BASAL BODIES OF BACTERIAL FLAGELLA IN PROTEUS MIRABILIS : II. Electron Microscopy of Negatively Stained Material

This paper investigates further the question of whether the flagella of Proteus mirabilis emerge from basal bodies. The bacteria were grown to the stage of swarmer differentiation, treated lightly with penicillin, and then shocked osmotically. As a result of this treatment, much of the cytoplasmic content and also part of the plasma membrane were removed from the cells. When such fragmented organisms were stained negatively with potassium phosphotungstate, the flagella were found to be anchored—often by means of a hook—in rounded structures approximately 50 mµ wide, thus confirming Part I of our study. In these rounded structures a more brilliant dot was occasionally observed, which we interpret as being part of the basal granule. A prerequisite for the demonstration of the basal granules within the cells was, however, the removal of both the cytoplasm and the plasma membrane from their vicinity. In some experiments, the chondrioids were "stained" positively by the incorporation into them of the reduced product of potassium tellurite. The chondrioids were here observed to be more or less circular areas from which rodlike structures extended. The chondrioids adhered so firmly to the plasma membrane that they were carried away with it during its displacement by osmotic shocking, while the basal bodies were left behind. This observation disproves our previous suggestion that the flagella might terminate in the chondrioids. The basal bodies often occur in pairs, which suggest that they could be self-reproducing particles.     

THE DEVELOPMENT OF BASAL BODIES AND FLAGELLA IN ALLOMYCES ARBUSCULUS

The development of basal bodies and flagella in the water mold Allomyces arbusculus has been studied with the electron microscope. A small pre-existing centriole, about 160 mµ in length, was found in an inpocketing of the nuclear membrane in the vegetative hypha. Thus, formation of a basal body does not occur de novo. When the hyphal tip started to differentiate into gametangia, the centrioles were found to exist in pairs. One of the members of the pair then grew distally to more than three times its original length, whereas the other remained the same size. The larger centriole would correspond to the basal body of a future gamete. Gametogenesis was usually induced by transferring a "ripe" culture to distilled water. Shortly after this was done, a few vesicles were pinched off from the cell membrane of the gametangium and came in contact with the basal body. Apparently, they fused and formed a large primary vesicle. The flagellum then started to grow by invaginating into it. Flagellar fibers were evident from the very beginning. As the flagellum grew so did the vesicle by fusion with secondary vesicles, thus coming to form the flagellar sheath. The different stages of flagellar morphogenesis are described and the possible interrelationships with other processes are discussed.     

REGULATION OF MICROTUBULES IN TETRAHYMENA : I. Electron Microscopy of Oral Replacement

The coupled resorption and redifferentiation of oral structures which occurs in Tetrahymena pyriformis under conditions of amino acid deprivation has been studied by transmission electron microscopy. Two patterns of ciliary resorption have been found, (a) in situ, and (b) after withdrawal into the cytoplasm. No autophagic vacuoles containing cilia or ciliary axonemes have been seen. Stomatogenic field basal bodies arise by a process of rapid sequential nucleation, with new ones always appearing next to more mature ones, even though the latter may not be fully differentiated. Accessory radial ribbons of microtubules develop immediately adjacent to oral field basal bodies as a late step in their maturation. It can be seen that the formation of basal bodies and their orientation within the oral complex are separate processes. This is true for about 130 of the approximately 170 oral basal bodies; the remaining 40 or so form within the patterned groups of ciliary units as a later event. Clusters of randomly oriented thin-walled microtubules are found surrounding oral basal bodies at all times during stomatogenesis. They may either represent stores of microtubule subunit protein, or serve as effectors of basal body movement during their orientation into pattern.     

Electron Microscopy of Mummified Material

It has proved possible to cut ultrathin sections of mummified material obtained from an American Indian burial (approximate age unknown). Small pieces of tissue were placed for 48 hr in a softening fluid consisting of 96% ethyl alcohol, 30 vol.; 1% aqueous formalin, 50 vol.; 5% aqueous Na₂CO₃, 20 vol. During this period the fluid was changed twice. The tissue was then cut with a razor blade into cubes of 1 mm per side or less, dehydrated in graded ethanols, infiltrated and embedded in methacrylate and the plastic polymerised by placing in the oven at 58°C overnight. The blocks were trimmed to a truncated cone leaving a surface area of 0.5 mm² or less, and cut on a Porter Blum ultramicrotome using a glass or a diamond knife.     

THE FORMATION OF BASAL BODIES (CENTRIOLES) IN THE RHESUS MONKEY OVIDUCT

Basal body replication during estrogen-driven ciliogenesis in the rhesus monkey (Macaca mulatta) oviduct has been studied by stereomicroscopy, rotation photography, and serial section analysis. Two pathways for basal body production are described: acentriolar basal body formation (major pathway) where procentrioles are generated from a spherical aggregate of fibers; and centriolar basal body formation, where procentrioles are generated by the diplosomal centrioles. In both pathways, the first step in procentriole formation is the arrangement of a fibrous granule precursor into an annulus. A cartwheel structure, present within the lumen of the annulus, is composed of a central cylinder with a core, spoke components, and anchor filaments. Tubule formation consists of an initiation and a growth phase. The A tubule of each triplet set first forms within the wall material of the annulus in juxtaposition to a spoke of the cartwheel. After all nine A tubules are initiated, B and C tubules begin to form. The initiation of all three tubules occurs sequentially around the procentriole. Simultaneous with tubule initiation is a nonsequential growth of each tubule. The tubules lengthen and the procentriole is complete when it is about 200 mµ long. The procentriole increases in length and diameter during its maturation into a basal body. The addition of a basal foot, nine alar sheets, and a rootlet completes the maturation process. Fibrous granules are also closely associated with the formation of these basal body accessory structures.     

