Evidence for a direct role of nascent basal bodies during spindle pole initiation in the green alga Spermatozopsis similis.

Basal body replication in the naked biflagellate green alga Spermatozopsis similis was analyzed using standard electron microscopy and immunogold localization of centrin, an ubiquitous centrosomal protein, and p210, a recently characterized basal apparatus component of S. similis. Fibrous disks representing probasal bodies appear at the proximal end of parental basal bodies at the end of interphase and development proceeds via a ring of nine singlet microtubules. Nascent basal bodies dock early to the plasma membrane but p210, usually present in basal body-membrane-linkers of S. similis, was already present on the cytosolic basal body precursors. In addition to the distal connecting fiber and the nuclear basal body connectors (NBBC) of the parental basal bodies, centrin was present on the fibrous probasal bodies, in a linker between probasal bodies and the basal apparatus, in the connecting fiber between nascent basal bodies and their corresponding parent, and, finally, a fiber linking the nascent basal bodies to the nucleus. This NBBC probably is present only in mitotic cells. During elongation a cartwheel of up to seven layers is formed, protruding from the proximal end of nascent basal bodies. Microtubules develop on the cartwheel indicating that it temporarily functions as a microtubule organizing center (MTOC). These microtubules and probably the cartwheels, touch the nuclear envelope at both sides of a nuclear projection. We propose that spindle assembly is initiated at these attachment sites. During metaphase, the spindle poles were close to thylakoid-free lobes of the chloroplast, and the basal bodies were not in the spindle axis. The role of nascent basal bodies during the initial steps of spindle assembly is discussed.     

Basal apparatus behaviour during cellular division (sporulation) in the coccoid green algaChlorosarcina

Summary We studied the basal body cycle (including basal body segregation, duplication, migration, and reorientation) in dividing cells of the colonial coccoid green algaChlorosarcina stigmatica using serial thin sections. Although flagella are lacking, all cells examined possess a rudimentary flagellar apparatus composed of two basal bodies linked by a distal striated fibre, two probasal bodies, and four cruciately arranged microtubular roots (2-4-2-4 type). Basal body segregation occurs at preprophase, during which two half-basal apparatuses (each consisting of one basal body, one probasal body, and a left and a right root) migrate into opposite directions. The segregation axis is defined by the two left roots which remain closely associated during segregation and slide along each other. The segregation axis is parallel to the axis of chromosome separation, and perpendicular to the plane of subsequent cell division. Duplication of basal apparatus components does not occur until telophase when daughter basal apparatuses migrate towards the plane of division. At cytokinesis which is effected by the unilateral ingrowth of a septum, each daughter basal apparatus rotates 90° and becomes associated with the new septum.Abbreviations BA basal (body) apparatus - NBBC nucleus-basal body connector     

Transformation of the flagella and associated flagellar components during cell division in the coccolithophoridPleurochrysis carterae

Summary Immunofluorescence microscopy, conventional and high voltage transmission electron microscopy were used to describe changes in the flagellar apparatus during cell division in the motile, coccolithbearing cells ofPleurochrysis carterae (Braarud and Fagerlund) Christensen. New basal bodies appear alongside the parental basal bodies before mitosis and at prophase the large microtubular (crystalline) roots disassemble as their component microtubules migrate to the future spindle poles. By prometaphase the crystalline roots have disappeared; the flagellar axonemes shorten and the two pairs of basal bodies (each consisting of one parental and one daughter basal body) separate so that each pair is distal to a spindle pole. By late prometaphase the pairs of basal bodies bear diminutive flagellar roots for the future daughter cells. The long flagellum of each daughter cell is derived from the parental basal bodies; thus, the basal body that produces a short flagellum in the parent produces a long flagellum in the daughter cell. We conclude that each basal body in these cells is inherently identical but that a first generation basal body generates a short flagellum and in succeeding generations it produces a long flagellum. At metaphase a fibrous band connecting the basal bodies appears and the roots and basal bodies reorient to their interphase configuration. By telophase the crystalline roots have begun to reform and the rootlet microtubules have assumed their interphase appearance by early cytokinesis.Abbreviations CR1, CR2 crystalline roots 1 and 2 - CT cytoplasmic tongue microtubules - DIC differential interference contrast light microscopy - H haptonema - HVEM high voltage transmission electron microscopy - IMF immunofluorescence microscopy - L left flagellum/basal body - M metaphase plate - MT microtubule - N nucleus - R right flagellum/basal body - R1, R2, R3 roots 1, 2, and 3 - TEM transmission electron microscopy     

