Showing papers in "Journal of Cell Science in 1981"

Ciliary activity of cultured rabbit tracheal epithelium: beat pattern and metachrony.

Abstract: The beat pattern of rabbit tracheal cilia has been investigated using high-speed cine photography and scanning electron microscopy, on cultured epithelia of known orientation. The cilia normally rest in the position reached at the end of the effective stroke, the ciliary tips pointing towards the oropharynx. Each beat begins with a recovery (or preparative) stroke in which a bend is propagated up the cilium causing the cilium to rotate backwards in a clockwise sweep, as viewed from above. At the end of its recovery stroke the cilium progresses immediately into the effective (or power) stroke, which is almost planar and in a cephalad direction. The active cilium describes an arc of almost 110 degrees before reaching the rest stage. This beat pattern is not significantly altered over an increase in frequency from 13-29 Hz; the relative duration of the 2 active phases of the beat remain similar over this range. Metachronal waves exist in the form of short erratic areas of coordinated beating which travel only short distances. Within each area, the non-planar recovery strokes initiate an antilaeoplectic wave of activity which recruits inactive cilia to extent the wave. As cilia perform their effective strokes, adjacent cilia in the plane of beating move in an antiplectic sequence. This pattern of coordination is related to the pattern of beat of the cilia and their distribution on the epithelium.

Ultrastructural analysis of hyphal tip cell growth in fungi: Spitzenkörper, cytoskeleton and endomembranes after freeze-substitution.

Abstract: The ultrastructure of freeze-substituted tip cells of Fusarium acuminatum was analysed by conventional and high-voltage transmission electron microscopy (HVEM). At least 2 morphologically distinct types of Golgi-like endomembrane cisternae were observed, each existing as single, fenestrated sheets and tubular elements that were often very closely associated with mitochondria. From HVEM observations of thick (0.25 and 0.5 micron) sections, the Spitzenkorper appeared to correspond to an apical mass of vesicles. A network of microfilaments was identified among component vesicles of the Spitzenkorper and adjacent to developing septa. Microtubules were oriented primarily parallel to the direction of hyphal growth and were located in all areas of the cytoplasm, including the tip cell apex. Cytoplasmic vesicles were closely associated with these microtubules. From these observations it is suggested that cytoskeletal elements play important roles in localized cell wall formation. The filasome, a previously unreported type of coated vesicle in fungi, might also be involved in wall synthesis.

Expression of intermediate filaments in cultured cells

Abstract: The occurrence of different types of intermediate filaments in primary cultures of cells and in cultured cell lines was studied by the indirect immunofluorescence (IFL) technique. The antibodies used were spontaneous monoclonal human antibodies of immunoglobulin M (IgM) class against vimentin-type intermediate filaments (fibroblast 58 x 10 mol. wt subunit protein), and experimental rabbit antibodies of IgG class against vimentin, desmin (muscle 55 x io mol. wt subunit protein), glial fibrillary acidic protein (GFA), 68 x io mol. wt neurofilament polypeptide and human keratin polypeptides. Cultured fibroblasts from different species, and both aortic and venous endothelial cells, showed a fibrillar cytoplasmic fluorescence when stained with antibodies against vimentin. On the other hand, in cultures of chicken embryonal fibroblasts, cells showing a bright desminspecific fluorescence, but lacking vimentin-specific staining, were seen even after several subcultivations. The presence of both desmin and vimentin polypeptides in these cultures was also confirmed by polyacrylamide gel electrophoresis. In chicken fibroblast cultures the 2 types of intermediate filaments were not expressed simultaneously in individual cells, whereas in baby hamster kidney (BHK-21) cells and human rhabdomyosarcoma cells a variable co-staining with anti-vimentin and anti-desmin antibodies could be seen. In contrast, cultured human fibrosarcoma cells and simian virus 40-transformed human fibroblasts showed only vimentinspecific fibrillar fluorescence. Glial cells from mouse embryonic spinal cord appeared to express only GFA-containing intermediate filaments in a primary culture, whereas both subcultured mouse glial cells and a cultured glioma cell-line also showed vimentin-specific staining. On the other hand, neuronelike cells in the primary cultures could only be stained with the antibodies to 68 x io mol. wt neurofilament polypeptide. Interestingly, clones ofmouseneuroblastoma(Ci 300) cells contained

Anomalous preferences of cultured macrophages for hydrophobic and roughened substrata.

