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First published online 8 August 2006
doi: 10.1242/jcs.03092

Journal of Cell Science 119, 3502-3512 (2006)
Published by The Company of Biologists 2006
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Recruitment dynamics of GAK and auxilin to clathrin-coated pits during endocytosis

Dong-won Lee, Xufeng Wu, Evan Eisenberg and Lois E. Greene*

Laboratory of Cell Biology, NHLBI, NIH, Bethesda, MD 20892-0301, USA


Figure 1
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  		Fig. 1. TIRF microscopy images of a CV1 cell co-transfected with GFP-GAK and mRFP-clathrin. (A) A CV1 cell was simultaneously imaged for GFP-GAK (left) and mRFP-clathrin (middle) and images were merged (right). (B) Magnified time-lapse sequence of boxed areas from GFP-GAK and mRFP-clathrin images of A showing the behavior of GAK and clathrin. GAK and its corresponding clathrin puncta are indicated by the arrow, arrowhead and pointer.

 

Figure 2
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  		Fig. 2. Recruitment of GAK or auxilin to CCPs. Fluorescence intensity of GFP-GAK and mRFP-clathrin observed under a TIRF microscope was analyzed by Metamorph as described in the Materials and Methods. (A) Time course of the change in fluorescence intensities of GFP-GAK (bullet) and GFP-auxilin ({circ}). (B) Change in fluorescence intensities of GFP-GAK ({circ}) and mRFP-clathrin (bullet) as a function of time; measured from 56 puncta in 9 cells. (C) Change in fluorescence intensities of GFP-auxilin ({circ}) relative to mRFP-clathrin (bullet) as a function of time; measured from 41 puncta in 7 cells. Sequential images were taken one frame per second for 2 minutes. The zero time point was set to the time at which the GAK or auxilin reached its maximum.

 

Figure 3
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  		Fig. 3. Histograms showing the time interval between the clathrin and the peaks of GAK and auxilin. (A) Time interval between clathrin and GAK peaks, calculated from the events used in Fig. 2B. (B) Time interval between clathrin and auxilin peaks, calculated from the events used in Fig. 2C. (C) Time interval between clathrin and dynamin1 peaks, calculated from the events used in Fig. 4A. (D) The time interval between clathrin and dynamin2 peaks, calculated from the events in Fig. 4B. In calculating the peak of maximum clathrin fluorescence in the histograms, the fluorescence data were fitted using Microsoft Excel and a moving average of four to reduce noise in the clathrin data.

 

Figure 4
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  		Fig. 4. Recruitment of dynamin1 or dynamin2 to CCPs in CV1 cells. Fluorescence intensity of GFP dynamin and mRFP-clathrin observed under TIRF microscopy was analyzed by Metamorph as described in the Materials and Methods. (A) The change in fluorescence intensities of GFP-dynamin1 and mRFP-clathrin as a function of time measured from 25 puncta in 7 cells. (B) The change in fluorescence intensities of GFP-dynamin2 and mRFP-clathrin as a function of time measured from 32 puncta in 5 cells. The zero time point was set to the time at which dynamin reached its maximum.

 

Figure 5
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  		Fig. 5. Recruitment of dynamin 1 and GAK to the plasma membrane. CV1 cells were co-transfected with GFP-GAK and mRFP-dynamin 1. Metamorph analysis was used to identify GAK and dynamin colocalizing at the same localization. (A) The changes in fluorescence intensities of dynamin1 ({circ}) and GAK (bullet) were plotted as a function of time. The difference in times between the maximum of the dynamin 1 and GAK peaks is the time interval plotted in the histogram. (B) Histogram of the time intervals between the maximum dynamin and GAK fluorescence obtained from 32 events in two cells.

 

Figure 6
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  		Fig. 6. GFP-GAK and mRFP-clathrin recruitment and disappearance from the plasma membrane imaged using evanescent and epifluorescence microscopy. CV1 cells, co-transfected with GAK and clathrin, were simultaneously imaged at a rate of 1 frame per second for a total of 2 minutes. (A) Epifluorescence images from time-lapse sequence of GFP-GAK and mRFP-clathrin. GAK and its corresponding clathrin puncta are shown by the arrow and arrowhead. (B) Time course of the change in fluorescence intensities of GFP-GAK ({circ}) and mRFP-clathrin (bullet) imaged in the evanescent field from 11 events in 5 cells. (C) Time course of the change in fluorescence intensities of GFP-GAK ({circ}) and mRFP-clathrin (bullet) imaged in the epifluorescent field using the same events that were identified in the evanescent field. The data were normalized so that the peak of GAK fluorescence was set to zero.

 

Figure 7
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  		Fig. 7. Mutational analysis of the flashing requirements of GAK and auxilin. Different GFP constructs of GAK and auxilin were transfected into CV1 cells and then imaged under the TIRF microscope. The degree of flashing was determined by Metamorph analysis. The domains of the proteins are as follows, K, kinase domain; N, N-terminal auxilin homology domain; P, PTEN-like domain; CB, clathrin-binding domain; J, J-domain. GAK and auxilin constructs are aligned so that their domain structures are homologous to one another. The number of GAK and auxilin flashes that occurred in a 300-mm2 area of the cell over a 10-second time period was counted to obtain the values for the mean and s.d. from at least four cells.

 

Figure 8
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  		Fig. 8. Protein lipid overlay obtained using GST-PTEN-like domain of GAK. (A) Binding of GST-PTEN-like domain of GAK to phosolipids was measured using the protein lipid overlay strips. (B) Quantification of the phosolipid overlay. The binding to the phospholipids strip was performed as described in Materials and Methods. The circles along the periphery of the strip indicate the different phospholipids that were tested in the binding assay.

 

Figure 9
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  		Fig. 9. Kymographs of GAK and mRFP-clathrin obtained under different experimental conditions. GFP-GAK and mRFP-clathrin were imaged simultaneously by TIRF microscopy. Kymographs were generated using Metamorph software by taking a rectangular region and assembling this region in a side-by-side chronological order to form a montage. TIRF images were acquired under following conditions: in medium (DMEM and FBS; Control) in K+-depletion medium (K+-depletion), in hypertonic sucrose solution (sucrose) and in medium containing 2.5 µM LatA.

 

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© The Company of Biologists Ltd 2006