scholarly journals Cellular and Subcellular Localisation of Kv4-Associated KChIP Proteins in the Rat Cerebellum

2020 ◽  
Vol 21 (17) ◽  
pp. 6403
Author(s):  
Rocío Alfaro-Ruíz ◽  
Carolina Aguado ◽  
Alejandro Martín-Belmonte ◽  
Ana Esther Moreno-Martínez ◽  
Rafael Luján
Keyword(s):  
Plasma Membrane ◽  
Purkinje Cells ◽  
Granule Cells ◽  
Distribution Patterns ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
K Channel ◽  
Subcellular Localisation ◽  
Electron Microscopic ◽  
Parallel Fibres

The K+ channel interacting proteins (KChIPs) are a family of cytosolic proteins that interact with Kv4 channels, leading to higher current density, modulation of channel inactivation and faster recovery from inactivation. Using immunohistochemical techniques at the light and electron microscopic level combined with quantitative analysis, we investigated the cellular and subcellular localisation of KChIP3 and KChIP4 to compare their distribution patterns with those for Kv4.2 and Kv4.3 in the cerebellar cortex. Immunohistochemistry at the light microscopic level demonstrated that KChIP3, KChIP4, Kv4.2 and Kv4.3 proteins were widely expressed in the cerebellum, with mostly overlapping patterns. Immunoelectron microscopic techniques showed that KChIP3, KChIP4, Kv4.2 and Kv4.3 shared virtually the same somato-dendritic domains of Purkinje cells and granule cells. Application of quantitative approaches showed that KChIP3 and KChIP4 were mainly membrane-associated, but also present at cytoplasmic sites close to the plasma membrane, in dendritic spines and shafts of Purkinje cells (PCs) and dendrites of granule cells (GCs). Similarly, immunoparticles for Kv4.2 and Kv4.3 were observed along the plasma membrane and at intracellular sites in the same neuron populations. In addition to the preferential postsynaptic distribution, KChIPs and Kv4 were also distributed presynaptically in parallel fibres and mossy fibres. Immunoparticles for KChIP3, KChIP4 and Kv4.3 were detected in parallel fibres, and KChIP3, KChIP4, Kv4.2 and Kv4.3 were found in parallel fibres, indicating that composition of KChIP and Kv4 seems to be input-dependent. Together, our findings unravelled previously uncharacterised KChIP and Kv4 subcellular localisation patterns in neurons, revealed that KChIP have additional Kv4-unrelated functions in the cerebellum and support the formation of macromolecular complexes between KChIP3 and KChIP4 with heterotetrameric Kv4.2/Kv4.3 channels.

scholarly journals Ultrastructural localization of guanylate cyclase in bone cells.

1991 ◽  
Vol 39 (4) ◽  
pp. 529-535 ◽  
Author(s):  
O Fukushima ◽  
C V Gay
Keyword(s):  
Plasma Membrane ◽  
Guanylate Cyclase ◽  
Bone Cells ◽  
Ultrastructural Localization ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Lead Citrate ◽  
Free Medium ◽  
Membrane Guanylate Cyclase ◽  
Electron Microscopic

Guanylyl imidodiphosphate (GMP-PNP) hydrolyzing enzyme activity as a means of detecting plasma membrane guanylate cyclase was demonstrated in osteoblasts of chicken tibial metaphysis using a lead citrate histochemical method at the electron microscopic level. Activity was not discerned in osteoclasts or osteocytes. The reaction product development was completely abolished when the sections were incubated with substrate-free or MnCl2-free medium. Guanylate-(beta, gamma-methylene) diphosphate (GMP-PCP) was a less effective substrate than GMP-PNP, and Mn++ was a stronger stimulator than Mg++. No reaction product was observed on the plasma membrane of osteoblasts when beta-glycerophosphate or p-nitrophenylphosphate was used as substrate instead of GMP-PNP. The results implicate guanylate cyclase as a significant effector of osteoblast regulation at the site of the plasma membrane.

scholarly journals Expression, Cellular and Subcellular Localisation of Kv4.2 and Kv4.3 Channels in the Rodent Hippocampus

2019 ◽  
Vol 20 (2) ◽  
pp. 246 ◽  
Author(s):  
Rocío Alfaro-Ruíz ◽  
Carolina Aguado ◽  
Alejandro Martín-Belmonte ◽  
Ana Esther Moreno-Martínez ◽  
Rafael Luján
Keyword(s):  
Plasma Membrane ◽  
Hippocampal Neurons ◽  
Granule Cells ◽  
Regional Distribution ◽  
Distribution Patterns ◽  
Adult Brain ◽  
Subcellular Localisation ◽  
Similar Distribution ◽  
Principal Cells ◽  
Development Levels

