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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.

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Novel touch-induced, Ca(2+)-dependent phobic response in a flagellate green alga.

The biflagellate green alga Spermatozopsis similis exhibits a remarkable avoidance reaction in addition to the photophobic or stop response characteristic of such algae. S. similis normally swims forward with its anteriorly attached flagella directed posteriorly and propagating sine-like waves base to tip. Upon contact with surfaces or other cells, S. similis responds with rapid backward swimming, covering distances of up to 50 microns in 140 to 220 msec. This reaction, which we term the mechanoshock response, also can be triggered by vigorous mechanical stimulation, but not by physiological light intensities. It consists of 3 phases: (1) a rapid acceleration phase with average duration of 31 msec; (2) a phase of about 66 msec with constant high speed (maximal velocities of > 600 microns.sec-1) or slow deceleration; and (3) a deceleration phase of approximately 83 msec, followed by a stop or short period of circling. The cells then resume forward swimming in a random direction. Prior to the mechanoshock response the flagella rapidly are brought together into a close parallel configuration extending anteriorly of the cell body. They then appear to propel the cell by undulatory beating, while the cell describes a pronounced helical path. Small decreases in the extracellular Ca2+ concentration, as well as low concentrations of Ba2+, strongly suppress the probability of this phobic reaction. We conclude that this mechanoshock response involves large Ca2+ influxes, probably mediated by mechanosensitive and/or stretch-activated ion-channel(s).

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Genes for components of the chloroplast translational apparatus are conserved in the reduced 73-kb plastid DNA of the nonphotosynthetic euglenoid flagellate Astasia longa.

The colourless, nonphotosynthetic protist Astasia longa is phylogenetically related to Euglena gracilis. The 73-kb plastid DNA (ptDNA) of A. longa is about half the size of most chloroplast DNAs (cpDNAs). More than 38 kb of the Astasia ptDNA sequence has been determined. No genes for photosynthetic function have been found except for rbcL. Identified genes include rpoB, tufA, and genes coding for three rRNAs, 17 tRNAs, and 13 ribosomal proteins. Not only is the nucleotide sequence of these genes highly conserved between A. longa and E. gracilis, but a number of these genes are clustered in a similar fashion and have introns in the same positions in both species. The results further support the idea that photosynthetic genes normally encoded in cpDNA have been preferentially lost in Astasia, but that the chloroplast genes coding for components of the plastid translational apparatus have been maintained. This apparatus might be needed for the expression of rbcL and also for that of still unidentified nonphotosynthetic genes of Astasia ptDNA.

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