THE MITOTIC APPARATUS: ISOLATION BY CONTROLLED pH

The isolation of the mitotic apparatus (MA) from the echinoderm egg was studied in detail, with particular attention given to the factors governing its stability. Successful isolation depends mainly on the pH of the isolation solution, slightly acid values being required. The use of a 1 M solution of hexanediol, buffered at pH 6.0 to 6.4, gives high yields of stable MA, while MA of poorer quality can be isolated in water buffered at pH 5.5 to 5.8. Isolation is possible only over a very narrow range of pH, as the cells become more difficult to break at lower values and the MA becomes unstable at higher values. Within this range the fibrous structure of the MA varies with the pH. The isolated MA disintegrates slowly when transferred to water at pH 7 and dissolves rapidly in solutions of high ionic strength.     

THE MITOTIC APPARATUS ISOLATED IN GLYCEROL-CONTAINING MEDIUM

The mitotic apparatus of the sea urchin egg was isolated at 30°C in an isolation medium containing glycerol which is known to stabilize microtubules. After isolation in the 1 m glycerol-isolation medium, the mitotic apparatus was stabilized on addition of glycerol to a final concentration of 3 to 4 m. Without the addition, the chromosomes were disjoined from the spindle and the interzonal region between separating chromosomes was fragile resulting in separation of half spindles. Lowering the temperature of the isolation medium to 20°C or below, the isolation procedure allowed to isolate spindles. The isolated spindle behaved in a manner similar to the mitotic apparatus on the effect of glycerol concentration.
The glycerol-mitotic apparatus contained tubulin which was extractable with the isolation medium containing Ca ions or an organic mercurial. Tubulin was also extracted upon lowering the temperature to 0°C in the presence of GTP. Addition of KCl to a final concentration of 0.6 m immediately dispersed the mitotic apparatus. The extract revealed a colchicine binding of 0.001 mole per 105,000 × g of protein. The colchicine binding complex was found to have a molecular weight of 105,000. The DEAE Sephadex column chromatography of the KCl extract allowed to elute tubulin fraction which bound 0.1 mole colchicine per 105,000 × g of protein. The mitotic apparatus tubulin was shown to contain α and β subunits with mobilities quite identical with those of brain tubulin subunits. The molecular weights of the α and β subunits were 55,000 ± 1,000 and 51,000 ± 1,000, respectively.     

FINE STRUCTURES OF INTRACYTOPLASMIC ORGANELLES OF MYCOBACTERIA

The fine structure of the intracytoplasmic organelles of mycobacteria was studied by means of electron microscopy of ultrathin sections. A well-preserved nuclear apparatus was obtained by fixation with OsO₄ in acetate-veronal buffer, containing calcium and tryptone, or in collidine-HCl buffer, followed by uranyl-acetate treatment and embedding in araldite. A low density nuclear region was filled with fine fibrils, 30 A in diameter, in parallel or concentric arrangement. A membranous organelle, tentatively designated as "lamellar structure," consists of unit membranes in lamellar arrangement. The thickness of each lamella in this membranous organelle coincides with that of the three-layered cytoplasmic membrane Moreover, the continuity of this unit membrane with the cytoplasmic membrane was demonstrated.     

TAME stabilizes the cortex and mitotic apparatus of the sea urchin egg during isolation

The synthetic substrate p-tosyl-L-arginine methyl ester (TAME) has been included in buffered EGTA media used for the isolation of the mitotic apparatus from clam eggs and also for the isolation of the cortex from sea urchin eggs. In the course of an investigation of the role of actin-fascin and actin-myosin interactions in cytokinesis, the isolation of the sea urchin egg cortex was re-examined and the stability of the cortex to lysis in a buffered EGTA medium near neutrality found to depend directly on the presence of TAME. Lysis of eggs at metaphase in this medium yielded a mixture of cortices and mitotic apparatuses (MA); MA stability under these conditions also required the presence of TAME, although a reduced pH allowed MA isolation in its absence. The action of TAME in stabilizing the actin-based structure of the cortex and the microtubule-based structure of the MA is not duplicated by other proteolysis inhibitors and this compound will also induce actin polymerization and gelation in extracts of the soluble cytoplasmic proteins of the egg under conditions where these are normally inhibited.     

