1. Reliable methods for establishing fetal mouse spinal cord (SC) and dorsal root ganglion (DRG) cells in long term (greater than 1 mo) dissociated cell cultures are described. These cells have been studied by morphologic and intracellular electrophysiologic techniques. 2. Cells studied electrophysiologically can be relocated after preparation for electron microscopy and examined in thin sections. The electron microscope shows that the surface membranes of these cells were directly accessible to the culture medium. The surfaces of SC cells were studded with synaptic boutons, whereas the DRG cell surfaces generally had none. 3. Current-voltage relationships and linear electrotonic properties of the neurons are described. Delayed and anomalous rectification were seen in both cell types. The length of SC cell dendrites was about one characteristic electrotonic length, while little or no contribution of the relatively sparse DRG cell processes was seen in the transient responses of the DRG cells. 4. Postspike and posttetanic hyperpolarizations in DRG cells were due to a surface membrane conductance increase; this was probably primarily an increase in K+ conductance. Post-activation hyperpolarization in SC cells was primarily due to activation of an electrogenic Na+ pump.

The structure of membranes at junctions between the plasma membrane and underlying cisterns of endoplasmic reticulum in amphioxus muscle and mouse cerebellar neurons was studied using the freeze-fracture technique. In amphioxus muscle, subsurface cisterns of sarcoplasmic reticulum form junctions with the surface membrane at the level of the sarcomere I bands. On the protoplasmic leaflet of the sarcolemma overlying these junctions were aggregates of large particles. On the protoplasmic leaflet of the membranes of cerebellar basket, stellate, and Purkinje cells there were similar aggregates of large particles. In both tissues, the corresponding external membrane halves had arrays of pits apparently complementary to the aggregates of large particles. Cross fractures through junctions showed that the particle aggregates in neuronal and muscle membranes were consistently located over intracellular cisterns closely applied to the plasma membrane. Thus, a similar plasma membrane specialization is found at subsurface cisterns in mammalian neurons and amphioxus muscle. This similarity supports the hypothesis that subsurface cisterns in neurons, like those in muscle, couple some intracellular activity to the electrical activity of the plasma membrane.Junctions between the surface or transverse tubule membrane and the endoplasmic, or sarcoplasmic, reticulum of muscle are thought to mediate excitation contraction coupling (12,34,39). Junctions between the surface membrane and regions of endoplasmic reticulum (ER), known as subsurface cisterns, are also present in neurons (38). Their similarity to the junctions in muscle has been noted (27, 38), but the functions of subsurface cisterns in neurons are unknown. We have used the freeze-fracture technique to compare the structure of the membranes participating in these junctions in striated muscle from the chordate, amphioxus, to their structure in neurons of the mammalian cerebellar cortex. The morphologic similarities apparent in thin sections are paralleled by similarities in the arrays of membrane particles revealed by freeze-fracturing the membranes at these junctions. These further similarities lend support to the suggestion (19,20,38) that subsurface cistern junctions may function in neurons, as in muscle, to couple excitatory events at the surface membrane with intracellular activities. A preliminary report of our results has been presented (25). MATERIALS AND METHODSAmphioxus Branchiostoma californiensis were obtained from Pacific Bio-marine Supply Co., Venice, Calif., and maintained in cold natural seawater for short periods

SUMMARY1. The electrical properties of the cell membrane of clonal cytotoxic T lymphocytes in the mouse were studied by using the whole cell variation of the patch electrode voltage-clamp technique.2. Outward currents were activated with an exponential time course of several milliseconds time constant when the membrane potential was made more positive than -50 to -40 mV. This current is not activated as a result of Ca2+ entry.3. The estimated reversal potential of the current indicates that the current is predominantly carried by K+. The activation kinetics depend only on membrane potential, not on [K+]O.4. The amplitude of the current decreases exponentially with time constants of several hundred milliseconds during a maintained voltage pulse, due mainly to a decrease in conductance.5. Recovery from inactivation roughly followed a single exponential time course with a time constant of tens of seconds; this time constant depended upon not only the membrane potential but also the amount of initial inactivation.6. The current is suppressed by quinidine and tetraethylammonium, their halfsuppression concentrations being 23 /LM and 14 mm respectively.

