CORTICAL ULTRASTRUCTURE OF PARAMECIUM AURELIA : Studies on Isolated Pellicles

Two methods have been devised for the isolation of large quantities of purified pellicles (cortical layers) of Paramecium aurelia. Pellicles isolated by both procedures, when examined by electron microscopy, were found to contain ciliary basal bodies, two types of cortical membranes, ribbons of microtubules, kinetodesmal fibers, and elements of the infraciliary lattice system. By electron microscopy, the extent of preservation of the various cortical structures when pellicles are isolated by each method has been characterized. Pellicles isolated in both ways have been utilized to investigate cortical morphology of Paramecium. Both phase-contrast and electron microscopic observations have been made. Many new ultrastructural features were observed and are reported herein. An interesting result of this study is the discovery in stock CD that the structure of cortical territories (the territory is the functional unit of cortical morphogenesis and physiology) may vary within a single organism. Features which show variation include number of parasomal sacs, microtubular ribbons, and basal bodies (and therefore cilia) per territory, number of microtubules per ribbon, and length of kinetodesmal fibers. The possible significance of these variations, with respect to territory replication, is discussed. In addition, preliminary observations on the solubility of various cortical organelles in the presence of a number of protein-denaturing agents are reported.     

BASAL BODY REPLICATION AND CILIOGENESIS IN A SUCTORIAN, TOKOPHRYA INFUSIONUM

Basal body replication and ciliogenesis in Tokophrya infusionum were studied in synchronized cultures. Basal body replication occurs during the 1st hr of reproduction, which in Tokophrya is by internal budding. The number of basal bodies increases from about 20 to over 300 within this period. New basal bodies develop in association with mature basal bodies; they are formed at right angles to the mature basal body as short "probasal" bodies, which elongate, slant upward, become parallel to the mature basal body, and elongate to the mature size. Ciliogenesis occurs only during reproduction; the nonreproducing adult is not ciliated, and has only 18–25 barren basal bodies. Cilia first appear as short bulges above the basal body. The axonemal structure is incomplete at first, with one or both central microtubules absent, and occasionally the B fibers of the outer doublets are missing. Several accessory fibers are associated with the basal bodies, both in the adult and during reproduction. One of the fibers appears only after the cilia have sprouted. The scheme of basal body replication and ciliogenesis in Tokophrya is compared to that reported in other organisms, and the role of the accessory fibers is discussed.     

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.     

Protein phosphorylation and dynamics of cytoskeletal structures associated with basal bodies in Paramecium

The presence of phosphorylated proteins associated with microtubule organizing centers in tissue culture cells during mitosis has been demonstrated by the use of monoclonal antibodies raised against mitotic HeLa cells [Vandre et al., Proc. Natl. Acad. Sci. U.S.A. 81:4439-4443, 1984]. We report here that in Paramecium two of the mitosis specific antibodies, MPM-1 and MPM-2, decorate throughout the cell cycle all the microtubule organizing centers (MTOCs) located in the cortex and in the oral apparatus (gullet). Immuno-electron microscopy showed that these antibodies labeled the electron-dense material surrounding basal bodies from which several microtubule networks as well as kinetodesmal fibers originate. During mitosis, these antibodies also stained other cortical cytoskeletal structures, the kinetodesmal fibers (MPM-1 and MPM-2) and the epiplasm (MPM-1). Among the different polypeptides recognized by the antibodies on immunoblots, three major ones of 60, 63, and 116 kDa were found to be common to the cortex (where several thousand ciliary basal bodies are anchored) and the oral apparatus (which comprises several hundred basal bodies around which various arrays of cytoplasmic microtubules are organized). Alkaline phosphatase treatment abolished the immunoreactivity of the polypeptides and the labeling observed by immunofluorescence. These results demonstrate that phosphorylated proteins are associated with all the known active microtubule organizing centers present in the cortex throughout the cell cycle of Paramecium. Furthermore they indicate that in Paramecium phosphorylation of proteins could also be involved in the cell cycle dependent dynamics of cortical cytoskeletal structures other than microtubules.     