FURTHER ELECTRON MICROSCOPE STUDIES ON FIBRILLAR ORGANIZATION OF THE GROUND CYTOPLASM OF CHAOS CHAOS

Further evidence for fibrillar organization of the ground cytoplasm of Chaos chaos is presented. Fixations with osmium tetroxide at pH 6 or 8 and with glutaraldehyde at pH 6 or 7 were used on two preparations: (a) single actively streaming cells; (b) prechilled cells treated with 0.05% Alcian blue in the cold and returned to room temperature for 5–10 min. In addition, a 50,000 g pellet of homogenized cells was examined after fixation with glutaraldehyde-formaldehyde alone. In sections from actively streaming cells considerable numbers of filaments were observed in the uroid regions after glutaraldehyde fixation, whereas only traces of filaments were seen after osmium tetroxide fixation at either pH 6 or 8. Microtubules were not seen. In sections from dye-treated cells, filaments (4–6 mµ) and fibrils (12–15 mµ) were found with all three fixatives. The 50,000 g pellet was heterogeneous but contained both clumps of fibrils and single thick fibrils like those seen in the cytoplasm of dye-treated cells. Many fibrils of the same dimensions (12–15 mµ wide, 0.5 µ long) were also seen in the supernatant above the pellet. Negative staining showed that some fibrils separated into at least three strands of 4–6 mµ filaments.     

Mitotic Asters Separate, although Chromosomes Do Not Separate at Slightly Acidic pHi in the Fertilized Egg of the Sea Urchin, Hemicentrotus pulcherrimus

The effect of slightly acidic intracellular pH (pHi) on the development of the sea urchin, Hemicentrotus pulcherrimus was investigated. At first cleavage, the fertilized eggs were treated with artificial sea water containing sodium acetate (Ac-pHSW) at pH 6.8 or 7.0 at the onset of nuclear envelope breakdown, and their pHi decreased from 7.30 to 6.68 or 6.78, respectively. When the eggs were observed after fixation by indirect immunofluorescence and differential interference contrast microscopy, the mitotic stage of the treated eggs was arrested at metaphase and the mitotic apparatus was maintained until more than 50 min after the treatment, although it was smaller in size than that of non-treated eggs. On the other hand, the number of the mitotic asters increased from 2 to 3-4, and further to 6-8 following prolonged exposure, suggesting that the centrosomes had divided and replicated. These results suggest that the centrosome cycle advanced at slightly acidic pHi, even when the mitotic cycle did not advance beyond metaphase.     

The ultrastructure of human mitotic chromosomes and interphase nuclei treated by Giemsa banding techniques

Total preparations of mitotic chromosomes and interphase nuclei prepared as for Giemsa banding techniques were investigated by standard transmission electron microscopy and by a method of a three dimensional representation. Chromosomes as well as interphase nuclei appear to be composed of irregularely folded fibrils of at least 300 Å thickness. In the G-band regions the chromosomes are thicker containing more foldings of fibrils. Also the fibrils are darker stained in the G-band regions. Loops of fibrils stick out from chromosomes as well as from interphase nuclei. When chromosomes or interphase nuclei come to lie close enough, such loops may stick together and form fibrillar bridges between them. These as well as interchromatid bridges are considered to be artefacts. The fibrils seem to be built up either of one or of several finer fibrils. No further conclusions regarding the fine structure of the fibrils can be drawn.     

THE MECHANISM OF ACTION OF COLCHICINE : Colchicine Binding to Sea Urchin Eggs and the Mitotic Apparatus

Colchicine forms a complex in vivo with a protein present in fertilized or unfertilized sea urchin eggs; similar binding was obtained in vitro with the soluble fraction from egg homogenates. Kinetic parameters and binding equilibrium constant were essentially the same in vivo and in vitro. The binding site protein was shown to have a sedimentation constant of 6S by zone centrifugation. The protein was present in extracts of the isolated mitotic apparatus at a concentration which was several times higher than in whole-egg homogenates. It was extracted from the mitotic apparatus at low ionic strength under conditions which lead to the disappearance of microtubules. No binding could be detected to the 27S protein, previously described by Kane, which is a major protein component of the isolated mitotic apparatus. The properties of the colchicine-bindinG protein, (binding constant, sedimentation constant, Sephadex elution volume) are similar to those obtained with the protein from mammalian cells, sea-urchin sperm tails, and brain tissue, and thus support the conclusion that the protein is a subunit of microtubules.     

Cycle specific association of nascent chromatin with nuclear envelope components in Physarum polycephalum.

