Uncoupling ofChlamydomonas Flagellar Gene Expression and Outgrowth from Flagellar Excision by Manipulation ofCall

Chlamydomonas cells respond to certain environmental stimuli by shedding their flagella . Flagellar loss induces a rapid, transient increase in expression of a specific set of genes encoding flagellar proteins, and assembly of a new flagellar pair. While flagellar gene expression and initiation of flagellar outgrowth are normally tightly coupled to flagellar excision, our results demonstrate that these processes can be uncoupled by manipulating Cal+ levels or calmodulin activity. In our experiments, wild-type cells were stimulated to excise their flagella using mechanical shearing, and at times after deflagellation, flagellar lengths were measured and flagellar mRNA abundance changes were determined by Sl nuclease protection analysis . When extracellular Caz+ was lowered by adELLULAR responses to environmental cues often involve complex interactions between signal transduction pathways, gene regulatory circuits, and changes in cell morphology. In many cases, these responses are integrated through an intracellular messenger, such as Caz+ (5) . Although the role of Caz+ in signaling mechanisms, physiological responses, and cell movements has been well studied, relatively little is known about the role of Caz+ in regulating gene expression . The flagellar regeneration system of the unicellular green alga Chlamydomonas reinhardtii serves as a key model for studying the complexities of these types of interactions . A variety of extracellular treatments cause Chlamydomonas to excise their flagella (19a, 19, 21, 32) . When flagella are shed, expression ofa specific set offlagellargenes, 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) . The involvement of Caz+ in several aspects of flagellar regeneration has been demonstrated in previous studies . For example, some studies suggest that flagellar excision itself requires Caz+ (19a, 24 and references therein) . In addition, outgrowth of flagella after excision is dependent on Caz+ . Outgrowth is delayed or prevented by lowering the extracellular Caz+ concentration ([Caz+]e) to <106M, and occurs when the [Caz+], is restored (18, 23) . In other studies, low [Caz+] e/high salt medium causes flagellated cells to resorb © The Rockefeller University Press, 0021-9525/91/12/1651/9 $2 .00 TheJournalof Cell Biology, Volume 115, Number6, December 1991 1651-1659 1651 dition of EGTA to cultures before excision, flagellar mRNA abundance changes and flagellar outgrowth were temporally uncoupled from flagellar excision . When extracellular Cal+ was lowered immediately after excision or when calmodulin activity was inhibited with W-7, flagellar outgrowth was uncoupled from flagellar excision and flagellar mRNA abundance changes . Whenever events in the process of flagellar regeneration were temporally uncoupled, the magnitude of the flagellar mRNA abundance change was reduced . These results suggest that flagellar gene expression may be regulated by multiple signals generated from these events, and implicate Caz+ as a factor in the mechanisms controlling flagellar regeneration . and disassemble their flagella in the cytoplasm ; upon readdition of Caz+ to resorbed cells, flagellar outgrowth occurs, and flagellar protein synthesis is also induced (11) . Because the effects of Cat+ on flagellar gene expression at a control level preceding translation have not been examined, we began our studies by examining the effects of Caz+ on flagellar mRNA abundances and the interdependence offlagellar outgrowth and flagellar gene expression . Flagellar gene induction and flagellar outgrowth, the cellular responses that are the focus of this study, are normally tightly coupled to the stimulus of flagellar excision during flagellar regeneration . We report here that these responses can be temporally uncoupled from flagellar excision by manipulating the [Caz+], or by inhibiting calmodulin activity with W7, suggesting that the process offlagellar regeneration is composed ofdiscrete, separable events that are regulated by Call . In addition, we find that the magnitude of the flagellar mRNA abundance change is reduced whenever events during flagellar regeneration are temporally uncoupled, suggesting that the normal flagellar mRNA abundance change is composed of several overlapping gene regulatory responses contributed by these events . Materials and Methods