The Uni2 phosphoprotein is a cell cycle regulated component of the basal body maturation pathway in Chlamydomonas reinhardtii

Mutations in the UNI2 locus in Chlamydomonas reinhardtii result in a "uniflagellar" phenotype in which flagellar assembly occurs preferentially from the older basal body and ultrastructural defects reside in the transition zones. The UNI2 gene encodes a protein of 134 kDa that shares 20.5% homology with a human protein. Immunofluorescence microscopy localized the protein on both basal bodies and probasal bodies. The protein is present as at least two molecular-weight variants that can be converted to a single form with phosphatase treatment. Synthesis of Uni2 protein is induced during cell division cycles; accumulation of the phosphorylated form coincides with assembly of transition zones and flagella at the end of the division cycle. Using the Uni2 protein as a cell cycle marker of basal bodies, we observed migration of basal bodies before flagellar resorption in some cells, indicating that flagellar resorption is not required for mitotic progression. We observed the sequential assembly of new probasal bodies beginning at prophase. The uni2 mutants may be defective in the pathways leading to flagellar assembly and to basal body maturation.     

THE CYTOSKELETON OF THE NAKED GREEN FLAGELLATE SPERMATOZOPSZS SIMILIS (CHLOROPHYTA): ANALYSIS OF ISOLATED BASAL APPARATUSES IN THE PARALLEL CONFIGURATION1,2

The biflagellate green alga Spermatozopsis similis is demonstrated to be a model organism for the biochemical and functional analysis of the basal apparatus. Basal apparatuses were isolated in the presence of 10⁻⁶ M Ca²⁺, which induces the reorientation of the basal bodies into the parallel state. Serial thin sectioning of enriched basal apparatuses stained with tannic acid reveals several novel details of the structure of the basal bodies, the distal connecting fiber, and the striated microtubule-associated fibers. We observed a pronounced difference in size of a striated fiber connecting the basal bodies to the five-stranded microtubular roots depending on its association with the developmentally older or younger basal body. Instead of a proximal connecting fiber, the proximal end of each basal body is associated with a striated triangular plate; these plates appear to serve as spacers for the basal bodies in the parallel and antiparallel configurations. We suggest that the plates play a role in maintaining basal body orientation during forward and backward swimming. The results are summarized in representative drawings of the basal apparatus.     

The centriole cycle in the amoebae of the myxomycetePhysarum polycephalum

Summary In the amoebae of the myxomycetePhysarum polycephalum, procentrioles are formed on the anterior and posterior centrioles in early prophase. Although the relative position of the parental and procentrioles is fixed, all relative positions of the daughter and parental centrioles were observed. During the different stages of mitosis daughter centrioles elongate and acquire anterior satellites, one of the characteristic features of the anterior centrioles. All other anterior morphological characteristics appear only in telophase and early reconstruction stages. In contrast to the parental posterior centrioles, which do not change morphologically during the successive mitotic stages, the parental anterior centrioles lose their morphological characteristics in late prophase and early prometaphase and then acquire the morphological features characteristic of the posterior centrioles. Thus, the following maturation scheme is suggested: a procentriole becomes an anterior centriole during the first mitosis and a posterior centriole during the second mitosis. Since posterior features are maintained during mitosis, the posterior centriole corresponds to the final state of centriole maturation.     

Basal body replication in green algae--when and where does it start?

