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Science 329 (5990): 436-439

Copyright © 2010 by the American Association for the Advancement of Science

A Septin Diffusion Barrier at the Base of the Primary Cilium Maintains Ciliary Membrane Protein Distribution

Qicong Hu1, Ljiljana Milenkovic2,3, Hua Jin4, Matthew P. Scott2,3,5–,8, Maxence V. Nachury4, Elias T. Spiliotis9,*, and W. James Nelson1,4,*

1 Department of Biology, Stanford University, Stanford, CA 94305, USA.
2 Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
3 Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA.
4 Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA.
5 Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA.
6 Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
7 Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
8 Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA.
9 Department of Biology, Drexel University, Philadelphia, PA 19104, USA.


Figure 1
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Fig. 1. (A) FRAP of proteins in the whole cilium 24 hours after serum starvation. IMCD3 cells stably (a) or transiently (b) expressing Htr6SEP or IFT88YFP (d), and SmoYFP stably expressed in MEFs treated with 100 nM SAG for 24 hours (c); arrowheads mark one end of the cilium. Scale bars, 5 µm. (B) Kinetics of average (±SEM) fluorescence recovery of proteins photobleached in the whole cilium (a, b; n = 8 to 12). Summary of fluorescence recovery of ciliary membrane proteins as a percentage of the initial unbleached fluorescence level compared with IFT88YFP distribution (c; ***P < 0.0001). (C) FRAP of Htr6SEP (a), SmoYFP (b), and IFT88YFP (c) represented as heat-map images after photobleaching part of the cilium in IMCD3 cells; dotted lines mark the photobleached/unbleached boundary. Scale bars, 5 µm. (D) Representative example of kinetics of fluorescence recovery of photobleached region (orange curve), unbleached region (blue curve), and the two regions combined (red curve) of a primary cilium (n = 12 to 13).

 

Figure 2
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Fig. 2. (A) SEPT2 localization at the cilium of IMCD3 cells stably expressing FlHtr6SEP (a), MEFs stably expressing SmoYFP treated with 100 nM SAG for 24 hours (b), and IMCD3 cells stained for acetylated {alpha}-tubulin (AcTub) (c; higher magnification of boxed region shown at the bottom), and imaged with a super-resolution Optical Microscope Experimental (OMX) system using three-dimensional structured illumination (d). Scale bars, 2 µm. (B) SEPT2 and AcTub localization at the cilium of IMCD3 cells transiently expressing CEP164LAP (a), Odf2LAP (b) or PeriCTRFP (c). Scale bars, 2 µm. (Lower panels) Fluorescence intensity profiles of protein staining from the basal body to the tip of the cilium. (C) IMCD3 cells grown for 24 hours without (–) or with (+) serum and stained for SEPT2, {alpha}-tubulin ({alpha}-tub), and {gamma}-tubulin ({gamma}-tub). Scale bars, 2 µm. (D) Percentage of cells with SEPT2 localized at the base of primary cilia (–serum) or pericentriolar region (+serum). Error bars represent SD of three independent experiments (n = 60 to 178 each; ***P < 0.0001).

 

Figure 3
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Fig. 3. (A) IMCD3 cells transfected with scrambled (scramble) or SEPT2 siRNA (siSEPT2), serum-starved to induce ciliogenesis, and stained for SEPT2 and AcTub. Scale bars, 10 µm. (B) Whole-cell lysates of scramble and siSEPT2 cells immunoblotted for SEPT2 and glyceraldehyde phosphate dehydrogenase (GAPDH); lower panel, SEPT2 band intensities normalized to GAPDH. (C) Percentage of scramble and siSEPT2 cells with a cilium >1 µm long (±SD of three independent experiments; n = 101 to 205 each; **P = 0.0098). (D) IMCD3 cells (Parental), and IMCD3 cells stably depleted of SEPT2 using shRNA1, shRNA2, or shRNA3, stained for SEPT2, AcTub, and 4',6'-diamidino-2-phenylindole (DAPI). Scale bars, 10 µm. (E) Whole-cell lysates of parental IMCD3 cells and three arbitrarily chosen clones of shRNA1, 2, or 3 cells immunoblotted for SEPT2 and GAPDH; lower panel, SEPT2 band intensities normalized to GAPDH, and percentage of parental and shRNA1, 2, and 3 IMCD3 cells with a cilium (n = 115 to 282).

 

Figure 4
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Fig. 4. (A and B) FRAP of the whole cilium of parental IMCD3 (Parental) and shRNA1 IMCD3 cells transiently expressing Htr6SEP or SmoYFP and treated with 100 nM SAG for 24 hours. Scale bars, 5 µm. (B) Kinetics of average (±SEM) fluorescence recovery of photobleached proteins (n = 9 to 12). (C) Summary of percentage of fluorescence recovery of photobleached ciliary membrane proteins in parental and shRNA1 cells (**P < 0.002; ***P < 0.0005). (D) Summary of barrier index in parental and shRNA1 cells (n = 31 to 59; ***P < 0.0001). (E) Gli1 (left) and Ptc1 (right) mRNA induction in parental and shRNA1 cells after 100 nM SAG or Shh treatment for 24 hours (average of three independent experiments ±SD; **P = 0.005; ***P < 0.0007). (F) Whole-cell lysates of parental and shRNA1 cells immunoblotted for Smo, SEPT2, and GAPDH; normalized Smo levels in parental and shRNA1 cells (right).

 


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