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First published online 12 September 2006
doi: 10.1242/jcs.03167

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Visualisation of macropinosome maturation by the recruitment of sorting nexins

¹ Institute for Molecular Bioscience and ARC Centre in Bioinformatics, University of Queensland, St. Lucia, QLD 4072, Australia
² The Russell Grimwade School of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC 3010, Australia

View larger version (104K): [in a new window]   Fig. 1. EGF stimulation rapidly induces highly dynamic SNX5-positive tubule formation on newly formed macropinosomes. (A) HEK-GFP-SNX5 cell monolayers were serum starved overnight before being exposed to 100 ng/ml recombinant EGF. The interval between image capture was 10 seconds. EGF was added 1 minute and 40 seconds into the recording (+EGF). Selected frames are presented with the time captured relative to the first frame recorded at the top right (minutes:seconds). The periphery of the cell is marked in yellow. Asterisks indicate the lumen of an individual macropinosome. (B) HEK-GFP-SNX5 cell monolayers were serum starved overnight before being incubated with dextran conjugated to tetramethylrhodamine (dextran-TR) and 100 ng/ml EGF for 3 minutes before fixation with 0.9% PFA at 4°C. SNX5-positive macropinosomes were then analysed by immunofluorescence microscopy. (C) HEK293 cell monolayers were co-transfected with pEYFP-Rabankyrin 5 and pCMU-myc-SNX5 as described in the Materials and Methods, fixed with 0.9% PFA and analysed by indirect immunofluorescence using an anti-myc monoclonal antibody followed by Cy3-conjugated goat anti-mouse IgG. Bars, 10 µm.

View larger version (101K): [in a new window]   Fig. 2. Ultrastructural characterisation of growth-factor-induced pinocytic structures and localisation of SNX5 in HEK293 cells. (A-D) HEK-GFP-SNX5 cell monolayers were serum-starved overnight before being exposed to 100 ng/ml EGF and HRP for 3 minutes. The cells were then washed, fixed, and processed for plastic embedding. Semi-thick sections (200 nm) were cut parallel to the culture substratum. Asterisks indicate large HRP-labelled structures with the morphology of macropinosomes close to the plasma membrane. HRP-positive tubular structures (arrows) can be seen extending from the macropinosome-like structures. n, nucleus; pm, plasma membrane. (E) HEK293 cell monolayers were transiently transfected with pEGFP-SNX5 and serum starved overnight before being exposed to 100 ng/ml EGF for 2 minutes, fixed in 4% PFA and then processed for frozen sectioning. SNX5 in thawed sections was detected with a rabbit anti-GFP antibody followed by Protein A 10 nm gold. GFP-SNX5 labelling (arrowheads) was readily observed in the cytoplasm and on large electron-lucent vesicular structures underlying the plasma membrane. The specificity of GFP-SNX5 immunogold labelling was determined by examining random areas from 25 transfected cells and 25 untransfected cells at a magnification of 30,000x. The total number of gold particles and the number of gold particles associated with specific cellular features were compared with untransfected cells labelled in parallel under identical conditions. Total number of cytoplasmic gold particles in transfected cells is 242 and in untransfected cells is 27. The gold particle distribution was as follows (expressed as a % of total cellular labelling in transfected cells with the expected proportion contributed from background as determined from observed immunolabelling of untransfected cells in parentheses); plasma membrane, 12.0% (0%); cytosol, 67.4% (9.9%); electron-lucent vacuoles (putative macropinosomes, E), 4.1% (0%); associated tubules and small vesicles, 13.6% (0%); multivesicular bodies, 1.2% (0%); other, 1.7% (1.2%). Bars, 5 µm (A); 1 µm (B,D); 500 nm (C,E).

View larger version (83K): [in a new window]   Fig. 3. SNX5-positive tubule extension and departure plays a role in the maturation of the macropinosome. HEK-GFP-SNX5 cell monolayers were serum starved overnight before being exposed to 100 ng/ml recombinant EGF 1 minute before the videomicroscopy was started. Time-lapse movies were recorded as in Fig. 1A. The interval between image capture was 10 seconds. Selected frames are presented with the time captured relative to the first frame recorded on the top right (minutes:seconds). Bar, 10 µm.

View larger version (117K): [in a new window]   Fig. 4. Extension of SNX5-positive tubules is dependent on microtubules. (A) HEK-GFP-SNX5 cell monolayers were fixed in 0.9% PFA and analysed by indirect immunofluorescence using a monoclonal antibody against ß-tubulin followed by Cy3-conjugated goat anti-mouse IgG. (B) HEK-GFP-SNX5 cell monolayers were serum starved overnight before being exposed to the microtubule-destabilising agent nocodazole (10 µM) for 30 minutes, and then to EGF as in Fig. 3. Time-lapse movies were recorded for a total period of 15 minutes with a 10-second interval between image capture. The total capture period was subsequently extended to 90 minutes with little apparent loss of fluorescent signal of GFP-SNX5 from the macropinosome. Asterisks indicate the lumen of an individual macropinosome. Bars, 5 µm (A); 10 µm (B).

