Stacks of Flattened Smooth Endoplasmic Reticulum Highly Enriched in Inositol 1,4,5-Trisphosphate(InsP3)Receptor in Mouse Cerebellar Purkinje Cells.

Abstract

ABSTRACT. By immunogoldelectron microscopy we have shown that in mouse cerebellar Purkinje cells fixed by per fusion with formaldehyde-glutaraldehyde solution, the InsP3 receptor are numeously detected on the stacks of flattened cisterns (OTSUet al, (1990) CellStruct. Funct. , 15: 163-173). In the present experiment we investigated distribution, structure and properties of the stacks by conventional electronmicroscopy, lectin cytochemistry and immunoelectron microscopy. The size and number of stacks were variable depending on their intracellular localization; short stacks with 2-4 parallel cisterns predominate in the perikaryon, long stacks with 4-15 cisterns in the proximal dendrite, and long stacks with 3-4 cisterns in the distal dendrites. The flattened cisterns bind with concanavalin A but not with wheat-germ agglutinin and may contain KDELproteins loaded with Lys-Asp-Glu-Leu at their C-terminin in their lumens, indicating that the cisterns are derived from ER membranes. The electron dense materials sandwiched between the cisternal membranesare composed of small particles, short cylindrical in shape and -20 nm in diameter, and markedly labeled with anti InsP3R antibody. We suggest that they correspond to the tetramer of the InsP3R or their related molecules. It is not clear whether the stacks of flattened cisterns exist per se in the Purkinje cells or smooth ER existing in singlet in vivo in the Purkinje cells forms stacks during fixation. It is strongly suggested, however, that the smooth ER membranes covered by the InsP3R or their related molecules can easily interact and stack each other in the Purkinje cells.

Conservation of localization patterns of IP3 receptor type 1 in cerebellar Purkinje cells across vertebrate species

“…Mammalian Purkinje cells show a high level of IP 3 binding activity (Worley et al, 1989) and express a high density of IP 3 R protein associated with the ER (Mignery et al, 1989;Otsu et al, 1990;Nakanishi et al, 1991;Yamamoto et al, 1991;Furuichi et al, 1993;Suburo et al, 1993;Sharp et al, 1993;Miyata et al, 1999). Typically, Purkinje cells of the cerebellum show the highest label intensity for IP 3 R throughout the central nervous system and express the IP 3 R protein intracellularly in all compartments (axon, soma, dendrites) with the exception Fig.…”

“…Studies that used mammalian cerebella clearly showed the presence of all major components of IP 3 -mediated Ca release using a variety of different techniques (Nakanishi et al, 1991;Watras et al, 1991;Eilers et al, 1996;Finch and Augustine, 1998). Indeed, the IP 3 R is expressed at very high levels on the ER of mammalian cerebellar Purkinje neurons (Otsu et al, 1990;Yamamoto et al, 1991;Yamada et al, 1992). The present study had three aims: 1) to characterize further the distribution of IP 3 R type 1 (IP 3 R1) in mammalian cerebella by determining its localization in hamster and comparing it to that of two other rodent species, rat and mouse; 2) to localize the IP 3 R1 in cerebella of zebrafish Danio rerio, claw frog Xenopus laevis and skate Raja erinacea, vertebrate species that are important model animals in neurogenetics or neurophysiology; and 3) to compare the cerebellar localization pattern of IP 3 R1 immunoreactivity in different vertebrate phyla.…”

Subcellular Localization and Physiological Significance of Intracellular Mannan-binding Protein

“…Immunoelectron Microscopy-Cryo-ultramicrotomy and immunogold staining were followed as described previously (14) with a slight modification. Briefly, cells were fixed in 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4, for 30 min.…”

GFP-like Proteins Stably Accumulate in Lysosomes

“…For immunoelectron microscopy, the post-embedding immunogold method was used to label cultured cells embedded in LR white resin (London Resin Co.) as previously described (Yamamoto et al, 1991).…”