SDS-digested freeze-fracture replica labeling electron microscopy to study the two-dimensional distribution of integral membrane proteins and phospholipids in biomembranes: practical procedure, interpretation and application

Fujimoto, Kazushi

doi:10.1007/s004180050092

Cited by 120 publications

(113 citation statements)

References 25 publications

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“…After freeze fracture, samples were bonded in Lexan plastic to gold index grids (22), thawed, photomapped, and cleaned in chromic͞sulfuric acid. For freeze-fracture immunolabeling, samples were cleaned with 2.5% SDS before immunogold labeling (22,26,27).…”

Section: Methodsmentioning

confidence: 99%

Aquaporin-4 square array assembly: Opposing actions of M1 and M23 isoforms

Furman

Gorelick-Feldman

Davidson

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

285

287

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Osmotic homeostasis in the brain involves movement of water through aquaporin-4 (AQP4) membrane channels. Perivascular astrocyte end-feet contain distinctive orthogonal lattices (square arrays) assembled from 4-to 6-nm intramembrane particles (IMPs) corresponding to individual AQP4 tetramers. Two isoforms of AQP4 result from translation initiation at methionine residues M1 and M23, but no functional differences are known. In this study, Chinese hamster ovary cells were transfected with M1, M23, or M1؉M23 isoforms, and AQP4 expression was confirmed by immunoblotting, immunocytochemistry, and immunogold labeling. Square array organization was examined by freeze-fracture electron microscopy. In astrocyte end-feet, >90% of 4-to 6-nm IMPs were found in square arrays, with 65% in arrays of 13-30 IMPs. In cells transfected with M23, 95% of 4-to 6-nm IMPs were in large assemblies (rafts), 85% of which contained >100 IMPs. However, in M1 cells, >95% of 4-to 6-nm IMPs were present as singlets, with <5% in incipient arrays of 2-12 IMPs. In A quaporins are specialized water transport channels in plasma membranes of water-permeable tissues (1). Aquaporins 1 and 4 (AQP1 and AQP4) are most important to fluid movements in mammalian brain. AQP4 exists as two isoforms, differing at their N termini, because of translation initiation at the first methionine (M1, 323 aa) or the second methionine (M23, 301 aa) (2, 3). Both isoforms are present in brain, but M23 is at least 3-fold more abundant (4, 5). Endogenous AQP4 is a tetramer usually containing M1 and M23 subunits. The water permeabilities of M1 and M23 are similar, and functional differences are not known (3, 4).Fluid movements are precisely orchestrated within the rigid cranium to prevent physical damage from swelling or shrinkage. Interfaces between brain parenchyma and cerebrospinal fluid occur around the ventricles, surrounding blood vessels, and at the brain surface. AQP1 is expressed in rat choroid plexus, the site of cerebrospinal fluid secretion (6), whereas AQP4 is enriched in rat astrocyte end-feet surrounding brain capillaries (7,8). Astrocyte processes forming the glia limitans at brain surfaces, ependymal cells lining brain ventricles, and Müller cells facing the vitreous body and retinal blood vessels all have abundant AQP4 (9). AQP4 in perivascular membranes of astrocyte end-feet has been implicated in neurological disorders, including acute hyponatremic edema, postischemic injury, and epileptic seizures (10-13).Perivascular membranes of astrocyte end-feet contain numerous strikingly regular arrays of intramembrane particles (IMPs) in freeze-fracture electron micrographs. These IMP arrays have been referred to as square arrays, assemblies, or orthogonally arranged particles (OAPs) (14). In early freeze-fracture images of astrocyte end-feet (15), square arrays were resolved as 6-nm IMP protrusions in P-face images (protoplasmic leaflets) or as smaller pits in E-faces (extraplasmic leaflets). The sizes and shapes of square arrays vary, but the IMPs and pits have un...

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Section: Methodsmentioning

confidence: 99%

Aquaporin-4 square array assembly: Opposing actions of M1 and M23 isoforms

Furman

Gorelick-Feldman

Davidson

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

285

287

View full text Add to dashboard Cite

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“…Freeze-fracture Immunogold Labeling-To survey for TIP47 and adipophilin in lipid bodies in the electron microscope, macrophages were lipid-laden for 2 days and freeze-fractured and replicated using metal evaporation and immunogold labeling as outlined in detail elsewhere (21)(22)(23). Briefly, chemically unfixed macrophages were scraped from the culture flasks, incubated in 30% glycerol (for a maximum of 2 min), collected by mild centrifugation, frozen in Freon at Ϫ200°C, and fractured in a vacuum coating unit (BA310, Balzers, Liechtenstein).…”

