Studies on the transition of the cristal membrane from the orthodox to the aggregated configuration. I: Topology of bovine adrenal cortex mitochondria in the orthodox configuration

Bovine adrenal cortex mitochondria examined by electron microscopyin situ orin vitro in 0·25 M sucrose have an unusual cristal membrane structure. The cristae usually appear as unconnected vesicles within a double membrane system. A few of the vesicles appear to be attached to the inner boundary membrane or to one or more other vesicles. The configuration of such mitochondria will be defined as the orthodox configuration. In this communication we will provide evidence that the inner membrane is not composed of multiple vesicles, but is one continuous membrane with tubular invaginations, and that these invaginations alternately are ballooned out and squeezed down. A mechanism has been proposed to account for the differentiated structure of the cristae of adrenal cortex mitochondria.     

In Vitro Cellular Division of Trypanosoma abeli Reveals Two Pathways for Organelle Replication

Since the observation of the great pleomorphism of fish trypanosomes, in vitro culture has become an important tool to support taxonomic studies investigating the biology of cultured parasites, such as their structure, growth dynamics, and cellular cycle. Relative to their biology, ex vivo and in vitro studies have shown that these parasites, during the multiplication process, duplicate and segregate the kinetoplast before nucleus replication and division. However, the inverse sequence (the nucleus divides before the kinetoplast) has only been documented for a species of marine fish trypanosomes on a single occasion. Now, this previously rare event was observed in Trypanosoma abeli, a freshwater fish trypanosome. Specifically, from 376 cultured parasites in the multiplication process, we determined the sequence of organelle division for 111 forms; 39% exhibited nucleus duplication prior to kinetoplast replication. Thus, our results suggest that nucleus division before the kinetoplast may not represent an accidental or erroneous event occurring in the main pathway of parasite reproduction, but instead could be a species‐specific process of cell biology in trypanosomes, such as previously noticed for Leishmania. This “alternative” pathway for organelle replication is a new field to be explored concerning the biology of marine and freshwater fish trypanosomes.     

Further Observations on the Fine Structure of Acanthamoeba palestinensis (Reich, 1933). The Golgi Complex,Microbodies, and Mitochondria1

The fine structure of the trophozoite of Acanthamoeba palestinensis with a special emphasis on the Golgi complex, microbodies, and mitochondria has been examined. Golgi complexes are distributed throughout the cytoplasm but are most abundant in the perinuclear region. Usually two Goigi complexes are found in the same plane on opposite sides of the nucleus. One of them appears to be in an intimate association with the nuclear membrane. The region of contact contains compact cisternae, vesicles of various sizes, as well as granular and amorphous electron-dense material. Structural changes in the nuclear envelope are also observed in this area. A structure consisting of a Golgi complex and electron-dense microtubule organizing center, comparable to the centrosphere of other Acanthamoeba species, has been observed. Microbodies, surrounded by a single unit membrane and containing a granular matrix and tubular inclusions, are scattered throughout the cytoplasm. These organelles, circular (～1 μm in diameter) or ovoidal (～1 μm in length and ～0.5 μm in width) in section, have often an irregular outline. These microbodies are probably the morphological equivalent of peroxisomes and glyoxysomes. Most mitochondria show a typical structure including tubular cristae and intracristal inclusions. Occasionally mitochondria with two apposed double membranes running through the midline are found. Such atypical cristae have never been reported in small amoebae before.     

Multiplication of Trypanosoma pacifica (Euglenozoa: Kinetoplastea) in English sole, Parophrys vetulus, from Oregon coastal waters

Multiplication of Trypanosoma pacifica was common in the fish host from observations of live flagellates and Giemsa-stained blood smears. Multiplication began with the elongation of the kinetoplast, thickening of the posterior portion of the body, and appearance of a new flagellum near the kinetoplast. The new flagellum was very rigid when less than 3 microm in length, but it became flexible as it elongated. When the new flagellum was approximately 12 microm in length, cell division began and the kinetoplast also began to divide. The timing of nuclear division was variable. Generally, it did not occur until division of the kinetoplast had begun, but occasionally binucleate individuals were observed before cell or kinetoplast division was apparent. As division continued, 1 nucleus migrated past the dividing kinetoplast into the future daughter trypanosome. Finally, the kinetoplast completed division and the trypanosomes separated. Cell division was unequal, with the daughter trypanosome being smaller than the parent and with a more weakly developed undulating membrane.     

