A Review of Reagents for Fluorescence Microscopy of Cellular Compartments and Structures, Part II: Reagents for Non‐Vesicular Organelles

Kilgore, Jason A.; Dolman, Nick J.; Davidson, Michael W.

doi:10.1002/0471142956.cy1231s66

Cited by 19 publications

(30 citation statements)

References 81 publications

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“…A plethora of commercial antibodies exist to detect specific proteins in cells. In addition, several specific compartment or cellular organelle markers exist and can be used to provide a reference in infected cells . The nonexhaustive list comprises chloromethyl‐X‐rosamine known as MitoTracker for mitochondria , BODIPY or ER‐Tracker for the endoplasmic reticulum , or LysoTracker for lysosomes .…”

Section: General Considerations When Imaging Admentioning

confidence: 99%

Imaging the adenovirus infection cycle

Pied

Wodrich

2019

FEBS Letters

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Edited by Urs GreberIncoming adenoviruses seize control of cytosolic transport mechanisms to relocate their genome from the cell periphery to specialized sites in the nucleoplasm. The nucleus is the site for viral gene expression, genome replication, and the production of progeny for the next round of infection. By taking control of the cell, adenoviruses also suppress cell-autonomous immunity responses. To succeed in their production cycle, adenoviruses rely on wellcoordinated steps, facilitated by interactions between viral proteins and cellular factors. Interactions between virus and host can impose remarkable morphological changes in the infected cell. Imaging adenoviruses has tremendously influenced how we delineate individual steps in the viral life cycle, because it allowed the development of specific optical markers to label these morphological changes in space and time. As technology advances, innovative imaging techniques and novel tools for specimen labeling keep uncovering previously unseen facets of adenovirus biology emphasizing why imaging adenoviruses is as attractive today as it was in the past. This review will summarize past achievements and present developments in adenovirus imaging centered on fluorescence microscopy approaches.Adenoviruses (Ads) are nonenveloped icosahedral viruses with a diameter of~90 nm with slight structural differences between genotypes. The majority of the Ad capsid shell is composed of 240 hexon trimers, and each of the 12 vertices of the icosahedron is occupied by a penton. Pentons are involved in cell attachment and receptor recognition and are composed of the pentameric penton base from which the trimeric fiber molecule extends. Minor capsid proteins IIIa, VI, VIII, and IX are embedded in the capsid and contribute to capsid stability. Protein VI is located at the inner surface of the capsid, and biochemical data suggest that it may connect the capsid to the core containing the viral genome via protein V. The genome is ã 36-kb double-stranded linear DNA molecule with the terminal protein (TP) covalently bound to each 5 0end. Adenovirus genomes are highly condensed and organized into chromatin by several hundred copies of Abbreviations ADP, adenovirus death protein

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Section: General Considerations When Imaging Admentioning

confidence: 99%

Imaging the adenovirus infection cycle

Pied

Wodrich

2019

FEBS Letters

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“…hBMSCs were cultured at 378C under 5% by volume CO 2 in a-minimum essential media (Invitrogen) containing 16.5% by volume fetal bovine serum (FBS, Atlanta Biologicals), 4 mmol/L L-glutamine (Invitrogen), and penicillin/streptomycin (100 U/mL penicillin and 100 lg/mL streptomycin, Cellgro). 13 25,26 MitoTracker Red CMXRos accumulates in mitochondria of live cells through a chargebased interaction and remains after cell fixation (Table I). 26 Plates were incubated for 1.5 h in the cell culture incubator, washed with phosphate buffered saline (PBS), and replenished with fresh serum-free medium.…”

Section: Cell Culturementioning

confidence: 99%

“…The samples were stained with Alexa Fluor 546-phalloidin (F-actin stain, 20 nmol/ L in PBS, Invitrogen) 28 and DAPI (4' ,6-diamindo-2-phenylindole, dihydrochloride, 300 nmol/L in PBS, Invitrogen). 26 Finally, samples were washed in the PBS, washed in water, and air dried.…”

Section: Cell Culturementioning

confidence: 99%

Nanofiber scaffolds influence organelle structure and function in bone marrow stromal cells

