Genes & Development



Figure S1: Characterization of SCLT1 and FBF1. (A & B) Cell cycle profile of SCLT1 and FBF1. RPE-1 were stained with an antibody to SCLT1 (A), or FBF1 (B) (red), centrin 3 (green) and DAPI (blue). Cell cycle stages are indicated. In ciliated cells (lower panels) where primary cilia are marked by antibodies against acetylated α-tubulin (green), both DAP proteins localize to the ciliary base. (C & D) RPE-1 cells stably expressing HA-tagged CEP83 were stained with antibodies to SCLT1 (red), HA (c) (green) or Odf2 (D) (green) and centrin (blue) and examined with super-resolution microscopy (OMX DeltaVision, Applied Precision, Inc.). Note that SCLT1 colocalizes with HA- CEP83 both in top view (upper panels) and side view (lower panels), but does not colocalize with the subdistal appendage protein Odf2. Small bar indicates 200 nm. (E & F) RPE-1 cells stably expressing HA-tagged CEP83 were stained with antibodies to FBF1 (red), HA (E) (green) or Odf2 (F) (green) and centrin (blue) and examined with an OMX super-resolution microscope. Note that FBF1 colocalizes with HA- CEP83 both in top view (upper panels) and side view (lower panels), but does not colocalize with the subdistal appendage protein Odf2. Small bar indicates 200 nm. Figure S2: Odf2 is not required for the centriolar recruitment of DAP components. (A-D) RPE1 cells were retrovirally transduced with control shRNA or an Odf2-targeting shRNA as indicated, and stained with antibodies to CEP83 (A), or Cep164 (B), or CEP89 (C), or FBF1 (D) (red), Odf2 (green), and Centrin 3 (blue). Note that DAP proteins are unchanged in the absence of Odf2 and that they maintain their ring like pattern. Bar indicates 1 ?m. (E & F) RPE1 cells were retrovirally transduced with control shRNA or an CEP83-targeting shRNA as indicated, and stained with antibodies to CEP83 (red), Odf2 (E) or centriolin (F) (green) and centrin 3 (blue). Bar = 1 ?m. (G) Western blot indicating CEP83 protein levels in control shRNA and CEP83 shRNA cells, upper panel, and protein levels for the other DAPs examined. Note that depletion of CEP83 does not change total levels of other distal appendage proteins. Actin loading control is shown in the lower panel. Figure S3: Loss of DAP proteins blocks ciliogenesis prior to TZ assembly. (A-C) Both branches of DAP components are required for the recruitment of IFT88 to mother centrioles. (A) RPE-1 cells stably expressing centrin-GFP (green) were transfected with control siRNA (upper panels) or SCLT1 siRNA (lower panels), serum-starved for 48 hrs and stained with antibodies to IFT88 (red) or Cep164 (green). Small bar=1 ?m. Large bar=5 ?m (B) RPE-1 cells stably expressing centrin-GFP (blue) were transfected with control siRNA (upper panels) or CEP89 siRNA (lower panels), serum-starved for 48 hrs and stained with antibodies to IFT88 (red) or CEP89 (green). Small bar=1 ?m. Large bar=5 ?m. (C) Western blot indicating IFT88 protein levels (left upper panel) and protein levels for CEP83 (right upper panels) in control shRNA and CEP83 shRNA cells. ?-tubulin loading control is shown in the lower panels. Note that depletion of CEP83 does not change total levels of IFT88. (D & E) Ciliogenesis defect can be rescued by restoring expression of distal appendage proteins. (D) RPE-1 cells with (right) or without (left & center) inducible expression of RNAi-resistant form of HA-tagged CEP83 (HA-CEP83R) were retrovirally transduced with control shRNA (left) or CEP83 shRNA (center and right panels). Cells were grown in serum free media for 48 hrs and then fixed and stained with antibodies to CEP83 (red) or HA (red) and acetylated tubulin (green). (E) RPE-1 cells with (right) or without (left & center) inducible expression of rat HA-tagged SCLT1 (HA-rat SCLT1) were transfected with control siRNA (left), or SCLT1 siRNA (center and right panels). Cells were grown in serum free media for 48 hrs and then fixed and stained with antibodies to SCLT1 (red) or HA (red) and acetylated tubulin (green). DNA staining with DAPI is shown in blue. A quantification of this result is shown on the right.Figure S4: CEP83 is required for centriole-to-membrane docking in IMCD3 murine epithelial cells. (A) IMCD3 cells were retrovirally transduced with either a luciferase control shRNA, or a CEP83-targeting shRNA. 48 hours after infection, cells were split and plated on coverslips, and serum-starved 24 hours later. Maximum projection of a confocal Z stack of an IMCD3 monolayer, adquired with a LEICA SP5 point scanner. Cilia are marked with acetylated tubulin (green); CEP83 is shown in red, DNA with DAPI in blue, and γ-tubulin is shown in the insets in blue. Note that CEP83 is not present in CEP83 knockdown cells, and that these cells do not have cilia. Bar indicates 10 ?m and small bar indicates 1 ?m. (B) Quantification of IMCD3 ciliated cells from the experiment shown in (a). (C) Western blot indicating CEP83 knockdown in IMCD3 cells. (D) Additional EM images of control and CEP83-depleted centrioles. IMCD3 cells were retrovirally transduced with either a luciferase control shRNA (i-vi), or a CEP83-targeting shRNA (vii-xi), three days after infection cells were processed as described for Fig. 4A. (i) Thin section showing a centriole pair and cilia in IMCD3 control cells, 25K, scale bar indicates 1 ?m. (ii) Centriole pair. Scale bar indicates 500 nm. (iii) Consecutive section showing the same centriole pair as in (b) and the beginning of the ciliary axoneme. Note that the mother centriole is still buried and only its distal appendages can be clearly seen. (iv) Consecutive section showing both centrioles and the ciliary axoneme. Note that microtubules can be seen attached to one of the subdistal appendages. (v) Consecutive section showing both centrioles and the ciliary axoneme. Two more distal appendages can be seen. (vi) Following section showing the centriole pair as well as two more subdistal appendages. (vii) Centriole pair in IMCD3 cell depleted of CEP83. 25K, scale bar indicates 1 ?m. (viii) Higher magnification of pair of centrioles shown in centriole in (vii). Note that the centriole is close to the membrane but has not docked. (ix) (x) (xi) Continuous sections following the same centriole pair. Figure S5: CEP83 depletion does not disrupt apical-basal polarity in IMCD3 cells. IMCD3 cells were retrovirally transduced with either a luciferase control shRNA or a CEP83-targeted shRNA. Three days after infection, cells were plated on polystyrene Transwell filters and allowed to polarize for 72 hours. (A) control (left) or CEP83-depleted cells (right) were stained for acetylated tubulin to mark cilia (red), or ?-tubulin to mark the position of the centrioles (green). CEP83 staining is shown in middle panels. DNA is marked by DAPI (blue). The XZ projection shows that centrosomes remained positioned near the apical cortex in the absence of CEP83. (B) control (left) or CEP83-depleted cells (right) were stained for the basolateral marker E-Cadherin, and the tight junction marker ZO-1, as indicated . DNA is marked by DAPI. The XZ projection shows that the apical-basal polarity is normal in the absence of CEP83. (C) Maximum intensity projection of a confocal z-stack showing F-actin in red, acetylated tubulin in green and DAPI in blue. Note that the actin cytoskeleton (and apical actin microvilli) distribution does not change upon depletion of CEP83 (right) compared to control (left). Table S1: Evolutionary profile of novel DAP proteins in ciliated organisms ................
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