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First published online 13 June 2006
doi: 10.1242/jcs.02999

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Microtubule plus-end loading of p150^Glued is mediated by EB1 and CLIP-170 but is not required for intracellular membrane traffic in mammalian cells

Department of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol, BS8 1TD, UK

View larger version (74K): [in a new window]   Fig. 1. siRNA-mediated depletion of lamin A/C, p150Glued, EB1 or CLIP-170. (A) Cells depleted of lamin A/C, p150Glued, EB1 or CLIP-170 were probed with antibodies specific for lamin A/C, p150Glued, EB1 or CLIP-170 as indicated. (B) RNAi-mediated depletion of EB1 is seen with siRNAs designed to target EB1 but also with CLIP-170b designed to target CLIP-170. This was also seen when we resynthesized this siRNA to eliminate cross-contamination as a possible explanation (CLIP-170b2). EB1 is not depleted by other CLIP-170 targeted siRNA duplexes (CLIP-170a, c or d). Notice that one lane of the lower panel has been removed to align the top and bottom panels with one another (shown by the black vertical line). (C) Use of non-fluorescent (top panels) or fluorescent (lower panels) siRNA duplexes targeting lamin A/C shows the efficiency of transfection with the modified calcium phosphate protocol. Both fluorescence images were acquired with the same microscope settings; fluorescence is only seen when fluorescent siRNA duplexes were used (bottom right) and does not result from cellular autofluorescence. (D) Immunofluorescence localization of EB1 in cells depleted of lamin A/C (top) or EB1 (bottom) confirms efficiency of target depletion. Bars, 10 µm.

View larger version (75K): [in a new window]   Fig. 2. Localization of +TIPs following siRNA-mediated depletion of lamin A/C, p150Glued, EB1 or CLIP-170. (A) RNAi-mediated depletion of p150Glued, EB1 or CLIP-170 all cause a loss of p150Glued from MT plus-ends. Lamin A/C depletion does not affect the localization of p150Glued to MT plus-ends. (B) Enlargements of the boxed regions from (A). (C) Depletion of lamin A/C or CLIP-170 does not affect the localization of EB1. (D) Depletion of EB1 causes a loss of CLIP-170 localization to MT plus-ends. Boxed regions in C and D are shown magnified in the right bottom corner of the images. Bars, 10 µm.

View larger version (40K): [in a new window]   Fig. 3. Quantification of plus-end p150Glued. Example quantification of plus-end labelling with anti-p150Glued. (A) Localization of p150Glued at MT plus-ends was determined by immunofluorescence and quantified by analysis of fluorescence intensity of tubulin and p150Glued along a line as shown in the enlargement. Box, 4 µm square. (B) Localization of p150Glued in siRNA transfected cells as indicated. (C) Overlays of the images from B with the corresponding tubulin localization. All panels 42x35 µm in B and C. (D) Graphical representation of fluorescence intensity at plus-ends, calculated as in (A). (E) Quantification of >150 plus-ends for each siRNA transfection (at least ten plus-ends from five cells from three coverslips taken from two independent experiments). The histogram shows that average (mean) intensity values (arbitrary units) with the individual points showing the scatter of the data (notice the close clustering of data points close to the x-axis in EB1 and CLIP-170-depleted cells).

View larger version (36K): [in a new window]   Fig. 4. Depletion of p150Glued, EB1 or CLIP-170 disrupts polarization of cells in response to scratch wounding. A549 cells transfected with siRNAs that target lamin A/C, p150Glued, EB1 or CLIP-170 were grown for 72 hours and subsequently scratched to wound the monolayer. After a further 2-hour incubation, the position of the Golgi and centrosome was determined. (A) Example wounded monolayer (lamin A/C siRNA) in which cells have reoriented towards the wound edge (white line). Also shown are dots showing the calculated cell centroid and the mask used to score reorientation. Area shown, 86x69 µm. (B) Percentage of cells showing orientation towards the wound edge was scored for each siRNA as well as for a mock-transfected control and also a scratch control (in which cells were scratched and immediately fixed). Notice that a `random' orientation, as found in the scratch control, would mean 33% of cells polarized towards the edge. Data from three experiments, >100 cells for each transfection are shown; error bars show the standard error of the mean (s.e.m.); *, significance determined using ANOVA (P<0.05).

