Analysis of Chlamydomonas SF-assemblin by GFP tagging and expression of antisense constructs

Karl-Ferdinand Lechtreck Jutta Rostmann Andrea Grunow Botanisches Institut Universitt zu Kln Germany Author for correspondence (e-mail: ) - Striated fiber assemblin (SF-assemblin or SFA) is the major component of the striated microtubule-associated fibers (SMAFs) in the flagellar basal apparatus of green flagellates. We generated nuclear transformants of Chlamydomonas expressing green fluorescent protein (GFP) fused to the C-terminus of SFA. SFA-GFP assembled into striated fibers that exceeded those of wild-type cells in size by several fold. At elevated temperatures ( 32 C) SFAGFP was mostly soluble and heat shock depolymerized the SMAFs. C-terminal deletions of 18 or only six residues disturbed the ability of SFA-GFP to polymerize, indicating an important role of the C-terminal domain for fiber formation. The exchange of the penultimate Ser275 with alanine made SFA-GFP highly insoluble, causing aberrant fiber formation and conferring heat stability to the fibers. By contrast, a replacement with glutamic acid increased the solubilty of the molecule, indicating that phosphorylation The eukaryotic flagellar apparatus commonly consists of the protruding flagellum and the flagellar basal apparatus, a cytoskeletal complex that organizes and anchors the flagellum. The ultrastructure of the flagellar basal apparatus varies considerably among species and cell types. Microtubules surround the basal bodies in various patterns and the basal bodies are attached to a variety of fibrous structures (Pitelka, 1969). Immunological and biochemical analyses indicated that the fibrous elements in the basal apparatus are heterogeneous in composition but, in protists, at least two common types have been identified, namely striated fiber assemblin (SFA)- and centrin-fibers. The latter are contractile and include connections between basal bodies or between basal bodies and the cell nucleus. Centrin and centrin homologues were also observed in the lumen of basal bodies and centrioles, at spindle poles or in the half bridge of the yeast spindle pole body, suggesting a broad range of functions for this calcium-binding protein (Schiebel and Bornens, 1995). The second class of fibrous elements in the basal apparatus includes the microribbons of Giardia, the kinetodesmal fibers of ciliates and the green algal SMAFs (striated microtubule-associated fibers) (Holberton et al., 1988; Sperling et al., 1991; Lechtreck and Melkonian, 1991). These fibers are noncontractile, narrowly striated (~30 nm axial repeat) and often attached to microtubules (reviewed by Lechtreck and Melkonian, 1998). Although microribbons and kinetodesmal fibers are complex in composition (consisting of several proteins on Ser275 might control solubility of SFA. In vivo observation of GFP fluorescence showed that SFA-GFP fibers were disassembled during mitosis. In cells overexpressing full-length or truncated SFA-GFP, the amount of wild-type protein was reduced. Elevated temperatures dissolved SFA-GFP fibers and induced the synthesis of SFA, suggesting that cells control both the amount of soluble and polymeric SFA. By expressing constructs consisting of cDNA and genomic DNA for parts of SFA in antiparallel configuration, the amount of SFA was severely reduced. In these strains we observed defects in flagellar assembly, indicating an important role for noncontractile striated roots in the flagella apparatus. of ~30 kDa), SMAFs can be assembled in vitro solely from the 34 kDa protein SFA, making them an ideal system to study noncontractile striated fibers. In Chlamydomonas and other green flagellates, the SMAFs form a cross-like pattern and run alongside the proximal parts of the four bundles of flagellar root microtubules. Striated fiber assemblins from flagellate green algae are about 20% identical to b -giardin, one of the proteins in the microribbons (Weber et al., 1993). Both proteins share a similar domain structure with a short, nonhelical, proline-rich head domain and a coiled-coil domain of approximately 250 residues. Striated fiber assemblin is phosphorylated in vivo and the head domain of green algal SFAs contains several potential phosphorylation sites for the p34cdc2 kinase. It is not known whether phosphorylation at these sites is responsible for the disassembly of the SMAFs observed during mitosis (Lechtreck and Silflow, 1997). The rod domain of b -giardin/SFA is characterized by a distinctive series of heptads arranged into blocks of four, followed by a skip residue interrupting the heptad pattern (Holberton et al., 1988; Weber et al., 1993). A study of the in vitro assembly properties of ChlamydomonasSFA showed that headless molecules were assembly incompetent, whereas large deletions and insertions in the rod domain were tolerated and resulted in a shift in the axial repeat of the striated fibers (Lechtreck, 1998). SFA polymers are polar and consist of 2 nm protofilaments that are thought to be composed of parallel dimers overlapping with the N- and Cterminal parts of their rod domains (Patel et al., 1992; Lechtreck, 1998). Because SMAFs and similar fibers are rigid, they are thought to function as stabilizing elements in the basal apparatus. Noncontractile striated fibers, often with differing periodicities to the SMAFs, have been described in a broad range of organisms, including mammals, suggesting that these fibers have an important function in the basal apparatus. Detailed studies on in vivo function of SMAFs and similar roots are lacking, mainly because of the absence of mutants with defects in these fibers. Here, we present in vivo data on the expression of green fluorescent protein (GFP)-tagged SFA molecules and the repression of SFA expression by RNA interference. Our results indicate that the amount of SFA in Chlamydomonas is controlled by a complex mechanism balancing synthesis, degradation and polymerization of the protein, and that SMAFs are needed for correct flagellar assembly. Chlamydomonas reinhardtii strain CC-3395 (cwd, arg7.8) was maintained in TAP media supplemented with 0.02% ariginine in batch cultures (Gorman and Levine, 1965). If not otherwise noted, cells were maintained in a light:dark cycle of 14:10 hours and at 25C. Onset of light was at 6 oclock Middle European Summer Time (MESZ) and experiments were performed or started during the first six hours of the light phase. In heat-shock experiments cells were allowed to recover at room temperature unless otherwise indicated. All in vivo observation were carried out at room temperature. Plasmids and transformation A genomic library of Chlamydomonas (Schnell and Lefebvre, 1993) was screened using a cDNA-clone coding for SFA of C. reinhardtii (or CRA8) (see Lechtreck and Silflow, 1997). The screen resulted in three clones (BA1, BA5 and BA10); BA1 was used as a template for subsequent PCR reactions. The coding region of SFA including 88 bp of the 5 -UTR was amplified using Taq polymerase (GeneCraft, 488163 Mnster, Germany), a forwar (...truncated)