Journal: EMBO Reports

Article Title: BBSome-deficient cells activate intraciliary CDC42 to trigger actin-dependent ciliary ectocytosis

doi: 10.1038/s44319-024-00326-z

Figure Lengend Snippet: ( A , B ) Representative micrographs of cilia ( A ) and quantification of the cilia length ( B ), stained with antibody to acetylated tubulin (Ac-tub), in the WT and BBS4 KO/KO RPE1 cells. Scale bar, 5 µm. Medians with interquartile range from three independent experiment ( n = 96 cilia). ( C ) Expansion microscopy of the cilia axoneme of the WT and BBS4 KO/KO RPE1 cells visualized via staining with antibody to acetylated tubulin. Enlarged insets and white arrows point to bulges at cilia tips observed in BBS4 KO/KO cells. Scale bar, 2 µm. ( D ) Representative micrographs of cilia visualized by staining with antibody to acetylated tubulin (Ac-tub) and staining of F-actin (Phalloidin) in WT, BBS1 KO/KO , and BBS4 KO/KO RPE1 cells treated with the ROCK1 inhibitor Y27632 or CDC42 inhibitor ML141 for 2 h. Cilia base at bottom. Scale bar, 5 µm. ( E ) Quantification of the cilia length based on Ac-tub signal in the WT, BBS1 KO/KO , and BBS4 KO/KO RPE1 cells non-treated (NT) or treated with Y27632 for 2 h. Medians with interquartile range from three independent experiments ( n = 160–290 cilia). ( F ) Quantification of the cilia length based on Ac-tub signal in the WT, BBS1 KO/KO , BBS4 KO/KO , and BBS9 KO/KO RPE1 cells treated with vehicle or ML141 for 2 h. Medians with interquartile range from three independent experiments ( n = 145–270 cilia). ( G ) Merged micrographs (left) show representative cilia stained with antibodies against ARL13B (green) and Ac-tubulin (red), along with the corresponding intensity line scans (dashed line next to the cilium) from the cilia base (B) to the cilia tip (T) in WT RPE1 cells. The arrow indicates the foci at the tip of the top cilium, defined as the ARL13B signal extending (~0.5 μm) beyond the Ac-tubulin signal, as illustrated in the intensity plot on the right. The bottom cilium does not exhibit foci based on this criterion. Scale bar, 5 µm. ( H ) Quantification of the frequency of ciliary tip foci observed in ( G ). Mean and SD of three independent experiments ( n = 100–200 cilia). ( I , J ) Representative micrographs ( I ) and quantification of the cilia length ( J ), in WT and Bbs4 KO/KO MEFs treated with vehicle or ML141 and stained with antibodies to acetylated tubulin (Ac-tub) and ARL13B. Cilia base at bottom. Scale bar, 5 µm. Medians with interquartile range from three independent experiments ( n = 300–400 cilia). ( K ) Representative Western blots from total cell lysates (TCL, left blots) and EVs purifications (right blots) prepared from GPR161-mCherry WT and Bbs4 KO/KO MEFs treated with SAG and vehicle or ML141 for 2.5 h probed with antibodies to IFT88 (red star—middle band), mCherry, TSG101, CD9 and Calnexin. ( L , M ) Quantification of the GPR161-mCherry ( L ) and IFT88 ( M ) present in the ciliary EV fraction purified by ultracentrifugation of cell supernatants of GPR161-mCherry WT and Bbs4 KO/KO MEFs treated with SAG and vehicle or ML141 for 2.5 h. Mean and SD of four independent experiments. Data information: Statistical significance was calculated using the two-tailed Mann–Whitney test ( B , E , F , J ), two-tailed paired t-test ( H ), and one-tailed paired t-test ( L , M ) and the obtained p -values are indicated. Merged micrographs show nuclei staining by DAPI—blue ( D , G , I ). .

Article Snippet: ML141 , Tocris Biosciences , 4266.