The action of heparin on isolated nuclei derived from different phases of the mitotic cycle in plasmodia of Physarum polycephalum was studied. Heparin addition at two-fold excess over DNA concentration to nuclei in Mg-free low ionic strength buffer (10 mM TRIS-HC1, 10 mM Na2 HPO4, pH = 8) releases 60-80% of chromatin from S, G2, and mitotic phase nuclei. The RNA/protein ratio of herparin-solubilized cromatin is constant through S and G2 phases, but rises about two-fold at early prophase coincident with nucleolar breakdown. Purified nuclear envelopes were obtained from heparin-treated nuclei by sedimentation according to Bornens procedures (Nature 244, 28, 1973), and examined by transmission electron microscopy. Residual chromatin is seen at all stages with fine network of DNA fibrils in contact with the envelop. Regardless of time in S, 80% of 3H-labeled DNA was released into soluble chromatin with identical 3H/14C ratios. The residual chromatin in nuclear envelopes exhibited a preferential association of early S-DNA in nuclei engaged in early S replication, and late S preferential association in nuclei engaged in late S replication.     

NUCLEAR EXTRUSION AND INTRACISTERNAL INCLUSIONS IN THE RABBIT BLASTOCYST

Observations have been made on the role of a divalent cation (calcium ion) during OsO₄ fixation of nuclei of frog erythrocytes, mainly after isolation from cells. The volume of the nucleus depends partly on the molecular interaction of charged macromolecules, is controlled by the ionic strength of the medium, and hence may be used as a guide in attempts to preserve structure. When the isolation and fixation media contain 0.01 M calcium at pH 6.3 the volume changes, in the light microscope, during processing are small. When the fixative does not contain these ions, reversible volume changes occur during fixation and dehydration. The chromatin of nuclei processed with minimal volume change appears, in the electron microscope, to contain fine dots and lines about 20 to 40 A in diameter, relatively close together. The chromatin structure of nuclei in which volume changes have occurred consists of dense irregularly shaped patches, relatively far apart, and ranging in diameter from about 200 A down to the limits of visibility (20 to 30 A). It is suggested that the latter structure is a precipitation artefact.     

FIBRILLAR DIFFERENTIATION IN MYXOMYCETE PLASMODIA

Myxomycete plasmodia of four different types (not including Physarum polycephalum) were studied in thin sections viewed in the electron microscope. In the cytoplasm of the protoplasmodia of Clastoderma debaryanum and the phaneroplasmodia of Fuligo septica fixed in situ, fibrillar differentiations of three rather distinct kinds were observed. One of these is filamentous and closely resembles the filaments (or "microtubules") of the mitotic apparatus of other species. The larger phaneroplasmodia of two species belonging to the Physarales and the plasmodium of Hemitrichia vesparium showed fewer and less well defined fibrils, and no fibrils were seen in the aphanoplasmodium of Stemonitis fusca. Good stabilization of such fibrils in larger plasmodia may require fixation methods more rigidly controlled than those which succeed with microscopic protoplasmodia. The function of the observed fibrils cannot yet be determined. Their presence in cytoplasm fixed in situ, however, lends support to those theories of protoplasmic movement which are dependent on integral cross-bonding of one or a few molecular species.     

THE MITOTIC APPARATUS : Structural Changes after Isolation

The fibrous structure of the mitotic apparatus (MA) isolated from dividing sea urchin eggs undergoes no changes visible in phase contrast during extended storage, but the solubility of the MA rapidly decreases after isolation. Polarization microscopy shows that a decrease in the birefringence of the MA also occurs after isolation and is correlated with the loss of solubility. This loss of birefringence indicates that some structural change takes place during this period, and such a change was demonstrated by means of electron microscopy. The tubular filaments which form the spindle of the intracellular MA and of the freshly isolated MA were found to break down during storage to rows of dense granules, this loss of continuity presumably accounting for the loss of birefringence. The interrelations of the observed changes and the significance of these observations for investigations on the isolated MA are discussed.     