Basal body duplication in the green alga Spermatozopsis similis was reinvestigated using GT335, an antibody binding to polyglutamylated tubulins, and antibodies directed to p210, a component of the flagellar transition region which represents the distal border of the basal body. p210 was also detected in small spots at the base of each basal body which increased in size prior to mitosis. The presence of p210 on one of the microtubular flagellar roots suggested a transport of basal body material along these tracks. Immunogold electron microscopy confirmed the presence of p210 in the probasal bodies. Further, small probasal bodies are apparently connected to the mature basal bodies by centrin fibers as observed after artificially induced basal body separation in Xenopus egg extract. While basal bodies grew, most of the p210 remained at the tip of elongating basal bodies, but two or four additional spots were observed in distinct patterns near the base of the basal bodies. In cytokinesis, basal body pairs separated and p210 was observed in a strong signal at the tip and a weaker one in the vicinity of the proximal end of each basal body. We interpret the data as indicating that a new p210-containing structure forms near the proximal end of the basal bodies during basal body elongation, representing the precursor of the next generation of basal bodies. Thus, basal bodies appear to seed the succeeding generation already during their own development, a mechanism which could ensure the correct number and position of basal bodies.     

Ultrastructure of the flagellar apparatus inPleurochrysis (classPrymnesiophyceae)

Summary The ultrastructure of the flagellar apparatus of aPleurochrysis, a coccolithophorid was studied in detail. Three major fibrous connecting bands and several accessory fibrous bands link the basal bodies, haptonema and microtubular flagellar roots. The asymmetrical flagellar root system is composed of three different microtubular roots (referred to here as roots 1,2, and 3) and a fibrous root. Root 1, associated with one of the basal bodies, is of the compound type, constructed of two sets of microtubules,viz. a broad sheet consisting of up to twenty closely aligned microtubules, and a secondary bundle made up of 100–200 microtubules which arises at right angles to the former. A thin electron-dense plate occurs on the surface of the microtubular sheet opposite the secondary bundle. The fibrous root arises from the same basal body and passes along the plasmalemma together with the microtubular sheet of root 1. Root 2 is also of the compound type and arises from one of the major connecting bands (called a distal band) as a four-stranded microtubular root and extends in the opposite direction to the haptonema. From this stranded root a secondary bundle of microtubules arises at approximately right angle. Root 3 is a more simple type, composed of at least six microtubules which are associated with the basal body. The flagellar transition region was found to be unusual for the classPrymnesiophyceae. The phylogenetic significance of the flagellar apparatus in thePrymnesiophyceae is discussed.     

FINE STRUCTURE STUDIES OF PHLEUM ROOT MERISTEM CELLS. II. MITOTIC ASYMMETRY AND CELLULAR DIFFERENTIATION

Avers , Charlotte J. (Douglass Coll., Rutgers—The State U., New Brunswick, N.J.) Fine structure studies of Phleum root meristem cells. II. Mitotic asymmetry and cellular differentiation. Amer. Jour. Bot. 50(2): 140–148. Illus. 1963.—An electron microscopical study showed that ultrastructural differences distinguished the cell dividing symmetrically from that undergoing asymmetrical division in timothy grass epidermis. The spindle orientation led to cytokinesis which produced either equal- or unequal-sized sister cells, but the mitotic apparatus itself varied in the mitoses. In asymmetrical cells, the basal pole showed more extensive endoplasmic reticulum (ER) polarizations, which intruded into the spindle area during metaphase and anaphase. Such ER polarity was not obvious in symmetrical mitosis or in the apical end of asymmetrically dividing cells. The mitotic sequence is described photographically. Foci of ER were observed as early as prophase in the polar region, and it is suggested that there is a resemblance to astral ray foci seen in prophase of animal cell mitosis. Cell plate formation could be detected in anaphase by accumulations of vesicles and ER fragments along the spindle equator. Phragmosomes apparently were not involved in cell plate formation in Phleum, unlike Allium, cytokinesis. The mitotic asymmetry is discussed as a consequence of an intracellular gradient separate from the intercellular gradient of differentiation along the entire length of growing root tip epidermis.     