THE MORPHOGENESIS OF BASAL BODIES AND ACCESSORY STRUCTURES OF THE CORTEX OF THE CILIATED PROTOZOAN TETRAHYMENA PYRIFORMIS

Dividing cells of Tetrahymena pyriformis were observed by transmission electron microscopy for signs of morphogenesis of cortical structures. The earliest stage of basal body development observed was of a short cylinder of nine single tubules connected by an internal cartwheel structure. This is set perpendicular to the mature basal body at its anterior proximal surface under the transverse microtubules and next to the basal microtubules. Sequential stages show that the single tubules become triplet tubules and that the "probasal bodies" then elongate and tilt toward the organism's surface while maintaining a constant distance of 75–100 mµ with the "parent." The new basal body after it is fully extended contacts the pellicle, and then assumes a parallel orientation with and moves anterior to the parent basal body. The electron-opaque core in the lumen of the basal body and accessory structures around its outer proximal surface appear after the developing basal body has elongated. These accessory structures associating with their counterparts from other basal bodies and with the longitudinal microtubules may play a role in the final positioning of basal bodies and thus in the maintenance of cortical patterns. Observations on a second sequence of basal body formation suggest that the oral anlage arises by multiple duplication of somatic basal bodies.     

Fine Structure, Reconstruction and Possible Functions of Components of the Cortex of Tetrahymena pyriformis

SYNOPSIS. Improved methods of preparing cells for electron microscopic study have permitted a more complete examination of the tubular and fibrous elements already reported in the cortex of Tetrahymena pyriformis. Three of these structures, a kinetodesmal fiber, a band of postciliary microtubules and a band of transverse microtubules, are all associated at one of their ends with the proximal end of a basal body-cilium complex. From this position they radiate out as tho from one corner of a tetrahedron, each passing separately to one of the other 3 corners. Available evidence is presented and discussed for a structural function for these elements. A firm bonding of these fibers and bands to the basal body at one of their ends and to the amorphous material under the pellicle at their other ends is thought to provide for this support. Connecting the proximal ends of the left side of the basal bodies of a kinety is another, previously undescribed, set of tubules. Their diameter, 24 mμ, and cross sectional structure are similar to those of the other microtubules. However, their more sinuous longitudinal appearance, small number of tubules per kinety and different reaction to fixatives suggest a different function. Because of their location it is proposed that they may be a communication line between basal body-cilium complexes of a kinety. A 3-dimensional drawing shows the positioning of the above structures in the cortex. Bodies with an internal tubular structure appear anterior to the proximal end of some basal bodies. They are referred to as probasal bodies due to their resemblance to procentrioles; they may be immature basal bodies. Smooth-membraned cisternae which have bristle-coated pits and bear a resemblance to the outer pellicular membrane appear in the cytoplasm. Their origin is discussed.     

Ultrastructure of the Somatic Cortex of the Gymnostome Ciliate Spathidium spathula (O. F. M.)1

The somatic cortex of Spathidium spathula is described ultrastructurally. The pellicle consists of an outer membrane and an underlying alveolar system. Numerous membrane-bound mucocysts and spherical electron-opaque bodies have similar circular sites of attachment to the outer membrane. Below these are a microfibrillar zone and an underlying region of rough ER. Mitochondria are lined up under the rough ER in longitudinal cortical ridges. Parasomal sacs are found near the basal bodies and are associated with cytoplasmic membranous sacs. Various microtubular and fiber systems are associated with single basal bodies: (1) a short kinetodesmal fiber; (2) two transverse microtubular ribbons and a transverse fiber; (3) a postciliary microtubular ribbon, initially sandwiched by two fibers, which gives rise to longitudinal subpellicular microtubules extending posteriorly for a distance of some four or five basal bodies; and (4) a system of overlapping subkinetal microtubules. A three-dimensional reconstruction is included. The somatic cortex of S spathula is similar to that reported for other Haptorida of the ciliate subclass Gymnostomata.     

FLAGELLAR MOTION AND FINE STRUCTURE OF THE FLAGELLAR APPARATUS IN CHLAMYDOMONAS

The biflagellate alga Chlamydomonas reinhardi was studied with the light and electron microscopes to determine the behavior of flagella in the living cell and the structure of the basal apparatus of the flagella. During normal forward swimming the flagella beat synchronously in the same plane, as in the human swimmer's breast stroke. The form of beat is like that of cilia. Occasionally cells swim backward with the flagella undulating and trailing the cell. Thus the same flagellar apparatus produces two types of motion. The central pair of fibers of both flagella appear to lie in the same plane, which coincides with the plane of beat. The two basal bodies lie in a V configuration and are joined at the top by a striated fiber and at the bottom by two smaller fibers. From the area between the basal bodies four bands of microtubules, each containing four tubules, radiate in an X-shaped pattern, diverge, and pass under the cell membrane. Details of the complex arrangement of tubules near the basal bodies are described. It seems probable that the connecting fibers and the microtubules play structural roles and thereby maintain the alignment of the flagellar apparatus. The relation of striated fibers and microtubules to cilia and flagella is reviewed, particularly in phytoflagellates and protozoa. Structures observed in the transitional region between the basal body and flagellar shaft are described and their occurrence is reviewed. Details of structure of the flagellar shaft and flagellar tip are described, and the latter is reviewed in detail.     