The Kv4 family of voltage-gated K+ channels underlie the fast transient (A-type) outward K+ current. Although A-type currents are critical to determine somato-dendritic integration in central neurons, relatively little is known about the precise subcellular localisation of the underlying channels in hippocampal circuits. Using histoblot and immunoelectron microscopic techniques, we investigated the expression, regional distribution and subcellular localisation of Kv4.2 and Kv4.3 in the adult brain, as well as the ontogeny of their expression during postnatal development. Histoblot demonstrated that Kv4.2 and Kv4.3 proteins were widely expressed in the brain, with mostly non-overlapping patterns. During development, levels of Kv4.2 and Kv4.3 increased with age but showed marked region- and developmental stage-specific differences. Immunoelectron microscopy showed that labelling for Kv4.2 and Kv4.3 was differentially present in somato-dendritic domains of hippocampal principal cells and interneurons, including the synaptic specialisation. Quantitative analyses indicated that most immunoparticles for Kv4.2 and Kv4.3 were associated with the plasma membrane in dendritic spines and shafts, and that the two channels showed very similar distribution patterns in spines of principal cells and along the surface of granule cells. Our data shed new light on the subcellular localisation of Kv4 channels and provide evidence for their non-uniform distribution over the plasma membrane of hippocampal neurons.

scholarly journals Immunohistochemical characterization of a differentiation-specific carbohydrate epitope in the plasma membrane of the epithelial cells of rat small intestine.

1993 ◽  
Vol 41 (1) ◽  
pp. 71-79 ◽  
Author(s):  
H Schiechl
Keyword(s):  
Small Intestine ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Basolateral Membrane ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Lateral Part ◽  
Rat Small Intestine ◽  
Electron Microscopic

The monoclonal antibody (MAb) SI/EC1 was produced by immunization of Balb/c mice with an antigen prepared from the isolated basolateral membrane (BLM) of rat small intestine epithelial cells by trypsin cleavage. Immunohistochemical labeling at the light and electron microscopic level shows that the SI/EC1 epitope is localized in the plasma membrane (PM) of the small intestine epithelial cells and is expressed around Day 21 after birth (weaning time). There are, however, differences in the labeling between crypt and villous cells. In the crypt cells, the microvillous membrane (MVM) and the lateral part of the BLM are strongly labeled, whereas the basal part of the BLM is unlabeled. In the villous cells, both the MVM and the basal and lateral part of the BLM are labeled, but the labeling is not as intense as in the crypts. In immunoblotting experiments with the isolated BLM, three protein bands (125 KD, 110 KD, and 90 KD) were labeled specifically with the MAb. Enzymic cleaving of the BLM with exo- and endoglycosidases and subsequent immunoblotting, as well as other findings, suggest that the specific structure of the SI/EC1 epitope consists mainly of carbohydrates (CH) (oligosaccharides). This finding points out the possibility that this epitope may have something to do with the variable adhesion of the small intestine epithelial cells along the crypt-villus axis.

A simplified method of emulsion coating and development for quantitative electron microscopic radioautography

1978 ◽  
Vol 36 (2) ◽  
pp. 64-65
Author(s):  
K. Yoshida ◽  
F. Murata ◽  
S. Ohno ◽  
T. Nagata
Keyword(s):  
Mass Production ◽  
Identical Condition ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Simplified Method ◽  
Complicated Process ◽  
Critical Problems ◽  
Electron Microscopic Radioautography ◽  
Quantitative Radioautography

IntroductionSeveral methods of mounting emulsion for radioautography at the electron microscopic level have been reported. From the viewpoint of quantitative radioautography, however, there are many critical problems in the procedure to produce radioautographs. For example, it is necessary to apply and develop emulsions in several experimental groups under an identical condition. Moreover, it is necessary to treat a lot of grids at the same time in the dark room for statistical analysis. Since the complicated process and technical difficulties in these procedures are inadequate to conduct a quantitative analysis of many radioautographs at once, many factors may bring about unexpected results. In order to improve these complicated procedures, a simplified dropping method for mass production of radioautographs under an identical condition was previously reported. However, this procedure was not completely satisfactory from the viewpoint of emulsion homogeneity. This paper reports another improved procedure employing wire loops.

X-Ray Microanalysis of Nickel and Cobalt Intensified Peroxidase- Antiperoxidase For Verification of Reaction Sites

1985 ◽  
Vol 43 ◽  
pp. 468-469
Author(s):  
A. Angel ◽  
K. Miller ◽  
V. Seybold ◽  
R. Kriebel
Keyword(s):  
Reaction Mechanisms ◽  
Visual Discrimination ◽  
Osmium Tetroxide ◽  
Ultrastructural Level ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
X Ray ◽  
Insoluble Polymer ◽  
Cytochemical Reaction

Localization of specific substances at the ultrastructural level is dependent on the introduction of chemicals which will complex and impart an electron density at specific reaction sites. Peroxidase-antiperoxidase(PAP) methods have been successfully applied at the electron microscopic level. The PAP complex is localized by addition of its substrate, hydrogen peroxide and an electron donor, usually diaminobenzidine(DAB). On oxidation, DAB forms an insoluble polymer which is able to chelate with osmium tetroxide becoming electron dense. Since verification of reactivity is visual, discrimination of reaction product from osmiophillic structures may be difficult. Recently, x-ray microanalysis has been applied to examine cytochemical reaction precipitates, their distribution in tissues, and to study cytochemical reaction mechanisms. For example, immunoreactive sites labelled with gold have been ascertained by means of x-ray microanalysis.

scholarly journals Murine endodermal cytokeratins Endo A and Endo B are localized in the same intermediate filament.

