Purification of basal bodies and basal body complexes from Chlamydomonas reinhardtii.
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Cytoskeletal discontinuities in the cell body cortex initiate basal body assembly and oral development in the ciliate Stentor.
My previous work has shown that disconnecting the oral apparatus of Stentor into two parts induces mass assembly of basal bodies on the ventral cell surface and thus initiates oral development. This operation severs the extensive microtubule tracts joining the oral membranelles at their bases. To determine whether basal body assembly and oral development are also induced by permanently disconnecting the longitudinal microtubule fibre tracts (mt fibre tracts) of the cell body cortex, I interposed a ring of inverted (heteropolar) cortex between the anterior and posterior halves of interphase stentors. When successful, this operation made it impossible for these fibre tracts to rejoin at the heteropolar boundaries and always induced basal body assembly and oral development in the graft complex. By contrast, tripartite homopolar graft complexes rarely initiated oral development; when they did, it was apparently in response to the presence of disproportionately small oral structures, which is the normal stimulus for oral development in Stentor. The mt fibre tracts of tripartite homopolar grafts also eventually became continuous. These results support the hypothesis that permanent, extensive discontinuities anywhere within the cortical cytoskeleton can trigger basal body assembly and oral development. Since the onset of these processes is known to initiate cell division in Stentor, the results also suggest that development of discontinuities within the cortical cytoskeleton during interphase growth may be the endogenous stimulus initiating cell division in Stentor.
Animals↗
A nucleus-basal body connector in Chlamydomonas reinhardtii that may function in basal body localization or segregation.
We have isolated a nucleus-basal body complex from Chlamydomonas reinhardtii. The complex is strongly immunoreactive to an antibody generated against a major protein constituent of isolated Tetraselmis striata flagellar roots (Salisbury, J. L., A. Baron, B. Surek, and M. Melkonian, J. Cell Biol., 99:962-970). Electrophoretic and immunoelectrophoretic analysis indicates that, like the Tetraselmis protein, the Chlamydomonas antigen consists of two acidic isoforms of approximately 20 kD. Indirect immunofluorescent staining of nucleus-basal body complexes reveals two major fibers in the connector region, one between each basal body and the nucleus. The nucleus is also strongly immunoreactive, with staining radiating around much of the nucleus from a region of greatest concentration at the connector pole. Calcium treatment causes shortening of the connector fibers and also movement of nuclear DNA towards the connector pole. Electron microscopic observation of negatively stained nucleus-basal body complexes reveals a cluster of approximately 6-nm filaments, suspected to represent the connector, between the basal bodies and nuclei. A mutant with a variable number of flagella, vfl-2-220, is defective with respect to the nucleus-basal body association. This observation encourages us to speculate that the nucleus-basal body union is important for accurate basal body localization within the cell and/or for accurate segregation of parental and daughter basal bodies at cell division. A physical association between nuclei and basal bodies or centrioles has been observed in a variety of algal, protozoan, and metazoan cells, although the nature of the association, in terms of both structure and function, has been obscure. We believe it likely that fibrous connectors homologous to those described here for Chlamydomonas are general features of centriole-bearing eucaryotic cells.
Calcium↗
Nucleus-basal body connector in Chlamydomonas: evidence for a role in basal body segregation and against essential roles in mitosis or in determining cell polarity.