STRUCTURE AND PHYSIOLOGY OF THE HAPTONEMA IN CHRYSOCHROMULINA (PRYMNESIOPHYCEAE). I. FINE STRUCTURE OF THE FLAGELLAR/HAPTONEMATAL ROOT SYSTEM IN C. ACANTHA AND C. SIMPLEX1

The intracellular structural relationships between the flagella and haptonema in Chrysochromulina acantha Leadbeater & Manton (Prymnesiophyceae) were studied in detail and a reconstruction is presented. Three micro-tubular roots are associated with the flagellar apparatus. The largest, consisting of a sheet of approximately 20 microtubules, has its origins at the base of the left basal body. The main body of microtubules passes over the surface of a mitochondrion toward the left chloroplast and apparently terminates at a pair of microtubules oriented perpendicularly to it. Four microtubules diverge from the sheet and pass behind the left basal body. Two other roots–one consisting of a 2 + 2 + 1 arrangement of microtubules, the other of a single microtubule only—are associated with the right basal body. The two basal bodies are connected by distal and proximal fibers, and they are linked also to the base of the haptonema, three fibers extending from the haptonemal base to the right basal body, one only to the left. An additional fiber extending from the right basal body passes between the left basal body and the base of the haptonema, terminating at the largest microtubular root. Lateral extensions link this fiber to both the left basal body and the haptonematal base. Negative staining of isolated root systems of C. simplex Estep et al. shows that the arrangement of microtubules and fibrous connections is similar to that in C. acantha. The root system of C. acantha is compared to those of other members of the Prymnesiophyceae.     

The Resorption of Cilia in Cyathodinium piriforme*

SYNOPSIS. The fine structure of the cilium, kinetosome, kinetodesmal fiber, and basal microtubules has been described in Cyathodinium piriforme. The ciliary axoneme is encased in an electron-dense jacket termed the axonemal jacket. This jacket surrounds the axoneme and is found midway between the axoneme and the ciliary membrane when viewed in cross section. Before division or reorganization the cilia are withdrawn into the cell. Intact cilia surrounded by their jackets are found in the cytoplasm during the early phases of retraction. Degradation of the axonemal microtubules precedes the dissolution of the axonemal jacket. Profiles of the jackets are observed after the microtubules have been resorbed. The cilia appear to detach from the kinetosomes. Barren kinetosomes are seen below the cell surface frequently with kinetodesmal fibers still attached. Whether all or some of these barren kinetosomes contribute to the formation of the new ciliary anlage cannot be ascertained.     

ENTOSIPHON SULCATUM (EUGLENOPHYCEAE): FLAGELLAR ROOTS OF THE BASAL BODY COMPLEX AND RESERVOIR REGION1,2

The flagellar root system of Entosiphon sulcatum (Dujardin) Stein (Euglenophyceae) is described and compared with kinetoplastid and other euglenoid systems. An asymmetric pattern of three microtubular roots, one between the two flagellar basal bodies and one on either side (here called the intermediate, dorsal, and ventral roots), is consistent within the euglenoid flagellates studied thus far. The dorsal root is associated with the basal body of the anterior flagellum (F₁) and lies on the left dorsal side of the basal body complex. Originating between the two flagellar basal bodies, and associated with the basal body of the trailing flagellum (F₂), the intermediate root is morphologically distinguished by fibrils interconnecting the individual microtubules to one another and to the over lying reservoir membrane. The intermediate root is often borne on a ridge projecting into the reservoir. The ventral root originates near the F₂ basal body and lies on the right ventral side of the cell. Fibrillar connections link the membrane of F₂ with the reservoir membrane at the reservoircanal transition level. A large cross-banded fiber joins the two flagellar basal bodies, and a series of smaller striated fibers links the anterior accessory and flagellar basal bodies. Large nonstriated fibers extend from the basal body complex posteriorly into the cytoplasm.     

