17-AAG, an Hsp90 inhibitor, causes kinetochore defects: a novel mechanism by which 17-AAG inhibits cell proliferation.

The Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG), which is currently in clinical trials, is thought to exert antitumor activity by simultaneously targeting several oncogenic signaling pathways. Here we report a novel mechanism by which 17-AAG inhibits cell proliferation, and we provide the first evidence that HSP90 is required for the assembly of kinetochore protein complexes in humans. 17-AAG caused delocalization of several kinetochore proteins including CENP-I and CENP-H but excluding CENP-B and CENP-C. Consistently, 17-AAG induced a mitotic arrest that depends on the spindle checkpoint and induced misalignment of chromosomes and aneuploidy. We found that HSP90 associates with SGT1 (suppressor of G2 allele of skp1; SUGT1) in human cells and that depletion of SGT1 sensitizes HeLa cells to 17-AAG. Overexpression of SGT1 restored the localization of specific kinetochore proteins and chromosome alignment in cells treated with 17-AAG. Biochemical and genetic results suggest that HSP90, through its interaction with SGT1 (SUGT1), is required for kinetochore assembly. Furthermore, time-course experiments revealed that transient treatment with 17-AAG between late S and G2/M phases causes substantial delocalization of CENP-H and CENP-I, a finding that strongly suggests that HSP90 participates in kinetochore assembly in a cell cycle-dependent manner.

Electron tomographic and other approaches for imaging molecular machines.

Two new methods, single-particle cryo-electron microscopy reconstruction and electron tomography, are increasingly used to visualize molecular machines in vitro and in the cellular context, respectively. Current efforts focus on the development of methods capable of visualizing molecular signatures in the cell, and first progress in this direction has now been made.

CENP-E is essential for reliable bioriented spindle attachment, but chromosome alignment can be achieved via redundant mechanisms in mammalian cells.

CENP-E is a kinesin-like protein that when depleted from mammalian kinetochores leads to mitotic arrest with a mixture of aligned and unaligned chromosomes. In the present study, we used immunofluorescence, video, and electron microscopy to demonstrate that depletion of CENP-E from kinetochores via antibody microinjection reduces kinetochore microtubule binding by 23% at aligned chromosomes, and severely reduces microtubule binding at unaligned chromosomes. Disruption of CENP-E function also reduces tension across the centromere, increases the incidence of spindle pole fragmentation, and results in monooriented chromosomes approaching abnormally close to the spindle pole. Nevertheless, chromosomes show typical patterns of congression, fast poleward motion, and oscillatory motions. Furthermore, kinetochores of aligned and unaligned chromosomes exhibit normal patterns of checkpoint protein localization. These data are explained by a model in which redundant mechanisms enable kinetochore microtubule binding and checkpoint monitoring in the absence of CENP-E at kinetochores, but where reduced microtubule-binding efficiency, exacerbated by poor positioning at the spindle poles, results in chronically monooriented chromosomes and mitotic arrest. Chromosome position within the spindle appears to be a critical determinant of CENP-E function at kinetochores.

The emergence of electron tomography as an important tool for investigating cellular ultrastructure.

Electron tomography has emerged as the leading method for the study of three-dimensional (3D) ultrastructure in the 5-20-nm resolution range. It is ideally suited for studying cell organelles, subcellular assemblies and, in some cases, whole cells. Tomography occupies a place in 3D biological electron microscopy between the work now being done at near-atomic resolution on isolated macromolecules or 2D protein arrays and traditional serial-section reconstructions of whole cells and tissue specimens. Tomography complements serial-section reconstruction by providing higher resolution in the depth dimension, whereas serial-section reconstruction is better able to trace continuity over long distances throughout the depth of a cell. The two techniques can be combined with good results for favorable specimens. Tomography also complements 3D macromolecular studies by offering sufficient resolution to locate the macromolecular complexes in their cellular context. The technology has matured to the point at which application of electron tomography to specimens in plastic sections is routine, and new developments to overcome limitations due to beam exposure and specimen geometry promise to further improve its capabilities. In this review we give a brief description of the methodology and a summary of the new insights gained in a few representative applications.(J Histochem Cytochem 49:553-563, 2001)

Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.