Techniques: Staining, Microscopy, Western Blot, Purification, Two Tailed Test, MANN-WHITNEY, One-tailed Test

Journal: EMBO Reports

Article Title: BBSome-deficient cells activate intraciliary CDC42 to trigger actin-dependent ciliary ectocytosis

doi: 10.1038/s44319-024-00326-z

Figure Lengend Snippet: ( A ) Expansion microscopy of the cilia axoneme of the BBS1 KO/KO and BBS7 KO/KO RPE1 cells visualized by staining with antibody to acetylated tubulin. White arrows point to bulges at the cilia tips. Scale bar, 2 µm. ( B ) The graph shows the frequency of bulges at the cilia tips visualized by expansion microscopy of cilia in WT and BBS4 KO/KO RPE1 cells. The number of observed bulges out of the total count of imaged cilia is shown. Statistical analysis was done using the contingency table and one-sided Chi-square test. ( C ) Representative micrographs of the cilia visualized by staining with antibody to acetylated tubulin (Ac-tub) and staining of actin cytoskeleton with Phalloidin-TexasRed in the WT RPE1 cells treated with inhibitors of ROCK1/RHOA (Y27632), CDC42 (ML141), RAC1 ( CAS 1177865-17-6 (CAS)), ARP2/3 (CK666), and actin polymerization (Cytochalasin D) for 2 h. Scale bar, 5 µm. ( D ) Quantification of the cilia length based on acetylated tubulin signal in the WT and BBS4 KO/KO RPE1 cells treated with DMSO or CK666 or Cytochalasin D (CytoD) for 2 h as in ( C ). Medians with interquartile range from three independent experiments ( n = 220–320 cilia). ( E ) Quantification of the cilia length based on acetylated tubulin signal in the WT and BBS4 KO/KO RPE1 cells treated with vehicle or CAS for 2 h as in ( C ). Medians with interquartile range from three independent experiments ( n = 250–280 cilia). ( F ) Representative micrographs of the cilia visualized by staining with antibody to acetylated tubulin (Ac-tub) and ARL13B in the WT and BBS4 KO/KO RPE1 cells treated with DMSO or ML141 for 2 h. Scale bar, 5 µm. ( G ) Quantification of the cilia length in WT, BBS4 KO/KO , and BBS4 KO/KO RPE1 cells expressing YFP-BBS4 treated with DMSO or ML141 for 2 h and stained with antibodies to acetylated tubulin (Ac-tub) and ARL13B. Medians with interquartile range from three independent experiments ( n = 200–250 cilia). ( H ) Quantification of the frequency of ARL13B foci at the cilia tips in WT, BBS4 KO/KO , and BBS4 KO/KO RPE1 cells expressing YFP-BBS4 treated with DMSO or ML141 for 2 h in ( G ). Mean and SD of three independent experiments ( n = 200–250 cilia). ( I ) Intensity plot profiles of the Ac-tub and ARL13B signal measured from the cilia base (B) to the cilia tip (T) in Bbs4 KO MEFs treated with ML141 in Fig. . The ARL13B signal extends (~0.5 μm) beyond the Ac-tub signal. ( J ) Representative chromatogram analysis of DNA sequencing data for WT ST2 and Ift88 KO ST2 cell clones reveals a homozygous mutation characterized by the insertion of a T nucleotide between bases 248 and 249 of the Ift88 gene (NM_009376.3, arrow). The position of the sgRNA and PAM sequence used for targeting is indicated. ( K ) Representative Western blot from total cell lysates prepared from WT MEFs, SAG/DMSO treated GPR161-mCherry WT MEFs (experiments #1–4 in Fig. ), WT and Ift88 KO ST2 cells and Ift88 KO ST2 cells expressing the IFT88-mNeonGreen and probed with antibodies to IFT88 and β-actin. The arrow highlights the IFT88 band (red star) corresponding to the expected size of 100 kDa, which is absent in the Ift88 KO ST2 cells. Data information: Statistical significance was calculated using the one-sided Chi-square test ( B ), two-tailed Mann–Whitney test ( D , E , G ) and two-tailed paired t-test ( H ) and the obtained p -values are indicated. Merged micrographs show nuclei staining with DAPI—blue ( F ).