Abstract: Peritoneal macrophages were plated out on a series of artificial haptotactic substrata consisting of grid patterns of vacuum-evaporated palladium metal alternating with hydrophobic untreated polystyrene and with hydrophilic sulphonated polystyrene. By their active locomotion the macrophages accumulated preferentially onto the less hydrophilic of either pair of these alternative substrata. This order of substratum preference is precisely the opposite to that shown by fibroblastic cells. Macrophages were also found to accumulate preferentially onto roughened culture surfaces as opposed to smooth ones, which is again opposite to the behaviour of fibroblasts. These opposite substratum preferences shown by macrophages may reflect physical as well as functional differences in their surfaces, and could serve as assay criteria for macrophages and cell types putatively equivalent to macrophages.

Contraction and organization of collagen gels by cells cultured from periodontal ligament, gingiva and bone suggest functional differences between cell types.

Abstract: Monkey periodontal ligament fibroblasts (MPLF cells), human gingival fibroblasts (HGF cells), rat embryonic calvaria cells (REC cells), porcine periodontal ligament epithelial cells (PPLE cells) and rat osteosarcoma 17/2 cells (ROS cells) were incorporated into 3-dimensional collagen gels plated in 60 mm Petri dishes in order: first, to measure the capacity of these cell types to contract; second, to investigate cell-collagen and intercellular relationships during contraction; and third, to define the cellular contribution to tissue contraction in an in vitro system. Measurements at times up to 72 h on 3 ml gels containing 5 x 10(5) cells and with a collagen concentration of 1.20 mg/ml showed that MPLF cells contracted the gels at a significantly greater rate (P less than 0.001) than did the other cell types. In addition, contraction started sooner and was of greater extent than with the other cells. HGF cells contracted the gels more rapidly than REC and PPLE cells, while ROS cells caused no contraction. Several stages of gel compaction could be defined: (1) the attachment of cells to collagen; (2) cellular spreading within the collagen fibre matrix; (3) organization and alignment of collagen fibres by cell processes; (4) cell migration; (5) establishment of intercellular contacts; and (6) the development of a cellular reticular arrangement within the gel and the extension of this arrangement into a 3-dimensional, tissue-like, honeycomb network. Electron microscopic observations on 0.1 ml gels containing MPLF cells showed that, in the early contractile phase, numerous cell processes attached to or enclosed collagen fibrils. These processes contained microfilamentous material and few organelles. In compacted gels, the cells contained an increased amount of distended rough endoplasmic reticulum and Golgi membranes. Since MPLF cells have the capacity for vigorous contraction of the collagen gels and since they develop a reticular, 3-dimensional structure in compacted gels that is reminiscent of the relationship of periodontal ligament fibroblasts to collagen fibres in vivo, it is suggested that they could provide the major force necessary for tooth eruption in vivo. This system also provides a well-defined in vitro model to study the sequential stages that occur during contraction processes.

Chemotactic reorientation of granulocytes stimulated with micropipettes containing fMet-Leu-Phe.

Abstract: Human granulocytes were stimulated by means of a micropipette, with an orifice of about 0.2 micrometer in diameter, which contained fMet-Leu-Phe at a concentration of 10(-5) M. The cells were reorientated by extending lamellipodia towards the source of the attractant, often within less than 10 s. Any part of the granulocyte, from the front to the tip of the tail, could be stimulated to produce new lamellipodia. Usually, but not always, this response occurred at the side of the cell nearest to the micropipette. Cells stimulated from behind responded in one of the following ways: (1) Cells that maintained their polarity extended new lamellipodia at one side of the leading front and reorientated by moving in a U-turn towards the micropipette. Occasionally, the leading front was split because one part of the front tried to make a left-hand and the other a right-hand turn. (2) Formation of lamellipodia at the leading front was arrested and new lamellipodia were formed at the tail instead, indicating reversal of polarity. The result was an immediate change in the direction of locomotion by about 180 degrees. (3) A combination of the first 2 forms of behaviour was observed occasionally. Transiently, lamellipodia were extended from cell surface areas both close to and distant from the micropipette. These observations show that parts of a cell can respond independently to chemotactic gradients by extending lamellipodia towards the source of the attractant. The phenomenon can easily be explained by assuming that a temporal change of attractant concentration is recognized.

