A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusible ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A. V. Somlyo, and A. P. . Ultramicros. 1:317-339.) of freeze-dried ultrathin cryo sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of C1 in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition of striated muscle.There was no evidence of sequestered C1 in the terminal cisternae of resting muscles, although calcium (66 mmol/kg dry wt -+ 4.6 SE) was detected. The values of [C1]l determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles and over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial C1 ratio of 2.4 -+ 0.88 SD.The elemental concentrations (mmol/kg dry wt -+ SD) of muscle fibers measured with 0.5-9-/zm diameter electron probes in normal frog striated muscle were: P, 302 -4.3; S, 189 -+ 2.9; C1, 24 -1.1; K, 404 +-4.3, and Mg, 39 +-2.1. It is concluded that: (a) in normal muscle the "excess Cl" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic Cl in low [K]o solutions exceeds that predicted by a passive electrochemical distribution.Hypertonic 2.2 x NaC1, 2.5 • sucrose, or 2.2 • Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P -~(6 Ca + 1 Mg)/6 P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules. 828THe JOURNAL OF CELL BIOLOGY" VOLUME 74, 1977" pages 828-857 on May 11, 2018 jcb.rupress.org Downloaded from http://doi.org/10. 1083/jcb.74.3.828 Published Online: 1 September, 1977 KEY WORDS electron probe analysis 9 cryoultramicrotomy 9 striated muscle hypertonicity 9 sarcoplasmic reticulum The distribution of diffusible ions in, respectively, the extracellular space, cytoplasm, and cellular organelles, represents one of the most general problems of cell function. The main techniques available for determining the concentrations of ions in different compartments are the measurement of radio isotope ion fluxes in intact tissues and organeUe fractionation. These techniques are, however, subject to the respective uncertainties of the ultrast...

Muscle cultures treated with cytochalasin B yield mono-and oligonucleated cells of two kinds: (i) arborized, replicating precursor myogenic cells and fibroblasts; and (ii) round, post-mitotic, terminally differentiating myoblasts and myotubes. The arb6rized cells do not bind fluorescein-labeled antibody against myosin, do not contract rhythmically, and do not display hexagonally stacked thick and thin filaments. The round, mononucleated myoblasts and round, oligonucleated myotubes bind the fluorescein-labeled antibody against myosin, contract rhythmically, and display clusters of hexagonallystacked thick and thin filaments. When cytochalasin B is removed and replaced by colcemide, the arborized cells, but not the post-mitotic muscle cells, acquire a radial symmetry and are induced to assemble massive, meandering cables that may occupy over 25% of the cell volume. These tortuous cables are positively birefringent and consist exclusively of enormous numbers of 100-A, intermediate-sized filaments.After 3 days in vitro primary cultures of embryonic chick muscle contain immature, post-mitotic, multinucleated myotubes, post-mitotic mononucleated myoblasts, replicating myogenic cells, replicating fibrogenic cells, and replicating mesenchyme cells (1,2,40). The addition of cytochalasin B to these cultures promptly blocks fusion of myoblasts into myotubes. Cytochalasin B also rapidly blocks locomotion of all replicating cells, induces them to "retract," and gradually transforms them into fully arborized cells (3-6). Cytochalasin B does not arborize immature myotubes, but it does cause them to retract from the clot, and subsequently the myotubes degenerate. Because cytocholasin B blocks, not the initiation, but the completion, of cytokinesis (6, 7), these cultures show an increase in oligonucleated cells with time. Cytocholasin B does not significantly impair either the initiation or continued synthesis of myosin, actin, or tropomyosin, for by day 6 the post-mitotic cells generated in these cultures contract spontaneously (3, 6).The addition of colcemide to 3-day-old muscle cultures results in the metaphase-arrest of many mononucleated myogenic and fibrogenic cells. Colcemide fragments the elongated, immature myotubes into multinucleated myosacs and dismantles their microtubules. The myosacs are rich in "lakes" of i00-A, intermediate-sized filaments, and it has been proposed that there is an inverse correlation between the microtubules in myotubes and the 100-A filaments in myosacs (6,8). Colcemide has no detectable effect on the synthesis of contractile proteins, their organization into hexagonally-stacked thick and thin filaments, or their interaction required for spontaneous contractions (9). We wish to report that muscle cultures exposed to cytochalasin B gen-513 erate large numbers of post-mitotic, mononucleated myoblasts rich in scattered stacks of thick and thin filaments. Although Z. H, and I bands are atypical, these scattered stacks of thick and thin filaments permit the cell to contract spontaneously. W...

