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First published online 12 September 2006
doi: 10.1242/jcs.03192

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Trimerisation is important for the function of clathrin at the mitotic spindle

Stephen J. Royle^*, and Leon Lagnado

MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

View larger version (18K): [in a new window]   Fig. 1. Overview of the organisation of clathrin and of the constructs used in this study. (A) Model of a clathrin triskelion proposed by Fotin et al. (Fotin et al., 2004). The triskelion is viewed looking down onto the vertex. Coloured regions show the features of a CHC molecule (see key, right). (B) Schematic representations of each CHC construct used in the study. Variable region (residues 1631-1675) is shown in grey, GFP has been omitted for clarity. Short names used in the paper are in black and full descriptive names are in grey. Trimerisation was predicted based on previous publications (Fotin et al., 2004; Liu et al., 1995; Nathke et al., 1992; Ybe et al., 2003). X22 epitope is between residues 1109-1128 of CHC (Liu et al., 1995). Constructs were compared with `GFP', GFP expressed on a CHC RNAi background and with `Control', GFP expressed on a control RNAi background.

View larger version (43K): [in a new window]   Fig. 2. Knockdown of endogenous CHC and replacement with GFP-tagged CHC constructs. (A) Representative confocal images of cells expressing CHC constructs (left) that were immunostained for CHC (middle) using antibody X22. Nucleic acids are shown in blue in the merged panels (right). Bar, 10 µm. Note the knockdown of endogenous CHC in GFP and 1-479 and the X22 immunoreactivity in 1-1675. (B) Histogram to compare the amount of X22 immunoreactivity in cells expressing each of the CHC constructs. Cells were outlined using the GFP channel as a guide and the mean greyscale pixel value for the red signal (X22/Alexa Fluor 546) was measured. Results are mean ± s.e.m. of 26-47 cells per construct and are normalised to control.

View larger version (16K): [in a new window]   Fig. 3. Stunted is a trimeric molecule. (A) Sedimentation analysis of 1-1639 and 1-1597. Quantification of a typical experiment where cell lysates were separated on a 15-40% glycerol gradient. Note the second peak at fraction 8 in 1-1639 that is absent in 1-1597. (B) Sedimentation analysis of Stunted and Stuntedtripod. Quantification of a typical experiment where cell lysates were separated on a 10-35% glycerol gradient. Note the presence of a second peak in Stunted that is absent in Stuntedtripod. The sedimentation of molecular mass standards is shown for each gradient (arrows). The calculated molecular masses for monomers of 1-1639, 1-1597, Stunted and Stuntedtripod were 215, 210, 117 and 108 kDa respectively. Fractions were analysed by SDS-PAGE and immunoblotting for GFP. Following densitometry, the signal in each fraction was expressed as a percentage of the total GFP signal from all fractions on the blot.

View larger version (41K): [in a new window]   Fig. 4. CME was only supported in cells expressing functional triskelia. (A) Representative confocal micrographs of transferrin uptake in cells expressing some of the CHC constructs on a CHC knockdown background. Panels show GFP (left), transferrin-Alexa-Fluor-546 (middle) and merge (right). Bar, 20 µm. (B) Quantification of transferrin uptake. Each cell was outlined using the GFP channel as a guide, the transferrin-Alexa-Fluor-546 image was then thresholded and the number of puncta within the ROI was counted. Results are mean ± s.e.m. of 18-38 cells per construct, normalised to control; **P<0.01 compared with transferrin uptake in GFP cells.

View larger version (100K): [in a new window]   Fig. 5. Constructs containing the N-terminal domain were recruited to the mitotic spindle. (A) Representative confocal micrographs of each construct in HEK293 cells in metaphase. Bar, 10 µm. (B) A cell expressing 1-1639 on a knockdown background. ROIs (grey, 1.04x1.04 µm) were placed over the spindle (black outline) and in the cytoplasm (white outline). Spindle regions and non-spindle areas were defined by staining for -tubulin using DM1A/Alexa Fluor 546 (left). Measurements were taken from the GFP channel (right). (C) Histogram to show the recruitment to the spindle of CHC constructs on a CHC-knockdown background. Spindle recruitment is the GFP fluorescence measured in a 1.04x1.04 µm ROI at the spindle divided by that measured in a same-sized ROI in the cytoplasm (Fspindle/Fcytoplasm). Results are mean ± s.e.m. of 12-27 cells per construct; **P<0.01 compared with levels in GFP cells.

View larger version (21K): [in a new window]   Fig. 6. Rescue of mitotic defects by trimeric CHC constructs. Quantification of (A) mitotic index and (B) the frequency of metaphase-like cells with misaligned chromosomes in cells expressing CHC constructs on a CHC-knockdown background. Mitotic index was assessed by counting the number of GFP-positive cells in mitosis as a proportion of the total number of GFP-positive cells, per unit area. The number of GFP-positive cells in a metaphase-like state with misaligned chromosomes was also counted. Hoechst 33342 staining was used to identify mitotic cells and misaligned chromosomes. Results are mean ± s.e.m.; **P<0.01 compared with levels in GFP cells.

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