The role of the cytoskeleton in the motility of coccidian sporozoites

Abstract: The sporozoites of Eimeria tenella and Eimeria acervulina show bending, pivoting and gliding motility. All these types of motility occur intermittently and with decreasing frequency during the life of a sporozoite. Gliding is the only locomotive action expressed by these sporozoites and is only seen when the sporozoites are in contact with the substratum. All gliding sporozoites adopt a set pattern of body 'attitudes', which suggests that locomotion involves a fixed body shape. The microtubule inhibitors, colchicine, griseofulvin, vinblastine sulphate and nocodazole, have no effect on sporozoite motility. Ultrastructural examination reveals, in addition, that they have no effect on the subpellicular microtubules. The microfilament inhibitor, cytochalasin B, completely, and reversibly, inhibits pivoting and gliding but bending is only slightly depressed by the drug. High magnesium ion concentration inhibits all motility completely. The cell membrane was readily labelled with fluorescein isothiocyanate-conjugated cationized ferritin, the label was rapidly capped and shed from the posterior of the sporozoite. This capping reaction takes place only during sporozoite locomotion. The membrane label was seen to 'move' backwards realtive to the sporozoite at the same rate as the sporozoite moved forwards relative to the substratum. The substratum and the leading edge of the cap remained static relative to each other. Both capping and locomotion are sensitive to low temperature and cytochalasin B. From these results a theory of sporozoite motility is postulated. The sporozoites adhere to the substratum by surface ligands. This ligand/substratum complex is then capped along the fixed spiral of the sporozoite body by a microfilament-based contractile system. This proposed model for motility of coccidia sporozoites is consistent with all current observations on cell invasion by the sporozoa and therefore suggests that locomotion is an integral component of host cell invasion in this group of parasites.

Determination of secretory vesicle production rates by dictyosomes in pollen tubes of Tradescantia using cytochalasin D.

Abstract: Pollen tubes of Tradescantia were grown in vitro and exposed to 0.3 microgram/ml cytochalasin D for 5 or 10 min. Fine-structural observations revealed no visible effect of the drug on the organelles. Stereological analysis, using a method recently developed by Rose (1980) to obtain sphere size-distributions corrected for section thickness, revealed substantial increase in the number of secretory vesicles present in the cytoplasm around the dictyosomes. Equating the rate of vesicle accumulation with the rate of vesicle production, a total of 5388 vesicles per minute are formed by a growing tube. This corresponds to 2.4 vesicles per minute per dictyosome, and a turnover rate of 3.7 min for a single dictyosome cisterna, or about 15-18.5 min for a complete dictyosome. The calculated vesicle production rate agrees well with that required to sustain the observed growth rate of such tubes, based on the addition of membrane or wall material to the tube tip.

Inhibition of leukocyte locomotion by hyaluronic acid

Abstract: The effect of hyaluronate on neutrophil motility in vitro was studied by the micropore filter technique and by direct visual analysis of the locomotion of neutrophils on glass. Both directed and random locomotion of neutrophils was inhibited by physiological concentrations (0.5-6.0 mg ml(-1)) of hyaluronate in a dose- and molecular weight-dependent manner. Inhibition of cell movement was more pronounced for high molecular weight chemoattractants such as casein than for small chemotactic peptides such as f-Met-Leu-Phe. Chemotactic factor gradient formation in filter chambers was profoundly retarded by hyaluronate, which may partly explain the inhibitory effects of hyaluronate on directed neutrophil locomotion. In addition, hyaluronate inhibited the binding of chemotactic factor to the neutrophil surface. This effect, together with a reduction in cell-to-substratum adhesion, may provide an additional explanation for hyaluronate-induced inhibition of random neutrophil locomotion. Inhibition of locomotion by hyaluronate was easily reversed by washing the cells free of hyaluronate; thus competition by hyaluronate for cell-surface binding sites is unlikely, and physical effects such as steric exclusion or molecular sieving by the large hyaluronate polymer provide the most probable explanations of its inhibitory effect on cell locomotion. Since hyaluronate is a major constituent of tissue matrices, these results draw attention to the importance of the extracellular environment in regulating inflammatory cell movement in vivo.

Variability in individual cell cycles of Saccharomyces cerevisiae.

Abstract: The kinetics of cell proliferation of Saccharomyces cerevisiae were studied at 4 growth rates using time-lapse cinephotomicrography. Cells were grown on media with a high refractive index to reveal greater intracellular detail under the phase-contrast microscope. The morphological cell-cycle events scored were: bud emergence, nuclear migration, nuclear division, onset of cytokinesis and cell separation. Cell size was measured at cell separation and at bud emergence. The daughter-cycle time was always longer than the parent-cycle time mainly due to the large difference in the lengths of the unbudded phases. Parent cells had a shorter budded period than daughter cells. The large variance in daughter-cycle times was accounted for by the large variance in the lengths of the unbudded phase of daughter cells. The duration and variability of the periods in the cyclc from nuclear migration onwards were equivalent for parent and daughter cells. Daughter cells were always smaller than parent cells at division. There was wide variation in cell size at both division and bud emergence. The results indicated that a modified deterministic model could best explain cell proliferation kinetics in yeast. The data were used to evaluate 2 different models. The 'sloppy size control' model of Wheals (1981 a) was more consistent with the data than the 'tandem' model of Shilo, Shilo & Simchen (1976). The distribution of unbudded periods of daughter cells suggested that there was an additional incompressible period not present in parent cells.

