@article {48416, title = {Diverse protocols for correlative super-resolution fluorescence imaging and electron microscopy of chemically fixed samples.}, journal = {Nature Protocols}, volume = {12}, year = {2017}, month = {2017 May}, pages = {916-946}, abstract = {

Our groups have recently developed related approaches for sample preparation for super-resolution imaging within endogenous cellular environments using correlative light and electron microscopy (CLEM). Four distinct techniques for preparing and acquiring super-resolution CLEM data sets for aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. The choice of the best protocol for a given application depends on a number of criteria that are discussed in detail. Tokuyasu cryosectioning is relatively rapid but is limited to small, delicate specimens. Whole-cell mount has the simplest sample preparation but is restricted to surface structures. Cell unroofing and platinum replication creates high-contrast, 3D images of the cytoplasmic surface of the plasma membrane but is more challenging than whole-cell mount. Resin embedding permits serial sectioning of large samples but is limited to osmium-resistant probes, and is technically difficult. Expected results from these protocols include super-resolution localization (\~{}10-50 nm) of fluorescent targets within the context of electron microscopy ultrastructure, which can help address cell biological questions. These protocols can be completed in 2-7 d, are compatible with a number of super-resolution imaging protocols, and are broadly applicable across biology.

}, issn = {1750-2799}, doi = {10.1038/nprot.2017.017}, author = {Kopek, Benjamin G and Paez-Segala, Maria G and Shtengel, Gleb and Sochacki, Kem A and Sun, Mei G and Wang, Yalin and Xu, C Shan and Van Engelenburg, Schuyler B and Taraska, Justin W and Looger, Loren L and Hess, Harald F} } @article {47424, title = {Movement and structure of mitochondria in oligodendrocytes and their myelin sheaths.}, journal = {Glia}, volume = {64}, year = {2016}, month = {2016 May}, pages = {810-25}, abstract = {

Mitochondria play several crucial roles in the life of oligodendrocytes. During development of the myelin sheath they are essential providers of carbon skeletons and energy for lipid synthesis. During normal brain function their consumption of pyruvate will be a key determinant of how much lactate is available for oligodendrocytes to export to power axonal function. Finally, during calcium-overload induced pathology, as occurs in ischemia, mitochondria may buffer calcium or induce apoptosis. Despite their important functions, very little is known of the properties of oligodendrocyte mitochondria, and mitochondria have never been observed in the myelin sheaths. We have now used targeted expression of fluorescent mitochondrial markers to characterize the location and movement of mitochondria within oligodendrocytes. We show for the first time that mitochondria are able to enter and move within the myelin sheath. Within the myelin sheath the highest number of mitochondria was in the cytoplasmic ridges along the sheath. Mitochondria moved more slowly than in neurons and, in contrast to their behavior in neurons and astrocytes, their movement was increased rather than inhibited by glutamate activating NMDA receptors. By electron microscopy we show that myelin sheath mitochondria have a low surface area of cristae, which suggests a low ATP production. These data specify fundamental properties of the oxidative phosphorylation system in oligodendrocytes, the glial cells that enhance cognition by speeding action potential propagation and provide metabolic support to axons. GLIA 2016;64:810-825.

}, issn = {1098-1136}, doi = {10.1002/glia.22965}, author = {Rinholm, Johanne E and Vervaeke, Koen and Tadross, Michael R and Tkachuk, Ariana N and Kopek, Benjamin G and Brown, Timothy A and Bergersen, Linda H and Clayton, David A} } @article {47242, title = {Synthesis of a far-red photoactivatable silicon-containing rhodamine for super-resolution microscopy.}, journal = {Angewandte Chemie (International ed. in English)}, volume = {55}, year = {2015}, month = {2015 Dec 11}, pages = {1723-7}, abstract = {

The rhodamine system is a flexible framework for building small-molecule fluorescent probes. Changing N-substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si-containing analogue of Q-rhodamine. This probe is the first example of a "caged" Si-rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red-shifted to allow multicolor imaging. The dye is a useful label for super-resolution imaging and constitutes a new scaffold for far-red fluorogenic molecules.