Electron Microscopy of Herpes Simplex Virus: I. Entry

Although capsids of herpes simplex virus were encountered within phagocytic vesicles, they were more commonly observed free within the cytoplasm. Stages in the release of virus from vesicles were not seen. There appeared to be five distinct steps in the process whereby the virus initiates infection: attachment, digestion of the viral envelope, digestion of the cell wall, passage of the capsid directly into the cytoplasm, and digestion of the capsid with release of the core. Antibody probably interferes with the first two stages.     

STUDY OF MITOCHONDRIA IN RAT LIVER : Quantitative Electron Microscopy

The electron microscope has been used to determine the weight distribution of isolated subcellular particles from normal rat liver. The following results are reported: (1) There exist at least two well defined weight populations of subcellular particles; their respective median weights are 1.3 x 10^-14 and 11 x 10^-14 gm. The lighter fraction is considered to consist of lysosomes, the heavier of mitochondria. (2) The mitochondrial fraction shows a log-normal distribution of the particle weight. (3) By the introduction of morphologic criteria, the mitochondrial fraction is divided into two groups, one consisting of a spherical, the other of an oblong type of particle. The data found support the following concepts: (a) Mitochondria increase their weight from a certain size up by linear growth. (b) Mitochondria divide. The division is not necessarily symmetric; in all cases, however, one part of the division product is a spherical particle. It is felt that these results constitute a valuable demonstration of the general capabilities of quantitative electron microscopy and may stimulate many other useful applications of this technique in cytology, bacteriology, and virology.     

ULTRASTRUCTURAL ORGANIZATION OF CILIA AND BASAL BODIES OF THE EPITHELIUM OF THE CHOROID PLEXUS IN THE CHICK EMBRYO

Ultrastructural studies were performed on normal and abnormal cilia and basal bodies associated with the choroidal epithelium of the chick embryo. Tissues were prepared in each of several fixatives including: 1% osmium tetroxide, in both phosphate and veronal acetate buffers; 2% glutaraldehyde, followed by postfixation in osmium tetroxide; 1% potassium permanganate in veronal acetate buffer. Normal cilia display the typical pattern of 9 peripheral doublets and 2 central fibers, as well as a system of 9 secondary fibers. The latter show distinct interconnections between peripheral and central fibers. Supernumerary fibers were found to occur in certain abnormal cilia. The basal body is complex, bearing 9 transitional fibers at the distal end and numerous cross-striated rootlets at the proximal end. The distal end of the basal body is delimited by a basal plate of moderate density. The tubular cylinder consists of 9 triple fibers. The C subfibers end at the basal plate, whereas subfibers A and B continue into the shaft of the cilium. The 9 transitional fibers radiate out from the distal end of the basal body, ending in bulblike terminal enlargements which are closely associated with the cell membrane in the area of the basal cup. One or 2 prominent basal feet project laterally from the basal body. These structures characteristically show several dense cross-bands and, on occasion, are found associated with microtubules.     

POLYPHYLETIC ORIGIN OF PARALLEL BASAL BODIES IN SWIMMING CELLS OF CHLOROPHYCEAN GREEN ALGAE (CHLOROPHYTA)1

The evolutionary affinities of Heterochlamydomonas Cox and Deason and Dictyochloris Vischer ex Starr were investigated using phylogenetic analyses of a combined data set of 18S and 28S rDNA sequences with those from 38 additional green algae. Previous ultrastructural studies have shown that motile cells of Heterochlamydomonas and Dictyochloris have an unusual flagellar apparatus organization in that the two flagella are of unequal length and the basal bodies are persistently parallel. Because of this similarity these taxa, along with Bracteacoccus Tereg, a third taxon with this same flagellar apparatus arrangement, are hypothesized to be closely related. We show, with maximum parsimony and Bayesian analyses, that the parallel basal bodies are not homologous in the three genera. Rather, Heterochlamydomonas is most closely related to Chlamydomonas baca in the clockwise flagellar apparatus clade, and Dictyochloris and Bracteacoccus are nested within the Sphaeropleales, which has the directly opposite flagellar absolute orientation. Surprisingly, Dictyochloris and Bracteacoccus are not supported as closest relatives. These relationships are supported by morphological features such as the presence or absence of a walled motile cell but not by the orientation of the basal bodies. In addition, our data are derived from multiple isolates of each study genera, and the analyses show that Heterochlamydomonas and Dictyochloris are each monophyletic.     

