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Figures
- Fig. 1.
Loss of BBS proteins and genetic interaction with endosomal components enhances Notch pathway activation. (A) GFP fluorescence in representative Tp1blgob:eGFP transgenic embryos at 48 hours post fertilization (hpf) in bbs1 morphant (MO) embryos and nicd-mRNA-injected embryos. Scale bar: 0.5 mm (B) qRT-PCR quantification of GFP mRNA levels relative to β-actin in 24 hpf Tp1blgob:eGFP embryos. *P≤0.01 compared with standard control morpholino; **P≤0.005 compared with deltaA morpholino (Student's t-test). (C) qRT-PCR analysis of relative HES5 expression in HEK293 cells expressed as the fold change relative to control cells. Data represent the mean±s.d. (seven separate experiments). *P≤0.01 compared with control; **P≤0.01 compared with either short hairpin alone (Student's t-test). (D) Double immunofluorescent labeling of either early endosomes (EEA1) or recycling endosomes (RAB11) and primary cilia (ARL13B or acetylated tubulin) in hTERT-RPE1 cells. Histograms represent the intensity of red fluorescence across the image. Scale bars: 5 µm. (E) qRT-PCR analysis showing relative GFP expression in Tp1bglob:eGFP zebrafish embryos at 24 hpf. Data show the mean±s.d. *P≤0.005 compared with standard control morpholino; **P≤0.005 compared with morpholinos alone (Student's t-test).
- Fig. 2.
Notch receptor localization shifts away from plasma membrane in BBS-depleted cells. (A) Sucrose-gradient fractionation of HEK293 cells transfected with HA-NOTCH1 alone or HA-NOTCH1+shBBS4 probed for HA, N-cadherin (plasma membrane marker), and EEA1 (endosome marker). (B) Quantification of the p180 band in A displayed as the proportion in each fraction relative to the total across all fractions. (C) Sucrose-gradient fractionation of HEK293 cells transfected with HA-NOTCH1 alone or HA-NOTCH1+shBBS1 probed for HA, N-cadherin and EEA1. (D) Quantification of p180 from C displayed as the proportion in each fraction relative to the total across all fractions. (E–G) hTERT-RPE1 cells immunostained with antibody against endogenous NOTCH1 (green). Scale bars: 10 µm. (H,I) hTERT-RPE1 cells coimmunostained for endogenous NOTCH1 (green) and plasma membrane marker N-cadherin (red) showing colocalization (arrowheads, inset). Histograms represent the intensity of green fluorescence across the white line. Arrows above histograms represent regions of plasma membrane fluorescence. Scale bars: 5 µm (J) Quantification of all intensity values within individual membrane peaks. Data represent the mean±s.d. (≥50 cells per treatment, two cross-sections per cell). *P≤0.001 compared with control (Student's t-test).
- Fig. 3.
NOTCH1 accumulates in late endosomes but is decreased in recycling endosomes. (A–H) hTERT-RPE1 cells immunostained with antibodies against endogenous NOTCH1 (green) and markers of late endosomes (RAB7, red) or multi-vesicular bodies (TSG101, red). Scale bars: 2 µm. (I,J) Quantification of the proportion of either RAB7-positive or extra-nuclear TSG101-positive endosomes that are also positive for NOTCH1 staining shown as a percentage of total RAB7- or TSG101-positive endosomes. Data show the mean±s.d. (K,L) hTERT-RPE1 cells coimmunostained for endogenous NOTCH1 (green) and recycling endosomes (RAB11, red). All immunofluorescence images represent a single plane of focus at the denoted endosomes and are at 100× magnification, with enlarged regions outlined by white boxes and enlarged individual vesicles outlined by dashed boxes and shown as insets. Scale bars: 2 µm (M) Quantification of the proportion of RAB11-positive endosomes that are also positive for NOTCH1 shown as a percentage of total RAB11-positive endosomes. Data show the mean±s.d. *P≤0.001 compared with control (Student's t-test).
- Fig. 4.