In the unicellular biflagellate green alga Chlamydomonas reinhardtii each basal body is linked to the nucleus by a fibrous nucleus-basal body connector (NBBC) that contains the calcium-binding protein centrin. (Wright et al.: Journal of Cell Biology 101:1903-1912.; Salisbury et al.: Journal of Cell Biology 107:635-642; Huang et al.: Journal of Cell Biology 107:121-131). In order to explore the cellular function of the NBBC we used antiserum directed against centrin to examine a number of mutants known to be defective for basal body assembly and/or localization. Of three variable flagella-number mutants examined, one, vfl-2, is dramatically defective with respect to the NBBC in that 1) the union between basal bodies and nucleus is very labile, 2) there is no detectible centrin in the NBBC region, and 3) total cellular centrin levels are reduced 75-80% relative to wild type. The existence of these defects in a mutant incapable of maintaining normal flagellar number supports the view that the NBBC plays an important role in determining proper basal body localization and/or segregation. In contrast to vfl-2, the mutants vfl-1, vfl-3, uni-1, and bald-2 contain approximately normal levels of centrin and possess stable NBBCs. The observation of NBBCs in the mutant bald-2, which lacks all but very rudimentary basal bodies, indicates that the assembly of the NBBC does not require fully formed basal bodies and that such assembly may not require basal bodies at all. Finally, the possibility that the NBBC is required for induction of gene expression following deflagellation was tested by examining vfl-2 for such induction. Results indicate that the connector does not play a necessary role in the induction process.
Animals↗
Evaluation of the basal body temperature; an analysis of 1012 basal body temperature recordings.
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Basal Bodies↗
Prediction of ovulation with basal body temperature.
Basal body temperature (BBT) is used extensively to monitor ovulation in the treatment of infertility. There are well-defined methods of interpreting the BBT graph retrospectively that identify a presumptive day of ovulation. The prediction of ovulation through utilization of the BBT graph (strictly concurrent interpretation) is adversely influenced by temperature fluctuation and cycle length variability. The BBT graph can be very useful when its interpretation is well defined and its limitations understood.
Body Temperature↗
Electrophoretic analysis of basal body (centriole) proteins.
Purified basal bodies isolated from the chicken oviduct were analyzed by using several different electrophoretic techniques. For comparison, oviduct cilia proteins were also analyzed. Prominent among the basal body proteins were the tubulin subunits (representing approximately 20% of the protein) and a low molecular weight protein (approximately 17,400 daltons). In addition, major bands were present with molecular weights of approximately 180,000 and approximately 90,000. Electrophoretically purified basal body tubulin subunits had isoelectric points of 5.45 (alpha subunit) and 5.1 (beta subunit). In addition, these isoelectric focus gels contained at least four other proteins that had higher isoelectric points, which indicates that tubulin purified by one-dimensional electrophoresis contains other proteins. On the basis of several different electrophoretic techniques, it was found that basal body tubulin differed from cilia tubulin even though they both had similar isoelectric focusing points. Whereas basal bodies did not contain any proteins that corresponded to the cilia dynein ATPase, five different sets of proteins were common to both cilia and basal bodies. Basal bodies did not contain significant amounts of actin, myosin, or desmin.
Animals↗
Assembly of chick brain tubulin onto isolated basal bodies of Chlamydomonas reinhardi.
Basal bodies isolated from Chlamydomonas reinhardi will serve as initiation centers for the assembly of chick brain microtubule protein subunits (tubulin) into microtubules. The rate of microtubule assembly is tubulin-concentration dependent; this assembly occurs onto both distal and proximal ends of the basal body mnicrotubules, with distal assembly greatly favored. In vitro assembly of brain tubulin also occurs onto the mid-lateral aspects of the basal bodies, presumably onto the fiber connecting the two basal bodies.
Animals↗
Centrin deficiency in Paramecium affects the geometry of basal-body duplication.
BACKGROUND: Ciliary or flagellar basal bodies and centrioles share the same architecture and remarkable property of duplicating once per cell cycle. Duplication is known to proceed by budding of the daugther organelle close to and at right angles to the mother structure, but the molecular basis of this geometry remains unknown. Among the handful of proteins implicated in basal-body/centriole duplication, centrins seem required in all eukaryotes tested, but their mode of action is not clear. We have investigated centrin function in Paramecium, whose cortical organization allows detection of any spatial or temporal alteration in the pattern of basal-body duplication. RESULTS: We have characterized two pairs of genes, PtCEN2a and PtCEN2b as well as PtCEN3a and PtCEN3b, orthologs of HsCEN2 and HsCEN3, respectively. GFP tags revealed different localization for the two pairs of gene products, at basal bodies or on basal-body-associated filamentous arrays, respectively. Centrin depletion induced by RNAi caused mislocalization of the neoformed basal bodies: abnormal site of budding (PtCen2ap) or absence of separation between mother and daughter organelles (PtCen3ap). Over successive divisions, new basal bodies continued to be assembled, but internalization of the mispositionned basal bodies led to a progressive decrease in the number of cortical basal bodies. CONCLUSIONS: Our observations show that centrins (1) are required to define the site and polarities of duplication and to sever the mother-daughter links and (2) play no triggering or instrumental role in assembly. Our data underscore the biological importance of the geometry of the duplication process.