The elemental composition of individual cells of rapidly frozen and cryosectioned Escherichia coli B was measured with electron optical microanalytic methods. The Ca content was high (26.2 mmol/kg) in a 10-nm-wide region of the cell envelope. Amounts of cytoplasmic Ca in actively dividing cells were significantly higher (32.6 mmol/kg [dry weight]) than in the log-phIse (1.5 mmol/kg) cells. Cellular Mg was 205 mmol/kg (dry weight) and it was uniformly distributed throughout the cell. Cells washed in distilled water before freezing lost monovalent ions (Na, Cl, and K), but the membrane-bound Ca and cellular Mg were not reduced, indicating that cellular Mg and membrane Ca are more tightly bound.The ability of bacteria to actively transport and concentrate inorganic ions is well known (11,19), but uncertainty exists about their intracellular distribution and exchange ability. For example, recent authors (17) found that the Mg content of Escherichia coli is higher than had been previously reported (14, 16) and suggested that it was bound to DNA in the nucleoid. Until now, bacterial electrolytes have been measured with bulk chemical methods, primarily atomic absorption spectroscopy. However, with such methods, it is not possible to determine the intracellular distribution and proportion of membrane-bound ions nor to detect fluxes in putative ionic messengers, such as Ca2+, that may occur in a small proportion of a partially synchronized population. In contrast, electron probe microanalysis (EPMA), X-ray mapping, and electron energy loss spectroscopy (EELS) of ultrathin cryosections are techniques uniquely suitable for measuring the subcellular concentration and distribution of various elements (28) and have been used previously to demonstrate the distribution of calcium and other elements in bacterial spores (1,30,31). We used these latter methods to quantitate and determine the subcellular distribution of Mg2+, Ca2+, Na+, K+, and other elements (P, S, and Cl) in E. coli. In addition to these measurements of cell composition and elemental distribution, we demonstrated the high Ca concentration in E. coli envelopes and showed that the cytoplasmic Ca2+ was increased in dividing cells. A preliminary report of some of these findings has been published (4). Rapid freezing. The centrifuged pellets were scraped with a spatula, and the material was transferred from the spatula to a bamboo stick 0.4 mm in diameter. Upon transferring the material onto the stick, the stick was immediately frozen by plunging it manually into supercooled Freon 22 (E. I. du Pont de Nemours & Co., Inc., Wilmington, Del.) at -164°C. Frozen specimens were sectioned and were freeze-dried as described in detail by Somlyo and Silcox (29). A schematic diagram of the experiment described above is shown in Fig. 1. MATERIALS AND METHODSA representative electron micrograph of a cryosection of a dense region of a pellet is shown in Fig. 2a, and images of freeze-substituted bacteria (the latter not used for analysis) are shown in Fig. 2b. The latter specimen wa...

SUMMARYStriated muscles from Rana pipiens have been exposed for variable periods of time to Ringer solutions made hypertonic by addition of either sucrose or sodium chloride. The muscles have been rapid-frozen and then prepared for electron microscopy by either freeze-substitution, freeze-fracture or cryoultramicrotomy.The only compartment greatly affected by hypertonicity is the transverse tubular system, which is visibly swollen. None of the elements of the sarcoplasmic reticulum increase in size.

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