Loss of NOTCH1 degradation and lysosomal localization in BBS-depleted cells. (A) Detection of full-length (p300) and post-site1-cleaved N-terminal (p180) NOTCH1 by HA detection in HA–NOTCH1-transfected cells, and detection of C-terminal domains (p120) of endogenous NOTCH1 in whole-cell lysates of HEK293 cells transfected with or without shBBS4. Graphs show the quantification of band intensity for each, measured using ImageJ software, relative to β-actin. (B,C) Double immunostaining of hTERT-RPE1 cells for NOTCH1 (green) and the lysosomal marker, LAMP2 (red) (D,E) Double immunostaining of hTERT-RPE1 cells for NOTCH1 (green) and the early endosomal marker, EEA1 (red). Immunofluorescence images are at 100× magnification, with enlarged regions denoted by white boxes and enlarged individual vesicles denoted by dashed boxes and shown as insets. Scale bars: 2 µm. (F) Quantification of the percentage of LAMP2-positive lysosomes that also contain NOTCH1. *P≤0.001 compared with control (Student's t-test). (G) Quantification of EEA1-positive early endosomes containing NOTCH1. n.s., non-significant. Data shown the mean±s.d.
- Fig. 5.
Enhancement of Notch signaling and accumulation of receptor in late endosomes with loss of BBS3 or ALMS1. (A) qRT-PCR analysis of relative HES5 expression in HEK293 cells shown as the fold change relative to control cells. *P≤0.001, **P≤0.05 compared with control (Student's t-test). (B) qRT-PCR analysis of expression of GFP mRNA relative to that of β-actin in Tp1bglob:eGFP zebrafish embryos at 24 hpf. *P≤0.01 compared with standard control morpholino (MO) (Student's t-test). (C) Quantification of double immunostaining experiments for RAB7 and NOTCH1 in hTERT-RPE1 cells. The graph represents the percentage of RAB7-positive endosomes that also contain NOTCH1. *P≤0.001 compared with control (Student's t-test). (D) Quantification of double immunostaining experiments for RAB11 and NOTCH1 in hTERT-RPE1 cells. The graph represents the percentage of RAB11-positive endosomes that also contain NOTCH1. *P≤0.005 compared with control (Student's t-test). (E) Quantification of plasma membrane staining intensity as measured by intensity of fluorescence. The graph represents the average across ≥5 cells of the average of intensity values for each membrane peak, measured by ImageJ analysis. *P≤0.001 compared with control (Student's t-test). All data show the mean±s.d.
- Fig. 6.
Ciliary localization of the NOTCH1 receptor is disrupted with loss of BBS proteins. (A) Control hTERT-RPE1 cells immunostained for NOTCH1 (green, arrowheads) and ARL13B (red). (B) Control hTERT-RPE1 cells immunostained for NOTCH1 (green) and γ-tubulin (red). Enlarged regions are outlined by white boxes. Scale bars: 0.5 µm. (C–N) hTERT-RPE1 cells transfected with the indicated constructs and coimmunostained for NOTCH1 (green) and either ARL13B (red; C,E,G,I,K,M) or γ-tubulin (D,F,H,J,L,N). Arrowheads, regions of co-localization. Arrows, lack of axonemal localization. Scale bars: 0.5 µm. (O) Quantification of NOTCH1 localization at basal bodies and axonemes. The graph represents the average proportion of basal bodies or axonemes for which NOTCH1 colocalization could be observed. Axonemal localization reflects localization throughout the axoneme. Data show the mean±s.d. *P≤0.001 compared with control (Chi-squared test).
- Fig. 7.
Model of Notch receptor trafficking in endosomes of cells deficient in basal body proteins. (A) In control cells, the Notch receptor (green lines) localizes to the cilium and plasma membrane, from where it is endocytosed into early endosomes (pink). The receptor is either recycled back to the membrane by recycling endosomes (yellow) or is sorted to the late endosomes and multi-vesicular bodies (MVBs, red) for degradation in the lysosomes (blue), a process that is mediated in part by proteins such as Shrub, Deltex (Dx), Suppressor of Deltex [Su(Dx)] and HOPS (also known as TMUB1), which are involved in endosomal trafficking and degradation of the receptor. (B) Loss of BBSome proteins does not disrupt endocytosis, but results in reduced membrane and ciliary localization as a result of reduced recycling (dashed arrow). The disruption in recycling results in increased trafficking to the late endosomes (heavy black arrow), from where active NICD can be produced. (C) Loss of ALMS1 does not disrupt recycling or membrane or ciliary localization, but increases the accumulation of receptor in late endosomes (heavy black arrow) and increases signaling.
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