Fig 1.
Flagellar asymmetry in C. reinhardtii.
A) Schematic representation of C. reinhardtii viewed from the side (left) and from the top (right). Flagella (red) extend from the two basal bodies (green), each of which is associated with two microtubular rootlets (purple). The eyespot (orange) is associated with the 4-standed microtubular rootlet (D4) linked to the daughter basal body. Thus, the cis-flagellum is closer to the eyespot than the trans-flagellum. The rootlets are named to indicate the number of microtubules (2 or 4) and their association with the mother (M) or daughter (D) basal body [12]. (B) Differential interference contrast (DIC) and brightfield (BF) images of a live cell captured at two different focal planes showing the flagella (left) and eyespot (right) indicated with an arrowhead. Bar = 2μm.
Fig 2.
Carbonic anhydrase 6 is active in flagella.
A) Schematic presentation of the CAH6 gene and the insertion in the cah6 mutant. The insertion of the APHVIII cassette caused a deletion (indicated by a red crossed rectangle) of 224 bp encompassing the start codon and first exon of CAH6. The positions of the primers (F1, R1, and R2) used to track the mutation by PCR are indicated (see S1A Fig). B) Western blot analysis of isolated flagella from wild-type (g1) and the cah6 mutant were probed with anti-CAH6. Antibodies to the IFT particle protein IFT81 were used to control for equal loading. C) 18O-exhange measurements (open circles) and models (solid lines) for control (g1) and cah6 flagella. Three 13CO2 isotopologues are tracked and analyzed: 13C16O16O (blue circles), 13C18O16O (green circles) and 13C18O18O (red circles). After determining the background rate of exchange (before time 0), isolated flagella from the strain indicated were added at time 0 and the exchange rate was measured for 400 seconds. CA activity is indicated by the accelerated conversion of 13C18O18O into 13C18O16O and ultimately 13C16O16O. The best fit line (kcf) of the model after addition of isolated flagella is shown. D) Flagellar and whole cell CA activity (kcf/kuf) of g1 and cah6; the values were normalized for the protein concentration (mg/mL) in the respective samples and are based on repeat measurements of the same samples (n = 1). E) Western blot analysis for CAH6 in wild-type (g1) flagella. Cells were treated for 24 hours prior to the flagellar isolation as follows: The cultures were either aerated with high CO2 (0.5%) or maintained without aeration both in the presence (+) or absence (-) of cycloheximide. Anti-IC2 was used as a loading control.
Fig 3.
The asymmetric localization of CAH6-mNG in flagella is lost in the bbs1 mutant.
A) Schematic representation of the CAH6-mNG expression vector. The sequence for mNG was inserted at the 3’ end of the genomic CAH6 coding region. B) Brightfield (BF) images of the flagella (left) and the eyespot (right, indicated with black arrowheads) and the corresponding TIRF images of live cah6 CAH6-mNG and bbs1 cah6 CAH6-mNG cells. The trans-flagella are indicated with red arrowheads and the cis-flagella with yellow arrowheads. Bar = 2μm. C) Histograms of the fluorescence intensity of CAH6-mNG in the trans- and cis-flagella of the cah6 CAH6-mNG rescue strain and the bbs1 cah6 CAH6-mNG strain. For each cell, the fluorescence intensity in its trans-flagellum was set to 100% and the fluorescence intensity of the corresponding cis-flagellum is displayed as % of that of the trans-flagellum. One cell, lacking detectable CAH6-mNG in the cis-flagellum and thus having an outlier trans/cis-ration of 158, was ignored for the statistical analyses. Error bars indicate the standard deviation. n = number of cells analyzed. D) Histogram of the ratio of the fluorescence intensity of CAH6-mNG between the trans- and the cis-flagellum in the cah6 CAH6-mNG and the bbs1 cah6 CAH6-mNG strain; shown is the mean of the ratios determined for the individual trans/cis pairs of individual cells. Error bars indicate the standard deviation and n the number of flagellar pairs analyzed. See S3C Fig for the individual data points.
Fig 4.
The asymmetric distribution of CAH6-mNG is established early during ciliary assembly and restored after photobleaching.
A) Partial kymograms showing IFT (open arrow) and diffusion (white arrowhead) of CAH6-mNG. Scale bars: 2 s and 2 μm. B) Kymograms showing the movements of CAH6-mNG in full-length cah6 CAH6-mNG and bbs1 cah6 CAH6-mNG flagella. CAH6-mNG moved mostly by diffusion (white arrowhead) but occasional transport by IFT (open arrows) was observed as well. Scale bars: 2 s and 2 μm. C, D) Analysis of CAH6-mNG in regenerating flagella. C) Still images showing the distribution of CAH6-mNG during flagella regeneration of cah6 CAH6-mNG and bbs1 cah6 CAH6-mNG cells. TIRFM images of different live cells were obtained at 30 minutes (T30), 60 minutes (T60) and 90 minutes (T90) after deciliation by a pH shock. Bar = 2μm. D) Quantification of the ratios of CAH6-mNG fluorescence intensity between regenerating trans- and cis-flagella at 30, 60 and 90 minutes after deciliation. The error bars, indicating standard deviation, and the number of cells analyzed (n) are indicated. E, F) FRAP analysis of CAH6-mNG in full-length flagella. E) Brightfield (BF) of cah6 CAH6-mNG and bbs1 cah6 CAH6-mNG cells showing optical section through the flagella and the eyespot (indicated with a black arrow). The corresponding TIRF images show the flagella before (pre), immediately after (T0) and 5 minutes after (T5) the photobleaching step. While partial, the recovery pattern indicated that CAH6-mNG cis/trans-asymmetry in wild-type cells is maintained dynamically, i.e., involves continuous entry and exit of CAH6-mNG from flagella. Bar = 2μm. F) Histograms with individual data points comparing recovery of CAH6-mNG fluorescence in trans- and cis-flagella at 5 minutes after photobleaching. Data are presented as percentage of the prebleach intensity of the trans-flagellum and were calculated for each flagella pair. The significance, based on a two-tailed t test, is indicated. n = number of flagella analyzed.
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.
Fig 6.
Schematic summary and model of expression regulation and flagellar localization of CAH6-mNG in C. reinhardtii.
A) CAH6 in flagella is actively converting carbon dioxide to bicarbonate and vice versa. CAH6 is predicted to attach to the flagellar membrane (pink) by a dual fatty acid modification (indicated by two wavy lines). B) In CO2-limiting conditions, the amount of CAH6 in flagella is upregulated 2- to 10-fold. C, D) The BBSome largely prevents the entry of CAH6-mNG into the cis-flagellum. In a hypothetical scenario, the younger basal body could recruit BBSomes to a specific location or activate the BBSome (indicated by the dark red color) to minimize entry of CAH6-mNG into the cis-flagellum; during maturation, the older basal body lost its ability to modulate BBSome function and CAH6-mNG will enter the flagella.