Section: Methodsmentioning

confidence: 99%

Spatial Integration of TIP47 and Adipophilin in Macrophage Lipid Bodies

Robenek

Lorkowski

Schnoor

et al. 2005

Journal of Biological Chemistry

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We studied the distribution of the PAT family proteins TIP47 and adipophilin in lipid bodies of THP-1 cell-derived macrophages using freeze-fracture immunolabeling and other techniques. Lipid bodies in macrophages comprise lipid droplets and extensive, previously scantily characterized sheet-like organelles, which we descriptively call "lipid sails." TIP47 and adipophilin are components of many, but not all, the lipid droplets. Both proteins are not confined to the surface of lipid droplets, as supposed, but are also inside lipid droplet cores. They are not codistributed stoichiometrically in lipid droplets. How TIP47 and adipophilin, which are polar proteins, enter the lipid droplets and are packaged among the hydrophobic neutral lipids of the core is unclear. However, in the lipid layers of the core, these proteins are directed sometimes inward and sometimes outward. Because TIP47 and adipophilin also localize to lipid sails, lipid sails are intimately involved in intracellular lipid metabolism.An entirely new view of the functions and composition of lipid bodies is currently emerging from studies on proteins associated with these organelles. Lipid bodies are clearly more than mere storage depots for superfluous neutral lipids in times of metabolic stress (1). Aside from their obvious involvement in lipid homeostasis, they appear also to partake exquisitely in cell signaling (2, 3), intracellular vesicle trafficking (3, 4), and disease (5, 6). Lipid bodies are metabolically active organelles in cells of virtually all animals, many plants, and even some prokaryotes (7,8).Several proteins with similar amino-terminal amino acid sequences and hence common structural motifs, the PAT (perilipin, adipophilin, tail-interacting protein of 47 kDa) family proteins, interact with lipid bodies in as yet unknown ways. PAT family proteins are believed to localize exclusively to the surface of lipid bodies and to be evolutionarily conserved (9 -12). Similarities in the structure of PAT family proteins and their targeting to and localization in lipid bodies are suggestive of a common underlying function in lipid homeostasis and/or lipid body biogenesis.Among the members of the PAT family proteins, the tailinteracting protein of 47 kDa (TIP47) is still of uncertain status. TIP47 seems to be involved in delivering mannose-6-phosphate receptors from endosomes to the trans-Golgi network (13,14) and, at the same time, to be a component of the surface of lipid bodies (10, 15). These two cellular compartments, lipid bodies and the late endosome sorting compartment, are currently thought to be completely divorced.Several controversial reports on the putative association of TIP47 with lipid droplets, the most common form of lipid body in -tagged TIP47 in Chinese hamster ovary cells transfected with a vector containing the coding sequence for murine TIP47. GFP-TIP47 appeared to be at the lipid droplet surface, despite the fact that TIP47 was also strongly expressed throughout the cytoplasm of the cells. Nevertheless, the authors conclud...

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“…A particular advantage of FRIL is that labeling specificity is assessed directly, based on restriction of immunogold labels to a single type of molecular array (e.g., gap junctions, tight junctions, postsynaptic densities, and AQP4 "square arrays") in ultrastructurally-identified cells, and consistent absence of that label from any other specific ultrastructural feature (Fujimoto, 1995(Fujimoto, , 1997 Rash et al, 2004b). In typical samples with low background, the "signal-to-noise ratios" ranged from 1000:1 to 15,000:1 [for details, see (Meier et al, 2004;Rash et al, 2005)].…”

Section: Replica Cleaning In Sds and Immunogold Labelingmentioning

confidence: 99%

Connexin36 vs. connexin32, “miniature” neuronal gap junctions, and limited electrotonic coupling in rodent suprachiasmatic nucleus

et al. 2007

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Suprachiasmatic nucleus (SCN) neurons generate circadian rhythms, and these neurons normally exhibit loosely-synchronized action potentials. Although electrotonic coupling has long been proposed to mediate this neuronal synchrony, ultrastructural studies have failed to detect gap junctions between SCN neurons. Nevertheless, it has been proposed that neuronal gap junctions exist in the SCN; that they consist of connexin32 or, alternatively, connexin36; and that connexin36 knockout eliminates neuronal coupling between SCN neurons and disrupts circadian rhythms. We used confocal immunofluorescence microscopy and freeze-fracture replica immunogold labeling to examine the distributions of connexin30, connexin32, connexin36, and connexin43 in rat and mouse SCN and used whole-cell recordings to re-assess electrotonic and tracer coupling. Connexin32-immunofluorescent puncta were essentially absent in SCN but connexin36 was relatively abundant. Fifteen neuronal gap junctions were identified ultrastructurally, all of which contained connexin36 but not connexin32, whereas nearby oligodendrocyte gap junctions contained connexin32. In adult SCN, one neuronal gap junction was >600 connexons, whereas 75% were smaller than 50 connexons, which may be below the limit of detectability by fluorescence microscopy and thin-section electron microscopy. Whole-cell recordings in hypothalamic slices revealed tracer coupling with Neurobiotin in <5% of SCN neurons, and paired recordings (>40 pairs) did not reveal obvious electrotonic coupling or synchronized action potentials, consistent with few neurons possessing large gap junctions. However, most neurons had partial spikes or spikelets (often <1 mV), which remained after QX-314 had blocked sodium-mediated action potentials within the recorded neuron, consistent with spikelet transmission via small gap junctions. Thus, a few "miniature" gap junctions on most SCN neurons appear to mediate weak electrotonic coupling between limited numbers of neuron pairs, Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2008 February 15. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript thus accounting for frequent detection of partial spikes and hypothetically providing the basis for "loose" electrical or metabolic synchronization of electrical activity commonly observed in SCN neuronal populations during circadian rhythms. Keywordsimmunocytochemistry; electrical synapse; freeze-fracture replica immunogold labeling; spikelet; metabolic coupling; syn...

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SDS-digested freeze-fracture replica labeling electron microscopy to study the two-dimensional distribution of integral membrane proteins and phospholipids in biomembranes: practical procedure, interpretation and application

Cited by 120 publications

References 25 publications

Aquaporin-4 square array assembly: Opposing actions of M1 and M23 isoforms

Aquaporin-4 square array assembly: Opposing actions of M1 and M23 isoforms

Spatial Integration of TIP47 and Adipophilin in Macrophage Lipid Bodies

Connexin36 vs. connexin32, “miniature” neuronal gap junctions, and limited electrotonic coupling in rodent suprachiasmatic nucleus

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