Pollen tube development in Petunia hybrida following compatible and incompatible intraspecific matings.

Abstract: Pollen tubes formed following compatible and incompatible intraspecific matings in Petunia have been examined with light and electron microscopes. Compatible and incompatible tubes develop in an identical fashion on the stigma but, on entry into the top 1 mm of the stylar transmitting tissue changes occur both to the cytology of the tubes and their rates of growth. The early cytological changes are common to tubes of both compatibilities but, although both types of tube accelerate on entry into the style, incompatible tubes grow more slowly than compatible. Cytological differences became apparent between compatible and incompatible tubes following a short period of growth in the style, the latter possessing thicker cell walls and a cytoplasm packed with both organelles and reserves. Incompatible tubes subsequently burst or simply cease growth and die. The characteristic image afforded by this cytoplasm resembles that or burst or dead compatible tubes, except in that proportions of the cell components may differ. These data are discussed in terms of current models proposed to explain pollen tube growth and the operation of the self-incompatibility response in Petunia.

Sequential alterations in the nuclear chromatin region during mitosis of the fission yeast Schizosaccharomyces pombe: video fluorescence microscopy of synchronously growing wild-type and cold-sensitive cdc mutants by using a DNA-binding fluorescent probe.

Abstract: Video-connected fluorescence microscopy was introduced to study the yeast nuclear chromatin region. It was defined as the nuclear area where a DNA-binding fluorescent probe 4′,6-diamidino-2-phenylindole specifically bound and fluoresced. The 3-dimensional feature of the mitotic chromatin region was deduced by analysing the successive video images of a cell viewed at different angles. By investigating synchronous culture of the wild-type fission yeast Schizosaccharomyces pombe, we found sequential structural alterations in the chromatin region during mitosis. The steps found include the compaction of the chromatin region from the regular hemispherical form, the formation of a U-shaped intermediate and the rapid segregation into 2 daughter hemispherical forms. Six cs cdc mutants, apparently blocked in mitosis, were observed by fluorescence microscopy. Under the restrictive conditions their chromatin regions exhibited either hemispherical, compact, disk-like, U-shaped or partially segregated chromatin regions. Two mutants showed anomalous nuclear locations. The results of the temperature shift-up experiments of the highly reversible KM52 and KM108 strains supported the above scheme of sequential alterations in the chromatin region.

Fibroblast adhesion on collagen substrata in the presence and absence of plasma fibronectin.

Abstract: We have used scanning electron microscopy and transmission electron microscopy to compare the initial adhesion of baby hamster kidney (BHK) cells and early passage human skin fibroblasts on glass, dried collagen, and hydrated collagen substrata. On glass or dried collagen substrata, BHK cell spreading required fibronectin and the cells were extensively flattened, with broad lamellipodia. In marked contrast, BHK cell spreading on hydrated collagen substrata did not require fibronectin and the cell bodies tended to be rounded, with one or several large filipodia. These filipodia generally penetrated into collagen lattice where they interwove with the collagen fibrils. Proteolysis of the collagen was not evident. In the presence of fibronectin, the morphology of BHK cell spreading on hydrated collagen substrata was similar, except that there was an increase in the number of cells that spread with lamellipodia that did not penetrate the collagen lattice. Human fibroblasts also demonstrated marked differences in cell spreading on glass or dried collagen compared to hydrated collagen substrata. Spreading was characterized by lamellipodia on glass or dried collagen but by multiple filipodia on hydrated collagen. In the latter case, the filipodia penetrated just beneath the surface of the collagen lattice. Added fibronectin was not required for spreading on human fibroblasts on any of the substrata and the presence of added fibronectin had no effect on cell morphology. Finally, the human fibroblasts were observed to form adhesion plaques in some cases where the fibroblasts made close contacts with individual collagen fibrils. The results are discussed in terms of connective tissue organization.

Isolation of the cytoskeleton from Giardia. Tubulin and a low-molecular-weight protein associated with microribbon structures

