JCB Article Intraﬂagellar transport balances continuous turnover of outer doublet microtubules: implications for ﬂagellar length control Wallace F. Marshall and Joel L. Rosenbaum Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520 Athe size of their organelles. Flagellar length control central question in cell biology is how cells determine is a convenient system for studying organelle size regulation. Mechanistic models proposed for ﬂagellar length regulation have been constrained by the assumption that ﬂagella are static structures once they are assembled. However, recent work has shown that ﬂagella are dynamic and are constantly turning over. We have determined that Introduction A fundamental question in cell biology that remains unan- swered is how cells regulate the size of their organelles. Does size regulation imply the existence of “size-measuring ma- chinery?” Answering such a question experimentally is challenging, in part because of the difficulty in measuring the size of an organelle that has a three-dimensional shape. However, the flagellum or cilium has a one-dimensional structure, and its size can be determined simply by measur- ing its length. One can easily measure flagellar length and screen for length mutations in the biflagellate alga Chlamydomonas, and this has enabled the isolation of several types of mutants with abnormal length flagella. Among these are the long flagella (lf )* (McVittie, 1972; Jarvik et al., 1976; Barsel et al., 1988; Asleson and Lefebvre, 1998), short flagella (shf ) (Jarvik et al., 1984; Kuchka and Jarvik, 1987), and flagellar assembly (fla)–defective mutants (Huang et al., 1977). It is not yet known how any of these genes act to regulate flagellar length. To understand how flagellar length is regulated, it is important to determine whether flagella are dynamic struc- Address correspondence to Wallace Marshall, KBT310 MCDB Dept., Yale University, New Haven, CT 06520-8103. Tel.: (203) 432-3473. Fax: (203) 432-6161. E-mail: wallace.marshall@yale.edu *Abbreviations used in this paper: fla, flagellar assembly; HA, hemagluti- nin; IFT, intraflagellar transport; lf, long flagella; shf, short flagella. Key words: ﬂagella; tubulin; Chlamydomonas; kinesin; intraﬂagellar transport  The Rockefeller University Press, 0021-9525/2001/10/405/10 $5.00 The Journal of Cell Biology, Volume 155, Number 3, October 29, 2001 405–414 http://www.jcb.org/cgi/doi/10.1083/jcb.200106141 this turnover occurs at the ﬂagellar tips, and that the assembly portion of the turnover is mediated by intraﬂagellar transport (IFT). Blocking IFT inhibits the incorporation of tubulin at the ﬂagellar tips and causes the ﬂagella to resorb. These results lead to a simple steady-state model for ﬂagellar length regulation by which a balance of assembly and disassembly can effectively regulate ﬂagellar length. tures, i.e., are the components of the flagellum turning over? One might think this is an unreasonable question, as all parts of a living cell are turning over to one extent or another. How- ever, historically, ciliary and flagellar microtubules have been regarded as “stable” and rather “static” structures (Behnke and Forer, 1967; Tilney and Gibbins, 1968), not changing in length in the presence of microtubule depolymerizing drugs such as colchicine and nocodazole, and maintaining their length in the cold. However, detailed radioactive pulse labeling studies (Gorovsky et al., 1970; Stephens, 1992, 1997, 2000; Song and Dentler, 2001) have shown quite clearly that all parts of the flagellum are turning over. More recently, it has been shown in Caenorhabditis elegans that genes required for the assembly of sensory cilia during embryogenesis continue to be expressed in the adult after cilia are fully assembled, and that they are required for sen- sory cilia maintenance (Fujiwara et al., 1999). Therefore, there is no question that the flagellar axonemal microtubules and their associated structures are dynamic. The phenomenon of intraflagellar transport (IFT) provides further evidence that flagella are dynamic structures. IFT is a motile process within flagella in which large protein com- plexes move from one end of the flagellum to the other (Kozminski et al., 1993b, 1995). Anterograde movement of these particles to the plus end of the flagellum is driven by a heterotrimeric kinesin-II, one motor subunit of which is en- coded by the flagellar assembly (FLA)10 gene in Chla- mydomonas. Temperature-sensitive fla10 mutants (Huang et 405