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doi: 10.1242/10.1242/jcs.00117

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Dual control of caveolar membrane traffic by microtubules and the actin cytoskeleton

¹ Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9039, USA
² Departments of Biology and Cell Biology, University of Virginia, Charlottesville, Virginia 22903, USA

View larger version (25K): [in a new window]   Fig. 1. Caveolin-1-GFP behaves like endogenous caveolin-1. CHO cells stably transfected with caveolin-1-GFP were grown for 2 days until confluent. (A) Triton X-100-insoluble caveolae were separated from soluble membranes on sucrose gradients and immunoblotted with anti-caveolin-1. Caveolin-1-GFP (Cav-GFP) co-fractionated with endogenous caveolin-1 (Caveolin-1). The intensity of the bands for the two proteins indicates that caveolin-1-GFP constitutes a minor amount of the total caveolin-1 pool. (B) Cells were incubated for 2.5 hours in the presence of 3H-palmitate to label acylated proteins. Caveolin-1 was immunoprecipitated, separated by PAGE and processed for autoradiography. Both the endogenous protein (caveolin-1) and the caveolin-1-GFP (cav-GFP) were covalently labeled with 3H-palmitate. (C) Caveolin was extracted from cells using a combination of Triton X-100 and deoxycholate to solubilize the caveolar membranes and then analyzed by velocity gradient sedimentation. Both the endogenous caveolin (caveolin-1) and caveolin-1-GFP (cav-GFP) form oligomers that migrate to the bottom of the gradient.

View larger version (123K): [in a new window]   Fig. 2. Caveolin-1-GFP is located in caveolae. (A) A mixed culture of parental CHO cells and CHO cells stably expressing the caveolin-1-GFP were plated on coverslips and grown for 2 days before they were fixed in methanol and labeled with anti-caveolin-1 followed by an Alexa-568-labeled goat anti-rabbit IgG. The green channel (GFP) shows only those cells that express caveolin-1-GFP, while the red channel shows anti-caveolin-1 staining. The anti-caveolin-1 antibody detects both endogenous caveolin-1 and caveolin-1-GFP. The merged image shows nearly perfect overlap indicating that all of the GFP signal is from caveolin-1-GFP and not from GFP that has been cleaved from the caveolin. The addition of a GFP moiety has not affected the distribution of caveolin (compare the caveolin distribution in the non-expressing cell at the top with one that is expressing the GFP construct below it). (B) Cells were grown for 2 days in 6-well tissue culture dishes and incubated for 1.75 hours in the presence of 10 µM nocodazole, which was used to enhance our ability to recognize caveolae at the cell surface of these cells. The samples were then fixed and processed for immunogold localization of GFP. Bars, 20 µm (A); 0.25 µm (B).

View larger version (121K): [in a new window]   Fig. 3. Caveolin-1-GFP is localized at the MTOC, in close proximity to recycling endosomes. CHO cells stably expressing caveolin-1-GFP were plated on coverslips and processed for confocal imaging. Cells were then fixed and labeled either with anti-mannosidase II to stain the Golgi (B), anti-tubulin to stain microtubules (E) or anti-transferrin receptor antibody to stain recycling endosomes (G). Secondary antibodies were either Alexa-568-conjugated goat anti-rabbit IgG or Alexa-595-conjugated goat anti-mouse IgG. The green channel shows the distribution of caveolin-1-GFP (A,D,G) and the red channel the distribution of mannosidase II (B), tubulin (E) or the transferrin receptor (H). Merged images are shown in panels C, F and I. Each picture is a single confocal image through the center of the cell. Bar, 10 µm.

View larger version (91K): [in a new window]   Fig. 4. Caveolin-1-GFP displays three different types of movement. Cells were plated 48 hours before use into culture dishes that had a glass coverslip glued to the bottom. Confocal sections were taken at 1.6 second intervals near the bottom of the cell, just under the nucleus in cells stably expressing caveolin-1-GFP. (A) Some of the caveolin-1-GFP in these cells showed only saltatory movements (yellow arrows). A 12.8 second recording showed, however, that other patches of caveolin-1-GFP in the same cell rapidly moved apart (follow white arrows). (B) A third population of caveolin-1-GFP was associated with dynamic tubular structures (yellow arrows), which can also be seen in the accompanying movie. (C) Stills from two videos showing examples of long range and saltatory movement in control cells. Bar, 10 µm (A); 5 µm (B). The movies that correspond to panels B and C can be viewed online (http://jcs.biologists.org/supplemental).

View larger version (96K): [in a new window]   Fig. 5. FRAP analysis showing that there is active exchange between caveolin-1-GFP located in the peri-centrosomal region and the other pools of caveolin. Cells stably expressing caveolin-1-GFP were plated 48 hours before viewing into culture dishes that had a glass coverslip glued to the bottom. Confocal images were taken at 1.6 second intervals for approximately 1 minute and then the entire peri-centrosomal region of the upper cell was bleached in 6 spots for 2 seconds each using the 488 laser at full power. Immediately following the bleach, images were again collected at 1.6 second intervals for another 5 minutes and 40 seconds. Panel A shows the prebleach image and peri-centrosomal accumulations of caveolin-1-GFP in two cells. The central accumulation in the top cell was then bleached (area within the white circle, B). Panel C shows that the accumulation at the center of the cell has recovered within 5 minutes, indicating that caveolin-1-GFP travels between the various caveolin positive compartments. The corresponding movie can be viewed online (http://jcs.biologists.org/supplemental).

