The basal bodies of Chlamydomonas reinhardtii. Formation from probasal bodies, isolation, and partial characterization

The assembly and composition of basal bodies was investigated in the single-celled, biflagellate green alga, Chlamydomonas reinhardtii, using the cell wall-less strain, cw15. In the presence of EDTA, both flagellar axonemes remained attached to their basal bodies while the entire basal body-axoneme complex was separated from the cell body, without cell lysis, by treatment with polyethylene glycol-400. The axonemes were then removed from the basal bodies in the absence of EDTA, leaving intact basal body pairs, free from particulate contamination from other regions of the cell. The isolated organelles produced several bands on sodium dodecyl sulfate-urea polyacrylamide gels, including two tubilin bands which co-electrophoresed with flagellar tubulin. The formation of probasal bodies was observed by electron microscopy of whole mount preparations. Synchronous cells were lysed, centrifuged onto carbon-coated grids, and either negatively stained or shadowed with platinum. The two probasal bodies of each cell appeared shortly after mitosis as thin "annuli," not visible in thin sections, each consisting of nine rudimentary triplet microtubules. Each annulus remained attached to one of the mature basal bodies by several filaments about 60 in diameter, and persisted throughout interphase until just before the next cell division. It then elongated into a mature organelle. The results revive the possibility of the nucleated assembly of basal bodies.     

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.     

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     

Assembly and fate of basal bodies in the colourless phytoflagellate Polytoma papillatum

Summary— The morphogenesis of basal bodies is described in the phytoflagellate Polytoma papillatum. The observations are based on the analysis of ultrathin serial sections through the flagellar apparatus of interphase, mitotic, and postmitotic cells using transmission electron microscopy. Formation of new basal bodies starts in prometaphase. Individual A-subfibres develop orthogonally to the long axis of mature basal bodies. The microtubules assemble at the surface of an annulus of amorphous material. By telophase, a complete cylinder of A-subfibres with a length of approximately 300 nm has formed. Although the proximal ends of these new probasal bodies are detached from the mature basal bodies, prominent reorientation of the probasal bodies does not occur. They remain with their proximal ends in the vicinity of mature basal bodies. In daughter cells with probasal bodies around 400 nm long, the assembly of microtubular triplets is initiated. B- and C-subfibres first show up distal from the mature basal bodies and may elongate towards them. Thus, A-subfibres on the one side and B- and C-subfibres on the other appear to growt with opposite polarity. If A-subfibres grow at their plus ends, B- and C-subfibres elongate at their minus ends. The latter is unusual in comparison with individual cytoplasmic and spindle microtubules. Possible the presence of a lateral template in the form of the A-subfibres is responsible for the deviating growth characteristics of the incomplete B- and C-subfibres. In interphase cells, the mature basal bodies extend into long flagella. The new basal bodies remain devoid of flagella and are less than 85 nm long. Thus, they have shortened relative to their precursors in mitotic and postmitotic cells. At the onset of a new division cycle, the flagellate basal badies shed their flagella. The breaking point is at the triplet-doublet transition of the flagellum.     

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.     

Katanin Knockdown Supports a Role for Microtubule Severing in Release of Basal Bodies before Mitosis in Chlamydomonas

Katanin is a microtubule-severing protein that participates in the regulation of cell cycle progression and in ciliary disassembly, but its precise role is not known for either activity. Our data suggest that in Chlamydomonas, katanin severs doublet microtubules at the proximal end of the flagellar transition zone, allowing disengagement of the basal body from the flagellum before mitosis. Using an RNA interference approach we have discovered that severe knockdown of the p60 subunit of katanin, KAT1, is achieved only in cells that also carry secondary mutations that disrupt ciliogenesis. Importantly, we observed that cells in the process of cell cycle-induced flagellar resorption sever the flagella from the basal bodies before resorption is complete, and we find that this process is defective in KAT1 knockdown cells.     

