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.

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.

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.

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.

A 210 kDa protein is located in a membrane-microtubule linker at the distal end of mature and nascent basal bodies.

A monoclonal antibody raised against purified flagellar basal apparatuses from the green flagellate Spermatozopsis similis reacted with a protein of 210 kDa (p210) in western blots. The protein was partially cloned by immunoscreening of a cDNA library. The sequence encoded a novel protein rich in alanine (25%) and proline (20%), which contained regions similar to proteins of comparable amino acid composition such as extracellular matrix components or the membrane-cytoskeletal linker synapsin. Using a polyclonal antibody (anti-p210) raised against the C-terminal part of p210, it was shown that the protein was highly enriched in the basal apparatuses. Immunogold electron microscopy of isolated cytoskeletons or whole cells revealed that p210 was located in the flagellar transition region. The protein was part of the Y-shaped fibrous linkers between the doublet microtubules and the flagellar membrane, as indicated by statistical analysis of post-labeled sections using anti-centrin and anti-tubulin as controls. In premitotic cells p210 was located in a fibrous layer at the distal end of nascent basal bodies, which was perforated by the outgrowing axoneme. During deflagellation the protein remained at the basal body but we observed changes in its distribution, indicating that p210 partially moved to the tip of the basal body. p210 can be used as a marker to determine basal body position, orientation (parallel or antiparallel) and number in S. similis by indirect immunofluorescence. We suppose that p210 is involved in linking basal bodies to the plasma membrane, which is an important step during ciliogenesis.

SF-assemblin, striated fibers, and segmented coiled coil proteins.

The flagellar basal apparatus of many flagellate green algae contains noncontractile striated microtubule-associated fibers (SMAFs). The SMAFs consist of 2-nm protofilaments and are predominantly built from striated fiber (SF)-assemblin, an acidic 33-kDa protein. In this review we summarize the present knowledge concerning the biochemical properties of SF-assemblin and the molecular architecture of the SMAFs, provide evidence for homologous proteins and similar filament systems in other eukaryotes, and, finally, discuss possible biological functions of SF-assemblin.

Analysis of striated fiber formation by recombinant SF-assemblin in vitro.

The basal bodies of green flagellates are often connected to striated microtubule-associated fibers (SMAFs), which are highly ordered bundles of 2 nm filaments. SF-assemblin (33 kDa) is the principal structural subunit of the SMAFs and consists of a non-helical head domain of approximately 32 residues and an alpha-helical rod domain that shows a pronounced coiled-coil forming ability. To investigate the functional role of the head domain we expressed N-terminally truncated molecules using a cDNA coding for SF-assemblin from Chlamydomonas reinhardtii. Recombinant wild-type SF-assemblin or molecules with an N-terminal deletion of ten residues formed striated fibers with an axial repeat of 28 nm. N-terminal truncations of 19 and 29 residues yielded assembly-incompetent molecules, revealing that the head domain is necessary for the constitution of striated fibers. Further, molecules with an internal deletion of 24 residues or with duplicated segments corresponding to insertions of 29 and 58 residues were constructed. The resulting fibers had altered cross-striation patterns and axial repeats. The observed shifts in the axial repeat corresponded well to the number of inserted or deleted residues, indicating a linear coherence between molecule length and axial repeat. The heptad pattern of the rod domain of SF-assemblin is regularly interrupted by skip residues. The removal of one or two skip residues had no significant effect on the ultrastructure of the striated fibers. Substitution of skip no. 2 with alanine resulted in a modified, asymmetric cross-striation pattern, indicating a polar architecture of the striated fibers. In summary, various mutations of SF-assemblin effected the solubility of the molecules, and the axial repeat, cross-striation pattern, or overall appearance of the fibers. Thus, analysis of SF-assemblin may represent a valuable system to study the interactions involved in the polymerization of fibrous coiled-coil proteins. A model of the SMAFs based on staggered protofilaments consisting of overlapping 36 nm subunits is presented.

A novel 95-kD protein is located in a linker between cytoplasmic microtubules and basal bodies in a green flagellate and forms striated filaments in vitro.

The flagellar basal apparatus comprises the basal bodies and the attached fibrous structures, which together form the organizing center for the cytoskeleton in many flagellated cells. Basal apparatus were isolated from the naked green flagellate Spermatozopsis similis and shown to be composed of several dozens of different polypeptides including a protein band of 95 kD. Screening of a cDNA library of S. similis with a polyclonal antibody raised against the 95-kD band resulted in a full-length clone coding for a novel protein of 834 amino acids (90.3 kD). Sequence analysis identified nonhelical NH2- and COOH-terminal domains flanking a central domain of approximately 650 residues, which was predicted to form a series of coiled-coils interrupted by short spacer segments. Immunogold labeling using a polyclonal antibody raised against the bacterially expressed 95-kD protein exclusively decorated the striated, wedge-shaped fibers, termed sinister fibers (sf-fibers), attached to the basal bodies of S. similis. Striated fibers with a periodicity of 98 nm were assembled in vitro from the purified protein expressed from the cloned cDNA indicating that the 95-kD protein could be a major component of the sf-fibers. This structure interconnects specific triplets of the basal bodies with the microtubular bundles that emerge from the basal apparatus. The sf-fibers and similar structures, e.g., basal feet or satellites, described in various eukaryotes including vertebrates, may be representative for cytoskeletal elements involved in positioning of basal bodies/centrioles with respect to cytoskeletal microtubules and vice versa.

