Rapid changes in tubulin RNA synthesis and stability induced by deflagellation in Chlamydomonas.

Abstract

Detachment of the flagella of Chlamydomonas induces a rapid accumulation of mRNAs for tubulin and other flagellar proteins. Measurement of the rate of alpha and beta tubulin RNA synthesis during flagellar regeneration shows that deflagellation elicits a rapid, 4-7-fold burst in tubulin RNA synthesis. The synthesis rate peaks within 10-15 rain, then declines back to the predeflagellation rate. Redeflagellation of cells at times before the first flagellar regeneration is completed (and when cells have already accumulated elevated levels of tubulin RNA) induces another burst in tubulin RNA synthesis which is identical to the first in magnitude and duration. This finding indicates that the induction signal may act to simply reprogram the tubulin genes for a transient burst of maximal synthesis. Evidence is presented that the stability of the tubulin RNAs changes during regeneration. Stability changes include both an apparent stabilization during regeneration and accelerated decay following regeneration.The eucaryotic biflagellate, Chlamydomonas reinhardtii, undergoes flagellar regeneration after mechanical detachment of its flagella. The response to deflagellation involves a large increase in the synthesis of flagellar proteins (14, 33) effected by an increased accumulation (6-40-fold) of their messenger RNAs (15,20,29,31). Flagella are rapidly assembled, within 60-90 min, and subsequently flagellar-specific protein synthesis and mRNA abundance return to basal levels. Thus the response to deflagellation involves both an induction and a deinduction, the entire process spanning <3 h.Recent evidence from this laboratory indicates that the increase in alpha and beta tubulin mRNA levels is due, at least in part, to a transcriptional activation of the tubulin genes. Nuclei isolated from deflagellated cells support an average fivefold more transcription oftubulin RNA sequences than do nuclei from nondeflagellated cells (12). We were interested in obtaining more detailed information about the extent and duration of the transcriptional induction and its termination. In particular, we wanted to know the maximum level of induction and when it occurs, the timing of the deinduction, and whether it can be correlated with completion of flagellar assembly, and whether the activation of tubulin RNA synthesis is sufficient to account for the large increase in abundance observed.In this report, we describe the results of in vivo labeling experiments designed to elucidate the tubulin RNA synthetic and decay changes occurring throughout the regeneration process. MATERIALS AND METHODS Cell Growth and Preparation: Chlamydomonas reinhardtii cells,strain 21gr, were grown in a low phosphate variant of medium I (27) in which potassium phosphate salts have been reduced 20-fold (0.065 raM) and the media buffered with 10 mM HEPES. Cells were grown to ~1 x 106 celis/ml except where noted. For deflagellation and labeling, cells were concentrated to 1-2 x l07 cells/ml in spent media or in fresh media containing no phosphate. Cells were def...

Light Regulation of β-Tubulin Gene Expression during Internode Development in Soybean (Glycine max [L.] Merr.)

“…Illumination of 10-day-old etiolated seedlings not only stopped first internode elongation, but also brought about a 80% decrease in the steady state level of ,-tubulin mRNA over the course of the subsequent 12 hours. This strong down regulation of j6-tubulin mRNA occurred without significant changes in the size of the soluble tubulin pool and it was accompanied by a marked increase in chlorophyll a/b binding protein mRNA.cases, the tubulin dimer pool is maintained through both transcriptional and post-transcriptional control of tubulin gene expression (1,17). Little is known about the regulation of tubulin synthesis in plants, but Cyr et al (8) observed changes of several fold in the levels of tubulin protein and mRNA during carrot somatic embryogenesis.…”

Coordinate and selective beta-tubulin gene expression associated with cilium formation in sea urchin embryos.

“…Deflagellation of Chlamydomonas has been shown to induce tubulin gene transcription directly (Baker et al 1984;Keller et al 1984). In the sea urchin embryo a transcriptional response to deciliation was suggested by results of actinomycin D (Merlino et al 1978).…”

“…The higher peak concentration in the animalized embryo (5 x 10 s, compared with 3 x l0 s molecules per embryo) indicates that any concentration-dependent feedback control mechanism responsible for such a regulation would be pitched at a different level of sensitivity from that in the control embryo. Such a mechanism, which can be expected to depend directly on the levels of protein generated by the ~-tubulin mRNA, has indeed been posited to depend on the pool size of unpolymerized tubulin in mammalian cells (Ben Ze'ev et al 1979;Cleveland et al 1981;Caron et al 1985a}, and to involve tubulin mRNA stability in response to microtubule depolymerization in enucleated fibroblasts (Caron et al 1985b) and during flagellum regeneration in Chlamydomonas (Baker et al 1984). Posttranscriptional regulation is also apparent during cilium regeneration in the animalized embryo, since f~-tubulin mRNA levels increase without a change in transcription rate.…”

“…The major proteins of axonemal microtubules, the ~-and ~-tubulins, need to be synthesized for complete flagellum regeneration in Chlamydomonas (Rosenbaum et al 1969) and may be drawn from a preexisting pool, as in the case of cilium regeneration in Tetrahymena (Guttman and Gorovsky 1979). Deflagellation induces the coordinately increased transcription of the two ~-and two f3-tubulin genes of Chlamydomonas (Brunke et al 1984;Youngblom et al 1984;Silflow and Rosenbaum 1985) as well as the stabilizaiton of tubulin mRNA (Baker et al 1984). In the case of the sea urchin embryo, cilium regeneration could be demonstrated to proceed through an initial phase that is independent of new protein synthesis and a second phage that depends on protein synthesis (Bums 1979).…”

Uncoupling of Chlamydomonas flagellar gene expression and outgrowth from flagellar excision by manipulation of Ca2+.

“…A variety of extracellular treatments cause Chlamydomonas to excise their flagella (19a, 19, 21, 32) . When flagella are shed, expression ofa specific set offlagellar genes, including tubulin, is stimulated (1,9), and cells begin to assemble a new pair of full-length flagella, using flagellar proteins translated from these newly synthesized mRNA templates, and preexisting flagellar proteins from a pool of unassembled precursors (21).…”