Localization of p210-related proteins in green flagellates and analysis of flagellar assembly in the green alga Dunaliella bioculatawith monoclonal anti-p210

Recently, p210 was identified as a component of the flagellar basal apparatus in the green flagellate Spermatozopsis similis. In a search for potential homologues to p210, isolated cytoskeletons of several green flagellates were probed with a monoclonal antibody, BAS4.13, against p210. In Western blots, cross-reacting bands in the molecular-mass range of 210 kDa were detected only in the quadriflagellate Spermatozopsis exsultans. As described earlier for S. similis, the flagellar transition region was decorated in Chlamydomonas reinhardtii and several other green flagellates, whereas in the marine alga Dunaliella bioculata the antigen was present in the proximal part of the axoneme. Double immunofluorescence of D. bioculata with an antitubulin antibody further revealed dotlike signals at sites where the probasal bodies are located. Since most of the antigen in D. bioculata was located in the axoneme, deflagellation offered a possibility to study the kinetics of its incorporation during flagellar regeneration. The antigen was only detected after a flagellum reached a length of 3-4 microm and its integration into the growing flagellar proceeded from proximal to distal. A similar delay in the incorporation of the antigen was also observed during flagellar assembly on new basal bodies during cell division. Thus, the antigen of BAS4.13 was incorporated late and from proximal to distal into the growing flagellum. We conclude that the pace and site by which individual proteins are integrated into the flagellum differ greatly.     

The UNI3 Gene Is Required for Assembly of Basal Bodies of Chlamydomonas and Encodes δ-Tubulin, a New Member of the Tubulin Superfamily

We have cloned the UNI3 gene in Chlamydomonas and find that it encodes a new member of the tubulin superfamily. Although Uni3p shares significant sequence identity with α-, β-, and γ-tubulins, there is a region of Uni3p that has no similarity to tubulins or other known proteins. Mutant uni3–1 cells assemble zero, one, or two flagella. Pedigree analysis suggests that flagellar number in uni3–1 cells is a function of the age of the cell. The uniflagellate uni3–1 cells show a positional phenotype; the basal body opposite the eyespot templates the single flagellum. A percentage of uni3–1 cells also fail to orient the cleavage furrow properly, and basal bodies have been implicated in the placement of cleavage furrows in Chlamydomonas. Finally when uni3–1 cells are observed by electron microscopy, doublet rather than triplet microtubules are observed at the proximal end of the basal bodies. We propose that the Uni3 tubulin is involved in both the function and cell cycle-dependent maturation of basal bodies/centrioles.     

An evolutionarily conserved coiled-coil protein implicated in polycystic kidney disease is involved in basal body duplication and flagellar biogenesis in Trypanosoma brucei

Trypanosoma brucei is a flagellated protozoan with a highly polarized cellular structure. TbLRTP is a trypanosomal protein containing multiple SDS22-class leucine-rich repeats and a coiled-coil domain with high similarity to a mammalian testis-specific protein of unknown function. Homologues are present in a wide range of higher eukaryotes including zebra fish, where the gene product has been implicated in polycystic kidney disease. Western blot analysis and immunofluorescence with antibodies against recombinant TbLRTP indicate that the protein is expressed throughout the trypanosome life cycle and localizes to distal zones of the basal bodies. Overexpression and RNA interference demonstrate that TbLRTP is important for faithful basal body duplication and flagellum biogenesis. Expression of excess TbLRTP suppresses new flagellum assembly, while reduction of TbLRTP protein levels often results in the biogenesis of additional flagellar axonemes and paraflagellar rods that, most remarkably, are intracellular and fully contained within the cytoplasm. The mutant flagella are devoid of membrane and are often associated with four microtubules in an arrangement similar to that observed in the normal flagellar attachment zone. Aberrant basal body and flagellar biogenesis in TbLRTP mutants also influences cell size and cytokinesis. These findings demonstrate that TbLRTP suppresses basal body replication and subsequent flagellar biogenesis and indicate a critical role for the LRTP family of proteins in the control of the cell cycle. These data further underscore the role of aberrant flagellar biogenesis as a disease mechanism.     