A Novel Basal Apparatus Protein of 90 kD (BAp90) from the Flagellate Green Alga Spermatozopsis similis is a Component of the Proximal Plates and Identifies the d-(dexter)Surface of the Basal Body.

The flagellar basal apparatus consists of the basal bodies and associated fibrous structures, and represents the organizing center for the microtubular cytoskeleton in many flagellate protists. To identify novel proteins associated with the basal bodies, basal apparatuses from the flagellate green alga Spermatozopsis similis (Chlorophyceae) were isolated and purified. A polyclonal antibody raised against a 95kD protein band enriched in purified basal apparatuses was used to screen a cDNA library of S. similis which resulted in the isolation of a full length clone coding for a novel protein of 812 amino acids (85.3kD). Sequence analysis of this clone identified extended a-helical regions and predicted several coiled-coil forming domains interrupted by spacer segments of variable lengths. A polyclonal antibody (anti-BAp90) raised against the bacterially expressed protein recognized a 90kD band (BAp90) in SDS-PAGE of isolated basal apparatuses of S. similis. Immunogold labeling using anti-BAp90 decorated the proximal plates (two striated, triangular fibers which serve as spacers between the basal bodies in their proximal region) and parts of the d-fibers (df) which interconnect the basal bodies with the microtubular d-roots and the striated microtubule-associated fibers (SMAFs). Thus, the 90kD basal apparatus protein of S. similis represents a biochemical landmark for the lateral asymmetry of the basal body identifying its d-(dexter)surface. Cytoskeletal elements containing BAp90 or structurally related proteins may be involved in the organization and/or maintenance of the positional relationship between basal bodies and the cellular cytoskeleton, and hence cell polarity.

SF-assemblin in Chlamydomonas: sequence conservation and localization during the cell cycle.

Previously, SF-assemblin has been identified as the filament-forming component of the striated microtubule-associated fibers (SMAFs), which emerge from the basal bodies in several green flagellates. We have sequenced cDNAs coding for SF-assemblin from Chlalmydomonas reinhardtii and C. eugametos. Comparison of the deduced amino acid sequences with the previously described green algal SF-assemblins shows identities between 54 and 71%, indicating a strong drift in sequence. Cells of C. reinhardtii were analyzed by double immunofluorescence using polyclonal anti-SF-assemblin and anti-alpha-tubulin. In interphase cells, SF-assemblin is associated with all four microtubular flagellar roots. During mitosis the SF-assemblin-based cytoskeleton is reorganized; it divides in prophase and is reduced to two dot-like structures at each spindle pole in metaphase. During anaphase, the two dots present at each pole are connected again. In telophase we observed an asymmetrical outgrowth of new fibers. These observations suggest a role for SF-assemblin in reestablishing the microtubular root system characteristic of interphase cells after mitosis.

Purification and characterization of basal apparatuses from a flagellate green alga.

Basal apparatuses consisting of two basal bodies and several attached fibers were isolated from the naked green flagellate Spermatozopsis similis by detergent extraction and mechanical disintegration. Sucrose density centrifugation yielded highly enriched basal apparatuses as shown by electron microscopy. SDS-PAGE revealed the absence of histones, indicating the removal of nuclear contaminations from the isolated basal apparatuses. A mass spectrometric analysis of the carboxyterminal peptides of alpha tubulin documented detyrosination and glutamylation as posttranslational modifications and showed that some 5% of the alpha tubulin carries a polyglutamyl side chain which can reach at least 17 residues in length. Monoclonal antibodies raised against the purified basal apparatuses were used to characterize novel components in the basal apparatus. A 210-kD component identified by mAB BAS (basal apparatus of Spermatozopsis) 1.4 was localized in the flagellar transitional region by immunogold electron microscopy. Antibody BAS 16.4 reacted with two high molecular weight bands (approximately 265 and 240 kD) in Western blotting and decorated a fiber attached to the proximal end of the basal bodies. Immunofluorescence staining of isolated cytoskeletons with these mABs demonstrated that the antigens are also present in the basal apparatuses of Chlamydomonas reinhardtii and Dunahella bioculata. These antibodies are useful tools for the molecular cloning of components from the basal apparatus.

The cruciated microtubule-associated fibers of the green alga Dunaliella bioculata consist of a 31 kDa SF-assemblin.