We discovered that many proteins located in the kinetochore outer domain, but not the inner core, are depleted from kinetochores and accumulate at spindle poles when ATP production is suppressed in PtK1 cells, and that microtubule depolymerization inhibits this process. These proteins include the microtubule motors CENP-E and cytoplasmic dynein, and proteins involved with the mitotic spindle checkpoint, Mad2, Bub1R, and the 3F3/2 phosphoantigen. Depletion of these components did not disrupt kinetochore outer domain structure or alter metaphase kinetochore microtubule number. Inhibition of dynein/dynactin activity by microinjection in prometaphase with purified p50 "dynamitin" protein or concentrated 70.1 anti-dynein antibody blocked outer domain protein transport to the spindle poles, prevented Mad2 depletion from kinetochores despite normal kinetochore microtubule numbers, reduced metaphase kinetochore tension by 40%, and induced a mitotic block at metaphase. Dynein/dynactin inhibition did not block chromosome congression to the spindle equator in prometaphase, or segregation to the poles in anaphase when the spindle checkpoint was inactivated by microinjection with Mad2 antibodies. Thus, a major function of dynein/dynactin in mitosis is in a kinetochore disassembly pathway that contributes to inactivation of the spindle checkpoint.

Three-dimensional transmission electron microscopy and its application to mitosis research.

Transmission electron microscopy produces images that are projections of the original object, with the consequence that features from different depths of the specimen overlap and give a confusing image. This problem is overcome by reconstructing the object in 3D from a series of 2D views using either serial thin section reconstruction or electron tomography. In the serial section approach, the series of 2D views is generated from images of successive serial sections cut thin enough to be effectively 2D slices of the specimen. For electron tomography the series of 2D views is generated by tilting a single, usually thicker, section in the electron beam. Resolution in the depth dimension is limited to twice the section thickness for serial section reconstruction and is determined by the number of tilt views collected (i.e., by the fineness of the angular interval between successive tilt views) for electron tomography. Both methods produce distorted 3D reconstructions because of missing material and alignment difficulties in the case of serial sections and the limited angular tilt range in the case of electron tomography. However, techniques have evolved for minimizing and circumventing these distortions and, as long as the user is aware of the limitations, misinterpretations can be avoided. Since electron tomography provides better resolution (generally 5-20 nm), it is the method of choice for determining detailed structural interactions such as the depth of kinetochore MT penetration into the kinetochore outer plate. On the other hand, serial section reconstruction is more effective for projects that require tracking through a complete object in the specimen, such as counting the number of kinetochore MTs on each kinetochore. If the project requires finding a relatively small object in a large specimen (e.g., finding centrioles in an oocyte), then it is sometimes advantageous to cut thicker plastic sections and analyze them via stereo viewing. The mitotic spindle, however, is generally too complex to be analyzed via stereo viewing. Currently, collapse of plastic sections in the electron beam limits the utility of serial section electron tomography. Once a 3D reconstruction is completed it must be analyzed with the 2D medium of the screen on a computer monitor. The easiest approach is usually to walk through the 3D reconstruction volume slice by slice. However, in order to appreciate 3D interactions, and to communicate the results to others, it is generally necessary to segment key components from the rest of the volume and use modeling and rendering techniques. Rendered surface views can easily be color coded and provided with a number of depth cues to simulate the surface viewing encountered in everyday life. In some instances, it is useful to look through a smaller portion of the reconstruction volume with "X-ray vision." This can accomplished by using volume rendering to create a series of semitransparent views from different tilt angles.

Relevance of kinetochore size and microtubule-binding capacity for stable chromosome attachment during mitosis in PtK1 cells.

Chromosomes attach to the mitotic spindle via their kinetochores. The average number of spindle microtubules binding to each kinetochore varies with species, the stage of mitosis, and the length of time that the kinetochore has been attached to the spindle. In this report, we investigate how kinetochore microtubule number varies with kinetochore size and chromosome size in PtK1 cells. From an analysis of serial-section electron micrographs, we determined that the average surface area of metaphase, taxol-treated metaphase, and anaphase kinetochores is 0.16 +/- 0.05 microm2 (N = 181). Surprisingly, kinetochore microtubules are packed more densely on the smaller kinetochores, as seen by a reduction in the average spacing between kinetochore microtubules from 89 nm to 59 nm. Our interpretation of this result is that PtK1 cells require a minimum kinetochore microtubule-binding capacity for survival during repeated rounds of mitotic division. We estimate the lower limit to be 23 kinetochore microtubules and suggest that this capacity is required to ensure stable attachment during the dynamic and highly stochastic process of kinetochore fiber formation. There is a modest but statistically significant increase in kinetochore microtubule number with chromosome size, indicating that chromosome size is a minor determinant of kinetochore microtubule number.

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

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 electron-translucent 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 (PtK1) 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.

