Paramecium BBS genes are key to presence of channels in Cilia

Abstract

Background: Changes in genes coding for ciliary proteins contribute to complex human syndromes called ciliopathies, such as Bardet-Biedl Syndrome (BBS). We used the model organism Paramecium to focus on ciliary ion channels that affect the beat form and sensory function of motile cilia and evaluate the effects of perturbing BBS proteins on these channels.

Methods: We used immunoprecipitations and mass spectrometry to explore whether Paramecium proteins interact as in mammalian cells. We used RNA interference (RNAi) and swimming behavior assays to examine the effects of BBS depletion on ciliary ion channels that control ciliary beating. Combining RNA interference and epitope tagging, we examined the effects of BBS depletion of BBS 7, 8 and 9 on the location of three channels and a chemoreceptor in cilia.

Results: We found 10 orthologs of 8 BBS genes in P. tetraurelia. BBS1, 2, 4, 5, 7, 8 and 9 co-immunoprecipitate. While RNAi reduction of BBS 7 and 9 gene products caused loss and shortening of cilia, RNAi for all BBS genes except BBS2 affected patterns of ciliary motility that are governed by ciliary ion channels. Swimming behavior assays pointed to loss of ciliary K+ channel function. Combining RNAi and epitope tagged ciliary proteins we demonstrated that a calcium activated K+ channel was no longer located in the cilia upon depletion of BBS 7, 8 or 9, consistent with the cells' swimming behavior. The TRPP channel PKD2 was also lost from the cilia. In contrast, the ciliary voltage gated calcium channel was unaffected by BBS depletion, consistent with behavioral assays. The ciliary location of a chemoreceptor for folate was similarly unperturbed by the depletion of BBS 7, 8 or 9.

Conclusions: The co-immunoprecipitation of BBS 1,2,4,5,7,8, and 9 suggests a complex of BBS proteins. RNAi for BBS 7, 8 or 9 gene products causes the selective loss of K+ and PKD2 channels from the cilia while the critical voltage gated calcium channel and a peripheral receptor protein remain undisturbed. These channels govern ciliary beating and sensory function. Importantly, in P. tetraurelia we can combine studies of ciliopathy protein function with behavior and location and control of ciliary channels.

Figure 1

Immunoprecipitation (IP) of FLAG-BBS9 or FLAG-BBS8 proteins from whole cell extracts (WCEs). Proteins were immunoprecipitated using anti-FLAG affinity beads and western blots were developed (AP) using polyclonal anti-FLAG. (A) IP from cells expressing FLAG-BBS9 (Test) and control cells expressing the FLAG plasmid (Control). Closed arrow indicates FLAG-BBS9. (B) IP from cells expressing FLAG-BBS8 (Test) and control cells expressing the FLAG plasmid (Control). Open arrow indicates FLAG-BBS8. Both blots show a molecular weight marker (M) and a 37 kDa FLAG-fusion protein as a positive control (P). Below is an example of a loading control from the FLAG-BBS9 IP. Protein concentrations were determined using a Pierce assay before solubilization to ensure equal amounts of protein were used for both the Test and Contol IP. The loading control blot was probed with anti-tubulin (50 kD).

Figure 2

Scanning electron microscopy images of control and BBS depleted cells. (A) Control cell, (B) BBS2-depleted cell, (C) BBS7-depleted cell and (D) BBS9-depleted cell. Scale bars are 10 μm; these are representative images.

Figure 3

Scanning electron micrographs of cilia and cell surfaces. (A) Control cell; yellow dotted hexagon and arrows emphasize cortical units with a cilium arising from the center. (B) BBS7- depleted cell. (C) BBS9-depleted cell. Red dotted hexagons and red arrows indicate cortical units with short cilia arising, or no cilia. Note these cells contain cortical units with average length cilia arising (yellow dotted hexagons and yellow arrows). These are representative images, scale bars represent 2 μm.

Figure 4

Normalized ciliary lengths of BBS-depleted cells. Data are averages of 138 to 798 cilia on 15 to 66 cells from three replicate experiments ± standard error of the mean (SEM). The control value before normalization was 11.72 μm ± 0.08 μm (average ± SEM, n = 798). Significant changes determined by the Mann–Whitney U-test are denoted as *P < 0.05, **P < 0.0001.