LIGHT MICROSCOPE AND ULTRASTRUCTURAL STUDIES OF THE MALE GAMETOPHYTE IN GINKGO BILOBA: THE SPERMATOGENOUS CELL

A light microscope and ultrastructural study was made of the pollen tube of Ginkgo biloba, with special emphasis given to the spermatogenous cell that gives rise to two motile sperms. Just prior to the mitotic division that results in the formation of two sperms, the spermatogenous cell consists of a large nucleus, two blepharoplasts, two large osmiophilic globules, and a conspicuous lipo-protein body. Other organelles in the cytoplasm include numerous electron-dense proplastids (with some lamellar development), mitochondria, small vacuoles, and lipid bodies. Ribosomes are present in abundance, but endoplasmic reticulum and dictyosomes are sparse. The nucleus, prior to mitosis, is relatively Feulgen-negative, due undoubtedly to the diffuse distribution of DNA. Each blepharoplast, the main organelle of interest, is nearly spherical, measures 3.5–4.5 μm in diam, and supports about 1,000 probasal bodies. The interior of a blepharoplast consists of an electron-dense matrix and of less dense regions which appear to be infiltrated by a network of microtubules. Each probasal body is composed of a cylinder of nine separate tubules (singlets) at the basal or proximal end. The cylinder becomes elaborated distally into nine pairs of subtubules (doublets) and then into nine sets of subtubules (triplets). A central tubule is present the entire length of the probasal body. Some of the subtubules, as well as microtubules from the interior of the blepharoplast, extend into the cytoplasm and probably constitute the “astral rays” as seen with the light microscope. Comparisons are made with other published accounts of the organization of blepharoplasts in plants and of centrosomes and centrioles in animals.     

Behavior of silver stainable material during mitosis inVicia faba

To reveal the behavior of silver stainable material localized mainly in the nucleoli and nucleolar organizing regions (NORs), the somatic cells ofVicia faba were investigated by silver staining throughout the mitotic cell cycle. Nucleoli of interphase and early prophase nuclei were darkly stained. From late prophase to anaphase the secondary constrictions were discriminated as silver stained NORs and many silver grains appeared throughout the cytoplasm. At late prophase the NOR condensed at the same rate as the chromosome arm. Small spherical bodies and two new nucleoli appeared in telophase nuclei and at the same time the cytoplasmic grains disappeared. On the basis of the above observations on the silver stainable material during each mitotic phase, the behavior of silver stainable material is interpreted.     

Purification and characterization of a basal body-associated Ca2+- binding protein

Isolated basal body complexes from the unicellular alga, Chlamydomonas reinhardtii were found to contain a low molecular mass acidic polypeptide, distinct from calmodulin, but with biochemical features in common with members of the calmodulin family of calcium-binding proteins. These common characteristics included a relative low molecular mass of 20 kD, an experimentally determined acidic pI of 5.3, an altered electrophoretic mobility in SDS-polyacrylamide gels in the presence of added calcium, and a calcium-dependent binding to the hydrophobic ligand phenyl-Sepharose which allowed its purification by affinity chromatography. The relatedness of the basal body-associated 20-kD calcium-binding protein (CaBP) to calmodulin was confirmed by amino acid compositional analysis and partial peptide sequencing of the isolated protein. A rabbit antibody specific for the 20-kD CaBP was raised and used to determine by indirect immunofluorescence the cellular localization of the protein in Chlamydomonas cells. In interphase cells the antibody stained intensely the region between the paired basal bodies, two fibers extending between the basal bodies and the underlying nucleus, and an array of longitudinal filaments surrounding the nucleus. The two basal body-nuclear connecting fibers were identified in thin-section electron micrographs to be narrow striated fiber roots. In mitotic cells the 20-kD CaBP was specifically associated with the poles of the mitotic spindle at the sites of the duplicated basal body complexes.     

The Centriole Cartwheel Protein SAS-6 in Trypanosoma brucei Is Required for Probasal Body Biogenesis and Flagellum Assembly