}, issn = {1521-3773}, doi = {10.1002/anie.201509649}, author = {Grimm, Jonathan B and Klein, Teresa and Kopek, Benjamin G and Shtengel, Gleb and Hess, Harald F and Sauer, Markus and Lavis, Luke D} } @article {6712, title = {Correlative photoactivated localization and scanning electron microscopy.}, journal = {PLoS One}, volume = {8}, year = {2013}, month = {2013 Oct 25}, pages = {e77209}, abstract = {

The ability to localize proteins precisely within subcellular space is crucial to understanding the functioning of biological systems. Recently, we described a protocol that correlates a precise map of fluorescent fusion proteins localized using three-dimensional super-resolution optical microscopy with the fine ultrastructural context of three-dimensional electron micrographs. While it achieved the difficult simultaneous objectives of high photoactivated fluorophore preservation and ultrastructure preservation, it required a super-resolution optical and specialized electron microscope that is not available to many researchers. We present here a faster and more practical protocol with the advantage of a simpler two-dimensional optical (Photoactivated Localization Microscopy (PALM)) and scanning electron microscope (SEM) system that retains the often mutually exclusive attributes of fluorophore preservation and ultrastructure preservation. As before, cryosections were prepared using the Tokuyasu protocol, but the staining protocol was modified to be amenable for use in a standard SEM without the need for focused ion beam ablation. We show the versatility of this technique by labeling different cellular compartments and structures including mitochondrial nucleoids, peroxisomes, and the nuclear lamina. We also demonstrate simultaneous two-color PALM imaging with correlated electron micrographs. Lastly, this technique can be used with small-molecule dyes as demonstrated with actin labeling using phalloidin conjugated to a caged dye. By retaining the dense protein labeling expected for super-resolution microscopy combined with ultrastructural preservation, simplifying the tools required for correlative microscopy, and expanding the number of useful labels we expect this method to be accessible and valuable to a wide variety of researchers.

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0077209}, author = {Kopek, Benjamin G and Shtengel, Gleb and Grimm, Jonathan B and Clayton, David A and Hess, Harald F} } @article {45187, title = {Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes.}, journal = {Proceedings of the National Academy of Science of the United States of America}, volume = {109}, year = {2012}, month = {2012 Apr 17}, pages = {6136-41}, abstract = {

Microscopic images of specific proteins in their cellular context yield important insights into biological processes and cellular architecture. The advent of superresolution optical microscopy techniques provides the possibility to augment EM with nanometer-resolution fluorescence microscopy to access the precise location of proteins in the context of cellular ultrastructure. Unfortunately, efforts to combine superresolution fluorescence and EM have been stymied by the divergent and incompatible sample preparation protocols of the two methods. Here, we describe a protocol that preserves both the delicate photoactivatable fluorescent protein labels essential for superresolution microscopy and the fine ultrastructural context of EM. This preparation enables direct 3D imaging in 500- to 750-nm sections with interferometric photoactivatable localization microscopy followed by scanning EM images generated by focused ion beam ablation. We use this process to "colorize" detailed EM images of the mitochondrion with the position of labeled proteins. The approach presented here has provided a new level of definition of the in vivo nature of organization of mitochondrial nucleoids, and we expect this straightforward method to be applicable to many other biological questions that can be answered by direct imaging.

}, keywords = {3T3 Cells, Animals, DNA, Mitochondrial, DNA-Binding Proteins, Fibroblasts, High Mobility Group Proteins, Imaging, Three-Dimensional, Luminescent Proteins, Mice, Microscopy, Electron, Microscopy, Fluorescence, Microscopy, Interference, Mitochondria, Mitochondrial Membranes, Mitochondrial Proteins, Reproducibility of Results}, issn = {1091-6490}, doi = {10.1073/pnas.1121558109}, author = {Kopek, Benjamin G and Shtengel, Gleb and Xu, C Shan and Clayton, David A and Hess, Harald F} } @article {4469, title = {Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction.}, journal = {Molecular and Cellular Biology}, volume = {31}, year = {2011}, month = {2011 Dec}, pages = {4994-5010}, abstract = {

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.

}, keywords = {3T3 Cells, Animals, Confocal, DNA, Fluorescence, Mice, Microscopy, Mitochondria, Mitochondrial, Mitochondrial Proteins, Plasmids, Sequence Alignment, Sequence Analysis}, issn = {1098-5549}, doi = {10.1128/MCB.05694-11}, url = {http://mcb.asm.org/content/31/24/4994.short}, author = {Brown, Timothy A and Tkachuk, Ariana N and Shtengel, Gleb and Kopek, Benjamin G and Bogenhagen, Daniel F and Hess, Harald F and Clayton, David A} } @article {1541, title = {Nodavirus-induced membrane rearrangement in replication complex assembly requires replicase protein a, RNA templates, and polymerase activity.}, journal = {Journal of Virology}, volume = {84}, year = {2010}, month = {2010 Dec}, pages = {12492-503}, abstract = {

