Glucose transporter recycling in response to insulin facilitated by myosin Myo1c (Supplemental Figure list- MS# C02163A)
Avirup Bose, Adilson Guilherme, Stacey I. Robida, Sarah M.C. Coulter, Qiong L. Zhou, Zhen Y. Jiang, Darcy P. Pomerleau and Michael P. Czech*
Program in Molecular Medicine, University of Massachusetts Medical School,
Worcester, MA 01605, U.S.A.
Methods:
Endogenous Myo1c and GLUT4 translocation assays
For detecting endogenous Myo1c and GLUT4 in 3T3-L1 adipocytes, the cells were serum starved for 4 hr in incomplete DMEM supplemented with 0.5% BSA and then treated with insulin for 15 min. They were then fixed, permeabilized with P-buffer26 and then stained with either Tu49 (5 g/ml ) or anti-Myo1c (5 g/ml) and goat anti-GLUT4 (C-20, 1 g/ml) overnight at 4C. They were then coupled to FITC-conjugated mouse anti-rabbit and CY3-conjugated mouse anti-goat secondary antibodies. The images were analyzed by confocal microscopy.
Rhodamine-Transferrin uptake in COS-1 cells
COS-1 cells were transfected with either GFP-Myo1c(Tail) or GFP-Myo1b(Tail). After 24 hr the cells were serum starved in KRH + 0.5% BSA + 2mM sodium pyruvate. They were then incubated in the presence of 5 g/ml of transferrin-rhodamine for 20 min. The cells were then fixed with 4% formaldehyde and observed using confocal microscopy. Arrows indicate transfected cells and arrowheads indicate untransfected cells in the same field.
Purification of GLUT4 vesicles from differentiated 3T3-L1 adipocytes and immunoblotting
Differentiated 3T3-L1 adipocytes from 16 -150 mm plates were serum starved overnight in DMEM + 0.5% BSA. The cells were then stimulated with or without 100 nM insulin.
The isolation and fractionation of GLUT4-containing vesicles were carried out as described before10. The proteins were resolved by SDS-PAGE and visualized by silver staining (Bio-Rad) and the bands were excised. The peptide sequences were determined by MALDI-TOF Mass spectrometry analysis as described10.
Confocal microscopy
The images analyzed by confocal microscopy were viewed with a 60X objective on a Nikon Diaphot 200 inverted microscope coupled to a Bio-Rad MRC1024 processing unit. Images were analyzed by the Lasersharp processing software. All experiments were done in triplicate and at least eight images were collected from each set.
FIGURE LEGENDS
Figure S1-supplement. Myosin I isoform, Myo1c, identified in GLUT4-containing membranes from differentiated 3T3-L1 adipocytes. a. Three peptide fragments corresponding to mouse Myo1c were identified in the insulin-sensitive GLUT4 fractions from differentiated 3T3-L1 adipocytes by mass spectrometry. b. Myo1c is associated in insulin sensitive GLUT4 containing membranes (fraction 7-11) from control (-) and insulin-stimulated (+) 3T3-L1 adipocytes. c. Myo1c is recruited to the insulin-sensitive Glut4 containing membranes. The arbitrary unit represents the ratio of the Myo1c protein level to Glut4 protein level in each fraction as measured by scanning densitometry.
Figure S2-supplement. Transferrin uptake in COS-I cells. Transferrin uptake in COS-1 cells expressing dominant negative cargo domains of Myo1b [GFP-Myo1b(Tail)] or Myo1c [GFP-Myo1c(Tail)]. Arrows indicate cells transfected with GFP-Myo1b(T)/GFP-Myo1c(T) and arrowheads indicate untransfected cells in the same field. The data represents results from four independent experiments.
Figure S3-supplement. Insulin recruits Myo1c to regions of cortical actin rearrangement in differentiated 3T3-L1 adipocytes. Insulin treated 3T3-L1 adipocytes were fixed and stained for endogenous Myo1c and with rhodamine phalloidin to visualize cortical actin rearrangement. The images were analyzed by confocal microscopy. The arrowheads indicate some of the areas where Myo1c colocalizes with cortical actin.
Figure S4-supplement. a. Insulin regulates movement of Myo1c and Glut4 to the plasma membrane in differentiated 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were serum starved (control) and then stimulated with insulin for 15 min. They were then fixed and stained for endogenous Myo1c with Tu49 and for GLUT4 with C-20. The images were analyzed by confocal microscopy. Arrows indicate cells with Myo1c staining at the plasma membrane. Arrowheads indicate the region of overlap of Glut4 and Myo1c staining after insulin stimulation. The data is representative of three independent experiments. b. Myo1c blot of plasma membrane fraction from serum starved (control) and insulin-stimulated adipocytes.
Figure S5-supplement. Whole-mount EM of the peripheral region of an insulin-stimulated 3T3-L1 adipocyte fixed and treated with goat anti-GLUT4 and rabbit anti-Myo1c and colloidal gold-tagged secondary antibodies. A. A larger area of the cell shown in Fig. 2 is shown at a higher magnification (930000X). B. A larger area of the cell labeled with goat and rabbit IgG in Fig. 2, panel B is shown here.