The BBSome restricts entry of tagged carbonic anhydrase 6 into the cis-flagellum of Chlamydomonas reinhardtii
Fig 5
Export of CAH6-mNG from bbs1-derived cis-flagella is slow.
A) Schematic representation of a mating between bbs1 cah6 gametes expressing CAH6-mNG (green dots) and cah6 gametes possessing intact BBSomes (indicated by yellow shading). After cell fusion, BBSomes and CAH6-mNG are present in the shared cytoplasm of the zygotes and will enter all four cilia, allowing us to study both the entry of CAH6-mNG into cah6-deficient flagella and the BBSome-dependent repair of CAH6-mNG distribution in the bbs1-derived flagella. B) Differential interference contrast (DIC), brightfield (BF) and TIRFM images of live zygotes from a mating between bbs1 CAH6-mNG and a rsp3 RSP3-mCherry strain; the RSP3-mCherry tag allows us to distinguish bbs1- (arrowheads) and wild type-derived flagella (arrows) after cell fusion. Red arrows and arrowheads indicate the trans-flagella and yellow arrows and arrowheads indicate the cis-flagella. Note that establishing CAH6-mNG cis/trans asymmetry in the rsp3 RSP3-mCherry-derived wild-type cilia precedes correcting the distribution of CAH6-mNG in the bbs1-derived flagella. Bar = 2 μm. C) Brightfield (BF) images of different focal planes showing the flagella and the two eyespots (indicated with arrowheads) and the corresponding TIRF image of live bbs1 cah6 CAH6-mNG × cah6 zygotes; the time passed since mixing of the gametes is indicated. The bbs1-derived flagella are marked by arrowheads (red for trans and yellow for cis) and the cah6-derived flagella are marked correspondingly with arrowheads. Bars = 2 μm. (D) Diagrams of the classification system of zygotes based on the distribution pattern of CAH6-mNG. Early stages possessed one flagellar pair containing ~82% of the total CAH6-mNG signal and a weaker pair accounting for ~18% of the signal. Intermediate stages possessed three flagella with strong signals and one with a weaker signal; accordingly, the signal strength of the stronger cis/trans-pair was reduced to 66% and that of the weaker pair increased to 34% of the total CAH6-mNG fluorescence in a given zygote. Late stages, only a few of which were detected, possessed two flagellar pairs with discernable cis/trans-asymmetry in the CAH6-mNG signal. (E) Cumulative bar chart showing the distribution of the three different patterns of flagellar CAH6-mNG distribution in zygotes (see panel D) at different time points.