Abstract: The sucking disk of Giardia is supported by a large, plate-like organelle: the ventral disk cytoskeleton. Extraction by Triton-X 100 of Giardia trophozoites from the mouse gut, or of G. duodenalis or G. lamblia grown from cultures, yields cell-free disk cytoskeletons. Up to 8 flagellar axonemes may be attached to an isolated disk. Disks are seen in the electron microscope to be composed of concentrically coiled microtubules bonded to microribbons. Microribbons are large, laminated structures, linked by dense networks of crossbridges. They are made up of regularly arranged subunits. Microtubules and microribbons are preserved in Triton for long periods, but crossbridges are slowly dissolved. Whereas the addition of ATP causes axonemes to resume bending, active movements were not detected in disks. It seems more likely that a disk is a passive effector, which may be acted upon by other contractile structures of the cytoplasm. It is highly specialized and quintessential cytoskeleton. Disks and axonemes will dissolve in sodium dodecyl sulphate (SDS), and after SDS-gel electrophoresis 2 prominent bands are apparent. One, corresponding to tubulin, migrates in low ionic strength, high pH buffers as 2 closely spaced bands of equal staining density. The other, a smaller protein, is a complex polypeptide band of molecular weight 30 000 Daltons. Allowing for staining differences, the 2 proteins are probably present in cytoskeletons in roughly equal amounts. Because of their size microribbons account for at least 75% of the structured material stained by electron stains in pellets of cytoskeletons. Disk and axoneme microtubules comprise the minority fraction. This result suggests that, like microtubules, microribbons are a source of structural tubulin and probably also contain the 30 000 mol. wt protein.

The effects of fibronectin on the migration of human foreskin fibroblasts and Syrian hamster melanoma cells into three-dimensional gels of native collagen fibres.

Abstract: The effects of fibronectin on the migration of human skin fibroblasts and Syrian hamster melanoma cells into 3-dimensional gels of native collagen fibres have been examined. Cell migration into the 3-dimensional gel was measured by plating cells on the gel surface and then determining the percentage of cells within the gel at various times thereafter by direct microscopic examination. We find that fibronectin bound to collagen inhibits the migration of human skin fibroblasts and stimulates the migration of melanoma cells into the gel matrix. Fibronectin had no apparent effect on cell adhesion to the collagen gels, proliferation or morphology under the conditions studied.

Effect of hyaluronic acid on neutrophil adhesion

Abstract: The effects of hyaluronate on rabbit neutrophil adhesion were studied using a variety of techniques. Exogenous hyaluronate inhibited neutrophil aggregation under conditions of both turbulent flow and constant shear rate. Hyaluronate also inhibited neutrophil adhesion to glass. Inhibition was dose-dependent above 100 micrograms ml-1 and a minimum molecular weight for hyaluronate of 1 x 10(4) was required. These effects were not simply the result of increased bulk viscosity of the hyaluronate-containing medium, nor did they appear to be mediated by putative cell-surface receptor mechanisms. Instead, physical factors such as hindrance and/or changes in the interfacial free-energy exchange at the cell surface due to the unusual hydrodynamic properties of the hyaluronate molecule were considered to be more important. Since neutrophil migration in vivo occurs through hyaluronate-rich connective tissue matrices, the relevance of these findings for processes such as inflammation and wound healing is clear.

The movement of cell clusters in vitro: morphology and directionality

Abstract: The movement of cells in small groups, or clusters, was studied in vitro using epithelioid cells from Gordon-Kosswig melanomas (from poecelid fish) and time-lapse cinemicrography. Tumour explants cultured on glass yield cell sheets from which groups of cells separate and become independently motile clusters. These clusters typically contain 3-30 cells, but may have as many as 50. They propel themselves at speeds of 0.2-4.0 micrometer/min by means of broad hyaline lamellae. The distribution of lamellae around the perimeter of each cluster correlates with both direction and speed of cluster movement, i.e. a cluster moves with its most lamellar region at its leading edge, and the greater the extent of the leading lamellar region the greater the speed. Also, a cluster tends to keep moving in the same direction. This persistence is due to a relatively constant distribution of lamellae. Cells on the trailing edge usually lack lamellae and most are very elongate and oriented perpendicular to the direction of cluster movement. In general, whenever a cell elongates, there is a loss of lamellar activity along its taut edges, parallel to the axis of elongation. Thus, any region with less lamellar activity would tend to be elongated by the outward pull of the more active regions to either side and would, in consequence, suffer a further reduction in lamellar activity. In this way, the distribution of regions of lamellar activity is self-reinforcing and the result is persistence of movement in a particular direction. This phenomenon could play an important role in giving directionality to certain morphogenetic movements, such as neural crest cell migration.