Cytoskeletons of Dunaliella bioculata, the biflagellate wallless green alga, were isolated and analyzed using a monoclonal and a polyclonal antibody raised against SF-assemblin, the major protein of the two striated microtubule-associated fibers of the alga Spermatozopsis similis. Indirect immunofluorescence showed antigenic structures associated with the four microtubular flagellar roots. SDS-PAGE followed by immunoblot analysis revealed a cross-reacting polypeptide of 31 kDa. This protein of D. bioculata was isolated using gel filtration chromatography in 8 M urea and in vitro reassembly of striated fibers. Microsequencing of the purified protein yielded various peptides, which could be aligned along the sequence of SF-assemblin from S. similis. A complete sequence of the Dunaliella protein was obtained by cDNA cloning. It documents the non helical head domain followed by a helical rod domain with a 29 residue repeat pattern based on four heptads followed by a skip residue. Compared to SF-assemblin of S. similis the SF-assemblin of Dunaliella has a shorter head and a slightly longer rod domain. The two algal SF-assemblins share only 57% sequence identity. We conclude that SF-assemblin and related proteins in various protists are representatives of a new class of alpha-helical proteins characterized by the ability to form a special segmented coiled coil and to assemble into striated fibers of 2 nm protofilaments in vivo and in vitro.

SF-assemblin, the structural protein of the 2-nm filaments from striated microtubule associated fibers of algal flagellar roots, forms a segmented coiled coil.

The microtubule associated system I fibers of the basal apparatus of the flagellate green alga Spermatozopsis similis are noncontractile and display a 28-nm periodicity. Paracrystals with similar periodicities are formed in vitro by SF-assemblin, which is the major protein component of system I fibers. We have determined the amino acid sequence of SF-assemblin and show that it contains two structural domains. The NH2-terminal 31 residues form a nonhelical domain rich in proline. The rod domain of 253 residues is alpha-helical and seems to form a segmented coiled coil with a 29-residue repeat pattern based on four heptads followed by a skip residue. The distinct cluster of acidic residues at the COOH-terminal end of the motifs (periodicity about 4 nm) may be related to tubulin binding of SF-assemblin and/or its self assembly. A similar structure has been predicted from cDNA cloning of beta-giardin, a protein of the complex microtubular apparatus of the sucking disc in the protozoan flagellate Giardia lamblia. Although the rod domains of SF-assemblin and beta-giardin share only 20% sequence identity, they have exactly the same length and display 42% sequence similarity. These results predict that system I fibers and related microtubule associated structures arise from molecules able to form a special segmented coiled coil which can pack into 2-nm filaments. Such molecules seem subject to a strong evolutionary drift in sequence but not in sequence principles and length. This conservation of molecular architecture may have important implications for microtubule binding.

Structure of striated microtubule-associated fibers of flagellar roots. Comparison of native and reconstituted states.

Several fiber systems are associated with the flagella and basal bodies of eukaryotic cells. Apart from the contractile and Ca(2+)-sensitive system-II fibers, these include the noncontractile system-I fibers that run parallel to flagellar root microtubules. Using electron microscopy and image reconstruction, we have investigated the structure of the system-I fibers of the flagellate green alga Spermatozopsis similis. The fibers were observed in three different states: (1) in situ, (2) after isolation of the intact fibers, (3) after disassembly and reconstitution of fibers in vitro from their 34 kDa subunit protein. The fibers are highly ordered; they show a constant repeat of 28 nm, they are polar, and they contain several transverse and longitudinal striations. A model is discussed showing the system-I fiber to be built from rod-like molecules with a staggered arrangement and identical polarities.

Striated microtubule-associated fibers: identification of assemblin, a novel 34-kD protein that forms paracrystals of 2-nm filaments in vitro.

Microtubule-associated fibers from the basal apparatus of the green flagellate alga Spermatozopsis similis exhibit a complex cross-striation pattern with 28-nm periodicity and consist of 2-nm filaments arranged in several layers. Fibers enriched by mechanical disintegration and high salt extraction (2 M NaCl) of isolated basal apparatuses are soluble in 2 M urea. Dialysis of solubilized fibers against 150 mM KCl yields paracrystals which closely resemble the native fibers in filament arrangement and striation pattern. Paracrystals purified through several cycles of disassembly and reassembly are greatly enriched (greater than 90%) in a single protein of 34 kD (assemblin) as shown by SDS-PAGE. A rabbit polyclonal antibody raised against assemblin labels the striated fibers as shown by indirect immunofluorescence of isolated cytoskeletons or methanol permeabilized cells and immunogold EM. Two-dimensional electrophoresis (isoelectric focusing and SDS-PAGE) resolves assemblin into at least four isoforms (a-d) with pI's of 5.45, 5.55, 5.75, and 5.85. The two more acidic isoforms are phosphoproteins as shown by in vivo 32PO4-labeling and autoradiography. Amino acid analysis of assemblin shows a high content of helix-forming residues (leucine) and a relatively low content of glycine. We conclude that assemblin may be representative of a class of proteins that form fine filaments alongside microtubules.