Abstract
The two flagella of Chlamydomonas reinhardtii are of the same size and structure but display functional differences, which are critical for flagellar steering movements. However, biochemical differences between the two flagella have not been identified. Here, we show that fluorescence protein-tagged carbonic anhydrase 6 (CAH6-mNG) preferentially localizes to the trans-flagellum, which is organized by the older of the two flagella-bearing basal bodies. The uneven distribution of CAH6-mNG is established early during flagellar assembly and restored after photobleaching, suggesting that it is based on preferred entry or retention of CAH6-mNG in the trans-flagellum. Since CAH6-mNG moves mostly by diffusion, a role of intraflagellar transport (IFT) in establishing its asymmetric distribution is unlikely. Interestingly, CAH6-mNG is present in both flagella of the non-phototactic bardet-biedl syndrome 1 (bbs1) mutant revealing that the BBSome is involved in establishing CAH6-mNG flagellar asymmetry. Using dikaryon rescue experiments, we show that the de novo assembly of CAH6-mNG in flagella is considerably faster than the removal of ectopic CAH6-mNG from bbs flagella. Thus, different rates of flagellar entry of CAH6-mNG rather than its export from flagella is the likely basis for its asymmetric distribution. The data identify a novel role for the C. reinhardtii BBSome in preventing the entry of CAH6-mNG specifically into the cis-flagellum.
Highlights
Cilia function, structure and composition varies considerably between different species and cell types
To eliminate possible second site mutations in cah6CLIP, we outcrossed the cah6CLIP mutant to the wild-type strain g1 resulting in strain cah6, which was used for all subsequent experiments
Western blot analysis of flagella isolated from control cells (g1) using a polyclonal antibody raised against the peptide encoded by the last exon of C. reinhardtii carbonic anhydrase 6 (CAH6) identified a band of ~25kDa, which is close to the predicted size of CAH6 (28 kDa) (Fig 2B)
Summary
Structure and composition varies considerably between different species and cell types. Multicellular organisms, for example, possess cells with motile cilia, primary cilia and specialized sensory cilia such as the outer segment of photoreceptor neurons [1]. Mammalian epithelial cells, such as those of the ependyma, first assemble a single nonmotile cilium ( the term primary cilium), followed by the development of numerous motile cilia [2]. Distinct types of motile cilia or flagella are present concomitantly on a given cell.
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