Article Snippet: ML141 , Tocris Biosciences , 4266.

Techniques: Microscopy, Staining, Expressing, DNA Sequencing, Clone Assay, Mutagenesis, Sequencing, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Reports

Article Title: BBSome-deficient cells activate intraciliary CDC42 to trigger actin-dependent ciliary ectocytosis

doi: 10.1038/s44319-024-00326-z

Figure Lengend Snippet: ( A ) Representative micrographs depict cilia visualized via staining with antibodies to acetylated tubulin (Ac-tub) and GPR161 in non-treated (nt) and SAG induced—2 h, WT and Bbs4 KO/KO MEFs. Cilia base at bottom. Scale bar, 5 µm. ( B , C ) Quantification of the frequency of GPR161 positive cilia ( B ) and of the frequency of GPR161 foci at the cilia tip ( C ) in non-treated (nt) and SAG induced—2 h, WT and Bbs4 KO/KO MEFs in ( A ). Mean and SD of three independent experiments ( n = 160–210 cilia). ( D ) Representative micrographs depict cilia visualized via staining with antibodies to acetylated tubulin (Ac-tub) and GPR161 in non-treated (nt) and SAG induced—2 h, WT and Bbs4 KO/KO MEFs concomitantly treated with ML141. Cilia base at bottom. Scale bar, 5 µm. ( E ) Quantification of the cilia length, in non-treated (nt) and SAG induced WT and Bbs4 KO/KO MEFs concomitantly treated with DMSO or ML141 and stained with antibodies to acetylated tubulin (Ac-tub) and GPR161. Medians with interquartile range from three independent experiments ( n = 170–190 cilia). ( F ) Representative micrographs depict cilia visualized via staining with antibodies to acetylated tubulin (Ac-tub) and GPR161 (Merge and insets), in non-treated (nt) and SAG induced—2 h, WT and Bbs4 KO/KO MEFs expressing the cilia targeted GFP-CDC42 WT or DN variant. Cilia base at bottom. Scale bar, 5 µm. ( G ) Quantification of the cilia length, in non-treated (nt) and SAG induced—2 h, WT, and Bbs4 KO/KO MEFs expressing cilia targeted GFP-CDC42 WT or DN variant stained with antibodies to acetylated tubulin (Ac-tub) and GPR161. Medians with interquartile range from three independent experiments ( n = 160–220 cilia). ( H ) Quantification of the frequency of GPR161 positive cilia in non-treated (nt) and SAG induced—2 h, WT and Bbs4 KO/KO MEFs expressing cilia targeted GFP-CDC42 WT or DN variant. Mean and SD of three independent experiments ( n = 160–190 cilia). ( I ) Quantification of the frequency of GPR161 tip foci in non-treated (nt) and SAG induced—2 h, WT and Bbs4 KO/KO MEFs expressing cilia targeted GFP-CDC42 WT or DN variant in ( H ). Mean and SD of three independent experiments ( n = 160–190 cilia). Data information: Statistical significance was calculated using two-tailed paired t-test ( B , C , H , I ), and two-tailed Mann–Whitney test ( E , G ) and the obtained p -values are indicated. Merged micrographs show nuclei staining with DAPI—blue ( A , D , F ). .

Article Snippet: ML141 , Tocris Biosciences , 4266.

Techniques: Staining, Expressing, Variant Assay, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Reports

Article Title: BBSome-deficient cells activate intraciliary CDC42 to trigger actin-dependent ciliary ectocytosis