The Ultrastructure of Crithidia fasciculata and Morphological Changes Induced by Growth in Acriflavin*

SYNOPSIS. Crithidia fasciculata is similar to other trypanosomatids in ultrastructure. Of considerable interest is the finding of a lamellar membrane formation attached to the mitochondrion. Some evidence is provided for the tentative hypothesis that this membrane formation is the precursor of mitochondrial membranes. The cellular origin of this structure is unknown. Growth in acriflavin results in a marked alteration of the mitochondrion and kinetoplast. Both structures are deficient in cristae. In addition, the kinetoplast loses its normal fibrillar appearance and becomes a smaller, more electron-dense organelle. These effects are discussed in relation to the proposed involvement of the kinetoplast in the elaboration of functional mitochondria.     

CYTOLOGY AND ULTRASTRUCTURE OF CHLORARACHNION REPTANS (CHLORARACHNIOPHYTA DIVISIO NOVA,CHLORARACHNIOPHYCEAE CLASSIS NOVA)1

The green amoeboid cells of Chlorarachnion reptans Geitler are completely naked and each contains a central nucleus, several bilobed chloroplasts each with a central projecting pyrenoid enveloped by a capping vesicle, several Golgi bodies, mitochondria with tubular cristae, extensive rough ER, and a distinct layer of peripheral vesicles. Complex extrusome-like organelles occur rarely in both the amoeboid and flagellate stages. The only organelles entering the reticulopodia are mitochondria, but microtubules are also present. The chloroplasts contain chlorophylls a and b, but histochemical tests suggest that the carbohydrate storage product probably is not a starch. The chloroplast lamellae are composed of one to three thylakoids or form deep stacks. A girdle lamella and interlamellar partitions are absent. Each chloroplast is bounded by either four separate membranes, a pair of membranes with vesicular profiles between them, or three membranes; all three arrangements may occur in the same chloroplast. A periplastidal compartment occurs near the base of the pyrenoid where there are always four surrounding membranes. The compartment has a relatively dense matrix and contains ribosome-like particles and small dense spheres; it extends over and into a deep invagination in the pyrenoid where its contents are enclosed in a double-membraned envelope which is penetrated by wide pores. The zoospores are ovoid and each bears a single laterally inserted flagellum which appears to be wrapped helically around the cell body during swimming. The flagellum lies in a groove in the cell surface and bears fine lateral hairs. Neither a second flagellum or vestige of one, nor an eyespot, is present. A single microtubular root and a larger homogeneous root run from the flagellar base parallel to the emerging flagellum, between the nuclear envelope and the plasmalemma. In the simple flagellar transition region, fine filaments connect adjacent axonemal doublets. A detailed comparison of C. reptans with all other algal taxa results in the conclusion that it must be segregated in the new class Chlorarachniophyceae, the only class in the new division Chlorarachniophyta. The possibility that C. reptans evolved from a symbiosis between a colorless amoeboid cell and a chlorophyll b- containing eukaryote is considered, but the possible affinities of the symbiont remain enigmatic. The implications of the unique chloroplast structure of C. reptans for current hypotheses concerning the origin of chloroplasts are discussed.     

Crystal structure of a conserved domain in the intermembrane space region of the plastid division protein ARC6

The chloroplast division machinery is composed of numerous proteins that assemble as a large complex to divide double‐membraned chloroplasts through binary fission. A key mediator of division‐complex formation is ARC6, a chloroplast inner envelope protein and evolutionary descendant of the cyanobacterial cell division protein Ftn2. ARC6 connects stromal and cytosolic contractile rings across the two membranes through interaction with an outer envelope protein within the intermembrane space (IMS). The ARC6 IMS region bears a structurally uncharacterized domain of unknown function, DUF4101, that is highly conserved among ARC6 and Ftn2 proteins. Here we report the crystal structure of this domain from Arabidopsis thaliana ARC6. The domain forms an α/β barrel open towards the outer envelope membrane but closed towards the inner envelope membrane. These findings provide new clues into how ARC6 and its homologs contribute to chloroplast and cyanobacterial cell division.     