Positive-strand RNA [(+)RNA] viruses invariably replicate their RNA genomes on modified intracellular membranes. In infected Drosophila cells, Flock House nodavirus (FHV) RNA replication complexes form on outer mitochondrial membranes inside \~{}50-nm, virus-induced spherular invaginations similar to RNA replication-linked spherules induced by many (+)RNA viruses at various membranes. To better understand replication complex assembly, we studied the mechanisms of FHV spherule formation. FHV has two genomic RNAs; RNA1 encodes multifunctional RNA replication protein A and RNA interference suppressor protein B2, while RNA2 encodes the capsid proteins. Expressing genomic RNA1 without RNA2 induced mitochondrial spherules indistinguishable from those in FHV infection. RNA1 mutation showed that protein B2 was dispensable and that protein A was the only FHV protein required for spherule formation. However, expressing protein A alone only "zippered" together the surfaces of adjacent mitochondria, without inducing spherules. Thus, protein A is necessary but not sufficient for spherule formation. Coexpressing protein A plus a replication-competent FHV RNA template induced RNA replication in trans and membrane spherules. Moreover, spherules were not formed when replicatable FHV RNA templates were expressed with protein A bearing a single, polymerase-inactivating amino acid change or when wild-type protein A was expressed with a nonreplicatable FHV RNA template. Thus, unlike many (+)RNA viruses, the membrane-bounded compartments in which FHV RNA replication occurs are not induced solely by viral protein(s) but require viral RNA synthesis. In addition to replication complex assembly, the results have implications for nodavirus interaction with cell RNA silencing pathways and other aspects of virus control.

}, keywords = {Animals, Blotting, Capsid Proteins, DNA-Directed RNA Polymerases, Drosophila melanogaster, Genome, Intracellular Membranes, Mitochondria, Nodaviridae, Northern, Plasmids, RNA, RNA Replicase, RNA Virus Infections, Small Interfering, Viral, Virus Replication, Western}, issn = {1098-5514}, doi = {10.1128/JVI.01495-10}, url = {http://jvi.asm.org/content/84/24/12492.short}, author = {Kopek, Benjamin G and Settles, Erik W and Friesen, Paul D and Ahlquist, Paul} } @article {4479, title = {Three-dimensional analysis of a viral RNA replication complex reveals a virus-induced mini-organelle.}, journal = {PLoS biology}, volume = {5}, year = {2007}, month = {2007 Sep}, pages = {e220}, abstract = {

Positive-strand RNA viruses are the largest genetic class of viruses and include many serious human pathogens. All positive-strand RNA viruses replicate their genomes in association with intracellular membrane rearrangements such as single- or double-membrane vesicles. However, the exact sites of RNA synthesis and crucial topological relationships between relevant membranes, vesicle interiors, surrounding lumens, and cytoplasm generally are poorly defined. We applied electron microscope tomography and complementary approaches to flock house virus (FHV)-infected Drosophila cells to provide the first 3-D analysis of such replication complexes. The sole FHV RNA replication factor, protein A, and FHV-specific 5-bromouridine 5{\textquoteright}-triphosphate incorporation localized between inner and outer mitochondrial membranes inside approximately 50-nm vesicles (spherules), which thus are FHV-induced compartments for viral RNA synthesis. All such FHV spherules were outer mitochondrial membrane invaginations with interiors connected to the cytoplasm by a necked channel of approximately 10-nm diameter, which is sufficient for ribonucleotide import and product RNA export. Tomographic, biochemical, and other results imply that FHV spherules contain, on average, three RNA replication intermediates and an interior shell of approximately 100 membrane-spanning, self-interacting protein As. The results identify spherules as the site of protein A and nascent RNA accumulation and define spherule topology, dimensions, and stoichiometry to reveal the nature and many details of the organization and function of the FHV RNA replication complex. The resulting insights appear relevant to many other positive-strand RNA viruses and support recently proposed structural and likely evolutionary parallels with retrovirus and double-stranded RNA virus virions.

