scholarly journals FerriTag: A Genetically-Encoded Inducible Tag for Correlative Light-Electron Microscopy

2016 ◽  
Author(s):  
Nicholas I. Clarke ◽  
Stephen J. Royle
Keyword(s):  
Electron Microscopy ◽  
Signal To Noise Ratio ◽  
Light And Electron Microscopy ◽  
High Signal ◽  
Subcellular Structure ◽  
Coated Pits ◽  
Interacting Protein ◽  
Protein Location ◽  
Huntingtin Interacting Protein ◽  
A Current

AbstractA current challenge is to develop tags to precisely visualize proteins in cells by light and electron microscopy. Here, we introduce FerriTag, a genetically-encoded chemically-inducible tag for correlative light-electron microscopy (CLEM). FerriTag is a fluorescent recombinant electron-dense ferritin particle that can be attached to a protein-of-interest using rapamycin-induced heterodimerization. We demonstrate the utility of FerriTag for CLEM by labeling proteins associated with various intracellular structures including mitochondria, plasma membrane, and clathrin-coated pits and vesicles. FerriTagging has a high signal-to-noise ratio and a labeling resolution of 10 ± 5 nm. We demonstrate how FerriTagging allows nanoscale mapping of protein location relative to a subcellular structure, and use it to detail the distribution of huntingtin-interacting protein 1 related (HIP1R) in clathrin-coated pits.

scholarly journals THE INDUCTION OF MELANIZATION IN GOLDFISH SCALES WITH ACTH, IN VITRO

1963 ◽  
Vol 18 (1) ◽  
pp. 181-194 ◽  
Author(s):  
Alden V. Loud ◽  
Yutaka Mishima
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Silver Nitrate ◽  
Light And Electron Microscopy ◽  
Subcellular Structure ◽  
Acth Stimulation ◽  
Cytoplasmic Bodies ◽  
Silver Nitrate Staining ◽  
Stimulation Of

The induction of melanization in xanthic goldfish scales with ACTH in vitro has been studied by light and electron microscopy utilizing ammoniated silver nitrate staining of premelanin and melanin. The melanized cells (melanophores and melanocytes) and the yellow pigmented cells (lipophores and the newly described lipocytes) were found to possess many similarities at the levels of cellular and subcellular structure. The latter cells contain characteristic cytoplasmic bodies which react positively to the premelanin stain. Changes accompanying ACTH stimulation of goldfish scales in tissue culture suggest that these bodies in the lipocytes and lipophores can become melanized. Electron micrographs illustrate the intermediate staining of newly formed melanin granules in an induced melanocyte and the appearance of a transitional melanolipophore. It is postulated that ACTH can promote the association of the enzyme tyrosinase with the preformed structure of unmelanized granules.

Clathrin-coated vesicle formation: a paradigm for coated-vesicle formation

2003 ◽  
Vol 31 (3) ◽  
pp. 736-739 ◽  
Author(s):  
E. Smythe
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Coated Pits ◽  
Permeabilized Cell ◽  
Cytosolic Proteins ◽  
Cell Assays

Clathrin-coated pits are the major ports of entry into the cell and are responsible for the internalization of a variety of biologically important macromolecules. These transport intermediates form as a result of the co-ordinated assembly of a number of cytosolic proteins on to the membrane which results in specific cargo recruitment. We have used a variety of approaches including permeabilized cell assays and light and electron microscopy to identify and characterize the proteins and enzymes involved in coated vesicle formation.

scholarly journals Clathrin light chain A drives selective myosin VI recruitment to clathrin-coated pits under membrane tension

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Matteo Biancospino ◽  
Gwen R. Buel ◽  
Carlos A. Niño ◽  
Elena Maspero ◽  
Rossella Scotto di Perrotolo ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Coated Vesicle ◽  
Apical Surface ◽  
Myosin Vi ◽  
Coated Pits ◽  
Interacting Protein ◽  
Vesicle Budding ◽  
Polarized Epithelia ◽  
Actin Motor ◽  
Huntingtin Interacting Protein

Abstract Clathrin light chains (CLCa and CLCb) are major constituents of clathrin-coated vesicles. Unique functions for these evolutionary conserved paralogs remain elusive, and their role in clathrin-mediated endocytosis in mammalian cells is debated. Here, we find and structurally characterize a direct and selective interaction between CLCa and the long isoform of the actin motor protein myosin VI, which is expressed exclusively in highly polarized tissues. Using genetically-reconstituted Caco-2 cysts as proxy for polarized epithelia, we provide evidence for coordinated action of myosin VI and CLCa at the apical surface where these proteins are essential for fission of clathrin-coated pits. We further find that myosin VI and Huntingtin-interacting protein 1-related protein (Hip1R) are mutually exclusive interactors with CLCa, and suggest a model for the sequential function of myosin VI and Hip1R in actin-mediated clathrin-coated vesicle budding.