Developmental features of calcium oxalate crystal sand deposition inBeta vulgaris L. Leaves

Summary Sugar beet (Beta vulgaris L.) leaf has a layer of cells extended laterally between the palisade parenchyma and spongy mesophyll that develop numerous small crystals (crystal sand) within their vacuoles. Solubility studies and histochemical staining indicate the crystals are calcium oxalate. The crystals are deposited within the vacuoles early during leaf development, and at maturity the cells are roughly spherical in shape and 2 to 3 times larger than other mesophyll cells. Crystal deposition is preceeded by formation of membrane vesicles within the vacuole. The membranes are synthesizedde novo in the vacuole and have a typical trilaminate structure as viewed with the TEM. The membranes are formed within paracrystalline aggregates of tubular particles (6–8nm outer diameter) as membrane sheets, but are later organized into chambers or vesicles. Calcium oxalate is then precipitated within the membrane chambers. The tubular particles involved in membrane synthesis are usually present in the vacuoles of mature crystal cells, but in very small amounts.     

Extra components in kinetoplast DNA preparations from Crithidiafasciculata

Kinetoplast DNA from the order Kinetoplastidae (trypanosomatids) exists as large associations (molecular weight 4 × 10¹⁰), made up of about 104 small, probably circular, molecules, commonly known as ‘minicircles’. These minicircles were originally thought to be identical in base composition, suggesting that the coding capacity of kinetoplast DNA is very restricted. However, linear molecules have also been observed in preparations of kinetoplast DNA, which, if they contain unique sequences, could represent additional genetic information. This linear DNA has been assumed to be derived from the kinetoplast, but the possibility of it being nuclear contamination has not been definitely ruled out. Work presented in this paper demonstrates that nuclear DNA contamination may indeed be present in kinetoplast DNA prepared by a commonly used method.     

Behavior of the Kinetoplast of Leishmania tarentolae upon Cell Rupture*

SYNOPSIS. The kinetoplast of L. tarentolae remains attached to the basal body upon cell rupture by detergent lysis, sonication, or hypotonic lysis in 0.02 M Tris buffer (pH 7.9) at 0–4 C. Hypotonic lysis in 0.02 M Tris-HCl-2 mM EDTA at 0–4 C and application of mild shearing forces bring about release of most of the swollen kinetoplasts. The kinetoplast DNA can be seen in phase contrast microscopy as a dark mass contiguous to the kinetoplast membrane directly opposite the basal body. Upon return to isotonic media, the kinetoplast shrinks; the membranes of such kinetoplasts are impermeable to added DNAase.     

Electron Microscopic Studies on the Basal Apparatus of the Flagellum in the Protozoon, Leishmania donovani

SYNOPSIS. The basal apparatus of the flagella and kinetoplast in Leishmania donovani have been studied with the electron microscope. The flagellar fibrils extend into the body of the protozoan to form the kinetosome. At the point of origin of the flagellum, the pellicle invaginates to form a kinetosomal vacuole around the kinetosome. The kinetoplast is formed by a transversely elongated banded structure, surrounded at some distance by a double layered kinetoplast membrane. There is no apparent connection between the kinetosome and the kinetoplast.     

MEMBRANES OF MESOPHYLL CELLS OF NICOTIANA RUSTICA AND PHASEOLUS VULGARIS WITH PARTICULAR REFERENCE TO THE CHLOROPLAST

Weier , T. E., and W. W. Thomson . (U. California, Davis.) Membranes of mesophyll cells of Nicotiana rustica and Phaseolus vulgaris with particular reference to the chloroplast. Amer. Jour. Bot. 49(8): 807–820. Illus. 1962.—The endoplasmic reticulum in mesophyll cells is represented by short lengths of irregularly disposed, paired membranes. It is occasionally associated with a typically double nuclear envelope. Groups of irregularly parallel, paired membranes suggesting disorganized dictyosomes occur infrequently. Mitochondria are unevenly distributed in mesophyll; they are large and have sparse tubular cristae around their periphery. In the great majority of instances the bounding membrane is diffusely stained with KMnO₄. When it is sharp and distinct, it may be double as usually pictured, or it may have well-delineated stretches of a single membrane bounding 25–50% of its circumference. The tonoplast and ectoplast are very fragile, the former appearing as a single dark line. In young leaves the ectoplast is visualized as a continuous single membrane adjacent to the cell wall, but in our micrographs of mature leaves it is always discontinuous. The plastid membrane sometimes is distinctly double, having 2 dark components bounding a light component. In the great majority of cases, however, this membrane is either a solid dark line, or the clear component of the double membrane is crossed by delicate dark lines giving the membrane a braided, or scalariform appearance. The various appearances of the membrane may intergrade with each other. The width of the plastid membrane is variable, ranging from 200 to 400 A. The inner component may invaginate into the stroma, and bodies may form in the clear space between the 2 outer membrane components. Micrographs suggest that these bodies, and others formed by small masses of stroma, may be expelled into the hyaloplasm, where they exist as spherical single-membraned particulates. The reality of the variable structure of the plastid membrane is discussed in light of concepts of membrane activity, molecular structure, and the relation of these factors to possible artifacts.     

