Figure 1.
(A) Putative domains of C11ORF24. SP: signal peptide, GFP: green fluorescent protein, TM: transmembrane domain, Tail: cytosolic tail. The numbers indicate the amino-acid position in the native protein. The putative signal peptide is underlined and colored in dark red and the putative transmembrane domain is underlined and colored in light blue. The tail domain is color-coded using Clustal W2 to highlight the similarities between different species. The position of the fragment used to raise the monoclonal antibody is indicated by the black bar. (B) The Homo sapiens (hs) C11ORF24 was aligned against its putative homologues in Mus musculus (mm, NP_082353.1) and Dano rerio (Dr, XP_690904.4) using Clustal W2 [13,14]. The cytosolic tail domain is well conserved in evolution.
Figure 2.
C11ORF24 is localized on the trans-Golgi Network.
(A) HeLa cells were transfected with a construct encoding human C11ORF24 tagged with GFP and fixed 18 hours later. Cells were co-stained with TGN46 (middle, red) and GM130 (right, blue). The second line represents a closer view of the Golgi apparatus outlined by a rectangle in the merge image. (B) HeLa cells were fixed and co-stained with TGN46, C11ORF24 and Giantin. Colocalization is very strong between TGN46 and C11ORF24 (column 4) whereas it is lower with Giantin (column 5). Line profiles of the C11ORF24 (green), TGN46 (red) and Giantin (blue) fluorescence intensities from the lines in the magnified views are shown in column 7. The colocalization between TGN46 and C11ORF24 is clearly visible throughout mitosis (lines 2-5). (C) HeLa cells were stained for C11ORF24 and imaged using dSTORM super-resolution microscopy. A Fire LUT was used to facilitate visualization. A magnified view of the boxed region of the left image is shown in the middle and a magnified view of the boxed region of the middle image is shown on the right.
The cell contour is outlined in red.
Figure 3.
C11ORF24 has a long luminal domain and a short cytosolic tail.
(A) Cartoon of a cell expressing GFP-tagged GalT or C11ORF24 and stained with an anti-GFP antibody and an anti-GM130 antibody. When the cells are not permeabilized (left) the antibodies don’t bind their targets. After saponin permeabilization (middle) all antibodies are able to reach their targets since all membranes are permeabilized. After digitonin permeabilization (right) only the cytosolic epitopes (GM130) are labeled whereas the luminal epitopes (GFP of GalT or C11ORF24) are protected by the intact Golgi membranes. Protocol modified from [18] (B) HeLa cells expressing GFP-GalT (a-c) or GFP-C11ORF24 (d-f) were stained with GFP and GM130 antibodies prior to fixation (a, d) or after fixation and permeabilization with saponin (b,e) or after fixation and permeabilization with digitonin (c,f). The GFP of C11ORF24 is accessible for the anti GFP antibody only after permeabilization of the Golgi membrane. (C) HeLa cells expressing GFP-GalT were stained with GFP and C11ORF24 antibodies prior to fixation (a) or after fixation and permeabilization with saponin (b) or after fixation and permeabilization with digitonin (c). The epitope of the C11ORF24 antibody is accessible only after permeabilization of the Golgi membrane. (D) HeLa cells were either transfected with GFP-RAB6 (line 1) or GFP-C11ORF24 (line 2) 18h prior to the incubation with the anti-GFP and internalization was performed at 37°C for 90 minutes. Cells were then fixed and stained with a secondary antibody and the localization of the anti-GFP antibody was compared with the GFP signal.
Figure 4.
C11ORF24 is a transmembrane protein.
(A) HeLa cells expressing GFP-C11ORF24 (line 1) or GFP-GalT (line 2) were bleached in a 3,5µm circular region of the Golgi apparatus (red circle) and then imaged for 2 minutes by spinning disk microscopy. The fluorescence recovery after photobleaching was measured in 3 independent experiments and plotted for GFP-GalT (red, n=18 cells) and GFP-C11ORF24 (black, n=22 cells). Error bars indicate the standard deviation. The recovery was similar for the two proteins.
Figure 5.
C11ORF24 is present on RAB6 positive transport carriers.
(A) HeLa cells expressing GFP-C11ORF24 were imaged every second for 5 minutes by spinning disk microscopy. The red arrow indicates a small transport carrier whereas the green arrow point to a long tube connected to the Golgi apparatus. A magnified view of these structures is shown in line 2 and 3. (B) HeLa cells were fixed and co-stained with TGN46, C11ORF24 and GTP-RAB6. Colocalization is very strong between TGN46, C11ORF24 and GTP-RAB6. A line profile of the C11ORF24 (green), TGN46 (red) and GTP-RAB6 (blue) fluorescence intensities from the lines in the magnified views are shown in column 7. (C) HeLa cells expressing GFP-C11ORF24 and mCherry-RAB6 were imaged every second for 5 minutes by spinning disk microscopy. The arrow indicates a transport carrier positive for GFP-C11ORF24 (green) and for mCherry-RAB6 (red). A magnified view of this structure is shown lines 4-6. (D) Transport carriers were visualized using a kymograph that was made along the line drawn on the merge image. All the dynamic elements positive for GFP-C11ORF24 (left and merge green) were positive for mCherry-RAB6 (middle and merge red).
Figure 6.
C11ORF24 is not necessary for the formation of RAB6 positive transport carriers.
(A) After a 48 hours knockdown by shRNA HeLa cells were transfected with GFP-RAB6 and the next day they were imaged every second for 30 seconds by spinning disk microscopy. Snap shots from the Movies S1 and S2 are presented for the control shRNA (upper lines) compared to the C11ORF24 shRNA (lower lines). (B) The number of RAB6 positive transport carrier per cell and the speed of theses carriers was then quantified for both treatments from 3 independent experiments and expressed as a percentage of the control. The number of cells is indicated for each treatment on the graphs (n). The error bars represent the SEM.