Tutak

Giri

Bajcsy

et al. 2016

J. Biomed. Mater. Res.

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Tutak, Wojtek; Jyotsnendu, Giri; Bajcsy, Peter; and Simon, Carl G. Jr., "Nanofiber scaffolds influence organelle structure and function in bone marrow stromal cells" (2016 Abstract: Recent work demonstrates that osteoprogenitor cell culture on nanofiber scaffolds can promote differentiation. This response may be driven by changes in cell morphology caused by the three-dimensional (3D) structure of nanofibers. We hypothesized that nanofiber effects on cell behavior may be mediated by changes in organelle structure and function. To test this hypothesis, human bone marrow stromal cells (hBMSCs) were cultured on poly(e-caprolactone) (PCL) nanofibers scaffolds and on PCL flat spuncoat films. After 1 dayculture, hBMSCs were stained for actin, nucleus, mitochondria, and peroxisomes, and then imaged using 3D confocal microscopy. Imaging revealed that the hBMSC cell body (actin) and peroxisomal volume were reduced during culture on nanofibers. In addition, the nucleus and peroxisomes occupied a larger fraction of cell volume during culture on nanofibers than on films, suggesting enhancement of the nuclear and peroxisomal functional capacity. Organelles adopted morphologies with greater 3D-character on nanofibers, where the Z-Depth (a measure of cell thickness) was increased. Comparisons of organelle positions indicated that the nucleus, mitochondria, and peroxisomes were closer to the cell center (actin) for nanofibers, suggesting that nanofiber culture induced active organelle positioning. The smaller cell volume and more centralized organelle positioning would reduce the energy cost of inter-organelle vesicular transport during culture on nanofibers. Finally, hBMSC bioassay measurements (DNA, peroxidase, bioreductive potential, lactate, and adenosine triphosphate (ATP)) indicated that peroxidase activity may be enhanced during nanofiber culture. These results demonstrate that culture of hBMSCs on nanofibers caused changes in organelle structure and positioning, which may affect organelle functional capacity and transport.

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“…In a modular approach, we used orthogonal DON functionalization and assembled multiprotein constructs bearing FLIP-HOB as a functional domain along with STV to tether Phalloidin (PL) or NLS as targeting domains to the supramolecular constructs. PL is a bicyclic heptapeptide toxin, which strongly binds to filamentous actin (f-actin) and its derivatives containing fluorescent dyes or biotin are routinely used for imaging f-actin in eukaryotic cells 39 . To study the targeting of protein-DON complexes to distinctive locations inside the cytosolic compartment, DON-4 containing six Cy5 labels as well as six CH and four biotin groups (Figure 1e…”

Section: Resultsmentioning

confidence: 99%

Cytosolic delivery of large supramolecular protein complexes arranged on DNA nanopegboards

Schilling

et al. 2017

Preprint

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A generic methodology for cytosolic delivery of large supramolecular multiprotein complexes into living cells is described that takes advantage of the highly-controllable bottom-up fabrication of protein-decorated DNA nanostructures and the microfluidic "cell squeezing" technique. Therein, cells are deformed upon passage through a narrow constriction leading to formation of transient holes in the cell membrane that enable the diffusion of the protein-DNA nanostructures from the surrounding buffer into the cytosol. A diverse set of multiprotein complexes was assembled on DNA origami nanostructures using streptavidin and the sensitive glucose sensor protein FLIP as model systems. We demonstrate that our approach allows for the direct cytosolic delivery of these multifunctional protein complexes into the cytosol of HeLa cells. We also demonstrate that targeting groups can be incorporated into the protein-DNA nanoassemblies to enable their intracellular targeting to cytosolic compartments, such as the cytoskeleton or nucleus. We believe that this methodology will open up novel strategies for research in fundamental cell biology, such as the reverseengineering of the supramolecular machinery involved in gene regulation, cell signalling, or cell division. Furthermore, direct applications in immunotherapy can be foreseen.

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A Review of Reagents for Fluorescence Microscopy of Cellular Compartments and Structures, Part II: Reagents for Non‐Vesicular Organelles

Cited by 19 publications

References 81 publications

Imaging the adenovirus infection cycle

Imaging the adenovirus infection cycle

Nanofiber scaffolds influence organelle structure and function in bone marrow stromal cells

Cytosolic delivery of large supramolecular protein complexes arranged on DNA nanopegboards

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