scholarly journals Cryo-Confocal Imaging for CLEM Mapping in Brain Tissues

2021 ◽  
Vol 29 (5) ◽  
pp. 34-39
Author(s):  
Connon I. Thomas ◽  
Nicolai T. Urban ◽  
Ye Sun ◽  
Lesley A. Colgan ◽  
Xun Tu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Laser Scanning ◽  
Signal To Noise Ratio ◽  
Light And Electron Microscopy ◽  
Brain Slices ◽  
Confocal Imaging ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Array Tomography ◽  
Biological Phenomena

Abstract:In correlative light and electron microscopy (CLEM) workflows, identifying the same sub-cellular features in tissue by both light (LM) and electron microscopy (EM) remains a challenge. Furthermore, use of cryo-fixation for EM is desirable to capture rapid biological phenomena. Here, we describe a workflow that incorporates cryo-confocal laser scanning microscopy into the CLEM process, mapping cells in brain slices to re-image them with serial section scanning electron microscopy (ssSEM) array tomography. The addition of Airyscan detection increased the signal-to-noise ratio (SNR), allowing individual spines in thick frozen tissue to be visualized at a sufficient spatial resolution, providing a new tool for a CLEM approach to capture biological dynamics.

scholarly journals Radial Carboxyterminal Peptides of Tubulin Directly Imaged in Microtubules by Electron Microscopy Tomography Exploiting Emplitude Contrast

2021 ◽  
Author(s):  
Andrea Fera ◽  
Thomas Reese ◽  
Joshua Zimmerberg ◽  
Dan Sackett
Keyword(s):  
Electron Microscopy ◽  
Outer Surface ◽  
Electron Beam Irradiation ◽  
Signal To Noise Ratio ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
High Signal ◽  
High Electron ◽  
Microscopy Image ◽  
Multiple Samples

Abstract Tubulin carboxyterminal tails (CTT) are peptides of 10-20 amino acids, unstructured and acidic, that vary in sequence between tubulin isotypes and are exposed on the outer surface of microtubules (MTs). These peptides have, so far, eluded direct visualization. In this report, electron microscopy tomography was applied to isolated MTs stained with Uranyl and tungstate salts demonstrated to resist sustained electron beam irradiation. Such resistance of high electron doses allows each electron microscopy image to be recorded with a high signal-to-noise ratio. Corresponding tomograms reconstructed from tilt series at high magnification show exceptional resolution of details, revealing features of average dimension ~ 1 nm without the need of averaging multiple samples. The known three-dimensional structure of the MT wall is apparent. But now images also reveal small stalks on the outer surface of MTs. Inspection of virtual sections demonstrates that the stalks are up to ~2.5 nm long and ~1 nm wide (at half length), protruding every 4 ± 0.8 (22) nm along the microtubule. This spacing corresponds to one stalk per tubulin monomer. The grafting point on each monomer is not random but is positioned at one end of each monomer, identifying that end as toward the (-) end of the MT. The stalks are not observed following CTT removal with subtilisin. We conclude that these stalks are the CTT peptides of tubulin.

scholarly journals An Actin-Binding Protein of the Sla2/Huntingtin Interacting Protein 1 Family Is a Novel Component of Clathrin-Coated Pits and Vesicles

1999 ◽  
Vol 147 (7) ◽  
pp. 1503-1518 ◽  
Author(s):  
Åsa E.Y. Engqvist-Goldstein ◽  
Michael M. Kessels ◽  
Vikramjit S. Chopra ◽  
Michael R. Hayden ◽  
David G. Drubin
Keyword(s):  
Actin Cytoskeleton ◽  
Binding Protein ◽  
Actin Binding ◽  
Cell Cortex ◽  
Sequence Alignments ◽  
Coated Pits ◽  
Interacting Protein ◽  
Actin Binding Protein ◽  
Huntingtin Interacting Protein 1 ◽  
Huntingtin Interacting Protein

The actin cytoskeleton has been implicated in endocytosis, yet few molecules that link these systems have been identified. Here, we have cloned and characterized mHip1R, a protein that is closely related to huntingtin interacting protein 1 (Hip1). These two proteins are mammalian homologues of Sla2p, an actin binding protein important for actin organization and endocytosis in yeast. Sequence alignments and secondary structure predictions verified that mHip1R belongs to the Sla2 protein family. Thus, mHip1R contains an NH2-terminal domain homologous to that implicated in Sla2p's endocytic function, three predicted coiled–coils, a leucine zipper, and a talin-like actin-binding domain at the COOH terminus. The talin-like domain of mHip1R binds to F-actin in vitro and colocalizes with F-actin in vivo, indicating that this activity has been conserved from yeast to mammals. mHip1R shows a punctate immunolocalization and is enriched at the cell cortex and in the perinuclear region. We concluded that the cortical localization represents endocytic compartments, because mHip1R colocalizes with clathrin, AP-2, and endocytosed transferrin, and because mHip1R fractionates biochemically with clathrin-coated vesicles. Time-lapse video microscopy of mHip1R–green fluorescence protein (GFP) revealed a blinking behavior similar to that reported for GFP-clathrin, and an actin-dependent inward movement of punctate structures from the cell periphery. These data show that mHip1R is a component of clathrin-coated pits and vesicles and suggest that it might link the endocytic machinery to the actin cytoskeleton.