Locomotion of Xenopus epidermis cells in primary culture

Abstract: The locomotion of single epidermis cells, grown out from Xenopus laevis tadpole tails has been investigated by time-lapse cinemicrography using phase-contrast and reflection-contrast optics. The cells develop a large, mostly 200-250 nm thick, lamella, which adheres homogeneously to the supporting coverglass and exceeds the projection area of the cell body. From the comparison of RIC-pictures taken at high (1.06) and low (0.62) numerical aperture of illumination (I.N.A.) we deduce that at low I.N.A. the embossment of the medium-facing side of the lamella is visualized. By this method microcolliculi are demonstrated, which form at the edge of the lamellipodium and move backward. They resemble ruffles, but are flatter and no membrane flow towards the perinuclear region is observed. Indirect immunofluorescence reveals an enhanced staining for actin and alpha-actinin in the lamellipodium and in the transition region of cell body and lamella. Tonofilaments do not participate in lamella formation, the relatively few microtubules seem to be oriented in the direction of cytoplasmic flow. Electron micrographs demonstrate the course of fibrils in the cell body and a meshwork of actin filaments and membranous tubules in the lamella. Based on these findings a model for cell locomotion is presented: the motive force is generated by the cell body causing a flow of cytoplasm towards the periphery and extension of the lamella at its edge. The activity of the lamellipodium has to ensure the flat form of the advanced edge; microcolliculi are assumed to represent a small membrane store for the extension of the lamella. The lamellipodium is not involved in the production of motive force. The cell body is anchored to the lamella by radiating fibrils and the fibrillar meshwork is inserted at the 'dorsal' membrane of the lamella and the basal filament cortex of the cell body. This anchorage provides the structural basis for the uptake of lamella material into the cell body in the transition region.

Ultrastructural organization, sites of transcription and distribution of fibrillar centres in the nucleolus of the mouse oocyte.

Abstract: The emergence of newly formed nucleoli and their development have been studied in mouse oocytes from pachytene to diplotene stages. At mid-pachytene, the nucleolus first appears as a fibrillar centre surrounded by a layer of electron-dense fibrils and penetrated by chromatin fibres emanating from the secondary constriction region of the nucleolar bivalent. Since this bivalent contains 2 paired nucleolar organizers, 2 nucleoli are formed in a symmetrical fashion. At advanced pachytene, the nucleoli are extended by strands of fibrillar component which become fibrillogranular distally. The 2 nucleoli fuse together at late pachytene. At diplotene, the nucleolus becomes large and reticulated. The development of the nucleolonema coincides with the appearance of numerous secondary fibrillar centres. Three-dimensional reconstruction of the reticulated nucleolus shows that the number of fibrillar centres largely exceeds that of nucleolar organizers. Radioautography after [3H]uridine incorporation demonstrates that during the first step of nucleologenesis the labelling is limited to the layer of electron-dense fibrils surrounding the fibrillar centre. Study of the time course of tritiated uridine incorporation from pachytene to diplotene shows that the labelling extends with the extending strands of fibrillar component. In the fully developed nucleolus, all fibrillar strands are labelled and contain, therefore, actively transcribed rDNA. These observations suggest that the rDNA, which is initially compacted in the primary fibrillar centre at the onset of nucleogenesis, progressively unravels and becomes distributed throughout the fibrillar parts of the nucleolonema. The lack of labelling of the secondary fibrillar centres suggests that they are zones of inactivity of the ribosomal genes where the rDNA remains locally compacted. A model of the ultrastructural organization of the nucleolus is proposed based on our observations.

Calcium requirement for the secretion of peroxidases by plant cell suspensions

Abstract: Spinach (Spinacia oleracea, L.) cells in liquid culture release peroxidases. This release is reduced by EGTA and promoted by calcium ions. In a medium deprived of calcium the rate of peroxidase release is low, but immediately after addition of I mM calcium there is a sudden increase of the extracellular peroxidase activity. Extracellular calcium apparently penetrates into the cultured cells rather freely and, as a consequence, the rate of peroxidase secretion by these cells is directly correlated with the concentration of calcium in the medium. Magnesium, at twice the concentration used for calcium, has no effect on the release of peroxidases. Cells treated with Na azide, Na hydrogenarsenate of fluphenazine secrete less peroxidase upon addition of calcium.

Hordeum and secale mitotic genomes lie apart in a hybrid

Abstract: In both unpretreated root tip metaphases and pretreated mitoses of Hordeum vulgare L. cv. Sultan x Secale africanum Stapf F1 hybrids, Hordeum chromosomes tended to be nearer the centre of the mitosis than Secale chromosomes. This was clear in 4 serially sectioned cells examined in the electron microscope. In Feulgen squashes of 38 of 40 cells studied in the light microscope, the mean distances in each cell from the mean centromere position for the cell was less for Hordeum centromeres than for Scale centromeres. Such spatial separation of parent genomes might prevent pairing of homoeologues in hybrids.

The interpretation of interference- reflection images of spread cells: significant contributions from thin peripheral cytoplasm

Abstract: In interference-reflection microscopy, used for investigating cell-substratum separation, it is commonly believed that cytoplasmic thickness can be ignored, provided a high illuminating numerical aperture (INA) is used. It is shown here that even when a maximal INA is used, cytoplasmic lamellae of I micrometer or less can be major determinants of the image. The leading lamella of spreading tissue cells and large peripheral areas of Dictyostelium discoideum amoebae on adhesive substrata are less than I micrometer thick and it is argued that hitherto unexplained features of the interference images of these cells may be interpreted in terms of the theory used here.

