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Membrane ruffling and macropinocytosis in A431 cells require cholesterol

Stine Grimmer1, Bo van Deurs2 and Kirsten Sandvig1,*

1 Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway
2 Structural Cell Biology Unit, Department of Medical Anatomy, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark



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  		Fig. 1. Effect of mßCD, mßCD/chol and a 1:1 mixture of mßCD and mßCD/chol (which does not change the cellular cholesterol content) on ricin endocytosis in the absence and presence of TPA to stimulate macropinocytosis. A431 cells were washed twice in Hepes medium, serum-starved for 4 hours and incubated with or without 5 mM mßCD, mßCD/chol or a 1:1 mixture of mßCD and mßCD/chol for 30 minutes at 37°C prior to addition of 1 µM TPA. After 10 minutes 125I-labeled ricin was added to the cells, and the amounts of endocytosed ricin were measured after 15 minutes, as described in Materials and Methods. The error bars show deviations (s.d.) between three independent experiments.

 


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  		Fig. 2. Effect of TPA and mßCD (A) or mßCD/chol (B) on the activity of PKC. A431 cells were washed twice with DMEM medium, serum-starved for 4 hours and preincubated with or without 5 mM mßCD or mßCD/chol for 30 minutes at 37°C before addition of 1 µM TPA. After 10 minutes at 37°C, the PKC activity was measured using the Protein Kinase C Assay System according to the manufacturer's instructions. The error bars show deviations between duplicates of a typical experiment.

 


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  		Fig. 3. TPA induces macropinocytosis in A431 cells. (A,B) EM micrographs of control A431 cells (Cont) serum-starved for 4 hours and then incubated with HRP (10 mg/ml) for 15 minutes at 37°C before processing for EM. Note the irregular membrane on the free surface (FS) of the cell in A. Moderate endocytosis of HRP into endosomes (En) has taken place. B shows the lateral membrane of a cell facing the intercellular space (IS). Here a few microvilli-like structures are seen. Arrowheads indicate the presence of caveolea. (C-F) Serum-starved A431 cells incubated with HRP as above but in the presence of 1 µM TPA. C and D show closure of membrane ruffles (arrows) and subsequent formation of large amounts of HRP-containing macropinosomes (Mp) at the free surface (FS). E and F show that TPA treatment also leads to an increased complexity of the lateral membranes at the intercellular spaces (IS) and that caveolea are present at the lateral membrane facing the intercellular space (arrowheads in F). Bars, 1 µm (A,C-E); 0.5 µm (B,F).

 


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  		Fig. 4. Cholesterol is required for the TPA-induced macropinocytosis. (A,B). A431 cells were serum-starved for 4 hours, preincubated for 30 minutes at 37°C with 5 mM mßCD to remove cholesterol and then for 15 minutes at 37°C with HRP (10 mg/ml) in the presence of both 5 mM mßCD and 1 µM TPA (CDx-TPA). It is evident how treatment with mßCD reduces the expression of membrane ruffles at the free surface (FS in B) and prevents the TPA-induced macropinocytosis of HRP from this surface as well as formation of a more complex membrane structure at the lateral membranes facing the intercellular spaces (IS). Moreover, only few caveolea are present. In C and D, serumstarved A431 cells were preincubated for 30 minutes at 37°C with a 1:1 mixture of 2.5 mM mßCD and 2.5 mM mßCD/chol (a mixture that does not change the cellular cholesterol content) and then for 15 minutes at 37°C with HRP in the presence of both TPA and the mßCD mixture (CDx-CDxCh-TPA). Under this condition closure of ruffles (arrows) at the free surface (FS) and the formation of HRP-containing macropinosomes (Mp) take place, complex membrane structures are formed at the lateral membranes at the intercellular spaces (IS), and caveolea are also present at these membranes (arrowheads in D). Bars, 1 µm.