View larger version (75K): [in a new window]   Fig. 6. Nocodazole blocks the movement of caveolin-1-GFP in live cells and organizes caveolin-1-GFP into linear arrays. CHO cells stably expressing caveolin-1-GFP were grown for 2 days before being viewed by confocal microscopy, as described in Materials and Methods. (A) Still from video of CHO cells pretreated with 10 µM nocodazole for 1 hour before viewing. Long range movements have disappeared. (B) Immunofluorescence micrograph of CHO cells pretreated with nocodazole taken at the bottom surface of the cell. (C) Still from video of stably expressing CHO cells pretreated with 10 µM nocodazole for 1 hour before the addition of 1 µM latrunculin A. Within 1 minute the arrays have disappeared and everything appears to be immobile. Bar, 10 µm. The movies that correspond to panels A and C can be viewed online (http://jcs.biologists.org/supplemental).

View larger version (56K): [in a new window]   Fig. 7. Association of caveolin-1-GFP with the actin cytoskeleton. Cells stably expressing caveolin-1-GFP were plated on coverslips, grown for 2 days and treated with 10 µM nocodazole for 30 minutes before fixing with paraformaldehyde. Panel A shows the distribution of caveolin-1-GFP; panel B the distribution of F-actin stained with Alexa-633-phalloidin; and panel C a merged image. The arrows indicate regions where caveolin is aligned along or adjacent to stress fibers.

View larger version (103K): [in a new window]   Fig. 8. Nocodazole reorganizes caveolin-1 and caveolae at the cell surface of normal human fibroblasts. (A-D) Normal human fibroblasts were grown on coverslips for 2 days. One set of cells was processed without further treatment for indirect immunofluorescence localization of caveolin-1 (A) and tubulin (B), while the other set was incubated in the presence of 40 µM nocodazole for 2 hours before staining for caveolin-1 (C) and tubulin (D). The linear arrays in the treated cells were both more extensive and thicker. (E) Normal human fibroblasts were grown in 6-well dishes for 2 days, incubated in the presence of 40 µM nocodazole for 2 hours, and processed for EM. This treatment induced a striking increase in the number of caveolae that could be detected by electron microscopy. Bars, 37 µm (A,B); 23 µm (C,D); 0.8 µm (E).

View larger version (111K): [in a new window]   Fig. 9. Disruption of the actin cytoskeleton blocks the formation of the linear arrays of caveolin-1-GFP induced by nocodazole. CHO cells stably expressing caveolin-1-GFP were plated on coverslips and grown for 2 days, as described in Materials and Methods. Cells were incubated for 30 minutes in the absence (A,B) or presence of either 2.5 µM cytochalasin D (C,D) or 1 µM latrunculin A (E,F) and then incubated for an additional 60 minutes with (B,D,F) or without (A,C,E) 10 µM nocodazole, before fixing with paraformaldehyde and processing for visualization of GFP. Note that both cytochalasin D and latrunculin A blocked the redistribution of caveolin into linear arrays (compare B with D and F) and that nocodazole blocked the latrunculin A induced redistribution of caveolin-1-GFP to the cell center (compare E with F). Each image is the projection of a stack of confocal images that extend through the entire cell. Bar, 20 µm.

View larger version (123K): [in a new window]   Fig. 10. Latrunculin A causes a redistribution of caveolin-1-GFP in CHO cells (A) and caveolae in normal human fibroblasts (B). (A) CHO cells stably expressing caveolin-1-GFP were treated with latrunculin A (1 µM) and the cells were imaged within 1 minute using confocal time-lapse microscopy. The three panels in A are stills from videos taken at 1.6 second intervals for a total of 6 minutes each after 1 minute, 10 minutes or 30 minutes of latrunculin A treatment. Within 1 minute there is a dramatic increase in the trafficking of caveolin-1-GFP in the cells. By 10 minutes the caveolin-1-GFP has formed larger aggregates and caveolin can be seen moving into and out of long processes that keep the cells attached to the substratum (arrows). By 30 minutes there was an accumulation of caveolin in the center of the cell. The videos taken at 1 minute and 10 minutes after latrunculin treatment can be viewed online. (B) Normal human fibroblasts were grown for 2 days and then incubated with 1 µM latrunculin A for 30 minutes before processing for immunogold localization of caveolin-1. Numerous large caveolin-positive structures were seen that appeared to have multiple invaginated caveolae all around them (*). The inset shows a higher magnification of these structures. Bars, 10 µm (A); 0.24 µm (B); 0.16 µm (inset). The corresponding movies can be viewed online (http://jcs.biologists.org/supplemental).

View larger version (31K): [in a new window]   Fig. 11. A model for trafficking in the caveolar membrane system of CHO cells. Taken together, our observations suggest that cells contain a more extensive caveolar membrane system than has been previously appreciated. In CHO cells it appears to consist of a population of fairly immobile and stable caveolae at the plasma membrane, peri-centrosomal caveosomes and a population of transport intermediates that may include both cavicles and tubules that serve as bi-directional transport intermediates between caveolae and caveosomes. The cavicles move along microtubules but cortical actin filaments restrict their inward mobility. Red, caveolin-1; green, caveosome; blue, actin; pink/purple, microtubule.

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