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First published online September 9, 2005
doi: 10.1242/10.1242/jcs.02548

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Depletion of GAK/auxilin 2 inhibits receptor-mediated endocytosis and recruitment of both clathrin and clathrin adaptors

Laboratory of Cell Biology, NHLBI, 50 South Drive, Rm 2537, MSC 8017, NIH, Bethesda, MD 20892-0301, USA

View larger version (42K): [in a new window]   Fig. 1. Depletion of GAK by vector based oligonucleotides. (A) Western blot and quantification of GAK in mock-depleted and GAK-depleted cells using the two different sequences as described in the Materials and Methods. Days are the number of days after transfection with vector-based RNAi. 10 µg of total protein was loaded per lane. The mock-depleted value of the GAK intensity band was normalized to 100% for comparison with the amount of GAK in the GAK-depleted cells. (B) Absence of GAK at Golgi in GAK-depleted cells. GAK was immunostained in mock-depleted (a) and GAK-depleted (b) cells.

View larger version (67K): [in a new window]   Fig. 2. Effect of GAK depletion on receptor-mediated endocytosis and MPR trafficking. (A) Uptake of transferrin is inhibited in GAK-depleted cells. Alexa Fluor 546-transferrin (10 µg/ml) was incubated with the cells for the times indicated. (a-d) Mock-depleted and (e-h) GAK-depleted cells. (B) Internalization of 125I-transferrin was measured as a function of time in mock-depleted (open circles) and GAK-depleted cells (solid circles). The total transferrin is the amount bound plus the amount endocytosed and later exocytosed. The initial surface values for both the control and GAK-depleted cells were about 3500 c.p.m. (C) Efflux of 125I-transferrin was measured as a function of time in mock-depleted (open circles) and GAK-depleted cells (solid circles). Uptake and efflux of transferrin was performed as described in the Materials and Methods. (D) Uptake of EGF is inhibited in GAK-depleted cells. Cells were incubated with Alexa Fluor 647 EGF (4 µM) for 30 minutes in mock-depleted (a) and GAK-depleted (b) cells. (E) Localization of MPR is altered after GAK-depletion. MPR was immunostained in mock-depleted (a) and GAK-depleted (b) cells.

View larger version (86K): [in a new window]   Fig. 3. GAK depletion alters distribution of CCPs on plasma membrane. Immunostained clathrin on the plasma membrane was imaged using confocal microscopy in mock-depleted (a) and GAK-depleted (b) cells. Transfected GFP-clathrin on the plasma membrane was imaged using confocal microscopy in mock-depleted (c) and GAK-depleted (d) cells. Transfected GFP-clathrin on the plasma membrane was imaged using TIR-FM in mock-depleted (e) and GAK-depleted (f) cells.

View larger version (56K): [in a new window]   Fig. 4. Localization of proteins on TGN is altered in GAK-depleted cells. Both mock-depleted (a,c,e,g) and GAK-depleted (b,d,f,h) cells were imaged using confocal microscopy, after being immunostained for clathrin (a,b), GM130 (c,d), TGN46 (e,f) and p230 (g,h). The identical cells were immunostained for GM130 and TGN46 proteins. The cells with no apparent cis or TGN Golgi proteins are indicated with asterisks.

View larger version (41K): [in a new window]   Fig. 5. Rescue of TGN-associated clathrin by mRFP-GAK in GAK-depleted cells. Both mock-depleted and GAK-depleted cells were transfected with mRFP-GAK. After immunostaining with clathrin antibodies, the cells were imaged using the confocal microscope to determine whether there was pronounced perinuclear clathrin staining associated with the TGN. Cells were GAK depleted using the 3'-UTR construct. (A) Mock-depleted (a,b) and GAK-depleted (c,d) cells were imaged for both mRFP-GAK (a,c) and clathrin (b,d). (B) Cells from two experiments (a minimum of 50 cells per experiment), were counted after being imaged using either clathrin immunostaining or GFP-clathrin to determine whether there was pronounced TGN-associated clathrin.

View larger version (39K): [in a new window]   Fig. 6. Clathrin exchange in GAK-depleted cells. (A) The exchange of GFP-clathrin on CCPs was determined in control (open circles), mock-depleted (solid circles) and GAK-depleted (solid triangles) HeLa cells by measuring the fluorescence recovery after photobleaching. (B) The exchange of cytosolic clathrin in GAK-depleted cells was measured in the presence (open triangles) and absence (solid triangles) of BFA. The inset shows the time course of recovery of cytosolic clathrin measured after treating mock-depleted cells with BFA (open circles). For comparison, the dashed line shows the rate of recovery of the cytosolic clathrin in GAK-depleted cells.

View larger version (103K): [in a new window]   Fig. 7. Colocalization of GFP-clathrin clusters in GAK-depleted cells with transferrin and EEA1. Confocal images of GFP-clathrin (a,d) and either transferrin (b) or immunostained EEA1 (e) in GAK-depleted cells. The cells internalized Alexa Fluor 546-transferrin for 30 minutes. (c,f) The merged images showing the overlaps.

View larger version (59K): [in a new window]   Fig. 8. Clathrin localization on the plasma membrane and TGN is altered in cells transfected with Hsp70(K71E). (A) TIR-FM images of GFP-clathrin in cells that were cotransfected with GFP-clathrin and either Hsp70 (a) or Hsp70(K71E) (b). (B) Confocal images of cells transfected with Hsp70(K71E) and immunostained with anti-flag (a) and anti-clathrin (b) antibodies. (C) The exchange of cytosolic clathrin clusters measured in cells cotransfected with GFP-clathrin and Hsp70(K71E). Data were collected from photobleaching 30 cells. One-third of the cells recovered with a half-life of 20 seconds (solid triangles), one-third showed no significant recovery (open triangles) and the remainder of the cells recovered with a time course between these extremes. The dashed line is the time course of recovery of the cytosolic clathrin measured in the GAK-depleted cells in Fig. 6B.

View larger version (79K): [in a new window]   Fig. 9. Localization of clathrin adaptors on the plasma membrane and TGN is altered in GAK-depleted cells. (A) TIR-FM images of mock-depleted (a,c) and GAK-depleted (b,d) cells cotransfected with DsRed-clathrin (a,b) and GFP-AP2 (b,d). (B) TIR-FM images of mock-depleted (a,c) and GAK-depleted (b,d) cells cotransfected with DsRed-clathrin (a,c) and GFP-epsin (b,d). In the inset, the brightness was increased to better visualize the pits of the dimmer cell (boxed region) that was expressing clathrin and epsin at lower levels than the neighboring cell. (C) Confocal images of immunostained TGN-associated clathrin adaptors, AP1 (a,b) and GGA3 (c,d) in mock-depleted (a,c) and GAK-depleted (b,d) cells.

View larger version (62K): [in a new window]   Fig. 10. Clathrin adaptors on the plasma membrane and TGN are altered in cells transfected with Hsp70(K71E). (A) TIR-FM images of GFP-AP2 (a,b) and GFP-epsin (c,d) in cells cotransfected with either Hsp70 (a,c) or Hsp70(K71E) (b,d). (B) Confocal images of cells transfected with Hsp70(K71E) stained with flag antibodies (a,c,e) and TGN-associated adaptors: AP1 (b), GGA3 (d) and GAK (f).

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