Ultrastructure of the quadriflagellate zoospores of the filamentous green algaeChaetophora incrassata andPseudoschizomeris caudata (Chaetophorales,chlorophyceae) with emphasis on the flagellar apparatus

Quadriflagellate zoospores ofChaetophora incrassata andPseudoschizomeris caudata have similar features including an appressed membrane between the pyrenoid matrix and the starch sheath, and identical flagellar apparatuses. Components of the flagellar apparatus include: directly opposed upper basal bodies, lower basal bodies in the clockwise absolute orientation, a grooved distal fiber, peripheral and terminal fibers between adjacent basal bodies, proximal fibers connecting the lower basal bodies to the X-membered rootlets two- and X-membered rootlets associated with electron-dense components, and at least one rhizoplast. The X-membered rootlets, are comprised of five microtubules inC. incrassata and four or five inP. caudata. These features of the flagellar apparatus suggest that the two algae are closely related, and together withStigeoclonium, Uronema, Draparnaldia andFritschiella, form a natural group, the Chaetophoraceae, Chaetophorales (sensu Mattox and Stewart).     

ENTOSIPHON SULCATUM (EUGLENOPHYCEAE): FLAGELLAR ROOTS OF THE BASAL BODY COMPLEX AND RESERVOIR REGION1,2

The flagellar root system of Entosiphon sulcatum (Dujardin) Stein (Euglenophyceae) is described and compared with kinetoplastid and other euglenoid systems. An asymmetric pattern of three microtubular roots, one between the two flagellar basal bodies and one on either side (here called the intermediate, dorsal, and ventral roots), is consistent within the euglenoid flagellates studied thus far. The dorsal root is associated with the basal body of the anterior flagellum (F₁) and lies on the left dorsal side of the basal body complex. Originating between the two flagellar basal bodies, and associated with the basal body of the trailing flagellum (F₂), the intermediate root is morphologically distinguished by fibrils interconnecting the individual microtubules to one another and to the overlying reservoir membrane. The intermediate root is often borne on a ridge projecting into the reservoir. The ventral root originates near the F₂ basal body and lies on the right ventral side of the cell. Fibrillar connections link the membrane of F₂ with the reservoir membrane at the reservoir-canal transition level. A large cross-banded fiber joins the two flagellar basal bodies, and a series of smaller striated fibers links the anterior accessory and flagellar basal bodies. Large nonstriated fibers extend from the basal body complex posteriorly into the cytoplasm.     

Three-dimensional organization of basal bodies from wild-type and delta-tubulin deletion strains of Chlamydomonas reinhardtii

Improved methods of specimen preparation and dual-axis electron tomography have been used to study the structure and organization of basal bodies in the unicellular alga Chlamydomonas reinhardtii. Novel structures have been found in both wild type and strains with mutations that affect specific tubulin isoforms. Previous studies have shown that strains lacking delta-tubulin fail to assemble the C-tubule of the basal body. Tomographic reconstructions of basal bodies from the delta-tubulin deletion mutant uni3-1 have confirmed that basal bodies contain mostly doublet microtubules. Our methods now show that the stellate fibers, which are present only in the transition zone of wild-type cells, repeat within the core of uni3-1 basal bodies. The distal striated fiber is incomplete in this mutant, rootlet microtubules can be misplaced, and multiflagellate cells have been observed. A suppressor of uni3-1, designated tua2-6, contains a mutation in alpha-tubulin. tua2-6; uni3-1 cells build both flagella, yet they retain defects in basal body structure and in rootlet microtubule positioning. These data suggest that the presence of specific tubulin isoforms in Chlamydomonas directly affects the assembly and function of both basal bodies and basal body-associated structures.     

REGULATION OF MICROTUBULES IN TETRAHYMENA : II. Relation Between Turnover of Microtubule Proteins and Microtubule Dissociation and Assembly during Oral Replacement

Experiments are reported which were designed to test for induced synthesis of microtubule proteins associated with the rapid proliferation of basal bodies and associated intracytoplasmic microtubules which occurs during oral replacement in Tetrahymena. None was found. Instead, it is shown that these structures can be formed with de novo synthesis of as little as 6% of their microtubule proteins. It is suggested that basal body proliferation may be controlled by synthesis of morphogenetic regulator proteins.     

Identification of the basal bodies and kinetodesmal fibers in living cells of Paramecium tetraurelia Sonneborn, 1975 and Paramecium sonneborni Aufderheide, Daggett & Nerad, 1983

Critical use of Nomarski DIC optics and a rotocompressor permits basal bodies and kinetodesmal fibers to be visualized in the cortices of living Paramecium tetraurelia and Paramecium sonneborni. The identification of these structures is confirmed by the correspondence of the images obtained by DIC optics of living cells and by brightfield optics of fixed cells stained by the Fernández-Galiano silver technique. Examination of cells carrying cortical inversions (portions of the cortex rotated 180 degrees) shows that inverted regions may be identified and distinguished from normal regions by the orientation of the kinetodesmal fibers of the kinetids (cortical units) within the kineties (ciliary rows). This demonstrates that both the asymmetry and the polarity of each cortical unit may be assessed in the living cell. This technique has useful applications in the study of morphogenesis and patterning in living cells and for the screening of mutations and variants conferring altered cortical phenotypes.     