View larger version (55K): [in a new window]   Fig. 5. Macropinosome-derived SNX5-positive tubules traffic to smaller SNX5-positive early endosomes. (A) HEK-GFP-SNX5 cell monolayers were treated and analysed using the same conditions as in Fig. 1A. The interval between images captured was 1 second for a total period of 3 minutes. Tif stacks were pseudocoloured to highlight variations in fluorescent intensity (black/blue, low intensity; white/yellow, high intensity). Arrows highlight the emergence and movement of a discrete SNX5-positive patch across the cytoplasmic face of the macropinosome. Asterisks indicate point of termination of tubules. Bar, 5 µm. (B) To visualise the kinetics of the structures presented in A, the movie was separated into individual frames using Image J v1.31 and associated Macro programs and stacked in the Z axis using software specifically developed for the task. The frames were arranged with the first frame in the foreground with each successive frame behind the first. Numbers in the image indicate frame numbers captured at 1-second intervals. Presented is a single view from Movie 6 in supplementary material. Red arrows highlight the path of an SNX5-positive patch across the cytoplasmic face of the macropinosome. Green arrows highlight the termination point of the tubules and asterisks indicate the acceptor endosome. (C) Fluorescent compaction was used to monitor the fusion event between the tubule and the acceptor endosome (asterisk). The relative fluorescent intensities of the tubule () and the acceptor endosome pre-fusion (ß) and post-fusion () were measured using Image Jv1.31.

View larger version (75K): [in a new window]   Fig. 6. SNX5 associates with discrete lipid microdomains on the surface of macropinosomes. (A) HEK-GFP-SNX5 cell monolayers were fixed in 0.9% PFA and analysed by indirect immunofluorescence using a monoclonal antibody against EEA1 followed by Cy3-conjugated goat anti-mouse IgG. (B) Surface rendered 3D reconstruction of the same macropinosome from A was generated from Z stacks using a LSM 510 META confocal laser-scanning microscope. Presented is a reconstruction rotated 45° on the y-axis. Optical sections used were 0.8 µm in depth.

View larger version (86K): [in a new window]   Fig. 7. SNX5 associates with early-stage macropinosomes. HEK-GFP-SNX5 and HEK293 cell monolayers were transfected as described in the Materials and Methods, serum starved overnight and exposed to 100 ng/ml EGF for 5 minutes before being fixed with 0.9% PFA. SNX5-positive macropinosomes were then analysed by indirect immunofluorescence. Images were collected as described in Fig. 1B. (A-C) HEK-GFP-SNX5 cell monolayers were labelled with monoclonal antibodies against SNX1, EEA1 and LAMP1, respectively, followed by Cy3-conjugated goat anti-mouse IgG secondary antibody. (D-F) HEK293 cell monolayers co-transfected with pCMU-myc-SNX5 and either GFP-Rab5wt (C), GFP-Rab7wt (D) or YFP-Rab11wt (E) as indicated were co-labelled with an anti-myc monoclonal antibody and Cy3-conjugated goat anti-mouse IgG. Bar, 5 µm.

View larger version (96K): [in a new window]   Fig. 8. SNX5-positive tubule departure is concurrent with macropinosome Rab5 to Rab7 conversion. (A,B) HEK293 cell monolayers were co-transfected with mCherry-SNX5 and either GFP-Rab5wt (A) or GFP-Rab7wt (B). Time-lapse movies were recorded with an interval between image capture of 2 seconds. Bars, 5 µm. (C) HEK-GFP-SNX5 cells and HEK293 cell monolayers transfected with either GFP-Rab5wt or GFP-Rab7wt were serum starved overnight before being incubated with dextran-TR and 100 ng/ml EGF for 3 minutes at 37°C before fixation with 0.9% PFA at 4°C. 50 dextran-positive macropinosomes (diameter >0.2 µm) were imaged and the presence of each marker was scored. Presented is the percentage of dextran-positive macropinosomes labelled with detectable levels of GFP-Rab5, GFP-SNX5 or GFP-Rab7.

View larger version (56K): [in a new window]   Fig. 9. SNX5 forms heteroligomers with SNX1. (A) Full-length SNX1 and SNX5 were fused to either the LexA DNA-binding domain in the pLexA vector or the B42 activation domain in the pYESTrp vector as indicated. Interactions were assessed using a liquid galactosidase assay and reported in Miller units (MU). Values are mean ± s.e.m. (B) Total extracts of HEK-GFP-SNX5 cell monolayers were prepared as described in the Materials and Methods, and lysates immunoprecipitated with an anti-GFP polyclonal antibody. Immune complexes were collected and subjected to SDS-PAGE under reducing conditions. After transfer to PVDF membranes, membranes were immunoblotted with monoclonal antibodies raised against SNX1 and HRP-conjugated anti-mouse immunoglobulin using a chemiluminescence detection system. Molecular mass in kDa is indicated on the left. (C) Total extracts of HEK-GFP-SNX5 cells treated with scrambled or SNX1-specific siRNA duplexes for 96 hours were prepared as described in the Materials and Methods and separated by SDS-PAGE. The relative amount of endogenous SNX1 and SNX2 in the samples was then determined by western immunoblotting with monoclonal anti-SNX1 and SNX2 antibodies. (D) HEK-GFP-SNX5 cell monolayers treated with scrambled or SNX1-specific siRNA duplexes for 96 hours were fixed in 0.9% PFA and labelled with a mouse anti-SNX1 monoclonal antibody followed by Cy3-conjugated goat anti-mouse IgG. Bar, 10 µm.

View larger version (41K): [in a new window]   Fig. 10. Sorting nexins in macropinosome maturation. Macropinosomes are formed at the base of actin-rich membrane ruffles in response to extracellular stimuli (EGF). The sorting nexins are rapidly recruited to discrete microdomains on the nascent organelle within the first few minutes following its formation. These microdomains traverse the surface of the macropinosome in a regulated and directed manner before being incorporated into highly dynamic tubular structures. The SNX-positive tubular structures extend from the macropinosome in a microtubule-dependent fashion eventually separating from the macropinosome and travelling along predefined paths towards the perinuclear region of the cell before fusing with acceptor early endosomes. Concurrent with the departure of the tubular structure is an increase in the apparent internal pressure of the donor macropinosome as well as a shift in its protein composition.