nucleoid DNA is transported to the daughter cell bud after initial formation of a bud without Nucleoid. The new bud Nucleoid is initially naked and not surrounded by membrane, but eventually acquires a complete Nucleoid envelope consisting of two closely apposed membranes as occurs in the mother cell. The membranes of the new Nucleoid envelope surrounding the bud Nucleoid are derived from intracytoplasmic membranes of both the mother cell and the bud. The cell division of G. obscuriglobus displays some unique features not known in cells of either prokaryotes or eukaryotes.
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the cell cycle of the planctomycete gemmata obscuriglobus with respect to cell compartmentalization
BMC Cell Biology, 2009Co-Authors: Kuo-chang Lee, Richard I Webb, John A. FuerstAbstract:Gemmata obscuriglobus is a distinctive member of the divergent phylum Planctomycetes, all known members of which are peptidoglycan-less bacteria with a shared compartmentalized cell structure and divide by a budding process. G. obscuriglobus in addition shares the unique feature that its Nucleoid DNA is surrounded by an envelope consisting of two membranes forming an analogous structure to the membrane-bounded Nucleoid of eukaryotes and therefore G. obscuriglobus forms a special model for cell biology. Draft genome data for G. obscuriglobus as well as complete genome sequences available so far for other planctomycetes indicate that the key bacterial cell division protein FtsZ is not present in these planctomycetes, so the cell division process in planctomycetes is of special comparative interest. The membrane-bounded nature of the Nucleoid in G. obscuriglobus also suggests that special mechanisms for the distribution of this nuclear body to the bud and for distribution of chromosomal DNA might exist during division. It was therefore of interest to examine the cell division cycle in G. obscuriglobus and the process of Nucleoid distribution and nuclear body formation during division in this planctomycete bacterium via light and electron microscopy. Using phase contrast and fluorescence light microscopy, and transmission electron microscopy, the cell division cycle of G. obscuriglobus was determined. During the budding process, the bud was formed and developed in size from one point of the mother cell perimeter until separation. The matured daughter cell acted as a new mother cell and started its own budding cycle while the mother cell can itself initiate budding repeatedly. Fluorescence microscopy of DAPI-stained cells of G. obscuriglobus suggested that translocation of the Nucleoid and formation of the bud did not occur at the same time. Confocal laser scanning light microscopy applied to cells stained for membranes as well as DNA confirmed the behaviour of the Nucleoid and Nucleoid envelope during cell division. Electron microscopy of cryosubstituted cells confirmed deductions from light microscopy concerning Nucleoid presence in relation to the stage of budding, and showed that the Nucleoid was observed to occur in both mother and bud cells only at later budding stages. It further suggested that Nucleoid envelope formed only after the Nucleoid was translocated into the bud, since envelopes only appeared in more mature buds, while naked Nucleoids occurred in smaller buds. Nucleoid envelope appeared to originate from the intracytoplasmic membranes (ICM) of both mother cell and bud. There was always a connecting passage between mother cell and bud during the budding process until separation of the two cells. The division cycle of the nucleated planctomycete G. obscuriglobus appears to be a complex process in which chromosomal DNA is transported to the daughter cell bud after initial formation of the bud, and this can be performed repeatedly by a single mother cell. The division cycle of the nucleated planctomycete G. obscuriglobus is a complex process in which chromosomal Nucleoid DNA is transported to the daughter cell bud after initial formation of a bud without Nucleoid. The new bud Nucleoid is initially naked and not surrounded by membrane, but eventually acquires a complete Nucleoid envelope consisting of two closely apposed membranes as occurs in the mother cell. The membranes of the new Nucleoid envelope surrounding the bud Nucleoid are derived from intracytoplasmic membranes of both the mother cell and the bud. The cell division of G. obscuriglobus displays some unique features not known in cells of either prokaryotes or eukaryotes.
