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Volume 8 issue 6 June 2007
Cholesterol sensitive Cdc42-activation regulates actin polymerization for endocytosis via the GEEC pathway
Rahul Chadda, Mark T. Howes†*, Sarah J. Plowman*, John F. Hancock*, Robert G. Parton*† and Satyajit Mayor
Supplementary Figure S1: Titration of effect of cholesterol depletion agents and actin drugs on uptake of fluid and transferrin FRαTb cells were treated with carrier or the drug at the concentrations indicated in duplicate dishes for 30minutes, MβCD; 15minutes, Filipin or 5minutes (Lat A, Cyto D, Jas). After treatment, cells were assayed for their endocytic activity, fixed and imaged. Histograms represent fluid and normalized transferrin uptake in cells, expressed relative to corresponding control cells (control values set to 1). Values represented are wt. mean ±SEM across two - three experiments
Figure 1(.jpg)
Supplementary Figure S2: Single molecule imaging of GFP tagged molecules
A) Characterization of single molecule imaging: 1 nM solution of GFP (in PBS) was taken on acid-cleaned glass coverslips. GFP molecules, which settled down on coverslip, were imaged under TIRF illumination and high on-chip multiplication gain, leading to single molecule sensitivity. Intensity traces of a single spot (montage) show that single molecules bleached in single step (Supplementary Video 3).
B) Bleaching profile of a single GFP molecule on coverslip. As has been described before single GFP molecules show large intensity fluctuations, which are attributed to light-driven spectral fluctuations in the GFP chromophore (1).
C) Histogram shows bleaching times of single molecules as a function of laser power applied, confirming that the disappearance of single molecules is a function of laser power and hence due to photobleaching (2) .
D) Cartoon shows how depth of TIRF field was calibrated. Dimensions of 1.2 mm bead were measured under wide field and evanescent field. Note we use objective type TIRF; beads, cells are observed using an inverted microscope setup with the sample being illuminated from beneath. A decrease in diameter of fluorescent bead indicates, when illumination is changed from wide-field to TIR mode, change in depth of illumination. An estimation of the change gives a means to calculate the depth of TIRF field.
E-F) Histograms show the comparison of distribution of fluorescence intensity from single molecules of GFP-Cdc42, single GFP molecules on coverslip, or cytosolically expressed GFP (pEGFP- N1). Note that GFP-Cdc42 when expressed at fleetingly low levels appeared as punctate structures on the plasma membrane under TIRF illumination. Intensity distributions of these punctuate structures matched with those of eGFP molecules on coverslip.
G) Cytosolically expressed eGFP molecules diffuse in cytosol, bounce off plasma membrane, residing for not more than 1-2 frames (~100ms). Intensity distribution of these events again matched with GFP-Cdc42, consistent with single molecules of GFP-Cdc42 being recruited to the plasma membrane. Moreover cells expressing GFP-Cdc42 were fixed and molecules were bleached. Single molecules of GFP-Cdc42 bleached in a single step in-situ (data not shown).
H) Maximum projection from a movie of GFP molecules expressed in single cell, shows all the molecules that ever visited the TIRF field (membrane) in the 10 sec. Notice that in absence of any membrane specific localization, the number of molecules entering TIRF field are very limited (compare with Cdc42 wt or N17; Figure 6 A, B; see also Supplementary Video 3). Scale bar, 5um.
Figure 2 (jpg)
Supplementary Figure S3: Actin and PH-domain distribution in cholesterol depleted cells
A) Images show cells transfected with PH-domain-GFP before and after filipin treatment. PH domain redistributes to cytosol and nucleus and recovers back onto membrane after cholesterol addition.
B) Distribution of polymerized actin in control cells or cells treated with 10mM MβCD or 15µg/ml filipin. Cells were depleted of cholesterol with either MβCD or Filipin and stained with A488 phalloidin to mark F-actin.
C-D) Dynamin2a-GFP transfected cells were imaged live at high sensitivity before and after cholesterol depletion. Numbers in the images denote dynamin events recorded in that cell. Many of these events colocalize with A568-Tf (data not shown). Histogram shows the time scale of these events. Note that the number or time scale of residence of dynamin 2a events does not change upon cholesterol depletion (Dynamin2a-GFP before {black} or after MβCD treatment {yellow}). Scale bar, 10um
Figure 3 (jpg)
Supplementary Video Legends
Note: The videos must be viewed with relatively low ambient lighting since the contrast of individual frames has been kept constant throughout the movie sequence.