Kinetochore fiber maturation in PtK1 cells and its implications for the mechanisms of chromosome congression and anaphase onset.

Kinetochore microtubules (kMts) are a subset of spindle microtubules that bind directly to the kinetochore to form the kinetochore fiber (K-fiber). The K-fiber in turn interacts with the kinetochore to produce chromosome motion toward the attached spindle pole. We have examined K-fiber maturation in PtK1 cells using same-cell video light microscopy/serial section EM. During congression, the kinetochore moving away from its spindle pole (i.e., the trailing kinetochore) and its leading, poleward moving sister both have variable numbers of kMts, but the trailing kinetochore always has at least twice as many kMts as the leading kinetochore. A comparison of Mt numbers on sister kinetochores of congressing chromosomes with their direction of motion, as well as distance from their associated spindle poles, reveals that the direction of motion is not determined by kMt number or total kMt length. The same result was observed for oscillating metaphase chromosomes. These data demonstrate that the tendency of a kinetochore to move poleward is not positively correlated with the kMt number. At late prometaphase, the average number of Mts on fully congressed kinetochores is 19.7 +/- 6.7 (n = 94), at late metaphase 24.3 +/- 4.9 (n = 62), and at early anaphase 27.8 +/- 6.3 (n = 65). Differences between these distributions are statistically significant. The increased kMt number during early anaphase, relative to late metaphase, reflects the increased kMt stability at anaphase onset. Treatment of late metaphase cells with 1 microM taxol inhibits anaphase onset, but produces the same kMt distribution as in early anaphase: 28.7 +/- 7. 4 (n = 54). Thus, a full complement of kMts is not sufficient to induce anaphase onset. We also measured the time course for kMt acquisition and determined an initial rate of 1.9 kMts/min. This rate accelerates up to 10-fold during the course of K-fiber maturation, suggesting an increased concentration of Mt plus ends in the vicinity of the kinetochore at late metaphase and/or cooperativity for kMt acquisition.

Chromosome fragments possessing only one kinetochore can congress to the spindle equator.

We used laser microsurgery to cut between the two sister kinetochores on bioriented prometaphase chromosomes to produce two chromosome fragments containing one kinetochore (CF1K). Each of these CF1Ks then always moved toward the spindle pole to which their kinetochores were attached before initiating the poleward and away-from-the-pole oscillatory motions characteristic of monooriented chromosomes. CF1Ks then either: (a) remained closely associated with this pole until anaphase (50%), (b) moved (i.e., congressed) to the spindle equator (38%), where they usually (13/19 cells) remained stably positioned throughout the ensuing anaphase, or (c) reoriented and moved to the other pole (12%). Behavior of congressing CF1Ks was indistinguishable from that of congressing chromosomes containing two sister kinetochores. Three-dimensional electron microscopic tomographic reconstructions of CF1Ks stably positioned on the spindle equator during anaphase revealed that the single kinetochore was highly stretched and/or fragmented and that numerous microtubules derived from the opposing spindle poles terminated in its structure. These observations reveal that a single kinetochore is capable of simultaneously supporting the function of two sister kinetochores during chromosome congression and imply that vertebrate kinetochores consist of multiple domains whose motility states can be regulated independently.

Mineralization of collagen may occur on fibril surfaces: evidence from conventional and high-voltage electron microscopy and three-dimensional imaging.

The interaction between collagen and mineral crystals in the normally calcifying leg tendons from the domestic turkey, Meleagris gallopavo, has been investigated at an ultrastructural level with conventional and high-voltage electron microscopy, computed tomography, and three-dimensional image reconstruction methods. Specimens treated by either aqueous or anhydrous techniques and resin-embedded were appropriately sectioned and regions of early tendon mineralization were photographed. On the basis of individual photomicrographs, stereoscopic pairs of images, and tomographic three-dimensional image reconstructions, platelet-shaped crystals may be demonstrated for the first time in association with the surface of collagen fibrils. Mineral is also observed in closely parallel arrays within collagen hole and overlap zones. The mineral deposition at these spatially distinct locations in the tendon provides insight into possible means by which calcification is mediated by collagen as a fundamental event in skeletal and dental formation among vertebrates.

Structural relations between collagen and mineral in bone as determined by high voltage electron microscopic tomography.