STUDY OF BULL SPERMATOZOA : Quantitative Electron Microscopy

The electron microscope has been used to determine the characteristic dimensions and the distribution of the dry mass in bull spermatozoa. The most important result is that all characteristic data are occurring in logarithmic distributions. Furthermore, no correlation between such parameters as head weight and tail weight or head length and tail length was found. The occurrence in logarithmic distributions and the non-correlation of parts in the assembly of a spermatozoon are considered to reflect significant biologic principles. Methodologically, a new procedure is added to quantitative electron microscopy permitting the recording of the mass cross-section (total mass per unit length) of an object. This approach makes possible determinations of the distribution and the total mass of very long and narrow structures.     

LPP-1 Infection of the Blue-Green Alga Plectonema boryanum: I. Electron Microscopy

One-step growth and intracellular growth experiments were performed at high multiplicities of the virus LPP-1 during the infection of the blue-green alga Plectonema boryanum. The eclipse period lasts until 4 hr after infection, the latent period terminates at 6 hr, and the rise period continues until 14 to 16 hr after infection. The burst size was independent of multiplicity of infection over the ranges from 1 to 50. The burst size was 3,000 to 5,000 plaque-forming units (PFU) per infectious center or about 200 PFU per cell. Samples for electron microscopy were taken at characteristic times during the lytic cycle. The first sign of viral infection was the invagination of the photosynthetic lamellae at 3 hr after infection. Mature virions were visible at 4 hr. By 6 to 7 hr, many mature intracellular viral particles could be seen, with lysis beginning at 7 hr. By 10 hr after infection, all infected cells contained mature virions. No evidence for mass migration of performed viral precursors was obtained. The invagination of the lamellae could be prevented by the early addition of chloramphenicol, which implies that this process requires protein synthesis.     

THE ORIGIN OF PROTEIN AND FATTY YOLK IN RANA PIPIENS : I. Phase Microscopy

Study of living frog oocytes with the phase microscope has shown that the early yolk appears in two forms. One of these, the protein yolk, consists of thin, dense, plate-like bodies which in face view are almost always regular hexagons. The other form, the fatty yolk, occurs as clusters of globules of varying sizes. The plate-like bodies occur both singly and in clusters. As the oocytes mature these plate-like bodies grow in size while retaining their hexagonal outline. Mitochondria have been observed to increase in length and numbers as the oocytes mature; they are rods or filaments at all stages of growth up to an oocyte diameter of 300 microns. The oocyte cytoplasm gradually becomes packed with long mitochondria, plate-like bodies, and clusters of globules.     

MITOSIS IN DUNALIELLA BIOCULATA (CHLOROPHYTA): CENTRIN BUT NOT BASAL BODIES ARE AT THE SPINDLE POLES

Centrin, a 20 kDa calmodulin-like protein, is located in various basal body-associated fibers in protists. We used indirect immunofluorescence of isolated cytoskeletons or methanol-fixed cells to analyze the distribution of centrin during mitosis of the biflagellate green alga Dunaliella bioculata (Butcher). The distance between the nucleus and the basal apparatus decreased in late interphase, presumably caused by the contraction of the two centrin-containing nucleus–basal body connectors (NBBCs). During prophase, centrin accumulated on the new basal bodies as shown by postembedding immunogold labeling of serial thin sections. The new basal bodies were in close contact with plaque-like structures on the nuclear envelope. In mitotic cells, basal body pairs were separated and positioned at a considerable distance from the poles of the mitotic spindle. At this stage, we observed four separated centrin dots, two associated with the pairs of basal bodies and two located at the spindle poles as shown by double immunofluorescence, including anti-tubulin staining. The latter signals corresponded to an accumulation of centrin between the plasma membrane and the nuclei, indicating that centrin could be involved in mitotic movements of the nuclei. In telophase, centrin was observed along the nuclear surface and one new NBBC developed in each cell half. Our results demonstrate that centrin is present at the acentriolar spindle poles of Dunaliella independently from its localization in the basal apparatus.     

CHARACTERIZATION OF VACUOLAR BODIES IN SPARTINA ALTERNIFLORA: I. FORMATION,DEVELOPMENT, MORPHOLOGY,AND ULTRASTRUCTURE

Spherical, golden bodies, 0.5 to 25 μm in diameter, were noted in outer bundle sheath and mesophyll of cells of fresh sections of Spartina alterniflora leaves. Attempts to further characterize these structures with light and electron microscope (EM) techniques after dehydration failed initially because these bodies were soluble in organic solvents such as alcohol. Subsequently, it was found that certain heavy metals would stabilize the structure so that dehydration techniques could be used. The resultant stabilized bodies as viewed with EM were found to reside in the cell vacuole. Two major structural components were recognized: (1) a granular matrix and (2) a non-electron dense internal vesicle. Internal vesicles were not always present. No membranes were visible with this technique. These vacuolar bodies also were found to occur in parenchyma cells of roots, peduncles, glumes, and rhizomes of S. alterniflora. The material which forms the vacuolar body matrix is probably produced in the cytoplasm and gradually accumulates in the vacuole, forming larger and more numerous vacuolar bodies during the growing season. The matrix material remains in the leaf cells following their senescence.