The Morphology of Vacuolated Cells in the Liver Following Administration of Vitamin A.

1973 ◽  
Vol 31 ◽  
pp. 408-409
Author(s):  
Odell T. Minick ◽  
Hidejiro Yokoo ◽  
Fawzia Batti
Keyword(s):  
Electron Microscopy ◽  
Body Weight ◽  
Vitamin A ◽  
Light And Electron Microscopy ◽  
Liver Cells ◽  
Prominent Feature ◽  
Vascular Space ◽  
Vacuolated Cell ◽  
Vacuolated Cells ◽  
Vacuolated Cytoplasm

Vacuolated cells in the liver of young rats were studied by light and electron microscopy following the administration of vitamin A (200 units per gram of body weight). Their characteristics were compared with similar cells found in untreated animals.In rats given vitamin A, cells with vacuolated cytoplasm were a prominent feature. These cells were found mostly in a perisinusoidal location, although some appeared to be in between liver cells (Fig. 1). Electron microscopy confirmed their location in Disse's space adjacent to the sinusoid and in recesses between liver cells. Some appeared to be bordering the lumen of the sinusoid, but careful observation usually revealed a tenuous endothelial process separating the vacuolated cell from the vascular space. In appropriate sections, fenestrations in the thin endothelial processes were noted (Fig. 2, arrow).

Control of Autogenous Vein Grafts Prior to Reimplantation, By Electron Microscopy

1972 ◽  
Vol 30 ◽  
pp. 106-107
Author(s):  
John H. L. Watson ◽  
John L. Swedo ◽  
M. Vrandecic
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Physiological Saline ◽  
Experimental Conditions ◽  
Vein Grafts ◽  
Arterial Blood ◽  
Saphenous Veins ◽  
Autogenous Vein ◽  
Control Of Parameters ◽  
Post Implantation

The ambient temperature and the nature of the storage fluids may well have significant effects upon the post-implantation behavior of venus autografts. A first step in the investigation of such effects is reported here. Experimental conditions have been set which approximate actual operating room procedures. Saphenous veins from dogs have been used as models in the experiments. After removal from the dogs the veins were kept for two hours under four different experimental conditions, viz at either 4°C or 23°C in either physiological saline or whole canine arterial blood. At the end of the two hours they were prepared for light and electron microscopy. Since no obvious changes or damage could be seen in the veins by light microscopy, even with the advantage of tissue specific stains, it was essential that the control of parameters for successful grafts be set by electron microscopy.

Ultrastructure of two neoplasms in culture compared with original biopsies

1984 ◽  
Vol 42 ◽  
pp. 50-51
Author(s):  
Joseph M. Harb ◽  
James T. Casper ◽  
Vlcki Piaskowski
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Biopsy Specimen ◽  
Cultured Cells ◽  
Light And Electron Microscopy ◽  
Human Tumor ◽  
Soft Agar ◽  
Human Tumor Cells ◽  
Morphological Distinction ◽  
Tumor Types

The application of tissue culture and the newer methodologies of direct cloning and colony formation of human tumor cells in soft agar hold promise as valuable modalities for a variety of diagnostic studies, which include morphological distinction between tumor types by electron microscopy (EM). We present here two cases in which cells in culture expressed distinct morphological features not apparent in the original biopsy specimen. Evaluation of the original biopsies by light and electron microscopy indicated both neoplasms to be undifferentiated sarcomas. Colonies of cells propagated in soft agar displayed features of rhabdomyoblasts in one case, and cultured cells of the second biopsy expressed features of Ewing's sarcoma.

The Effect of Vitamin A on the Intermittent Nitrogen Dioxide Gas Exposure in Hamsters

1976 ◽  
Vol 34 ◽  
pp. 176-177
Author(s):  
J.C.S. Kim ◽  
M.G. Jourden ◽  
E.S. Carlisle
Keyword(s):  
Electron Microscopy ◽  
Vitamin A ◽  
Nitrogen Dioxide ◽  
Chronic Exposure ◽  
Light And Electron Microscopy ◽  
Specific Effect ◽  
Deficient Diet ◽  
Intermittent Exposure ◽  
Hypovitaminosis A ◽  
Gas Exposure

Chronic exposure to nitrogen dioxide in rodents has shown that injury reaches a maximum after 24 hours, and a reparative adaptive phase follows (1). Damage occurring in the terminal bronchioles and proximal portions of the alveolar ducts in rats has been extensively studied by both light and electron microscopy (1).The present study was undertaken to compare the response of lung tissue to intermittent exposure to 10 ppm of nitrogen dioxide gas for 4 hours per week, while the hamsters were on a vitamin A deficient diet. Ultrastructural observations made from lung tissues obtained from non-gas exposed, hypovitaminosis A animals and gas exposed animals fed a regular commercially prepared diet have been compared to elucidate the specific effect of vitamin A on nitrogen dioxide gas exposure. The interaction occurring between vitamin A and nitrogen dioxide gas has not previously been investigated.

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