Flagellate Phylogeny: A Study in Conflicts*

SYNOPSIS. Information relating to the ultrastructure of 4 organellar systems of flagellates—nuclei (including mitosis), flagella, mitochondria and chloroplasts—is examined for bearing on the probable phylogeny of the principal flagellate groups, first considered singly and then in combination. The mitotic mechanism has not proved to be as conservative a character as might be hoped, but still remains characteristic for the average condition in many of the groups. Flagellar features are useful if allowance is made for the reduction or multiplication of the basic pair, and the loss of lateral and terminal hairs seems to have occurred independently several times. The presence of paraxial rods within flagella may be a useful indication of affinity. Rootlet systems are not dealt with in detail here, although the possible similarity between axial microtubular sheets in axostylar flagellates and some members of the green algae containing “manchettes” is noted. The basic patterns of chloroplast internal structure are summarized and their general agreement with other characters is affirmed, noting however that cryptomonads may be closer to the green flagellates (including euglenoids) than is generally accepted. Attention is drawn to the potential value of internal mitochondrial morphology as an indicator of large assemblages. Finally, a “tree” based on multiple cell organizational features is presented and discussed.     

Fine Structure of Trichomitus batrachorum (Perty)*

SYNOPSIS. Trichomitus batrachorum (Perty) shares with Trichomonadinae most of its fine-structural characteristics, especially those pertaining to the undulating membrane that consists of a high body fold, enclosing the loop-shaped, periodic marginal lamella, and of the recurrent flagellum applied to the fold. This flagellate has also certain structures, i.e. the costal base, comb-like organelle, and well developed marginal lamella, in common with Monocercomonas. It shares with Hypotrichomonas the costal base; large pelta; very fine fibers perpendicular to, and connecting the axostylar microtubules; structural details of the most proximal segment of the marginal lamella; and general relationships between dorsal body fold (poorly developed in Hypotrichomonas) and the recurrent flagellum. All these electronmicroscopic findings support the crucial position of Trichomitus in the evolutionary sequence Monocercomonas→Hypotrichomonas→Trichomitus→ Trichomonadinae suggested previously by lightmicroscopic observations. Further, Trichomitus shares with all Tritrichomonadinae the comb-like structure, not found either in Hypotrichomonas or Trichomonadinae; and it has in common with Tritrichomonas also the costa with Type A periodicity, being the only member of Trichomonadinae with this type of supporting organelle. It appears, therefore, that Trichomitus-type organisms represent an important link in the evolution of all Trichomonadidae from Monocercomonadidae.     

Order amidst animalcules: the Protoctista kingdom and its undulipodiated cells

The eukaryotic microorganisms have always been studied and described in the context of Zoology (as 'tiny animals), Botany (as tiny plants), Mycology (as water molds) or Microbiology (as disease agents). Because their extent (as a group of about 200,000 species) and differences from the rest of life (worthy of Protoctista kingdom status) only recently has been recognized, their classification within the Plant and Animal kingdoms remains in scandalous disarray. Admitting this problem, this paper represents a step toward achieving nomenclatural order based on ultrastructural, biochemical and evolutionary considerations.     

Physiology and ultrastructure of Leptothrix discophora SS-1

Leptothrix discophora strain SS-1 (ATCC 43182) is a Gram-negative, Mn²⁺-oxidizing, aerobic heterotroph which lost its sheath-forming ability after 18 months of cultivation on laboratory media. SS-1 possesses high 6-phosphogluconate dehydratase and KDPG aldolase activities, and a very low level of phosphofructokinase, indicating carbohydrate catabolism by the Entner-Doudoroff pathway. The strain is polarly flagellated, accumulates PHB up to 67% of its dry weight when grown in pyruvate-containing medium, and has a G+C content of 69.8 mol%. These properties indicate that L. discophora is essentially a pseudomonad which can form a sheath and oxidize Mn²⁺. Ultrastructural observations made before SS-1 lost its sheath-forming ability indicated two cell types. Short, flagellated, non-sheathed cells seen under the electron microscope probably corresponded to swarmer cells observed under phase-contrast microscopy. These cells contained plate organelles and PHB granules, and produced extracellular blebs approx. 25–50 nm in diameter. Larger sheathed cells also contained plate organelles, PHB granules, and blebs that were often sandwiched between the outer membrane and the sheath. Cells grown in the presence of added Mn²⁺ were surrounded by an extensive fibrillar matrix, rendered electron dense by precipitation of manganic oxide. The matrix was connected to various points of the cell by outer membrane evaginations or electron dense threads. We propose that the outer membrane blebs represent vehicles for excretion of unorganized sheath material and/or Mn²⁺-oxidizing protein produced by L. discophora.Parts of this work were presented previously (W.C. Ghiorse, Abst. Annu. Meet. Am. Soc. Microbiol., 1981, N 64, p 183)     

Structural changes induced in capillary endothelial cells by growth factors: Altered distribution of cytoskeletal elements and organelles in cells spreading in the presence of tumor conditioned medium and hypothalamus derived growth factor

Using high-voltage and conventional electron microscopy of cell whole mounts, we have investigated the effects of tumor-conditioned medium and hypothalmus-derived growth factor on the structure of capillary endothelial cells during their attachment and spreading in tissue culture. Cells were cultured in A, Dulbecco's Modified Eagle's Medium (DMEM) and 10% calf serum; B, equal parts of A and 48 hr mouse sarcoma conditioned medium; and C, A containing 10 units of hypothalamus-derived growth factor. Cells cultured in all three media were fully spread, and to the same extent, by 4 hr after plating. While spreading, cells cultured in DMEM alone developed prominent stress fibers and contained numerous bundles of microtubules which formed radical tracts along which mitochondria and other organelles rapidly moved to the cell periphery. Stress fibers were thinner and microtubule tracts fewer in number in cells cultured in tumor-conditioned medium. In 4 hr, organelles moved only part of the distance to the cell margin. Stress fibers were rudimentary and microtubules randomly orientated in cells exposed to hypothalamus-derived growth factor. Most organelles remained near the cell nucleus. The dramatic decrease in stress fibers and microtubule tracts in cells grown in tumor-conditioned medium and hypothalamus-derived growth factor and the subsequent decreased capacity of the cells to move organelles toward their periphery could have some functional significance relative to the growth-promoting activity of these substances.     