View larger version (50K): [in a new window]   Fig. 5. Analysis of membrane trafficking in +TIP-depleted cells. Cells were transfected with siRNA duplexes targeting lamin A/C, p150Glued, CLIP170 or EB1. After 72 hours, cells were fixed with methanol and processed for immunofluorescence. (A) Localization of golgin-97 (trans-Golgi network) and Sec24Cp (COPII-coated ER exit sites). (B) Localization of ERGIC-53 (ER-Golgi intermediate compartment) and giantin (Golgi). Bar, 10 µm.

View larger version (66K): [in a new window]   Fig. 6. Dynamics of fluorescent transferrin and LysoTracker labelled organelles in cells depleted of lamin A/C, p150Glued, CLIP-170 or EB1. (A) Live HeLa cells depleted of lamin A/C, p150Glued, CLIP-170 or EB1 were labelled with Alexa Fluor 568-transferrin for 10 minutes and subsequently imaged at three frames per second. The image shows enlarged regions of cells with tracks of moving objects overlayed onto them. Bar, 5 µm. See also supplementary material Movie 1. (B) Tracking of Alexa Fluor 568-transferrin-positive structures reveals that the speed of movement is unchanged by depletion of either Lamin A/C, p150Glued, CLIP-170 or EB1. Objects from at least 20 cells from two independent experiments were analyzed; histograms show the average of this data, the points plotted show the spread of velocities from 25 randomly selected objects of the >500 objects tracked. (C) Live HeLa cells depleted of lamin A/C, p150Glued, CLIP-170 or EB1 were labelled for 5 minutes with LysoTracker and subsequently imaged at two frames per second for 1 minute. The image shows cells with tracks of moving objects overlayed onto them. Bar, 10 µm. See also supplementary material Movie 2. (D) Tracking of LysoTracker-positive structures reveals that the speed of movement is unchanged by depletion of either Lamin A/C, p150Glued, CLIP-170 or EB1. Objects from at least ten cells for each depletion from two independent experiments were analyzed; histograms show the average of this data, the points plotted show the spread of velocities from 25 randomly selected objects of the >200 objects tracked for each depletion experiment. Stars in A and C indicate starting positions of the objects being tracked.

View larger version (48K): [in a new window]   Fig. 7. ER-to-Golgi transport is unperturbed in cells depleted of either lamin A/C, p150Glued, CLIP-170 or EB1. (A) tsO45G-YFP transport from the ER to Golgi was monitored by time-lapse microscopy. Cells were shifted to 32°C for 5 minutes before imaging. Bar, 10 µm. See supplementary material Movie 3. (B) Enlargements (2x) of the boxed regions (10x10 µm) in C show the presence of VTCs (arrows) that track to the Golgi (see supplementary material Movie 4). (C) Individual tsO45-G-YFP-labelled VTCs were tracked and grouped into bins of speeds of 0.2 µm s–1. The number of objects in each bin were plotted for experiments in which lamin A/C (x), p150Glued (), EB1 () or CLIP-170 () had been depleted.

View larger version (64K): [in a new window]   Fig. 8. Localization of human GFP-p150Glued in HeLa cells. (A) GFP-Hsp150Glued localizes to MT plus-ends in transiently transfected HeLa cells. (B) At intermediate expression level, p150Glued decorates the length of MTs as well as localizing to punctate structures that can be seen in time-lapse sequences to translocate along MTs. Bar, 10 µm; arrows in inset highlight two structures that translocate in the direction of the arrows. See supplementary movie 5. (C) Tracking of GFP-p150Glued in low expressing cells. Time-lapse imaging (see supplementary material Movie 7, upper panel) reveals punctate structures that translocate in a non-linear manner through the cytoplasm. Tracks are shown on the movie sequence that follow three such structures (supplementary material Movie 6, lower panel); one of these is overlayed on the still images taken from this movie (red). The lower panel shows a maximum-intensity projection of all time points from this sequence shows the trajectories taken by these structures as they translocate through the cytoplasm. (D) Density-gradient centrifugation shows that GFP-Hsp150Glued co-distributes across the gradient with endogenous Hsp150Glued. GFP and CLIP-170 distribute away from this complex whereas p50dynamitin co-distributes as expected, with p150Glued. We consistently observe a population of uncomplexed p50dynamitin for which we do not have a good explanation. (E) GFP-Hsp150Glued puncta colocalize with ERGIC-53 (ER-Golgi transport intermediates, top panels), known to localize to dynamic structures, but not with COPII-coated (Sec24Cp-positive, lower panels) ER exit sites, which are mainly immobile. Arrowheads indicate colocalizing structures, arrows indicate structures that are only positive for GFP-Hsp150Glued.

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