Abstract. Chlamydomonas reinhardtii cells shed their flagella in response to environmental stress . Under favorable conditions, flagella are quickly regrown . To learn more about the signals that trigger flagellar excision and regrowth we have investigated inositol phospholipid metabolites, molecules implicated in signal transduction in several other systems . After deflagellation by low pH or mastoparan, a potent activator of G proteins, there was a rapid increase in levels of inositol 1,4,5-trisphosphate measured by use of receptor-binding assays and HPLC. This increase was concomitant with a decrease in levels of phosphatidylinositol 4,5-bisphosphate and was followed by an increase in phosphatidic acid, results consistent with activation of phospholipase C and diacylglycerol kinase. Additional experiments suggest that this activated phospho-HE unicellular green alga Chlamydomonas reinhardtii sheds its two flagella when confronted with environmental stresses such as pH shock or mechanical shear (38, 39) . Deflagellation is quickly followed by increased synthesis of flagellar mRNA (2, 27, 31) and protein (29) and regrowth offlagella, which reach full length -90 min after deflagellation . Little is known about the biochemical pathways that couple environmental stress to deflagellation and the subsequent synthesis and assembly of flagellar components. Initially, the environmental stress (e.g ., pH shock) is communicated to the molecular apparatus responsible for deflagellation, and subsequently the deflagellated condition of the cell is communicated to the elements controlling relevant gene expression and flagellar assembly.The involvement of Caz+ in control of deflagellation (14,40, 42), flagellar mRNA accumulation (12a), and flagellar assembly (29) led us to postulate that products of inositol phospholipid hydrolysis might be components of signal transduction in this system. During signal transduction via the ino- lipase C is not important for flagellar regrowth but plays a role in informing the excision apparatus of the environmental stress . Addition of neomycin (an inhibitor of phospholipase C) before exposure of cells to low pH or mastoparan prevented the increase in inositol 1,4,5-trisphosphate and also prevented deflagellation . Addition of neomycin after deflagellation blocked increases in inositol 1,4,5-trisphosphate that normally followed deflagellation, but did not block flagellar assembly. Furthermore, a flagellar excision-defective mutant, fa-1, did not shed its flagella in response to low pH or mastoparan, yet both of these agents activated phospholipase C in these cells. The results suggest that activation of phospholipase C, possibly via a G protein, is a proximal step in the signal transduction pathway inducing deflagellation in Chlamydomonas.sitol phospholipid pathway in other systems, signal-activated phospholipase C hydrolyzes phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2)t yielding the "second messengers" inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) and 1,2-sn-diacylglycerol (DAG) . Th...

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Removal of the flagella of Chlamydomonas results in increases in both flagellar protein synthesis and tubulin messenger RNA accumulation. These observations led us to examine whether flagellar protein gene sequences are transcribed differentially in nuclei isolated before and after deflagellation. A nuclear isolation protocol was developed using the cell wall-less strain of Chlamydomonas, CW 15, after cell lysis with 0.5% Nonidet P-40. Transcriptional activity of isolated nuclei was determined by incorporating [32P]UTP into TCA-precipitable and phenol-extractable RNA, and by hybridizing newly transcribed RNA to complementary DNA clones containing alpha- and beta-tubulin sequences. Nuclei from deflagellated cells are more active in transcribing sequences that hybridize with alpha- and beta-tubulin complementary DNA probes than are nuclei from nondeflagellated cells. In addition, while total [32P]UTP incorporation is inhibited 45% by alpha-amanitin concentrations of 1.0 micrograms/ml, tubulin RNA synthesis in this system is completely inhibited by this concentration of alpha-amanitin. This demonstration of differential transcription in nuclei before and after cell deflagellation provides the means to study in vitro the mechanisms that signal and regulate flagellar protein gene activity.

Flagella are sensory organelles that interact with the environment through signal transduction and gene expression networks. We used microarray profiling to examine gene regulation associated with flagellar length change in the green alga Chlamydomonas reinhardtii. Microarrays were probed with fluorescently labeled cDNAs synthesized from RNA extracted from cells before and during flagellar assembly or disassembly. Evaluation of the gene expression profiles identified .100 clones showing at least a twofold change in expression during flagellar length changes. Products of these genes are associated not only with flagellar structure and motility but also with other cellular responses, including signal transduction and metabolism. Expression of specific genes from each category was further characterized at higher resolution by using quantitative real-time PCR (qRT-PCR). Analysis and comparison of the gene expression profiles coupled to flagellar assembly and disassembly revealed that each process involves a new and uncharacterized whole-cell response to flagellar length changes. This analysis lays the groundwork for a more comprehensive understanding of the cellular and molecular networks regulating flagellar length changes.