Gamma-tubulin in Chlamydomonas: characterization of the gene and localization of the gene product in cells

In addition to their role in nucleating the assembly of axonemal microtubules, basal bodies often are associated with a microtubule organizing center (MTOC) for cytoplasmic microtubules. In an effort to define molecular components of the basal body apparatus in Chlamydomonas reinhardtii, genomic and cDNA clones encoding gamma-tubulin were isolated and sequenced. The gene, present in a single copy in the Chlamydomonas genome, encodes a protein with a predicted molecular mass of 52,161 D and 73% and 65% conservation with gamma-tubulin from higher plants and humans, respectively. To examine the distribution of gamma-tubulin in cells, a polyclonal antibody was raised against two peptides contained within the protein. Immunoblots of Chlamydomonas proteins show a major cross-reaction with a protein of Mr 53,000. In Chlamydomonas cells, the antibody stains the basal body apparatus as two or four spots at the base of the flagella and proximal to the microtubule rootlets. During cell division, two groups of fluorescent dots separate and localize to opposite ends of the mitotic apparatus. They then migrate during cleavage to positions known to be occupied by basal bodies. Changes in gamma-tubulin localization during the cell cycle are consistent with a role for this protein in the nucleation of microtubules of both the interphase cytoplasmic array and the mitotic spindle. Immunogold labeling of cell sections showed that gamma-tubulin is closely associated with the basal bodies. The flagellar transition region was also labeled, possibly indicating a role for gamma-tubulin in assembly of the central pair microtubules of the axoneme.     

Centrin scaffold in Chlamydomonas reinhardtii revealed by immunoelectron microscopy

In the flagellate green alga Chlamydomonas reinhardtii the Ca(2+)-binding EF-hand protein centrin is encoded by a single-copy gene. Previous studies have localized the protein to four distinct structures in the flagellar apparatus: the nucleus-basal body connector, the distal connecting fiber, the flagellar transitional region, and the axoneme. To explain the disjunctive distribution of centrin, the interaction of centrin with as yet unknown specific centrin-binding proteins has been implied. Here, we demonstrate using serial section postembedding immunoelectron microscopy of isolated cytoskeletons that centrin is located in additional structures (transitional fibers and basal body lumen) and that the centrin-containing structures of the basal apparatus are likely part of a continuous filamentous scaffold that extends from the nucleus to the flagellar bases. In addition, we show that centrin is located in the distal lumen of the basal body in a rotationally asymmetric structure, the V-shaped filament system. This novel centrin-containing structure has also been detected near the distal end of the probasal bodies. Taken together, these results suggest a role for a rotationally asymmetric centrin "seed" in the growth and development of the centrin scaffold following replication of the basal apparatus.     

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.     

Distribution of polyglutamylated tubulin in the flagellar apparatus of green flagellates

Polyglutamylation is a widely distributed posttranslational modification of tubulin that can be demonstrated either by biochemical analysis or by the use of specific antibodies like GT335. Western blotting using GT335 demonstrated that polyglutamylated tubulin is enriched in isolated basal apparatus of Spermatozopsis similis. Single- and double-labeling experiments, using indirect immunofluorescence and immunogold electron microscopy of isolated cytoskeletons of S. similis and Chlamydomonas reinhardtii, revealed that polyglutamylated tubulin was predominately present in the basal bodies and the proximal part of the axonemes. Using immunogold labeling of whole mounts of Spermatozopsis cytoskeletons, we obtained evidence for a predominant occurrence of polyglutamylated tubulin in the B-tubule of the axonemal doublets. Polyglutamylation occurs early during premitotic basal body assembly in S. similis, whereas the probasal bodies of Chlamydomonas, which are present through interphase, showed a reduced staining with GT335 indicating that polyglutamylation is involved in basal body maturation. During flagella regeneration of C. reinhardtii, polyglutamylation preceded detyrosination and became visible shortly after the onset of flagellar regeneration. In C. reinhardtii and S. similis polyglutamylated tubulin was absent or highly reduced in the flagellar transition region, a specialized part of the flagellum linking the basal body to the axoneme. Furthermore, the transition region and the neighboring part of the axoneme showed reduced staining with L3, an antibody directed against detyrosinated tubulin. The results indicate that differences in the modification pattern can occur in a confined area of individual microtubules. The deficiency of polyglutamylated and detyrosinated tubulin in the transition region could have functional implications for flagellar turnover or excision.     

