Chapter 16 Electron Microscopy Immunocytochemistry Following Cryofixation and Freeze Substitution

Kiss, John Z.; McDonald, Kent L.

doi:10.1016/s0091-679x(08)60256-3

Cited by 32 publications

(23 citation statements)

References 51 publications

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“…However, even with HFP/FS the kinetochore is seen to consist of three defined domains that are intimately related: the corona that radiates from the surface of a mat of material, which is intimately associated with the chromosome surface. Thus in most cases those components demonstrated by immuno-EM studies to be located in the heterochromatin/inner plate (e.g., CENP A, B, C and G) or outer plate/corona region (CENP-E, cytoplasmic dynein) would likely have a comparable location in HFP/FS-prepared material, which may itself ultimately provide a superior method for detailing the location of many kinetochore antigens at the highest possible resolution (see Kiss and McDonald 1993). By contrast the location of those molecules reported to be in the electron-lucent middle layer, including the 3F3/2 epitopes (Campbell and Gorbsky 1995), needs to be reevaluated.…”

Section: Discussionmentioning

confidence: 99%

A new look at kinetochore structure in vertebrate somatic cells using high-pressure freezing and freeze substitution

et al. 1998

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Three decades of structural analysis have produced the view that the kinetochore in vertebrate cells is a disk-shaped structure composed of three distinct structural domains. The most prominent of these consists of a conspicuous electron opaque outer plate that is separated by a light-staining electrontranslucent middle plate from an inner plate associated with the surface of the pericentric heterochromatin. Spindle microtubules terminate in the outer plate and, in their absence, a conspicuous corona of fine filaments radiates from the cytoplasmic surface of this plate. Here we report for the first time the ultrastructure of kinetochores in untreated and Colcemid-treated vertebrate somatic (PtK 1 ) cells prepared for optimal structural preservation using high-pressure freezing and freeze substitution. In serial thin sections, and electron tomographic reconstructions, the kinetochore appears as a 50-75 nm thick mat of light-staining fibrous material that is directly connected with the more electron-opaque surface of the centromeric heterochromatin. This mat corresponds to the outer plate in conventional preparations, and is surrounded on its cytoplasmic surface by a conspicuous 100-150 nm wide zone that excludes ribosomes and other cytoplasmic components. High magnification views of this zone reveal that it contains a loose network of light-staining, thin (<9 nm diameter) fibers that are analogous to the corona fibers in conventional preparations. Unlike the chromosome arms, which appear uniformly electron opaque, the chromatin in the primary constriction appears mottled. Since the middle plate is not visible in these kinetochore preparations this feature is likely an artifact produced by extraction and coagulation during conventional fixation and/or dehydration procedures.

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Section: Discussionmentioning

confidence: 99%

A new look at kinetochore structure in vertebrate somatic cells using high-pressure freezing and freeze substitution

et al. 1998

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“…White resin (London Resin, Hampshire, England), and polymerized under vacuum at 45°C. Sections were cut and picked up as described above and then a 1 2 Ii b stained with BWD-1 antibodies to DNA (15) (kindly provided by Brian Kotzin, National Jewish Center for Immunology and Respiratory Medicine, Denver) or with MAb414 antibodies to the nuclear pore complex, followed by secondary antibodies conjugated to 10-nm gold particles (Biocell Laboratories) (16). Sections were poststained with uranyl acetate and lead citrate and imaged in a Philips CM10 electron microscope operating at 80 kV.…”

Section: Methodsmentioning

confidence: 99%

A mutation in the RCC1-related protein pim1 results in nuclear envelope fragmentation in fission yeast.

Demeter

Morphew

Sazer

1995

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

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Members of the RCC1 protein family are chromatin-associated guanine nucleotide exchange factors that have been implicated in diverse cellular processes in various organisms, yet no consensus has been reached as to their primary biological role. The fission yeast Schizosaccharomyces pombe, a single-celled eukaryote, provides an in vivo system in which to study the RCCl/Ran switch by using a temperature-sensitive mutant in the RCCl-related protein piml. Mitotic entry in the piml-dltS mutant is normal, but mitotic exit leads to the accumulation of cells arrested with a medial septum and condensed chromosomes. Although the yeast nuclear envelope normally remains intact throughout the cell cycle, we found a striking fragmentation of the nuclear envelope in the piml-dlts mutant following mitosis. This resulted in chromatin that was no longer compartmentalized and an accumulation of pore-containing membranes in the cytoplasm. The development of this terminal phenotype was dependent on the passage of cells through mitosis and was coincident with the loss of viability. We propose that piml is required for the reestablishment of nuclear structure following mitosis in fission yeast.RCC1 and Ran are human proteins forming the core of an evolutionarily conserved GTPase molecular switch: RCC1 is a chromatin-associated nucleotide exchange factor that interacts with the small GTPase Ran. This switch has been studied in a number of different organisms and has been implicated in a variety of cellular processes including chromatin conformation, nucleolar structure, chromosome stability, cell cycle progression, RNA processing, RNA export from the nucleus, protein import to the nucleus, and mating in budding yeast (reviewed in ref. 1; refs. 2-6). No consensus has been reached on the primary biological role of this GTPase switch.The piml protein (2, 6) of the fission yeast Schizosaccharomycespombe is a structural homologue of RCC1 and spil is nearly identical to human Ran. The piml gene was cloned by complementation of temperature-sensitive lethal mutations in the piml gene, and spil was cloned as a high-copy suppressor of these mutations (2, 6). When fission yeast cells enter mitosis they activate the p34cdc2 protein kinase, condense their chromosomes, depolymerize their cytoplasmic microtubules, and organize a mitotic spindle within the nucleus, because unlike the nuclear membrane in multicellular eukaryotes, the nuclear envelope does not break down at mitosis in fission yeast (7). At the exit from mitosis the p34cdc2 kinase is inactivated, the chromosomes decondense, the mitotic spindle is depolymerized, and the cytoplasmic microtubules are reformed.The fission yeast piml-dlts mutant is unable to properly reestablish the interphase state following mitosis. At the restrictive temperature the cells activate the p34cdc2 protein kinase and enter mitosis with normal kinetics. After mitosis the cells inactivate p34cdc2 and form a medial septum but failThe publication costs of this article were defrayed in part by page charge pay...

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“…A thermocouple to measure the temperature of the block, a heater, and an automatic temperature controller can maintain the desired temperature for freeze substitution. A very simple prototype of such a device is shown in Kiss and McDonald (1993). Commercially available freezesubstitution units include features for controlled, automatic warming, easier fluid exchanges, and UV polymerization at low temperature.…”

Section: Freeze-substitution Protocolsmentioning

confidence: 99%

Using rapid freeze and freeze-substitution for the preparation of yeast cells for electron microscopy and three-dimensional analysis

Giddings

O’Toole

Morphew

et al. 2001

Methods in Cell Biology

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Chapter 16 Electron Microscopy Immunocytochemistry Following Cryofixation and Freeze Substitution

Cited by 32 publications

References 51 publications

A new look at kinetochore structure in vertebrate somatic cells using high-pressure freezing and freeze substitution

A new look at kinetochore structure in vertebrate somatic cells using high-pressure freezing and freeze substitution

A mutation in the RCC1-related protein pim1 results in nuclear envelope fragmentation in fission yeast.

Using rapid freeze and freeze-substitution for the preparation of yeast cells for electron microscopy and three-dimensional analysis

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