Isolating toxic insulin amyloid reactive species that lack β-sheets and have wide pH stability

Amyloid diseases, including Alzheimer's disease, are characterized by aggregation of normally functioning proteins or peptides into ordered, β-sheet rich fibrils. Most of the theories on amyloid toxicity focus on the nuclei or oligomers in the fibril formation process. The nuclei and oligomers are transient species, making their full characterization difficult. We have isolated toxic protein species that act like an oligomer and may provide the first evidence of a stable reactive species created by disaggregation of amyloid fibrils. This reactive species was isolated by dissolving amyloid fibrils at high pH and it has a mass >100 kDa and a diameter of 48 ± 15 nm. It seeds the formation of fibrils in a dose dependent manner, but using circular dichroism and deep ultraviolet resonance Raman spectroscopy, the reactive species was found to not have a β-sheet rich structure. We hypothesize that the reactive species does not decompose at high pH and maintains its structure in solution. The remaining disaggregated insulin, excluding the toxic reactive species that elongated the fibrils, returned to native structured insulin. This is the first time, to our knowledge, that a stable reactive species of an amyloid reaction has been separated and characterized by disaggregation of amyloid fibrils.     

Identification of nonmitochondrial creatine kinase enzymatic activity in isolated sea urchin mitotic apparatus

ATP-dependent calcium sequestration was previously localized in vesicles of mitotic apparatus isolated from sea urchins. We now demonstrate that the mitotic apparatus contains an ATP-regenerative system characterized as creatine kinase (EC 2.7.3.2). Mitotic apparatus isolated with vesicles intact converted ADP to ATP if phosphocreatine was present. Omission of ADP or phosphocreatine gave negligible ATP. When mitotic apparatus were washed with detergent-containing buffer to remove vesicles, their ability to produce ATP from ADP and phosphocreatine was reduced. Assays of creatine kinase activity using NADP+:glucose-6-phosphate dehydrogenase indicated that 70% of the creatine kinase activity was extractable with 0.5% Triton X-100. The insoluble residue containing the skeleton of the mitotic apparatus had the rest of the activity. Experiments with a luciferin/luciferase assay showed that Triton removed above 82% of the activity. Preparations of intact mitotic apparatus were free of cytochrome c oxidase (EC 1.9.3.1) activity and therefore free of mitochondria. About 10(8) mitotic apparatus (total volume about 1 liter) could produce 17 mmol of ATP/min when substrates were not limiting. The creatine kinase enzyme activity described herein and the previously described membrane vesicular calcium sequestration system are nonmitochondrial, integral constituents of the sea urchin mitotic apparatus.     

Ultrastructure and ATPase activity of the centriolar body at the mitotic stage in neoblasts of the planarianDugesia sp.

The fine structure and ATPase activity of the mitotic spindle in neoblasts of planaria were examined. In neoblasts, the cells have a large nucleus and nucleolus. Mitochondria are aggregated around the nucleus with chromatoid bodies adjacent. The cytoplasm contains little endoplasmic reticulum (ER) and few Golgi bodies but many free ribosomes, forming polysomes, can be seen throughout the cytoplasmic and spindle ground areas. In addition, centriolar bodies, atypical centrioles, can also be recognized in the cytoplasm. Cells in the G₂ stage contain a pair of electron-dense bodies, both consisting of fibrogranules but differing from each other in fine structure and, in the mitotic stage, only one fibrogranular body can be recognized at each pole. ATPase activity was detected in the centriolar bodies in the G₂ and mitotic stages and in the ground area of the cytoplasm and spindle apparatus filled by free ribosomes. The activity associated with the microtubules differed with the developmental stage.     

THE DIRECT ISOLATION OF THE MITOTIC APPARATUS

A method for isolating the mitotic apparatus from dividing sea urchin eggs without the use of ethyl alcohol or of detergents is described. In the present method, the eggs are dispersed directly in a medium containing 1 M (to 1.15 M) sucrose, 0.15 M dithiodiglycol, and 0.001 M Versene at pH 6, releasing the visibly intact mitotic apparatus. The method is designed for studies of enzyme activities, lipid components, and the variables affecting the stability of the apparatus.     

SELECTIVE EXTRACTION OF ISOLATED MITOTIC APPARATUS : Evidence That Typical Microtubule Protein Is Extracted by Organic Mercurial

Mitotic apparatus isolated from sea urchin eggs has been treated with meralluride sodium under conditions otherwise resembling those of its isolation. The treatment causes a selective morphological disappearance of microtubules while extracting a major protein fraction, probably consisting of two closely related proteins, which constitutes about 10% of mitotic apparatus protein. Extraction of other cell particulates under similar conditions yields much less of this protein. The extracted protein closely resembles outer doublet microtubule protein from sea urchin sperm tail in properties considered typical of microtubule proteins: precipitation by calcium ion and vinblastine, electrophoretic mobility in both acid and basic polyacrylamide gels, sedimentation coefficient, molecular weight, and, according to a preliminary determination, amino acid composition. An antiserum against a preparation of sperm tail outer doublet microtubules cross-reacts with the extract from mitotic apparatus. On the basis of these findings it appears that microtubule protein is selectively extracted from isolated mitotic apparatus by treatment with meralluride, and is a typical microtubule protein.     