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.     

In vivo localization of centrin in the green alga Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii has been used as a model system to study flagellar assembly, centriole assembly, and cell cycle events. These processes are dynamic. Therefore, protein targeting and protein-protein interactions should be evaluated in vivo. To be able to study dynamic processes in C. reinhardtii in vivo, we have explored the use of the green fluorescent protein (GFP). A construct containing a fusion of centrin and GFP was incorporated into the genome as a single copy. The selected clone shows expression in 25-50% of the cells. Centrin-GFP was targeted in vivo to the nuclear basal body connectors and the distal connecting fibers. At the electron microscopic level, it was also localized to the flagellar transitional regions. EM data of transformants indicate that there are some abnormalities in the centrin-containing structures. The transitional region consists of only the transverse septum or has lesions in the H-piece. The distal connecting fibers are thinner and their characteristic crossbands seem to be incomplete. Deflagellation is not affected since more than 95% of the cells deflagellate. Also basal body segregation is not affected since cells with an abnormal flagellar number were not detected. Functional studies of the centrin-GFP fusion show the characteristic calcium-induced mobility shift in SDS-PAGE. Immunofluorescence revealed that during cell division, centrin-GFP remains associated with the basal bodies. In vivo localization of the fusion protein during cell division shows that in metaphase centrin-GFP appears as two opposing spots located close to the spindle poles. The distance between the spots increases as the cells progress through anaphase and then decreases during telophase. GFP is a useful tool to study dynamic processes in the cytoskeleton of C. reinhardtii.     

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.     

Localization of p210-related proteins in green flagellates and analysis of flagellar assembly in the green alga Dunaliella bioculata with monoclonal anti-p210

Summary.  Recently, p210 was identified as a component of the flagellar basal apparatus in the green flagellate Spermatozopsis similis. In a search for potential homologues to p210, isolated cytoskeletons of several green flagellates were probed with a monoclonal antibody, BAS4.13, against p210. In Western blots, cross-reacting bands in the molecular-mass range of 210 kDa were detected only in the quadriflagellate Spermatozopsis exsultans. As described earlier for S. similis, the flagellar transition region was decorated in Chlamydomonas reinhardtii and several other green flagellates, whereas in the marine alga Dunaliella bioculata the antigen was present in the proximal part of the axoneme. Double immunofluorescence of D. bioculata with an antitubulin antibody further revealed dotlike signals at sites where the probasal bodies are located. Since most of the antigen in D. bioculata was located in the axoneme, deflagellation offered a possibility to study the kinetics of its incorporation during flagellar regeneration. The antigen was only detected after a flagellum reached a length of 3–4 μm and its integration into the growing flagellar proceeded from proximal to distal. A similar delay in the incorporation of the antigen was also observed during flagellar assembly on new basal bodies during cell division. Thus, the antigen of BAS4.13 was incorporated late and from proximal to distal into the growing flagellum. We conclude that the pace and site by which individual proteins are integrated into the flagellum differ greatly. Received February 18, 2002; accepted May 17, 2002; published on line October 31, 2002 RID="*" ID="*" Correspondence and reprints: Botanisches Institut, Universit?t zu K?ln, Gyrhofstrasse 15, 50931 K?ln, Federal Republic of Germany     