doi: 10.1038/s44319-024-00326-z

Figure Lengend Snippet: ( A ) Quantification of the cilia length, in non-treated (nt) and SAG induced—2 h, WT and Bbs4 KO/KO MEFs concomitantly treated with DMSO or ML141 and stained with antibodies to acetylated tubulin (Ac-tub) and ARL13B. Medians with interquartile range from three independent experiments ( n = 90–130 cilia). ( B ) Quantification of the frequency of ARL13B foci at the cilia tips in non-treated (nt) and SAG induced—2 h, WT and Bbs4 KO/KO MEFs concomitantly treated with DMSO or ML141 in ( A ). Mean and SD of three independent experiments ( n = 90–130 cilia). ( C ) The donor lifetime values extracted from the FRET-FLIM analysis of cilia as a region of interest in non-treated (nt) and SAG induced—2 h, WT and Bbs4 KO/KO MEFs expressing N-Raichu-CDC42 treated with ML141 or DMSO and values measured for the no-FRET control expressed in WT MEFs treated with DMSO. Mean and SD of three (WT) and four (KO, no-FRET) independent experiments ( n = 13–20 cilia). ( D ) Representative micrographs of the cilia in non-treated (nt) and SAG induced WT and Bbs4 KO/KO MEFs expressing GPR161-mCherry, pre-treated for 30 min with DMSO or ML141 and imaged every 1 min for 100 min. Frames were extracted from the live cell videos (17–30 cilia per condition in two independent experiments). Scale bar, 2 μm. ( E ) Plots depict the variable dynamics of the length of the individual cilia in non-treated (nt) and SAG induced WT and Bbs4 KO/KO MEFs expressing GPR161-mCherry, pre-treated for 30 min with DMSO or ML141 and imaged every 1 min for 100 min in ( D ). The length of the cilium was normalized to the length measured at time 0 min. In total 17–30 cilia were monitored per condition in two independent experiments. Data information: Statistical significance was calculated using the two-tailed Mann–Whitney test ( A , C ) and two-tailed paired t-test ( B ) and the obtained p -values are indicated.

Article Snippet: ML141 , Tocris Biosciences , 4266.

Techniques: Staining, Expressing, Control, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Reports

Article Title: BBSome-deficient cells activate intraciliary CDC42 to trigger actin-dependent ciliary ectocytosis

doi: 10.1038/s44319-024-00326-z

Figure Lengend Snippet: ( A , B ) Representative micrographs ( A ) and quantification of the frequency ( B ) of GPR161-mCherry foci (arrows) in cilia visualized by staining with antibody to acetylated tubulin (Ac-tub) in non-treated (nt) and SAG induced—2 h, WT and Bbs4 KO/KO MEFs concomitantly treated with vehicle or ML141. Staining with antibody to SMO was employed to visualize the activation of the SHH pathway. Cilia base at bottom. Scale bar, 5 μm. Mean and SD of three independent experiments ( n = 100–160 cilia). Statistical significance was calculated using the two-tailed paired t-test. ( C ) Representative micrographs depicting ciliary ectocytosis detected by live cell imaging of WT and Bbs4 KO/KO MEFs expressing mCherry-GPR161 and induced with SAG for 2 h. The cells were pre-treated with DMSO (top) or ML141 (bottom) for 30 min and then imaged every 1 min for 100 min. White arrows point to the foci formation, ectocytosis and ectosomes. Cilia base at bottom. Scale bar, 2 μm. ( D ) The graph shows the frequency of cilia with ectocytosis events detected by live cell imaging of WT and Bbs4 KO/KO MEFs expressing mCherry-GPR161 non-treated or upon 2 h treatment with SAG and pre-treated with DMSO or ML141 before acquisition. The number of observed ectocytosis out of the total count of imaged cilia from two independent live cell imaging experiments is shown. Statistical analysis was done using the contingency table and Chi-square test. ( E ) Plots depict the average dynamics of the cilia length in non-treated (nt) and SAG induced—2 h, WT and Bbs4 KO/KO MEFs expressing GPR161-mCherry pre-treated with DMSO or ML141 for 30 min and then imaged for 100 min. The length of the cilium was normalized to the length measured at time 0 min. In total, 17–30 cilia were monitored per condition in two independent live cell imaging experiments. The data points (Mean ± SEM) were fitted using the linear regression and the statistical significance (two-tailed) of the slope of the regression lines was calculated for the indicated conditions. Data information: Statistical significance was calculated using the two-tailed paired t-test ( B ), Chi-square test ( D ), and two-tailed F-test for the difference between two slopes ( E ) and the obtained p -values are indicated. .