The centriole in eukaryotes functions as the cell''s microtubule-organizing center (MTOC) to nucleate spindle assembly, and its biogenesis requires an evolutionarily conserved protein, SAS-6, which assembles the centriole cartwheel. Trypanosoma brucei, an early branching protozoan, possesses the basal body as its MTOC to nucleate flagellum biogenesis. However, little is known about the components of the basal body and their roles in basal body biogenesis and flagellum assembly. Here, we report that the T. brucei SAS-6 homolog, TbSAS-6, is localized to the mature basal body and the probasal body throughout the cell cycle. RNA interference (RNAi) of TbSAS-6 inhibited probasal body biogenesis, compromised flagellum assembly, and caused cytokinesis arrest. Surprisingly, overexpression of TbSAS-6 in T. brucei also impaired probasal body duplication and flagellum assembly, contrary to SAS-6 overexpression in humans, which produces supernumerary centrioles. Furthermore, we showed that depletion of T. brucei Polo-like kinase, TbPLK, or inhibition of TbPLK activity did not abolish TbSAS-6 localization to the basal body, in contrast to the essential role of Polo-like kinase in recruiting SAS-6 to centrioles in animals. Altogether, these results identified the essential role of TbSAS-6 in probasal body biogenesis and flagellum assembly and suggest the presence of a TbPLK-independent pathway governing basal body duplication in T. brucei.     

Changes in the absolute configuration of the basal/flagellar apparatus and evidence of centrin during male gametogenesis in Chara contraria var. nitelloides (Charales, Charophyta)

The absolute configurations of the basal/flagellar apparatus during male gametogenesis of Chara contraria var. nitelloides (Charales, Charophyta) were carefully analysed. Emphasis was placed on the changes in the angles and lengths of the basal bodies, the microtubular root angles and the development of the distal as well the proximal connecting fibers. Six principal stages were recognized: a) parallel, non-axonemal, developing basal bodies connected by a non-striated, proximal fiber; b) non-parallel, non-axonemal, mature basal bodies connected by a developing, striated, distal fiber; c) non-parallel, axonemal basal bodies connected by a fully developed, striated, distal fiber; d) opposite, axonemal basal bodies not connected by fibers, e) axonemal basal bodies not connected by fibers and directed backwards and f) parallel, axonemal basal bodies not connected by fibers. A headpiece, a 3-membered root and a reduced multilayered structure developed during ontogeny. The initial parallel disposition of the basal bodies, the initial lack of MLS and the presence of only two microtubular roots from the very inception of the basal apparatus development, suggest a Mamiella-like ancestor for Charales. Ontogenetic evidence supports previous ideas in the sense that similarities of sperm morphology of charalean and bryophytan gametes are likely due to convergent evolution. In addition, the present study clearly reveals the presence of centrin in Charales.     

Mitotic reversion in prophase of PTK1 cells induced by argon laser microirradiation

In this paper, we report the effects of laser microirradiation of prophase nucleoli and mitotic chromosomes in cells of female rat kangaroo kidney epithelial cell line PTK₁. When the laser power delivered to sample surface was 90–190 mW, irradiation of one of the two nucleoli in the prophase cell did not inhibit the mitotic progress, but resulted in the loss of the irradiated nucleolus in daughter cells. When the laser power was increased to 360–420 mW, either irradiation of the nucleolus or chromosome in midprophase caused a blockage of mitosis at terminal midprophase. The irradiated cells returned morphologically to early prophase. No mitotic reversion occurred in the case of irradiation of chromosomes at late prophase, prometaphase, metaphase, and anaphase. Irradiation of the cytoplasm in prophase cells caused a 50–70 min mitotic delay at prophase. However, the irradiated cells underwent successive mitotic divisions. The mechanism of laser-induced mitotic prophase reversion is discussed.     

Transformation and development of the flagellar apparatus ofCryptomonas ovata (Cryptophyceae) during cell division

Summary InCryptomonas ovata, long, dorsal flagella are produced which transform during the following cell division into short, ventral flagella. At division there is a reorientation in cell polarity, and the parental basal apparatus, which comprises the basal bodies and associated roots, is distributed to the daughter cells via a complex sequence of events. Flagellar apparatus development includes the transformation of a four-stranded microtubular root into a mature root of different structure and function. Each newly formed basal body nucleates new microtubular roots, but receives a striated fibrous root from a parental basal body. The striated roots are originally produced on the transforming basal body and are transferred to the new basal bodies at each successive division. The development of the asymmetric flagellar apparatus throughout the cell cycle is described.     