THE CYTOSKELETON OF THE NAKED GREEN FLAGELLATE SPERMATOZOPSIS SIMILIS (CHLOROPHYTA):FLAGELLAR AND BASAL BODY DEVELOPMENTAL CYCLE1

Flagellar and basal body development during cell division was studied in the biflagellate green alga Spermatozopsis similis Preisig et Melkonian by light microscopy of immobilized living cells, statistical analysis of flagellar lengths during the cell cycle, and electron microscopy of cells and isolated cytoskeletons. Interphase cells display two flagella of unequal/subequal length. An eyespot located in an anterior lobe of the chloroplast is connected to the basal body bearing the shorter flagellum by means of a five-stranded microtubular root. Until cell division, the two parental flagella attain the same length. During cell division, each cell forms two new flagella that grow to a length of 1.5 μm before they are distributed in a semiconservative fashion together with the parental flagella to the two progeny cells at cytokinesis. During the following interphase, the flagella newly formed during the preceding cell division grow to attain the same length as the parental flagella until the subsequent cell division. The shorter of the two flagella of a cell thus represents the developmentally younger flagellum, which transforms to the mature state during two consecutive cell cycles. Interphase cells display only two flagella-bearing basal bodies; two nascent basal bodies are formed during cell division and are connected to the microtubular d-roots of respective parental basal bodies with which the newly formed basal bodies are later distributed to the progeny cells. During segregation, basal body pairs shaft into the 11/5 o'clock direction, thus conserving the 1/7 o'clock configuration of basal body pairs of interphase cells. Prior to chloroplast and cell division, an eyespot is newly formed near the cell posterior in close association with a 1s microtubular root, while the parental eyespot is retained. During basal body segregation, eyespot-root connections for both the old and newly formed eyespots are presumably lost, and new associations of the eyespots with the 2s roots of the newly formed basal bodies are established during cytokinesis. The significance of this “eyespot-flagellar root developmental cycle” for the absolute orientation of the progeny cells is discussed.     

Basal body reorientation mediated by a Ca2+-modulated contractile protein

A rapid, Ca2+-dependent change in the angle between basal bodies (up to 180 degrees) is associated with light-induced reversal of swimming direction (the "photophobic" response) in a number of flagellated green algae. In isolated, detergent-extracted, reactivated flagellar apparatus complexes of Spermatozopsis similis, axonemal beat form conversion to the symmetrical/undulating flagellar pattern and basal body reorientation (from the antiparallel to the parallel configuration) are simultaneously induced at greater than or equal to 10(-7) M Ca2+. Basal body reorientation, however, is independent of flagellar beating since it is induced at greater than or equal to 10(-7) M Ca2+ when flagellar beating is inhibited (i.e., in the presence of 1 microM orthovanadate in reactivation solutions; in the absence of ATP or dithiothreitol in isolation and reactivation solutions), or when axonemes are mechanically removed from flagellar apparatuses. Although frequent axonemal beat form reversals were induced by varying the Ca2+ concentration, antiparallel basal body configuration could not be restored in isolated flagellar apparatuses. Observations of the photophobic response in vivo indicate that even though the flagella resume the asymmetric, breaststroke beat form 1-2 s after photostimulation, antiparallel basal body configuration is not restored until a few minutes later. Using an antibody generated against the 20-kD Ca2+-modulated contractile protein of striated flagellar roots of Tetraselmis striata (Salisbury, J. L., A. Baron, B. Surek, and M. Melkonian, 1984, J. Cell Biol., 99:962-970), we have found the distal connecting fiber of Spermatozopsis similis to be immunoreactive by indirect immunofluorescence and immunogold electron microscopy. Electrophoretic and immunoblot analysis indicates that the antigen of S. similis flagellar apparatuses consists, like the Tetraselmis protein, of two acidic isoforms of 20 kD. We conclude that the distal basal body connecting fiber is a contractile organelle and reorients basal bodies during the photophobic response in certain flagellated green algae.