Fine Structure of Developing Spores of Thelohania bracteata (Strickland, 1913) (Microsporida,Nosematidae) Emphasizing Polar-Filament Formation*

SYNOPSIS. In the microsporidian, Thelohania bracteata, the polar filament, as it starts to develop in the sporoblast, apparently receives material synthesized by the granular endoplasmic reticulum and Golgi vesicles. In immature spores many dilated sacs are observed in areas where there is less endoplasmic reticulum. These sacs, that persist into the almost mature spore, are probably Golgi-type vesicles and may be related to the formation of the spore coat. The polar filament of the mature spore possesses 8 coils and in cross section or cross-fractured face the electron-dense central portion of the polar filament contains a tubular structure, ringed by 12–14 cylindrical structures. In thin sections, an electron-lucid zone is observed between the core and membrane of the polar filament. The polar filament runs through the highly laminated polaroplast which occupies the anterior portion of the spore. In cross-fractured face the lamellae of the polaroplast are arranged like the petals of a flower. The basal portion of the polar filament is enlarged, appearing arrow-shaped in thin sections and pear-shaped in frozen-etched preparations. Frozen-etched membranes differ in the size and distribution of the surface particles.     

The ultrastructural features and division of secondary plastids

Plastids in heterokonts, cryptophytes, haptophytes, dinoflagellates, chlorarachniophytes, euglenoids, and apicomplexan parasites derive from secondary symbiogenesis. These plastids are surrounded by one or two additional membranes covering the plastid-envelope double membranes. Consequently, nuclear-encoded plastid division proteins have to be targeted into the division site through the additional surrounding membranes. Electron microscopic observations suggest that the additional surrounding membranes are severed by mechanisms distinct from those for the division of the plastid envelope. In heterokonts, cryptophytes and haptophytes, the outermost surrounding membrane (epiplastid rough endoplasmic reticulum, EPrER) is studded with cytoplasmic ribosomes and connected to the rER and the outer nuclear envelope. In monoplastidic species belonging to these three groups, the EPrER and the outer nuclear envelope are directly connected to form a sac enclosing the plastid and the nucleus. This nuclear-plastid connection, referred to as the nucleus-plastid consortium (NPC), may be significant to ensure the transmission of the plastids during cell division. The plastid dividing-ring (PD-ring) is a conserved component of the division machinery for both primary and secondary plastids. Also, homologues of the bacterial cell division protein, FtsZ, may be involved in the division of secondary plastids as well as primary plastids, though in secondary plastids they have not yet been localized to the division site. It remains to be examined whether or not dynamin-like proteins and other protein components known to function in the division of primary plastids are used also in secondary plastids. The nearly completed sequencing of the nuclear genome of the diatom Thalassiosira pseudonana will give impetus to molecular and cell biological studies on the division of secondary plastids.     

Ultrastructure of Intraerythrocytic Babesia microti with Emphasis on the Feeding Mechanism*

SYNOPSIS. Babesia microti is a highly polymorphic organism. To unravel its fine structure and the function of organelles it was necessary to resort often to serial sections. A single plasma membrane covers the organism. In trophozoites approaching reproduction, segments of double membranes can be found below the plasma membrane. In electron micrographs of poor resolution these segments of double membranes look like pieces of thick membranes and they were often thought to be a thick 2nd membrane. Before the segments of double membranes appear 2 other organelles are formed in older trophozoites: micronemes and rhoptries. There are indications that these structures originate from vesicles of the Golgi apparatus. Large dense bodies of the same structure as the host cytoplasm are not food vacuoles but merely invaginations of host cytoplasm, as found in serial sections and in organisms removed from the host cell. Feeding in Babesia seems to take place by a special organelle composed of tightly coiled double membranes located partly inside and partly outside the parasite. It is assumed that extracellular digestion of host cytoplasm take place through this organelle. The nucleus remains undifferentiated throughout the whole intraerythrocytic stage. It becomes irregular, loboid, but does not divide and remains a single body until the late stage of reproduction when only a small portion, a bud, extends into the forming merozoite.     

The Fine Structure of Some Retinal Photoreceptors

An electron microscope study has been made of octopus and amphibian photoreceptors, after fixing with KMnO₄ and embedding in araldite. What has previously been seen as a single dense stratum bounding the tubular compartments (octopus) or the double membrane discs (rods and cones), now shows a double structure. We interpret this as showing that these tubules and discs have similar bounding surfaces, which are probably directly related to the cell membrane. This is confirmed by the finding that the tubules and discs are (at least occasionally) continuous with the cell membrane.     