Aspects of the ultrastructural interaction between collagen and mineral crystals in embryonic chick bone have been examined by the novel technique of high voltage electron microscopic tomography to obtain three-dimensional information concerning extracellular calcification in this tissue. Newly mineralizing osteoid along periosteal surfaces of mid-diaphyseal regions from normal chick tibiae was embedded, cut into 0.25 microns thick sections, and documented at 1.0 MV in the Albany AEI-EM7 high voltage electron microscope. The areas of the tissue studied contained electron dense mineral crystals associated with collagen fibrils, some marked by crystals disposed along their cylindrically shaped lengths. Tomographic reconstructions of one site with two mineralizing fibrils were computed from a 5 degrees tilt series of micrographs over a +/- 60 degrees range. Reconstructions showed that the mineral crystals were platelets of irregular shape. Their sizes were variable, measured here up to 80 x 30 x 8 nm in length, width, and thickness, respectively. The longest crystal dimension, corresponding to the c-axis crystallographically, was generally parallel to the collagen fibril long axis. Individual crystals were oriented parallel to one another in each fibril examined. They were also parallel in the neighboring but apparently spatially separate fibrils. Crystals were periodically (approximately 67 nm repeat distance) arranged along the fibrils and their location appeared to correspond to collagen hole and overlap zones defined by geometrical imaging techniques. The crystals appeared to be continuously distributed along a fibril, their size and number increasing in a tapered fashion from a relatively narrow tip containing smaller and infrequent crystals to wider regions having more densely packed and larger crystals. Defined for the first time by direct visual 3D imaging, these data describe the size, shape, location, orientation, and development of early crystals in normal bone collagen. The results suggest that platelet-shaped crystals are arranged in channels or grooves which are formed by collagen hole zones in register and that crystal sizes may exceed the dimensions of hole zones. Such data agree with those from mineral-matrix interaction in normally calcifying avian tendon obtained by similar high voltage tomographic means, but in addition they indicate a possible gradual and continuous deposition of crystals in collagen of bone unlike tendon and imply that individual collagen fibrils in local regions of osteoid are organized such that they all may be aligned in a coherent manner.

The relevance of dose-fractionation in tomography of radiation-sensitive specimens.

It is commonly assumed that the number of projections required for single-axis tomography precludes its application to most beam-labile specimens. However, Hegerl and Hoppe have pointed out that the total dose required to achieve statistical significance for each voxel of a computed 3D reconstruction is the same as that required to obtain a single 2D image of that isolated voxel, at the same level of statistical significance. Thus a statistically significant 3D image can be computed from statistically insignificant projections, as long as the total dose that is distributed among these projections is high enough that it would have resulted in a statistically significant projection, if applied to only one image. We have tested this critical theorem by simulating the tomographic reconstruction of a realistic 3D model created from an electron micrograph. The simulations verify the basic conclusions of the theorem and extend its validity to the experimentally more realistic conditions of high absorption, signal-dependent noise, varying specimen contrast and missing angular range. Individual projections in the series of fractionated-dose images could be aligned by cross-correlation because they contained significant information derived from the summation of features from different depths in the structure. This latter information is generally not useful for structural interpretation prior to 3D reconstruction, owing to the complexity of most specimens investigated by single-axis tomography. These results demonstrate that it is feasible to use single-axis tomography with soft X-ray and electron microscopy of frozen-hydrated specimens.

A marker-free alignment method for electron tomography.

In electron tomography of biological specimens, fiducial markers are normally used to achieve accurate alignment of the input projections. We address the problem of alignment of projections from objects that are freely supported and do not permit the use of markers. To this end we present a new alignment algorithm for single-axis tilt geometry based on the principle of Fourier-space common lines. An iterative scheme has been developed to overcome the noise-sensitivity of the common-line method. This algorithm was used to align a data set that was not amenable to alignment with fiducial markers.

Mineral and organic matrix interaction in normally calcifying tendon visualized in three dimensions by high-voltage electron microscopic tomography and graphic image reconstruction.