The Nucleolus in the Induced Amitosis

Nucleolar cycles were related to mitotic and amitotic phasesin meristematic cells of Allium cepa L. roots growing in controlconditions and in others where amitosis was induced by a 6-htreatment with ethidium bromide. The induced amitosis in these cells was followed by an unbalanceddistribution of chromosomal material between the two independentdaughter cells resulting from it. The first discrepancy between mitosis and amitosis was foundat the level of prophase nucleolar disorganization, which wasgreatly modified. The nucleolar cycle was permanently stoppedin amitosis even when recovery from treatment was allowed. Thekinetics of amitosis appearance in the meristem suggest a decisionpoint located round about mid-prophase. This decision pointseems to involve nucleolar function in prophase. These observationsconfirm the fact that prophase is one of the decisive stagesin the differentiation of a cell.     

Ultrastructure of the flagellar apparatus in the green flagellateSpermatozopsis similis

The ultrastructure of the flagellar apparatus of the naked, biflagellate green algaSpermatozopsis similis Preisig & Melkonian has been studied in detail using an absolute configuration analysis. The two basal bodies are displaced by 350 nm in the 1/7 o'clock direction and do not overlap proximally. They are interconnected by a principal distal connecting fibre consisting of a bundle of 5–8 nm filaments and possibly two proximal striated connecting fibres. The flagellar root system is cruciate (5-2-5-2 or 4-2-4-2 system) and contains a prominent continuous system I fibre overlying the two opposite two-stranded roots. A system II fibre is absent. Pronounced structural differences have been observed in the flagellar apparatus ultrastructure at two types of flagella orientation: During backward swimming basal bodies are parallel, the distal connecting fibre is extremely contracted; during forward swimming basal bodies assume various angles (from 20° to 180°) and the connecting fibre is about five times longer compared to the contracted state. The function of the connecting fibre as a contractile organelle and the mechanism of its contraction are discussed. On the basis of the flagellar apparatus ultrastructure,Spermatozopsis similis is related toChlamydomonas-type green algae.     

Temporal pattern of changes in mitotic frequency in the epidermis and other larval tissues of Bombyx mori

Temporal changes in mitotic frequency were examined in various tissues through late larval life of Bombyx mori. From the second larval ecdysis to the third and from the third larval ecdysis to the fourth, there was a definite temporal change of mitotic pattern in each tissue. In the epidermis as well as in the tracheal epithelium, mitoses began to appear about 1 day after an ecdysis, and showed a maximum 1 to 2 days after an ecdysis. In the fat body, mitoses were observed continuously through the instars, and the mitotic frequency showed a maximum state just before an ecdysis. In the abdominal muscle the frequency was highest at about the middle of the period between two successive ecdyses. Furthermore, epidermal mitoses coincided with the time when the density of epidermal nuclei per unit area decreased to a half. This suggests that epidermal mitoses may be initiated by some process related to the increase in cell size.     

Flagellar apparatus ultrastructure inMesostigma viride (Prasinophyceae)

The ultrastructure of the flagellar apparatus ofMesostigma viride Lauterborn (Prasinophyceae) has been studied in detail with particular reference to absolute configurations, numbering of basal bodies, basal body triplets and flagellar roots. The two basal bodies are interconnected by three connecting fibers (one distal fiber = synistosome, and two proximal fibers). The flagellar apparatus shows 180° rotational symmetry; four microtubular flagellar roots and two system II fibers are present. The microtubular roots represent a 4-6-4-6-system. The left roots (1s, 2s) consist of 4 microtubules, each with the usual 3 over 1 root tubule pattern. Each right root (1d, 2d) is proximally associated with a small, but typical multi-layered structure (MLS). The latter displays several layers corresponding to the S₁ (the spline microtubules: 5–7), and presumably the S₂—S₄ (the lamellate layers) of the MLS of theCharophyceae. At its proximal origin (near the basal bodies) each right root originates with only two microtubules, the other spline microtubules being added more distally. The structural and positional information obtained in this study strongly suggest that one of the right roots (1d) ofMesostigma is homologous to the MLS-root of theCharophyceae and sperm cells of archegoniate land plants. Thus the typical cruciate flagellar root system of the green algae and the unilateral flagellar root system of theCharophyceae and archegoniates share a common ancestry. Some functional and phylogenetic aspects of MLS-roots are discussed.Dedicated to Prof. DrLothar Geitler on the occasion of his 90th birthday.