Amino Acid Sequence↗
An updated basal body temperature method.
Basal body temperature (BBT) readings are handicapped as fertility predictors by exogenous influences on women's temperatures, many of which could be adjusted for by the presence of a non-cycling control. We, a married, cohabiting couple, tracked our BBTs for two months. We found substantial temperature covariability. When there were difficult-to-explain changes in the female partner's temperature, similar changes in the male partner's temperature suggested that these fluctuations were not attributable to ovulation. Additionally, a clear mid-cycle widening of our temperature gap suggested that ovulation had occurred. This is a limited trial of a new method. However, the potential for substantially improving the accuracy and usefulness of a globally utilized method, coupled with its inexpensiveness, ease, and painlessness, call for a larger study.
Adult↗
Basal body replication and cilogenesis 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.
Cell Membrane↗
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.
Animals↗
The formation of basal bodies (centrioles) in the Rhesus monkey oviduct.
Basal body replication during estrogen-driven ciliogenesis in the rhesus monkey (Macaca mulatta) oviduct has been studied by stereomicroscopy, rotation photography, and serial section analysis. Two pathways for basal body production are described: acentriolar basal body formation (major pathway) where procentrioles are generated from a spherical aggregate of fibers; and centriolar basal body formation, where procentrioles are generated by the diplosomal centrioles. In both pathways, the first step in procentriole formation is the arrangement of a fibrous granule precursor into an annulus. A cartwheel structure, present within the lumen of the annulus, is composed of a central cylinder with a core, spoke components, and anchor filaments. Tubule formation consists of an initiation and a growth phase. The A tubule of each triplet set first forms within the wall material of the annulus in juxtaposition to a spoke of the cartwheel. After all nine A tubules are initiated, B and C tubules begin to form. The initiation of all three tubules occurs sequentially around the procentriole. Simultaneous with tubule initiation is a nonsequential growth of each tubule. The tubules lengthen and the procentriole is complete when it is about 200 mmicro long. The procentriole increases in length and diameter during its maturation into a basal body. The addition of a basal foot, nine alar sheets, and a rootlet completes the maturation process. Fibrous granules are also closely associated with the formation of these basal body accessory structures.
Animals↗
Real-time observation of Ca2+-induced basal body reorientation in Chlamydomonas.
The two basal bodies of Chlamydomonas are connected by a bridge, the distal fiber, that contains a Ca2+-binding protein, centrin. Although various fibrous structures in many organisms containing centrin or similar proteins have been shown to contract at Ca2+ concentrations >10(-7)-10(-6) M, the contractility of the distal fiber in Chlamydomonas has not been demonstrated. To determine whether it undergoes Ca2+-dependent contraction, we isolated the flagella-basal body complex from the paralyzed-flagella mutant pf18 and measured the angle between the two axonemes at different Ca2+ concentrations. Use of a double mutant with the mutant fa1, deficient in the mechanism for Ca2+-dependent flagellar amputation, enabled the measurement at Ca2+ concentrations > or = 10(-4) M. The angle, 80-120 degrees at 10(-9) M Ca(2-), was found to decrease by about 20 degrees when the Ca2+ concentration was raised above 10(-6) M. The angle increased again when the Ca2+ concentration was lowered below 10(-7) M. The flagellar apparatuses isolated from the double mutant between pf18 and the mutant vfl2 deficient in the structural gene of centrin had an angle of 90-130 degrees at 10(-9) M Ca2+, but the angle did not change when the Ca2+ concentration was increased. Thus centrin must be involved in the basal body reorientation. In detergent-extracted cell models of the pf18fa1 mutant, the angle between the two axonemes was found to decrease transiently by about 15 degrees upon iontophoretic application of Ca2+. Hence, the Ca2+-induced basal body reorientation can take place even when the basal body is contained in the cell body covered by the cell wall. It may function as part of the mechanism for phobic responses wherein Chlamydomonas cells swim backward transiently upon reception of strong light or mechanical stimuli.