To define the ultrastructural accommodation of mineral crystals by collagen fibrils and other organic matrix components during vertebrate calcification, electron microscopic 3-D reconstructions were generated from the normally mineralizing leg tendons from the domestic turkey, Meleagris gallopavo. Embedded specimens containing initial collagen mineralizing sites were cut into 0.5-micron-thick sections and viewed and photographed at 1.0 MV in the Albany AEI-EM7 high-voltage electron microscope. Tomographic 3-D reconstructions were computed from a 2 degree tilt series of micrographs taken over a minimum angular range of +/- 60 degrees. Reconstructions of longitudinal tendon profiles confirm the presence of irregularly shaped mineral platelets, whose crystallographic c-axes are oriented generally parallel to one another and directed along the collagen long axes. The reconstructions also corroborate observations of a variable crystal length (up to 170 nm measured along crystallographic c-axes), the presence of crystals initially in either the hole or overlap zones of collagen, and crystal growth in the c-axis direction beyond these zones into adjacent overlap and other hole regions. Tomography shows for the first time that crystal width varies (30-45 nm) but crystal thickness is uniform (approximately 4-6 nm at the resolution limit of tomography); more crystals are located in the collagen hole zones than in the overlap regions at the earliest stages of tendon mineralization; the crystallographic c-axes of the platelets lie within +/- 15-20 degrees of one another rather than being perfectly parallel; adjacent platelets are spatially separated by a minimum of 4.2 +/- 1.0 nm; crystals apparently fuse in coplanar alignment to form larger platelets; development of crystals in width occurs to dimensions beyond single collagen hole zones; and a thin envelope of organic origin may be present along or just beneath the surfaces of individual mineral platelets. Implicit in the results is that the formation of crystals occurs at different sites and times by independent nucleation events in local regions of collagen. These data provide the first direct visual evidence from 3-D imaging describing the size, shape, orientation, and growth of mineral crystals in association with collagen of a normally mineralizing vertebrate tissue. They support concepts that c-axial crystal growth is unhindered by collage hole zone dimensions, that crystals are organized in the tendon in a series of generally parallel platelets, and that crystal growth in width across collagen fibrils may follow channels or grooves formed by adjacent hole zones in register.

Structure of the colcemid-treated PtK1 kinetochore outer plate as determined by high voltage electron microscopic tomography.

High voltage electron microscopic tomography was used to determine the organization of the kinetochore plate and its attachment to the underlying chromosome. Six reconstructions were computed from thick sections of Colcemid-treated PtK1 cells and analyzed by a number of computer graphics methods including extensive thin slicing, three-dimensional masking, and volume rendering. When viewed en-face the kinetochore plate appeared to be constructed from a scaffold of numerous 10-20-nm thick fibers or rods. Although the fibers exhibited regions of parallel alignment and hints of a lattice, they were highly variable in length, orientation and spacing. When viewed in stereo, groups of these fibers were often seen oriented in different directions at different depths to give an overall matted appearance to the structure. When viewed "on edge," the plate was 35-40 nm thick, and in thin slices many regions were tripartite with electron-opaque domains, separated by a more translucent middle layer, forming the inner and outer plate boundaries. These domains were joined at irregular intervals. In some slices, each domain appeared as a linear array of 10-20-nm dots or rods embedded in a less electron-opaque matrix, and adjacent dots within or between domains often appeared fused to form larger blocks. The plate was connected to the underlying chromosome by less densely arrayed 10-20-nm thick fibers that contacted the chromosome-facing (i.e., inner) surface of the plate in numerous patches. These patches tended to be arrayed in parallel rows perpendicular to the long axis of the chromosome. In contrast to connecting fibers, corona fibers were more uniformly distributed over the cytoplasmic-facing (i.e., outer) surface of the plate. When large portions of the reconstructions were viewed, either en-face or in successive slices parallel to the long axis of the chromosome, the edges of the plate appeared splayed into multiple "fingers" that partly encircled the primary constriction. Together these observations reveal that regions of the kinetochore outer plate contain separate structural domains, which we hypothesize to serve separate functional roles. Our three-dimensional images of the kinetochore are largely consistent with the hypothesis that the outer plate is composed of multiple identical subunits (Zinkowski, R. P., J. Meyne, and B. R. Brinkley. 1991. J. Cell Biol. 113:1091-1110).

A 360 degrees single-axis tilt stage for the high-voltage electron microscope.

A new type of specimen stage that permits more than 180 degrees of tilting about the axis of a side-entry rod has been developed for a high-voltage electron microscope (HVEM). Roughly cylindrical specimens, with radial dimensions of less than a few micrometers, that can be mounted on the tip of a microneedle or micropipette are applicable. For glass micropipettes, the energy of the 1-MeV beam of the HVEM is sufficient to image specimens through both walls. The stage employs a spindle mechanism that holds these needles or micropipettes coaxial with the tilt axis, allowing the specimen to be rotated without restriction. This arrangement, along with the cylindrical form of the specimen, is an important development for single-axis tomography, because it permits a complete 180 degrees set of projections to be recorded. The angular accuracy of the stage was demonstrated to be within +/- 0.20 degrees, with a cumulative error of less than 1.0 degrees over a 180 degrees span. The new stage was tested using puffball spores mounted on a micropipette. A 180 degrees tilt series was recorded and processed to yield a tomographic three-dimensional reconstruction which was displayed both as a cross-sectional view perpendicular to the tilt axis, and as a shaded surface viewed from different directions. The same computations were repeated using subsets of the tilt series to assess the effect of various amounts of missing information. Visual inspection of a selected cross-section from these reconstructions indicated that limiting the angular range to 160 degrees produced results nearly as good as the full data set. Limiting the range to 140 degrees, however, produced a noticeable geometric distortion, which became increasingly severe with ranges of 120 degrees and 100 degrees.