Identification of the Basal Bodies and Kinetodesmal Fibers in Living Cells of Paramecium tetraurelia Sonneborn, 1975 and Paramecium sonneborni Aufderheide,Daggett & Nerad, 19831

ABSTRACT. Critical use of Nomarski DIC optics and a rotocompressor permits basal bodies and kinetodesmal fibers to be visualized in the cortices of living Paramecium tetraurelia and Paramecium sonneborni. The identification of these structures is confirmed by the correspondence of the images obtained by DIC optics of living cells and by brightfield optics of fixed cells stained by the Fernández-Galiano silver technique. Examination of cells carrying cortical inversions (portions of the cortex rotated 180°) shows that inverted regions may be identified and distinguished from normal regions by the orientation of the kinetodesmal fibers of the kinetids (cortical units) within the kineties (ciliary rows). This demonstrates that both the asymmetry and the polarity of each cortical unit may be assessed in the living cell. This technique has useful applications in the study of morphogenesis and patterning in living cells and for the screening of mutations and variants conferring altered cortical phenotypes.     

The UNI1 and UNI2 Genes Function in the Transition of Triplet to Doublet Microtubules between the Centriole and Cilium in Chlamydomonas

One fundamental role of the centriole in eukaryotic cells is to nucleate the growth of cilia. The unicellular alga Chlamydomonas reinhardtii provides a simple genetic system to study the role of the centriole in ciliogenesis. Wild-type cells are biflagellate, but “uni” mutations result in failure of some centrioles (basal bodies) to assemble cilia (flagella). Serial transverse sections through basal bodies in uni1 and uni2 single and double mutant cells revealed a previously undescribed defect in the transition of triplet microtubules to doublet microtubules, a defect correlated with failure to assemble flagella. Phosphorylation of the Uni2 protein is reduced in uni1 mutant cells. Immunogold electron microscopy showed that the Uni2 protein localizes at the distal end of the basal body where microtubule transition occurs. These results provide the first mechanistic insights into the function of UNI1 and UNI2 genes in the pathway mediating assembly of doublet microtubules in the axoneme from triplet microtubules in the basal body template.     

A "microtubule" in a bacterium

A study of the anchorage of the flagella in swarmers of Proteus mirabilis led to the incidental observation of microtubules. These microtubules were found in thin sections and in whole mount preparations of cells from which most of the content had been released by osmotic shock before staining negatively with potassium phosphotungstate (PTA). The microtubules are in negatively stained preparations about 200 A wide, i.e. somewhat thicker than the flagella (approximately 130 A). They are thus somewhat thinner than most microtubules recorded for other cells. They are referred to as microtubules because of their smooth cylindrical wall, or cortex, surrounding a hollow core which is readily filled with PTA when stained negatively. Since this is probably the first time that such a structure is described inside a bacterium, we do not know for certain whether it represents a normal cell constituent or an abnormality, for instance of the type of "polysheaths" (16).     

FINE STRUCTURE OF CELL DIVISION IN CHLAMYDOMONAS REINHARDI : Basal Bodies and Microtubules

Cell division in log-phase cultures of the unicellular, biflagellate alga, Chlamydomonas reinhardi, has been studied with the electron microscope. The two basal bodies of the cell replicate prior to cytokinesis; stages in basal body formation are presented. At the time of cell division, the original basal bodies detach from the flagella, and the four basal bodies appear to be involved in the orientation of the plane of the cleavage furrow. Four sets of microtubules participate in cell division. Spindle microtubules are involved in a mitosis that is marked by the presence of an intact nuclear envelope. A band of microtubules arcs over the mitotic nucleus, indicating the future cleavage plane. A third set of microtubules appears between the daughter nuclei at telophase, and microtubules comprising the "cleavage apparatus" radiate from the basal bodies and extend along both sides of the cleavage furrow during cytokinesis. Features of cell division in C. reinhardi are discussed and related to cell division in other organisms. It is proposed that microtubules participate in the formation of the cleavage furrow in C. reinhardi.