Nuclear lamina assembly, synthesis and disaggregation during the cell cycle in synchronized HeLa cells.

Abstract: The pattern of assembly, synthesis and disaggregation of the nuclear envelope-associated lamina in synchronized HeLa cells has been examined by means of immunofluorescence microscopy of cell preparations treated with antibody to lamina polypeptide LP67 or lamin B, using the nomenclature of Gerace and Blobel. During the cell cycle the distribution of lamina polypeptide varies dramatically. Three distinct stages can be detected with respect to its location and synthesis: (1) Lamina polypeptide is cytoplasmically distributed during cell division particularly at the time of complete nuclear envelope breakdown. At telophase it is reassembled and becomes associated with regions of the chromosome surface. This process continues into early Glt lamina polypeptide associating progressively with the outer surface of the chromosomes; simultaneously its cytoplasmic concentration is reduced. The traffic of antigen from cytoplasm to the chromosomes is highest between telophase and Gt but continues at a reduced rate during G1. Inhibition of protein synthesis during division and G^ does not inhibit G1 lamina reformation, suggesting that the Gj lamina is constructed from depolymerized cytoplasmically-stored polypeptide derived from the previous cell cycle. (2) Lamin B is synthesized in S-phase. The protein is rapidly accumulated at the nuclear periphery during the doubling of the nuclear surface. There is very little if any cytoplasmic location of lamina polypeptide in

Surface changes during retraction-induced spreading of fibroblasts

Abstract: Retraction of the trailing edge of an embryonic chick heart fibroblast results in an abrupt increase in protrusive activity at the leading edge of the cell. This increase was studied with time-lapse cinemicrography and scanning electron microscopy. Increased spreading following retraction results primarily from an increase in the duration of the extension phase of lamellipodial spreading. Much ruffling accompanies this increased spreading, particularly during its earliest phase. Upon retraction of the trailing edge, folds appear on the surface of the retracted tail and adjacent cell body and, soon after, microvilli-like structures appear as well. Once the moving cell has fully respread, however, the upper surface is once again smooth and free of folds and microvilli. Artificial detachment of a spreading lamella with a microneedle, and its consequent retraction, also causes increased protrusive activity of the remaining lamellae of the cell. These results are consistent with the hypothesis that retraction of one part of the cell makes surface membrane and cytoplasm available for forming protrusions elsewhere.

Influence of molecular charge upon the endocytosis and intracellular fate of peroxidase activity in cultured arterial endothelium.

Abstract: The molecular charge of the macromolecule, horseradish peroxidase (HRPase, 40 000 mol. wt), was modified to yield highly anionic (PI less than 3.68) and cationic (PI = 9.5-10.5) derivatives. The effects upon the interactions between HRPase and arterial endothelium were then studied in vitro. The net rate of uptake of HRPase into endocytic vesicles and vacuoles of confluent endothelium was influenced by its molecular charge, there being less internalization of the anionic HRPase than of the native (pI = 7.9-8.2) and cationic derivatives. The molecular diameter was not significantly different between the cationic (Ae = 28.8 A), anionic (Ae = 31.2 A) and native (Ae = 29.6 A) HRPase. The rate of uptake of [U-14C]sucrose, a tracer of bulk fluid endocytosis, was unaffected by the presence of the differently charged HRPase, indicating that the volume of vesicles formed per cell per hour remained constant. The intracellular fate of HRPase of different charge was investigated biochemically and morphologically. The rate of loss of internalized HRPase activity in the endothelial cells approximated first-order kinetics. The rate of disappearance of intracellular HRPase activity was much greater for cationic (t1/2 = 8 h) and native (t1/2 = I 8 h) than for anionic HRPase (t1/2 = 80-100 h). By electron microscopy, all 3 forms of HRPase were restricted to intracellular membrane-bounded vesicles and vacuoles consistent with a vesicle-lysosomal pathway. Studies with purified lysosomal cathepsin D indicated that the differences in the intracellular half-lives of HRPase may be attributable in small part to decreased and increased rates of lysosomal proteolysis of anionic and cationic HRPase, respectively, in comparison with native HRPase. Pre-labelling of endothelial secondary lysosomes by inhibitors of phagosome-lysosome fusion (dextran sulphate, polyglutamate) lengthened the intracellular half-life of native HRPase, while introduction of cationic ferritin to cells pulsed with anionic HRPase greatly decreased its half-life. Thus an influence of molecular charge upon endosome-lysosome fusion cannot be excluded. The studies indicate that the net charge carried by exogenous HRPase influences both its internalization in endocytic vesicles and its subsequent intracellular fate, which in turn may be modified by the introduction of other differently charged macromolecules. These results are discussed in relation to macromolecular transport by vascular endothelium in vivo.