Trypanosomes in Wild California Sandflies, and Extrinsic Stages of Trypanosoma bufophlebotomi

SYNOPSIS. Trypanosomes were found in 94 of over 1,600 wild Lutzomyia vexatrix occidentis, a common phlebotomine sandfly in central California; 25% of the infected sandflies harbored T. bufophlebotomi, identifiable by its peculiar kinetoplast and body structure. The toad trypanosome was cultured from insect isolates and freeze-preserved. Growth in culture occurred at 23 C, but not at 15 C or 30 C. The other 75% of trypanosomes from wild sandflies remain unidentified, altho some were probably T. scelopori or T. gerrhonoti of lizards.     

Varying Kinetoplast Ultrastructure in Two Subspecies of Herpetomonas muscarum (Leidy)*

SYNOPSIS. The kinetoplast nucleoid of Herpetomonas muscarum muscarum is a disk-shaped mass of filaments with sharply delimited anterior and posterior margins. The kinetoplast nucleoid of Herpetomonas muscarum ingenoplastis is elongate, tapered, and made up of long, helically arranged filaments. Possible explanations of these structures on the basis of work by others on isolated kinetoplast DNA are given. The double flagellum of Herpetomonas is seen by electron microscope to be two complete flagella in close contact but with no visible connection to each other.     

L'Organisation Ultrastructurale Corticale de la Gregarine Lecudina pellucida; ses Rapports avec l'Alimentation et la Locomotion

SYNOPSIS. The structure of the cortical region (epicyte and ectoplasm) of the gregarine Lecudina pellucida , an intestinal parasite of the polychete worm Perinereis cultrifera was studied by electron microscopy.
The epicitary folds have 3 unit type membranes. Between the 1st and 2nd is a layer probably composed of fine longitudinal fibrils which has an arch-like or gutter-like structure at the crest of the folds. Inside these folds is cytoplasm without any noticeable differentiation or inclusion except for a granular (or finely fibrillar) layer under the limiting inner membrane and close to it.
The ectoplasmic zone of the entocyte is separated from the epicitary region by a lengthwise discontinuous cylindrical opaque layer, inwardly tangential to the folds. The ectoplasm lacks paraglycogen granules but has various organelles: apparently pinocytic vesicles against the wall between the folds, vesicles with myelinic membranes, opaque granules, a few mitochondria with blistered internal vesicles, and a few circular tubular fibers.
The superficial zone of the gregarine is supposed to contribute to nutrition, thru the extensive surface furnished by its folds and thru the pinocytic vesicles; but this alimentary intake is incomplete compared with that of the previously studied anterior region.
Insufficient mucus is discharged to account for locomotion. There are some circular ectoplasmic fibers, but locomotory myonemes are completely absent. However, there are deformations of the folds and corresponding waves that could account for locomotion by creeping or swimming. These movements of the folds might be due to the action of the contractile proteins and correspond with some of the layers seen in the wall.     

The kinetoplast ultrastructural organization of endosymbiont-bearing trypanosomatids as revealed by deep-etching, cytochemical and immunocytochemical analysis

The endosymbiont-bearing trypanosomatids present a typical kDNA arrangement, which is not well characterized. In the majority of trypanosomatids, the kinetoplast forms a bar-like structure containing tightly packed kDNA fibers. On the contrary, in trypanosomatids that harbor an endosymbiotic bacterium, the kDNA fibers are disposed in a looser arrangement that fills the kinetoplast matrix. In order to shed light on the kinetoplast structural organization in these protozoa, we used cytochemical and immunocytological approaches. Our results showed that in endosymbiont-containing species, DNA and basic proteins are distributed not only in the kDNA network, but also in the kinetoflagellar zone (KFZ), which corresponds to the region between the kDNA and the inner mitochondrial membrane nearest the flagellum. The presence of DNA in the KFZ is in accordance with the actual model of kDNA replication, whereas the detection of basic proteins in this region may be related to the basic character of the intramitochondrial filaments found in this area, which are part of the complex that connects the kDNA to the basal body. The kinetoplast structural organization of Bodo sp. was also analyzed, since this protozoan lacks the highly ordered kDNA-packaging characteristic of trypanosomatid and represents an evolutionary ancestral of the Trypanosomatidae family.