Figure 5

Backward swimming duration after depolarization in tetraethylammonium chloride (TEA) with Na⁺, TEA with Mg²⁺, or KCl. Durations of backward swimming (sec) in buffers with: (A) 25 mM TEA with 10 mM NaCl, (B) 25 mM TEA with 5 mM MgCl_2; and (C) 30 mM KCl. Data are normalized to the control backward swimming duration. Asterisks denote significant difference from control using the Mann–Whitney U-test (*P < 0.05; **P < 0.0001). Data are averages from three experiments; n = 45 to 418 (A); 45 to 165 cells (B) and 25 to 240 (C). Data are averages ± standard error of the mean (SEM).

Figure 6

Immunofluorescence of FLAG-SK1a channels or PKD2-FLAG channels in BBS7 - or BBS8 - or BBS9-depleted cells). (A) FLAG-SK1a channels. (B) PKD2-FLAG channels. FLAG-SK1a- expressing cells were stained with anti-FLAG antibody and as a contrast, anti-folate chemoreceptor (FBP) antibody in a series of control and RNAi conditions. Only the merged images are shown here. Complete immunofluorescence of the folate chemoreceptor and the FLAG-SK1a channel are shown separately in Additional file 9: Figure S5. The FLAG control is a cell microinjected with FLAG-pPXV vector and fed with RNAi empty vector (L4440) bacteria. The FLAG-SK1a control is a cell expressing FLAG-SK1a and fed bacteria with an RNAi empty vector (L4440). BBS7, BBS8 and BBS9 are the cells expressing FLAG-SK1a channel and also BBS7-, BBS8- or BBS9-depleted, respectively. Cells were immunostained with anti-FLAG (red) and anti-FBP (green) antibodies. Differential interference contrast (DIC) images are shown to document that cilia are present. Cells expressing PKD2-FLAG channel (B) were stained with anti-FLAG antibody (red) and anti-Tetrahymena centrin-1 antibody (green) in a series of control and RNAi conditions. Only the merged images are shown here; the staining of FLAG and centrin can be seen separately in Additional file 10: Figure S6. The FLAG control is a cell expressing the FLAG-pPXV vector and fed with RNAi empty vector (L4440) bacteria. The PKD2-FLAG control is a cell expressing the PKD2-FLAG channel and fed with bacteria with RNAi empty vector (L4440). BBS7, BBS8 and BBS9 are the cells expressing the PKD2-FLAG channel and are BBS7-, BBS8- or BBS9- depleted, respectively. Differential interference contrast (DIC) images are shown to document that cilia are present. All images were taken under 60× oil immersion objectives. Scales represent 15 μm. Images are representative of results of three experiments, n = 126 to 156 cells.

Figure 7

Immunoblots (ECL) of proteins immunoprecipitated from ciliary membranes. Ciliary membranes were solubilized in Triton X-114 and the proteins were immunoprecipitated using anti-FLAG affinity beads and separated on a 5-18% SDS-PAGE gel. (A) Control experiment where cells were expressing the empty FLAG vector (control) or FLAG-VGCC1c (TEST). The IP of the FLAG-VGCC1c can be seen in the TEST lane (arrow: 272 kD). (B) Cells expressing FLAG-VGCC1c were fed bacteria with the BBS8 RNAi plasmid (BBS8) or the empty RNAi vector (EV L4440) as a control. The immunoblots were developed using polyclonal anti-FLAG. Arrow indicates the band of the FLAG-VGCC1c proteins.

Figure 8

Cartoon of BBSome trafficking in Paramecium. Proteins are sorted from the endoplasmic reticulum (ER) to the Golgi and from the trans-Golgi network to the base of the cilia either in a BBSome-dependent pathway (red arrow with a dotted line) or a BBSome-independent pathway (blue arrow). The K⁺ channel (purple trapezoid) is trafficked to the cilia via the BBSome-dependent pathway while the Ca_v channel (green oval) is sorted via the BBSome-independent pathway. Structural proteins (not shown) traffic through cargo interactions with the BBSome to the cilia.