Purified cytoplasmic granules from cytotoxic rat large granular lymphocytes (LGL) tumors were cytolytic to erythrocytes, splenocytes, and a number of different lymphoid tumor cells. Granule concentrations of approximately 1 microgram/ml granule protein were adequate to lyse 100% of the erythrocytes, while the nucleated cells required up to 100 micrograms/ml granule protein to achieve complete lysis. Cytoplasmic granules purified from noncytotoxic lymphoid cells did not contain detectable cytolytic activity; purified granules from rat mast cells and rat liver lysosomes likewise failed to display cytolytic activity. However, granules prepared from normal rat peripheral blood LGL were cytolytic. Granule-mediated lysis of erythrocytes and nucleated cells was complete within 3 min at room temperature. The lytic activity required calcium at concentrations of 10(-4)-10(-2) M; magnesium or barium failed to replace calcium, while strontium could replace calcium at 10(-3)-10(-2) M when nucleated cells were the target. Exposure of LGL tumor granules to calcium before the addition of target cells resulted in an inactivation of granule cytolytic activity over the course of 20 min at room temperature. Granule cytolytic activity was heat and Pronase sensitive, and could be solubilized by 2 M salt. Examination of granules exposed to calcium in the electron microscope using negative staining showed that calcium treatment of granules results in the formation of ring-shaped structures previously described to be associated with LGL-mediated cytotoxicity. These results provide support for the hypothesis that the cytotoxic processes mediated by LGL are a secretory event characterized by the release of cytolytic material from the cytoplasmic granules after triggering by a surface receptor. The results further suggest that the ring structures visible in the electron microscope are associated with the lytic event.

Immunofluorescence microscopy reveals that both microtubule organizing center (MTOC) and Golgi apparatus are contained in the same perinuclear area of A549 cells in interphase. The cells display long microtubules stretching radially from the MTOC to the plasma membrane. Treatment of cells with taxol results in polymerization of microtubules without relation to the MTOC and formation of microtubule bundles predominantly localized in the cell periphery. After incubation with taxol, the Golgi apparatus is fragmented and is conspicuously present in areas of the cytoplasm enriched in microtubules. Incubation of cells with Colcemid results in complete depolymerization of microtubules and fragmentation of the Golgi into elements randomly distributed throughout the cytoplasm. Cells treated with taxol before being incubated with-Colcemid contain large numbers of Golgi-derived elements in close association with Colcemid-resistant microtubules. Microtubule depolymerization by vinblastine also is followed by fragmentation of the Golgi apparatus. These Golgi-derived elements show no association with the atypical polymers of tubulin induced by vinblastine. The codistribution of Golgi-derived elements with taxol-induced microtubule bundles can be reversed by microinjection of a monoclonal (YL 1/2) antibody reacting specifically with the tyrosylated form of a-tubulin.The processing of proteins that are synthesized in the endoplasmic reticulum before being secreted or sorted to the plasma membrane and cytoplasmic organelles takes place in the Golgi apparatus (for a review, see ref. 1). The Golgi apparatus consists of vesicles and several functionally related cysternae stacked in a defined order (1) organization and distribution of both microtubules and the Golgi apparatus were studied by dual indirect immunofluorescence microscopy as described (6). Cells were permeabilized by immersion in cold methanol (-20°C) for 2 min. The Golgi apparatus was studied by using a rabbit monospecific antibody raised against the Golgi enzyme ,B-galactosyltransferase and was a gift of E. G. Berger (7). Microtubules were studied by using the rat monoclonal antibody YL 1/2. Rhodamine-conjugated goat-anti-rabbit IgG and fluorescein-conjugated goat-anti-rat IgG were purified by affinity chromatography with, respectively, columns of rabbit IgG and rat IgG coupled to Sepharose 4B. These antibodies were used as second antibodies in indirect immunofluorescence microscopy studies. Microinjection of cells was performed as described (6, 8) with solutions of 20 mg of antibody per ml of phosphate-buffered saline (pH 7.0). The two rat monoclonal anti-a-tubulin antibodies, YL 1/2 and YOL 1/34, used in the microinjection experiments were provided by J. V. Kilmartin (9). Both antibodies, which were characterized as IgGs, were conjugated to fluorescein to study microtubules by direct immunofluorescence. YL 1/2 reacted specifically with the tyrosylated form of a-tubulin as shown by (i) immunoautoradiography of tyrosylated and detyrosylated a-tubulin resolve...