Ultrastructure of chromatin. II. Three-dimensional reconstruction of isolated fibers.

Electron-microscope tomography has been used to reconstruct isolated, negatively stained chromatin fibers from Necturus maculosus erythrocytes. Tilt series micrographs from +70 degrees to -70 degrees at 5 degrees intervals were obtained, allowing a reconstruction resolution of 3.3 nm for fibers lying parallel to the tilt axis. The fibers were found to be flattened in the plane of the carbon support, and also stained differentially according to the distance from the carbon. A number of methods of presenting the three-dimensional information were explored. Especially useful was an automatic peak search method for locating putative nucleosome positions coupled with the production of a computer-generated model. Other valuable techniques included the generation of projection stereograms and construction of solid models. A peripheral location of nucleosomes in the chromatin fiber was indicated, and helical arrangements of nucleosomes were observed over short regions. However, no long-range ordering of nucleosomes was apparent. The extent to which this lack of order may be the result of events occurring during the preparation of chromatin for electron microscopy is discussed.

Quantitative determination of the mineral distribution in different collagen zones of calcifying tendon using high voltage electron microscopic tomography.

High voltage electron microscopic tomography was used to make the first quantitative determination of the distribution of mineral between different regions of collagen fibrils undergoing early calcification in normal leg tendons of the domestic turkey, Meleagris gallopavo. The tomographic 3-D reconstruction was computed from a tilt series of 61 different views spanning an angular range of +/- 60 degrees in 2 degrees intervals. Successive applications of an interactive computer operation were used to mask the collagen banding pattern of either hole or overlap zones into separate versions of the reconstruction. In such 3-D volumes, regions specified by the mask retained their original image density while the remaining volume was set to background levels. This approach was also applied to the mineral crystals present in the same volumes to yield versions of the 3-D reconstructions that were masked for both the crystal mass and the respective collagen zones. Density profiles from these volumes contained a distinct peak corresponding only to the crystal mass. A comparison of the integrated density of this peak from each profile established that 64% of the crystals observed were located in the collagen hole zones and 36% were found in the overlap zones. If no changes in crystal stability occur once crystals are formed, this result suggests the possibilities that nucleation of mineral is preferentially and initially associated with the hole zones, nucleation occurs more frequently in the hole zones, the rate of crystal growth is more rapid in the hole zones, or a combination of these alternatives. All lead to the conclusion that the overall accumulation of mineral mass is predominant in the collagen hole zones compared to overlap zones during early collagen fibril calcification.

A critical evaluation of the use of filipin-permeabilized rat hepatocytes to study functions of the endoplasmic reticulum in situ.

We have used transmission (TEM) and scanning electron microscopy (SEM) and leakage of lactate dehydrogenase (LDH; EC 1.1.1.27) to evaluate two published procedures which use filipin to render isolated rat hepatocytes permeable to ionic substrates. Cells treated by the procedure of Jorgenson and Nordlie retained less than 10 per cent of their LDH. TEM revealed severe damage to the internal structure of these cells, which included swelling, disintegration and extensive vesicularization of the endoplasmic reticulum (ER). Hepatocytes treated with filipin by the procedure of Gankema et al. retained 65-75 per cent of their LDH and displayed incomplete but highly variable permeability to Trypan blue. SEM revealed the loss of microvilli, other signs of swelling, and the presence of large lesions in the plasma membrane. TEM revealed signs of cell swelling, but the nuclei and the mitochondria were only moderately altered. The rough ER was not swollen, but significant fragmentation was evident and characteristic stacks of lamellar ER were never seen. We conclude that useful information about the functions of the ER in situ cannot be obtained from studies of filipin-treated cells. Our results indicate that retention of LDH is not a sufficient criterion of preservation of cell morphology and that staining with Trypan blue may significantly underestimate the permeability of cells to small ionic metabolites.