 


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  		Fig. 5. TPA-induced reorganization of actin filaments at the cell periphery. (A-C) Serumstarved A431 cells (control) fixed, permeabilized and stained with rhodamine-labeled phalloidin to visualize filamentous actin. (D-I) Serum-starved A431 cells incubated with 0.5 µM TPA for 1, 2, 3, 4, 8 or 10 minutes at 37°C and then processed as the control cells to visualize filamentous actin. Note that distinct stress fibers at the free surface of the cells rapidly disappear and are replaced by actin associated with ruffles as well as an apparent increase in actin staining at the lateral membranes (D-I).

 


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  		Fig. 6. Cholesterol is required for the TPA-induced ruffling. (A,B) Serum-starved A431 cells were incubated with 5 mM mßCD for 30 minutes prior to addition of 0.5 µM TPA. After 4 or 10 minutes at 37°C the cells were fixed, permeabilized and stained with rhodamine-labeled phalloidin to visualize filamentous actin. In C and D, serum-starved A431 cells were incubated with a 1:1 mixture of mßCD and mßCD/chol (which does not change the cellular cholesterol content) and mßCD/chol, respectively, for 30 minutes prior to addition of 0.1 µM TPA. After 10 minutes at 37°C the cells were fixed, permeabilized and stained with rhodamine-labeled phalloidin to visualize filamentous actin.

 


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  		Fig. 7. Effect of TPA and mßCD on the activation state of Rac 1. A431 cells were serum-starved in the presence of 0.5 mCi/ml [32P]orthophosphate to label endogenous pools of GTP and GDP, and then incubated with or without 5 mM mßCD for 30 minutes at 37°C prior to addition of 0.5 µM TPA. After lysis of the cells with detergent, Rac 1 was immunoprecipitated and 32P-labelled nucleotides bound to Rac 1 analyzed by TLC as described in Materials and Methods. The error bars show deviations (s.d.) between three independent experiments.

 


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  		Fig. 8. (A) Cholesterol is required for the TPA-induced localization of Rac 1 to the free surface of the cells. (a-c) Serum-starved cells were untreated or incubated with or without 5 mM mßCD for 30 minutes at 37°C prior to addition of 0.1 µM TPA. After 10 minutes at 37°C the cells were fixed, permeabilized and stained with an antibody against Rac 1. Arrows in b indicate localization of Rac 1 to the free surface, where ruffling and macropinocytosis takes place. In d the cells were first incubated with 0.1 µM TPA for 10 minutes to localize Rac 1 to the cell periphery, followed by a 5 minute pulse with 15 mM mßCD to remove cholesterol. The cells were then processed to visualize endogenous Rac 1. (B) Dominant-negative Rac 1T17N inhibits TPA-induced actin reorganization required for membrane ruffling. Serum-starved A431 cells transiently transfected with wild-type Rac 1 (a,b) or Rac 1T17N (c,d) were incubated with 0.1 µM TPA for 10 minutes and processed for indirect immunofluorescence. Rac 1 was labeled with a mouse antibody against six amino acids in the EE-epitope, and F-actin was labeled with rhodamine-phalloidin. Arrows in `a' indicate localization of Rac 1 to the free surface (compare with c), while arrows in b indicate actin associated with ruffles in both transfected and untransfected cells. It should be noted that in d peripheral actin staining (arrows) is observed only in the cells not transfected with the dominant-negative mutant.

 


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  		Fig. 9. Removal of cholesterol affects the TPA-induced localization of ARF6 with Rac1 at the free surface of the cells. Serum-starved cells were untreated or incubated with or without 5 mM mßCD for 30 minutes at 37°C prior to addition of 0.1 µM TPA. After 10 minutes at 37°C the cells were fixed, permeabilized and stained with antibodies to visualize endogenous Rac1 and ARF6. Arrows indicate the localization of both Rac1 and ARF6 to the free surface where ruffling and macropinocytosis take place.

 

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