Animals↗
Atypical basal bodies in a cat with immotile-cilia syndrome.
Atypical basal bodies from the oviductal mucosa of a female, 2.5-years-old cat suffering from immotile-cilia syndrome are described. The microtubular pattern was observed in 250 cross-sectional basal bodies. Four basal bodies (1.6%) had a defective number of microtubular triplets resulting in the appearance of so-called 'half-centriole' type. Two out of 100 longitudinal basal bodies showed duplicating striated rootlets and only one had an additional set of nine subdistal appendages, so that two basal feet arose from a single basal body. Electron-dense material and isolated microtubules into the lumen of some basal apparatuses were occasionally seen. It is known that centrioles and basal bodies show an unusual degree of constancy in size, shape and geometrical configuration, so that their abnormalities are very rare.
Animals↗
Patterns of basal body addition in ciliary rows in Tetrahymena.
Most naked basal bodies visualized in protargol stains on the surface of Tetrahymena are new basal bodies which have not yet developed cilia. The rarity of short cilia is explained by the rapid development of the ciliary shaft once it begins to grow. The high frequency of naked basal bodies (about 50 percent) in log cultures indicates that the interval between assembly of the basal body and the initiation of the cilium is long, approximately a full cell cycle. Naked basal bodies are more frequent in the mid and posterior parts of the cell and two or more naked basal bodies may be associated with one ciliated basal body in these regions. Daughter cells produced at division are apparently asymmetric with respect to their endowment of new and old organelles.
Animals↗
Adenosine triphosphate induced change in light scattering of isolated basal bodies.
Suspensions of isolated basal bodies undergo a characteristic decrease in turbidity following their exposure to 10(-3) M ATP. Typically, turbidity changes range from 8% to 20%, depending on the preparation, with an average change of 12%. Nucleotides other than ATP did not cause a turbidity decrease. The reaction has a pH optimum of pH 8.5 and is inhibited by concentrations of divalent cations greater than 2 X 10(-3) M. These results indicate that ATP induces a conformational change in the basal body that may be related to its activity in the cell.
Adenosine Triphosphate↗
Image reconstruction of the flagellar basal body of Salmonella typhimurium.
The basal body is thought to be a part of the rotary motor of the bacterial flagellum. It consists of a central rod coaxial with a set of four rings, which are associated with the cell envelope. We used single-particle averaging methods to analyze images of negatively stained basal bodies of Salmonella typhimurium. Several averages were computed, so that the reliability of features could be assessed. We carried out the same analysis on electron micrographs of isolated, negatively stained L-P rings. In order to interpret the averages in terms of a three-dimensional structure, we carried out image reconstruction on them. The resulting three-dimensional map corresponds to the cylindrically averaged structure of the basal body. To show that the reconstruction procedure is legitimate, we demonstrate it on model data. The results of the modelling show that features very near to the axis of the reconstruction are not reliable but that broader, off-axis features are represented faithfully. The L ring is L-shaped, with the long arm of the L parallel to the axis of the rod, and the short arm pointing away from the rod. The P ring, on the other hand, appears to be a ring or disk. The position of the L-P ring complex on the rod seems to vary somewhat, consistent with its putative role as a bushing. The cross-sectional shape of the S ring is that of a frustum rather than a disk. The M ring, which is oval in cross section, sits atop the S ring, making contact with it at an outer radius. The S ring appears to make contact with the rod, whereas the M ring does not. This situation, if true, is difficult to reconcile with the common notion that the S ring is stationary and the M ring rotates. It seems more likely that the S ring and rod rotate as a unit.
Flagella↗