Article Snippet: ML141 , Tocris Biosciences , 4266.

Techniques: Staining, Activation Assay, Two Tailed Test, Live Cell Imaging, Expressing

Journal: EMBO Reports

Article Title: BBSome-deficient cells activate intraciliary CDC42 to trigger actin-dependent ciliary ectocytosis

doi: 10.1038/s44319-024-00326-z

Figure Lengend Snippet: ( A ) Representative micrographs depicting ciliary ectocytosis and actin polymerization detected by live cell imaging of WT and Bbs4 KO/KO MEFs expressing mNG-ARL13B and LifeAct-TagRFP. The cells were treated with SAG and DMSO (top) or ML141 (bottom) and imaged every 2 min for 2 h. White arrows point to the F-actin polymerization events. Red asterisks indicate the frames used for 3D visualization in ( B ). Cilia base at bottom. Scale bar, 2 μm. Maximum intensity projections of the z-stacks were done using Fiji ImageJ software and the intensities for both channels were adjusted post acquisition for better visualization. ( B ) 3D visualization of the F-actin patches and ciliary membrane in the WT and Bbs4 KO/KO MEFs at the indicated time points of the live cell imaging in ( A ). Left micrographs show the front view of the cilium and right micrographs show a 90° rotated view in the direction indicated by the arrows. Scale bar, 2 μm. ( C ) The graph shows the fraction of cilia with actin polymerization events observed in WT and Bbs4 KO/KO MEFs expressing mNG-ARL13B and LifeAct-TagRFP upon treatment with SAG and vehicle or ML141 during the live cell imaging in ( A ). The number of observed actin polymerization events out of the total count of imaged cilia from four independent live cell imaging experiments and mean with SD are shown. Statistical analysis was done using the contingency table and Chi-square test. ( D ) The plot depicts the duration of the observed actin polymerization events detected in cilia in the WT and Bbs4 KO/KO MEFs expressing mNG-ARL13B and LifeAct-TagRFP upon treatment with SAG and vehicle or ML141 during the live cell imaging in ( A ). Means with SD are shown. ( E ) The graph shows the fraction of cilia with actin polymerization events observed in Bbs4 KO/KO MEFs expressing mNG-ARL13B and LifeAct-TagRFP in the presence of WT or DN version of CDC42 upon treatment with SAG during the live cell imaging in (Fig. ). The number of observed actin polymerization events out of the total count of imaged cilia from six independent live cell imaging experiments and mean with SD are shown. Statistical analysis was done using the contingency table and two-sided Chi-square test. Data information: Statistical significance was calculated using the Chi-square test ( C ) and two-sided Chi-square test ( E ) and the obtained p -values are indicated. .

Article Snippet: ML141 , Tocris Biosciences , 4266.

Techniques: Live Cell Imaging, Expressing, Software, Membrane

Journal: EMBO Reports

Article Title: BBSome-deficient cells activate intraciliary CDC42 to trigger actin-dependent ciliary ectocytosis

doi: 10.1038/s44319-024-00326-z

Figure Lengend Snippet: ( A ) Representative micrographs show F-actin polymerization events observed in cilia in WT MEFs expressing mNeonGreen-ARL13B (green) and LifeAct-TagRFP (red) treated with SAG and DMSO and imaged for 2 h. Frames were extracted from time-lapse videos (51 in total, 6 actin polymerization events, four independent experiments). Scale bar, 2 μm. ( B ) Representative micrographs show F-actin polymerization events observed in cilia in WT MEFs expressing mNeonGreen-ARL13B (green) and LifeAct-TagRFP (red) treated with SAG and ML141 and imaged for 2 h. Frames were extracted from time-lapse videos (54 in total, 2 actin polymerization events, four independent experiments). Scale bar, 2 μm. Maximum intensity projections of the z-stacks were done using Fiji ImageJ software and the intensities for both channels were adjusted post acquisition for better visualization.

Article Snippet: ML141 , Tocris Biosciences , 4266.