}, keywords = {Animals, Drosophila, Electron, Imaging, Microscopy, Mitochondrial Membranes, Nodaviridae, Organelles, RNA, Three-Dimensional, Viral, Virus Replication}, issn = {1545-7885}, doi = {10.1371/journal.pbio.0050220}, url = {http://www.plosbiology.org/article/info\%3Adoi\%2F10.1371\%2Fjournal.pbio.0050220}, author = {Kopek, Benjamin G and Perkins, Guy and Miller, David J and Ellisman, Mark H and Ahlquist, Paul} } @article {1542, title = {Three-dimensional analysis of a viral RNA replication complex reveals a virus-induced mini-organelle.}, journal = {PLoS Biology}, volume = {5}, year = {2007}, month = {2007 Sep}, pages = {e220}, abstract = {

Positive-strand RNA viruses are the largest genetic class of viruses and include many serious human pathogens. All positive-strand RNA viruses replicate their genomes in association with intracellular membrane rearrangements such as single- or double-membrane vesicles. However, the exact sites of RNA synthesis and crucial topological relationships between relevant membranes, vesicle interiors, surrounding lumens, and cytoplasm generally are poorly defined. We applied electron microscope tomography and complementary approaches to flock house virus (FHV)-infected Drosophila cells to provide the first 3-D analysis of such replication complexes. The sole FHV RNA replication factor, protein A, and FHV-specific 5-bromouridine 5{\textquoteright}-triphosphate incorporation localized between inner and outer mitochondrial membranes inside approximately 50-nm vesicles (spherules), which thus are FHV-induced compartments for viral RNA synthesis. All such FHV spherules were outer mitochondrial membrane invaginations with interiors connected to the cytoplasm by a necked channel of approximately 10-nm diameter, which is sufficient for ribonucleotide import and product RNA export. Tomographic, biochemical, and other results imply that FHV spherules contain, on average, three RNA replication intermediates and an interior shell of approximately 100 membrane-spanning, self-interacting protein As. The results identify spherules as the site of protein A and nascent RNA accumulation and define spherule topology, dimensions, and stoichiometry to reveal the nature and many details of the organization and function of the FHV RNA replication complex. The resulting insights appear relevant to many other positive-strand RNA viruses and support recently proposed structural and likely evolutionary parallels with retrovirus and double-stranded RNA virus virions.

}, keywords = {Animals, Drosophila, Electron, Imaging, Microscopy, Mitochondrial Membranes, Nodaviridae, Organelles, RNA, Three-Dimensional, Viral, Virus Replication}, issn = {1545-7885}, doi = {10.1371/journal.pbio.0050220}, author = {Kopek, Benjamin G and Perkins, Guy and Miller, David J and Ellisman, Mark H and Ahlquist, Paul} } @article {1543, title = {An in vitro fluorescence screen to identify antivirals that disrupt hepatitis B virus capsid assembly.}, journal = {Nature Biotechnology}, volume = {24}, year = {2006}, month = {2006 Mar}, pages = {358-62}, abstract = {

Virus assembly has not been routinely targeted in the development of antiviral drugs, in part because of the lack of tractable methods for screening in vitro. We have developed an in vitro assay of hepatitis B virus (HBV) capsid assembly, based on fluorescence quenching of dye-labeled capsid protein, for testing potential inhibitors. This assay is adaptable to high-throughput screening and can identify small-molecule inhibitors of virus assembly that prevent, inappropriately accelerate and/or misdirect capsid formation to yield aberrant particles. An in vitro primary screen has the advantage of identifying promising lead compounds affecting assembly without the requirement that they be taken up by cells in culture and be nontoxic. Our approach may facilitate the identification of antivirals targeting viruses other than HBV, such as avian influenza and HIV.

}, keywords = {Antiviral Agents, Biological Assay, Boron Compounds, Capsid, Capsid Proteins, Dimerization, Fluorescence, Fluorescent Dyes, Hepatitis B virus, Kinetics, Light, Models, Molecular, Radiation, Scattering, Virus Assembly}, issn = {1087-0156}, doi = {10.1038/nbt1187}, author = {Stray, Stephen J and Johnson, Jennifer M and Kopek, Benjamin G and Zlotnick, Adam} } @article {4481, title = {Microwave oven synthesis of esters promoted by imidazole.}, journal = {Tetrahedron Letters}, volume = {44}, year = {2003}, month = {2003 Feb 24}, pages = {1831-3}, abstract = {

Using imidazole as promotion agent, primary, secondary and phenolic alcohol compounds were esterified with aliphatic and aromatic carboxylic acid anhydrides. Heating a ternary mixture of alcohol, anhydride and imidazole in an unmodified microwave oven produced esters in low to high yields, depending on the steric bulk of the alcohol.

}, url = {http://www.sciencedirect.com/science/article/pii/S0040403903001047}, author = {Hirose, Takuji and Kopek, Benjamin G and Wang, Zhao-Hui and Yusa, Ritsuko and Baldwin, Bruce W.} }