Recovery of sliding ability in arm-depleted flagellar axonemes after recombination with extracted dynein I.

Abstract: We compared sliding velocity between outer doublet tubules in demembranated axonemes of sea-urchin (Pseudocentrotus depressus) sperm flagella with that of arm-depleted axonemes recombined with extracted dynein I. The outer arm-depleted axonemes after extraction with 0.5 M NaCl had a velocity of 6.9 +/− I.0 micrometer/s, while the intact axonemes had a velocity of 14.3 +/− I.5 micrometer/s in the presence of I mM ATP and 2 microgram/ml trypsin at 25 degrees C. The sliding velocity was closely related to the number of remaining outer arms following the NaCl-extraction process. When the outer arm-depleted axonemes were recombined with dynein I, the sliding velocity increased to 11.3 +/− 1.3 micrometer/s. Electron microscopy confirmed the recovery of 94% of outer arms in the axonemes. After extraction with Tris-EDTA solution for 10 min, the axonemes lost their sliding ability completely, even in the presence of ATP and trypsin. Such axonemes lacked most of both inner and outer arms, although sometimes the basal segment of the arms appeared to remain. When the exogenous dynein I fraction extracted from other axonemes was added, the axonemes could extrude tubules, and both types of arms reappeared clearly and distinctly in the axonemes. The recombined axonemes with one-fold stoichiometric excess of dynein I recovered 58% of the total number of arms and had a velocity of 7.4 +/− 1.6 micrometer/s. Those with 2-fold stoichiometric excess had a velocity of 11.0 +/− 1.5 micrometer/s, up to 82% of the arms in these axonemes being restored. These results indicated that the exogenous dynein I fraction derived from the outer arms restored sliding ability to arm-depleted axonemes, recombining with th outer doublet tubules as inner and outer arms, and that the sliding velocity had a close relationship to the total number of arms in the axonemes, irrespective of their being inner or outer arms.

Control of cell shape by calcium in the euglenophyceae

Abstract: The euglenoid flagellates are able to change their shape rapidly in response to a variety of stimuli, or sometimes spontaneously. Two extremes of shape can be identified: the “relaxed” form is cylindrical; the contracted form is a somewhat distorted disc. These 2 forms can be interconverted by treatments that alter the Ca2+ concentration of the entire cell. The level of Ca2+ is believed to be normally controlled by a system of calcium-accumulating membranes, identified in Astasia longa by the technique of calcium oxalate precipitation. The system forms a set of parallel tubes of endoplasmic reticulum, one of which lies immediately below each of the ridges of the pellicle. The individual ridges, each with its associated reticulum, microtubules and other elements are suggested to be independent motor units. Local activation of a small number of these units by Ca2+ is made possible by the arrangement of Ca2+ -sequestering reticulum, producing the characteristic squirming euglenoid movement. Uniform activation or suppression of all units produces the 2 extremes of shape. The pellicle of A. longa with its associated microtubules has been purified and shown to contain a Ca2+ -binding site and ATPase activity.

Arrangement of subunits in microribbons from Giardia.

Abstract: Ultrasound has been used to disperse the cytoplasm of Giardia muris and Giardia duodenalis trophozoites, releasing disk cytoskeletons for negative staining and study by electron microscopy. Sonication also breaks down the corss-bridges uniting microribbons in disks. Individual ribbons and small bundles of these structures, are found in these preparations and have been imaged both from their edges and in flat face view. The outer layers of ribbons are 2 sheets of regularly arranged globular subunits, held apart by a fibrous inner core. The axial repeat of the microribbon is 15 nm, which is also the distance separating cross-bridge sites along ribbons. Pronounced striping at this interval is a feature of ribbon faces where they are joined in bundles. Subunits in the outer layer are arranged in vertical protofilaments that are set orthogonally to the long axis of the ribbon. Protofilaments bind tannic acid and are seen clearly in sectioned ribbons. Three protofilaments fit into the 15-nm longitudinal spacing. Optical diffraction patterns from ribbon images are dominated by orders of the 15-nm periodicity, including the third-order reflexions expected from protofilaments spacings. Fourth-order reflexions indicate that the ribbon core may also be structured. Ribbon face images give rise to a strong 4-nm layer line, corresponding to the vertical spacing of subunits in protofilaments. Neighbouring protofilaments are staggered by about 0.67 nm. The lattices found in ribbons are consistent with studies of cytoskeleton composition.