Video 1)Colocalization of GFP-Cdc42 with PLR-labeled FR-GPI: FRαTb cells were transfected with GFP-Cdc42 and labeled with PLR at low concentrations (1/20th of the saturation level for this probe), were imaged at 37°C with dual color TIR illumination (488 nm, and 543 nm laser lines). GFP-Cdc42 (green) and PLR (red) were recorded at 1.5 sec per frame, movie displayed at 2 fps to show dynamic regions at or near to the plasma membrane showing membrane regions enriched in both Cdc42 and FR-GPI. Scale bar, 2um.
Video 1 (mov)
Video 2) Colocalization of GFPCdc42 with actin: FRαTb cells transfected with GFP-Cdc42 and CherryTM-Actin were imaged at 37oC with dual color TIR illumination (488nm, and 543 nm laser lines). GFP-Cdc42 (green) and CherryTM-Actin (red) were recorded at 1.5 sec per frame, movie displayed at 2 fps to show dynamic regions at or near to the plasma membrane showing membrane regions containing both Cdc42 and CherryTM-Actin. Scale bar, 2um.
Video 2 (mov)
Video 3)Single molecules of GFP on coverslip and expressed in cell-cytosol: GFP molecules from a dilute solution were allowed to settle down on acid-cleaned glass coverslip or expressed in cytosol of FRαTb cells. Single molecules of GFP were imaged using TIR illumination; movies recorded at 12 fps and displayed at 2 fps. GFP molecules bleach in a single step, whereas the cytosolic GFP visits the membrane, appearing for a maximum time of one to two frames (from 10 to 90 ms). Scale bar, 2um.
Video 3 (mov)
Video 4) Movie of single molecules of GFP-tagged isoforms of Cdc42wt, N17, L61 and CRIB being recruited to membrane: FRαTb cells expressing different GFP tagged proteins were imaged with TIR illumination (488 nm laser line) at 37°C. Single molecules of GFP-tagged Cdc42 wt, N17, L61 and CRIB are recruited onto membrane where they show diffusive (N17) or localized activity. Movies were recorded at 12 fps (GFP-CRIB at 2 fps) and are displayed at 2 fps. Scale bar, 2um.
Video 4 (mov)
Video 5) Effect of cholesterol depletion and repletion on GFP-Cdc42wt dynamics at or near the plasma membrane: Cells expressing GFP-Cdc42 were imaged before and after cholesterol depletion and re-addition. Movies were recorded at 12 fps and displayed at 2 fps. Scale bar, 2um.
Video 5 (mov)
Video 6) Effect of cholesterol depletion on GTPase deficient Cdc42L61: Cells expressing GFP-Cdc42 L61 were imaged before and after cholesterol depletion. Movies were recorded at 12 fps and displayed at 2 fps. Scale
bar, 2um.
Video 6 (mov)
Video 7) Effect of cholesterol depletion of actin-GFP dynamics: Cells expressing actin-GFP were imaged before and after cholesterol depletion and re-addition. Movies were recorded at 12 fps and displayed at 2 fps. Scale
bar, 2um..
Video 7 (mov)
Video 8) Effect of cholesterol depletion on GFP-Rac1: Cells expressing GFP-Rac1 were imaged before and after cholesterol depletion. Movies were recorded at 12 fps and displayed at 2 fps. Scale bar, 2um.
Video 8 (mov)
Video 9) Effect of cholesterol depletion on PH domain GFP distribution: Cells expressing PH domain GFP were imaged before and after cholesterol depletion. Movies were recorded at 12 fps and displayed at 2 fps. Scale
bar, 2um.
Video 9 (mov)
Video 10) Effect of cholesterol depletion on GFP-dynamin 2aa: Cells expressing GFP-dynamin 2aa were imaged before and after cholesterol depletion. Movies were recorded at 0.5 fps and displayed at 2 fps. Scale bar, 2um.
Video 10 (mov)
References
1. Pierce DW, Hom-Booher N, Vale RD. Imaging individual green fluorescent proteins. Nature 1997;388(6640):338.
2. Douglass AD, Vale RD. Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells. Cell 2005;121(6):937-950.
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