Changes in glycosaminoglycan binding to collagen during desmoid mineralization as revealed by different electron-microscopic staining techniques.

Mineralization at collagen fibrils is regulated by glycosaminoglycans (GAG). Alterations in proteoglycan composition during mineralization as well as inhibition of mineralization by GAGs are well documented. Collagen-GAG interactions during desmoid osteogenesis in fetal rat calvariae were investigated ultrastructurally by means of different fixation techniques. Mineralization was restricted to the collagen of the osteoid at the ectocranial side. Beyond the osteoid, one layer containing degenerated cells was found, followed by sheets of healthy osteoblasts with nonmineralized collagen fibrils. These fibrils were ordered in bundles, but were irregularly arranged in the mineralized osteoid. After fixation in glutaraldehyde-ruthenium red (GA-RR), small RR-positive granules were periodically attached to the fibrils of the nonmineralized collagen. These granules were absent at collagen in the mineralized osteoid. Periodically bound granules (periodicity of 62 nm) could clearly be demonstrated along collagen fibrils by pretreatment with the positively charged protamine sulfate and subsequent fixation in GA-RR in the nonmineralized collagen. In the mineralized osteoid, however, these granules were present, but periodic binding was missing. Heparin pretreatment followed by fixation in GA-RR revealed periodically bound fine strands between collagen fibrils running parallel in the nonmineralized collagen; these threads were absent in the mineralizing osteoid. Restriction of mineralization to osteoid at the mineralization border may be reflected by the observed changes in GAG binding to collagen fibrils within the osteoid of developing fetal calvariae in contrast to binding to collagen in nonmineralized areas.     

THE ULTRASTRUCTURE OF FLAGELLAR FIBRILS

The tips of rat sperm tails were slightly frayed by mechanical agitation, thus exposing the fibrils, which were then studied by electron microscopy after negative staining. Only the fibrils survived this treatment. Each fibril proved to be a cylinder with a hollow core. The walls of the cylinders were made up of 10 longitudinally oriented filaments. The filaments had a markedly beaded appearance, with a repeating period of 88 A. The filament thickness (bead width) was approximately 35 to 40 A. Beads of neighboring filaments were in register with each other so that cross-linking bound the filaments together to complete the wall structure of each fibril. The center-to-center spacing from one filament to the next was 55 to 60 A. The periodicity and the diameters of the filaments make it unlikely that the filaments are related to either actin or myosin. From the way the fibrils kinked, it can be inferred that they possessed considerable mechanical strength. It is consistent with present knowledge that fibrils of the mitotic apparatus may have the same basic structure as the flagellar fibrils. Under some circumstances, pairs of fibrils separated from one another along their length, except at their extreme tips. It was apparent that there was special bridging material to be found there. In other preparations, however, the paired fibrils remained together, indicating a powerful coupling mechanism.     

STABILITY OF ISOLATED MITOTIC APPARATUS DURING STORAGE*

Mitotic apparatus (MA) were isolated from sea urchin zygotes using various isolation procedures, and various properties of the isolated MA were studied and compared. MA isolated using hexylene glycol had birefringences which depended on the pH of the isolation medium, the lower the pH the higher the MA birefringence. The stability of the MA Birefringence also depended on the pH of the hexylene glycol isolation medium (the lower the pH the slower the rate of decay of birefringence), as did the final birefringence reached after prolonged storage. MA isolated using glycerol-dimethylsulphoxide (MTME) had much more stable birefringence than MA isolated using hexylene glycol; their birefringence decay rates were about 1000 times slower than those of MA isolated using hexylene glycol. Birefringence which remained after extraction of MA with H₂O, or 0.5 M KC1 was also studied; the results depended on the MA isolation medium, on the medium the MA were stored in, and on the amount of time the MA were stored after isolation, as described in detail in the text. These results are discussed, and it is suggested that several components (including, perhaps, oriented ribosomes) contribute to birefringence of isolated MA.