1986 ◽  
Vol 34 (6) ◽  
pp. 785-793 ◽  
Author(s):  
W E Howe ◽  
F G Klier ◽  
R G Oshima
Keyword(s):  
Immunoelectron Microscopy ◽  
Intermediate Filament ◽  
Microscopic Level ◽  
Cytoskeletal Proteins ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Double Label ◽  
Secondary Antibodies ◽  
Endodermal Cells ◽  
20 Nm

The intracellular distribution of extra-embryonic endodermal, cytoskeletal proteins A (Endo A) and B (Endo B) was investigated by double-label immunofluorescent microscopy and double-label immunoelectron microscopy. In parietal endodermal cells, the immunofluorescent distribution of Endo B was always coincident with that of Endo A and could be distinguished from vimentin, particularly at the periphery of the cell. At the electron microscopic level, antibodies against both Endo A and Endo B recognized both bundles and individual intermediate filaments. Double-label immunoelectron microscopy was achieved by use of two sizes of colloidal gold particles (5 nm and 20 nm) that were stabilized with secondary antibodies. These results show that Endo A and B are found in the same intermediate filament and probably co-polymerize to form such structures.

scholarly journals FIXATION OF NEURAL TISSUES FOR ELECTRON MICROSCOPY BY PERFUSION WITH SOLUTIONS OF OSMIUM TETROXIDE

1962 ◽  
Vol 12 (2) ◽  
pp. 385-410 ◽  
Author(s):  
Sanford L. Palay ◽  
S. M. McGee-Russell ◽  
Spencer Gordon ◽  
Mary A. Grillo
Keyword(s):  
Electron Microscopy ◽  
Nervous System ◽  
Osmium Tetroxide ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Vascular Perfusion ◽  
Neural Tissues ◽  
Structural Relations ◽  
Cellular Components

This paper describes in detail a method for obtaining nearly uniform fixation of the nervous system by vascular perfusion with solutions of osmium tetroxide. Criteria are given for evaluating the degree of success achieved in the preservation of all the cellular components of the nervous system. The method permits analysis of the structural relations between cells at the electron microscopic level to an extent that has not been possible heretofore.

scholarly journals A New Method to Enhance Contrast of Ultrathin Cryosections for Immunoelectron Microscopy

2003 ◽  
Vol 51 (1) ◽  
pp. 31-39 ◽  
Author(s):  
Toshihiro Takizawa ◽  
Clark L. Anderson ◽  
John M. Robinson
Keyword(s):  
Immunoelectron Microscopy ◽  
Staining Method ◽  
Microscopic Level ◽  
New Method ◽  
Electron Microscopic Level ◽  
Positive Staining ◽  
Immunocytochemical Localization ◽  
Electron Microscopic ◽  
Morphological Detail ◽  
Ultrathin Cryosections

Adequate contrast of ultrathin cryosections is crucial for evaluating morphological detail to assess immunocytochemical localization at the electron microscopic level. We have developed a positive staining method for achieving contrast in ultrathin cryosections, from tissue fixed only in paraformaldehyde, that provides excellent contrast at the electron microscopic level.

scholarly journals GRAMP 100: a membrane protein concentrated on secretory granule membranes and the apical cell surface in exocrine acinar cells.

1992 ◽  
Vol 40 (12) ◽  
pp. 1827-1835 ◽  
Author(s):  
S M Laurie ◽  
M B Mixon ◽  
J D Castle
Keyword(s):  
Plasma Membrane ◽  
Apical Cell ◽  
Reduced Form ◽  
Electron Microscopic Level ◽  
Apical Plasma Membrane ◽  
Cell Fractionation ◽  
Immunocytochemical Localization ◽  
Electron Microscopic ◽  
Common Component ◽  
Rat Parotid

Using a monoclonal antibody (SG10A6) raised against secretion granule membranes of the rat parotid gland, we have identified an antigen that is a common component of both exocrine pancreatic and parotid granule membranes. SG10A6 (an IgM) immunoprecipitates antigen that migrates as a single band (M(r) approximately 80 KD unreduced; M(r) approximately 100 KD reduced) and immunoblots at least two polypeptides that are similar to the reduced and nonreduced immunoprecipitated antigen. This granule-associated membrane polypeptide (GRAMP 100; named for the apparent M(r) in reduced form) is also a prominent component of plasma membrane fractions. Immunocytochemical localization at the electron microscopic level demonstrates the presence of GRAMP 100 on granule membranes, especially condensing vacuoles and exocytotic figures, and the apical plasma membrane. Lower levels of antigen are detected on basolateral plasma membrane and on peri-Golgi membranes that may be part of the endosomal system. Both the cell fractionation and immunocytochemical localization indicate that GRAMP 100 differs in distribution from GRAMP 92 and 30K SCAMPs, two other components of exocrine granule membranes identified with monoclonal antibodies. To date, no polypeptides have been identified with this approach that are exclusive components of exocrine granule membranes.

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