Axons were loaded with calcium, rapidly frozen, and freeze-substituted. The endoplasmic reticulum, in addition to mitochondria, contained calcium deposits, as indicated by electron probe x-ray microanalysis. Oxalate injected into living axons helped to preserve calcium-containing deposits during preparation for microscopy. It is concluded that the endoplasmic reticulum is a calcium-sequestering compartment in the squid giant axon.

Purified cytoplasmic granules from rat large granular lymphocyte tumors having natural killer activity and/or antibody-dependent cell-mediated cytotoxicity induced a rapid, dose-dependent release of the water-soluble marker carboxyfluorescein from liposomes made of phosphatidylcholine. A solubilized, partially purified cytolytic preparation termed "cytolysin" from these granules showed identical properties. Marker release induced by granules or the cytolysin was strongly dependent on the presence of Ca2+ at a concentration of 0.1 mM or higher in the medium; Ca2+ could be replaced by higher concentration of Sr2' but not by Ba2+ or by Mg2+. These properties strikingly parallel the lytic effects that granules and granule cytolysin exert on cells. Marker release from liposomes was stopped instantaneously when an excess of EGTA was added to the medium. The remaining carboxyfluorescein inside the liposomes was present at the original internal concentration, indicating that marker release was all-or-none from individual liposomes. (approximately 30 nm in diameter) containing carboxyfluorescein were prepared as previously described (10,11) by sonication in the presence of 80 mM carboxyfluorescein (pH 7.4), followed by elution from a Sephadex G-25 PD-10 column (Pharmacia) into 145 mM NaCl/10 mM Hepes, pH 7.4. REVs were prepared by reverse-phase evaporation according to Szoka and Papahadjopoulos (12) with 80 mM carboxyfluorescein (pH 7.4) in the aqueous phase. The vesicles were extruded through polycarbonate membranes (Nuclepore) with 0.4-,m and 0.2-,um pores under argon pressure.The vesicles were separated from nonencapsulated carboxyfluorescein on a PD-10 column as described above. REVs prepared in this way are a few tenths of a micrometer in diameter.Carboxyfluorescein Release. The assay for release makes use of the self-quenching properties of the highly water-soluble fluorescent dye carboxyfluorescein (10, 11), which was purified as described (13 5551The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Organelle translocation in a number of cell types in tissue culture as seen by high-resolution Zeiss-Nomarski differential interference contrast optics was filmed and analyzed by computer. Principal cell types studied included primary chick spinal cord, chick dorsal root ganglion, rat brain, and various clones of continuous cell lines. Organelle translocations in all cell types studied exhibited frequent, large changes in velocity during any one translocation. The appearance of particles as seen with Nomarski optics was correlated with their fine structures in one dorsal root ganglion neurite by fixing the cell as it was being filmed and obtaining electron micrographs of the region filmed. This revealed the identity of several organelles as well as the presence of abundant neurotubules but no neurofilaments. Primary cell cultures exhibited more high-velocity organelle movements than continuous cell lines. The net progress of an organelle in a given direction was greater in primary neuronal cells than in fibroblasts or continuous cell lines. These findings are correlated with the literature on organelle translocation and axoplasmic transport.The literature on axoplasmic transport is composed chiefly of findings made with indirect methods of measuring the rate of displacement of axoplasmic components. Such methods have included the measurement of transport of radioactive isotopes (19,20,25) or of specific cytochemical markers (7,22). While cytoplasmic streaming, motility, and saltatory particle motion in a number of non-neuronal cell systems have been extensively studied (28), microscopic analysis of particle or organelle transport in vertebrate neuronal tissues has been the subject of relatively few reports. Even more scarce are studies on this type of transport in dissociated neurons in tissue culture. The purpose of this report is to provide quantitative data on organelle transport in several types of vertebrate neurons in tissue culture and to relate these data to current hypotheses of organelle translocation. Cultured cell types studied included chick embryo spinal cord, chick embryo dorsal root ganglion (DRG) cells, rat embryo brain cells, various clones of neuroblastoma, L cells, HeLa cells, and mouse embryo fibroblasts. MATERIALS AND METHODS Cell CulturesAll cultures were grown on round 25-mm diameter no. l lA Corning no. 2910 lead oxide-free cover glasses in Div. of Bioquest,