Techniques: Expressing, Software

Journal: EMBO Reports

Article Title: BBSome-deficient cells activate intraciliary CDC42 to trigger actin-dependent ciliary ectocytosis

doi: 10.1038/s44319-024-00326-z

Figure Lengend Snippet: ( A ) Representative micrographs show F-actin polymerization events observed in cilia in Bbs4 KO/KO MEFs expressing mNeonGreen-ARL13B (green) and LifeAct-TagRFP (red) treated with SAG and DMSO and imaged for 2 h. Frames were extracted from time-lapse videos (39 in total, 10 actin polymerization events, four independent experiments). Scale bar, 2 μm. ( B ) Representative micrographs show F-actin polymerization events observed in cilia in Bbs4 KO/KO MEFs expressing mNeonGreen-ARL13B (green) and LifeAct-TagRFP (red) treated with SAG and ML141 and imaged for 2 h. Frames were extracted from time-lapse videos (39 in total, 4 actin polymerization events, four independent experiments). Scale bar, 2 μm. Maximum intensity projections of the z-stacks were done using Fiji ImageJ software and the intensities for both channels were adjusted post acquisition for better visualization.

Article Snippet: ML141 , Tocris Biosciences , 4266.

Techniques: Expressing, Software

Journal: EMBO Reports

Article Title: BBSome-deficient cells activate intraciliary CDC42 to trigger actin-dependent ciliary ectocytosis

doi: 10.1038/s44319-024-00326-z

Figure Lengend Snippet: Reagents and tools table

Article Snippet: ML141 , Tocris Biosciences , 4266.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Staining, Modification, Protease Inhibitor, Software, Imaging, Bicinchoninic Acid Protein Assay, Microscopy

Journal: iScience

Article Title: Targeting dermatophyte Cdc42 and Rac GTPase signaling to hinder hyphal elongation and virulence

doi: 10.1016/j.isci.2024.110139

Figure Lengend Snippet: Mammalian Rac and Rac/Cdc42 inhibitors suppress conidial germination in Trichophyton rubrum (A) Structures of Rac inhibitors EHT1864 and NSC23766 and Rac/Cdc42 inhibitor AZA1. (B) Effects of mammalian small GTPase inhibitors on conidial germination in T. rubrum were observed. Conidia were stained with calcofluor white. n = 3 each. ∗∗, p < 0.01; ∗∗∗, p < 0.001. Mean ± SD. The lower panel showed representative fungal cell. (C) Effects of mammalian small GTPase inhibitors on mycelial growth in T. rubrum were observed. (D) Effects of mammalian Rac and Rac/Cdc42 inhibitors EHT1864, AZA1, and NSC23766 on conidial germination in T. rubrum were observed. Conidia were stained with calcofluor white. n = 3 each. ∗∗, p < 0.01; ∗∗∗∗, p < 0.0001. Mean ± SD.

Article Snippet: EHT1864, ML-141, EHop-016, ZCL-278, CASIN, Ketorolac, and Brefeldin A were obtained from Cayman Chemical Company, Germany.

Techniques: Staining

Journal: iScience

Article Title: Targeting dermatophyte Cdc42 and Rac GTPase signaling to hinder hyphal elongation and virulence

doi: 10.1016/j.isci.2024.110139

Figure Lengend Snippet: Effect of mammalian Rac and/or Cdc42 inhibitors against TrCdc42, TrRac, and conidial germination in T. rubrum

Article Snippet: EHT1864, ML-141, EHop-016, ZCL-278, CASIN, Ketorolac, and Brefeldin A were obtained from Cayman Chemical Company, Germany.

Techniques:

Journal: iScience

Article Title: Targeting dermatophyte Cdc42 and Rac GTPase signaling to hinder hyphal elongation and virulence

doi: 10.1016/j.isci.2024.110139

Figure Lengend Snippet:

Article Snippet: EHT1864, ML-141, EHop-016, ZCL-278, CASIN, Ketorolac, and Brefeldin A were obtained from Cayman Chemical Company, Germany.

Techniques: Virus, Recombinant, Software