A new type of photoreceptor in Anthopharynx sacculipenis (Plathelminthes,Solenopharyngidae)

The unpaired eye of A. sacculipenis consists of two sensory cells and one pigmented mantle cell. The light-sensing organelles are formed by flattened and rolled lamellae, which are not of ciliary origin but derive from the surface membrane of the sensory cells. The pigment of the mantle cell appears black in living animals, but electron-lucent in the EM picture. The significance of these special features is discussed.     

Murine GFP-Mx1 forms nuclear condensates and associates with cytoplasmic intermediate filaments: Novel antiviral activity against VSV

Type I and III interferons induce expression of the “myxovirus resistance proteins” MxA in human cells and its ortholog Mx1 in murine cells. Human MxA forms cytoplasmic structures, whereas murine Mx1 forms nuclear bodies. Whereas both HuMxA and MuMx1 are antiviral toward influenza A virus (FLUAV) (an orthomyxovirus), only HuMxA is considered antiviral toward vesicular stomatitis virus (VSV) (a rhabdovirus). We previously reported that the cytoplasmic human GFP-MxA structures were phase-separated membraneless organelles (“biomolecular condensates”). In the present study, we investigated whether nuclear murine Mx1 structures might also represent phase-separated biomolecular condensates. The transient expression of murine GFP-Mx1 in human Huh7 hepatoma, human Mich-2H6 melanoma, and murine NIH 3T3 cells led to the appearance of Mx1 nuclear bodies. These GFP-MuMx1 nuclear bodies were rapidly disassembled by exposing cells to 1,6-hexanediol (5%, w/v), or to hypotonic buffer (40–50 mosm), consistent with properties of membraneless phase-separated condensates. Fluorescence recovery after photobleaching (FRAP) assays revealed that the GFP-MuMx1 nuclear bodies upon photobleaching showed a slow partial recovery (mobile fraction: ∼18%) suggestive of a gel-like consistency. Surprisingly, expression of GFP-MuMx1 in Huh7 cells also led to the appearance of GFP-MuMx1 in 20–30% of transfected cells in a novel cytoplasmic giantin-based intermediate filament meshwork and in cytoplasmic bodies. Remarkably, Huh7 cells with cytoplasmic murine GFP-MuMx1 filaments, but not those with only nuclear bodies, showed antiviral activity toward VSV. Thus, GFP-MuMx1 nuclear bodies comprised phase-separated condensates. Unexpectedly, GFP-MuMx1 in Huh7 cells also associated with cytoplasmic giantin-based intermediate filaments, and such cells showed antiviral activity toward VSV.     

Studies with digitonin-treated rat hepatocytes (nude cells)

Isolated rat hepatocytes were treated with digitonin to strip the plasma membrane. The effect of digitonin concentration and exposure time on the recovery of marker enzymes for cell organelles was examined. Hepatocytes treated at room temperature for 1-2 min with 1 mg/ml of digitonin lose some 40% of their protein but retain over 95% of their intact mitochondria and peroxisomes, 90-95% of their endoplasmic reticulum, and about 80% of their lysosomal enzymes. There is little loss of the mitochondrial intermembrane content, and both oxygen uptake and phosphorylation are unimpaired by the treatment. Electron microscopy reveals a complete loss of the plasma membrane, in spite of limited loss of marker enzymes for this membrane. Scanning electron microscopy revealed the interior of the cells to be made up of a dense network of fibers and lamellae attached to the nucleus, mitochondria, and small organelles. The treated cells were stable for many hours when kept in 0.25 M sucrose containing 25 mM monovalent salts. In salt-free sucrose the cells broke up very rapidly into nuclei and other single organelles. Addition of 5 mM NaCl or KCl retards breakup, and 15-20 min were required for dissolution. Intermediate stages, illustrated by scanning electron micrographs, show structure and chains made up mainly of mitochondria held together by a lamellar network. The rapid breakdown occurred at a pH above 7.5 in an oxygen atmosphere and in the presence of phosphate and apparently is an energy-requiring process. It is slow below a pH of 7.2, and at a pH of 6.8 the treated cells remain completely stable in salt-free sucrose. Our results suggest that endoplastic reticulum is a major component of the cytostructure holding together nuclei and organelles.     

Nuclear and Organelle DNA Replication during Spore Germination in Bryophytes and Equisetum

In contrast to ferns, the spore nuclei of the moss, Funaria hygrometrica, are in the G₂ phase. They do not incorporate the thymidine analogue BUdr before the first mitosis. When spores germinate in the presence of aphidicolin, an inhibitor of the (nuclear) DNA polymerase alpha, they give rise to two-celled, negatively phototropic protonemata, consisting of relatively long and thin cells. Determinations of nuclear DNA content further confirm the presence of G₂-nuclei in Funaria spores. Spores of Pogonatum urnigerum and of Equisetum hyemale likewise contain G₂-nuclei, as shown by BUdr incorporation or experiments with aphidicolin, respectively. In germinating Funaria spores, the organelle DNA is synthesized very early and independent of nuclear DNA. A gradient in replication of organelle DNA is found only in the long tip cells which are formed in the presence of aphidicolin.