Figure 1.
Photo-Activation of GLUT4-EOS in the Plasma Membrane Clusters.
(a) Simultaneous TIRF/FPALM imaging of GLUT4-EOS. Non-activated GLUT4-EOS is detected in the green channel and shows abundant plasma membrane clusters. Individual activated GLUT4-EOS molecules are detected in the red channel. Bar, 5 µm. See also Movie S1 (b) Zoomed region from (A) depicting the sequential activation of a GLUT4-EOS. Bar, 2 µm. (c) A plot of mean fluorescence of activated GLUT4-EOS from region shown in (b) (d) Series of frames depicting single GLUT4-EOS molecule photo-bleaching event (upper panel); lower panel shows corresponding step-wise drop in fluorescence intensity measured within circular region. Bar, 1 µm.
Figure 2.
Trajectories of GLUT4-EOS molecules exhibiting different types of motion: (a) directed motion corresponding to vesicular transport (see Movie S2); (b) free lateral diffusion (see Movie S3); (c) constrained diffusion within plasma membrane cluster (see Movie S4). All bars, 2 µm. (d) Graphs of the Mean Square Displacement (MSD) of GLUT4-EOS molecules for three distinct types of motion: directed movement (red), free lateral diffusion (blue), and constrained diffusion within a cluster (orange). Dashed lines correspond to 95% confidence intervals obtained from simulation. (e) Percentage of trajectories categorized into three types of motion observed, based on the analysis of MSD. Trajectories containing at least 30 time points were scored as directed motion or constrained diffusion if five or more points were above or below 95% confidence interval bounds. At least 150 trajectories were analyzed for each condition. Error bars are SEM, N = 15 cells. *p<0.05; **p<0.01.
Figure 3.
Elongated Shape of GLUT4 Clusters.
(a) FPALM reconstruction of GLUT4-EOS positions detected during 600 frames. Clustered positions were associated with GLUT4 molecules trapped inside the clusters. Bar, 1 µm. (b) An example of GLUT4 trajectory within the cluster; sequential positions where GLUT4 molecule was localized are shown in pseudocolor; the bounding ellipse is shown in green. Behavior of GLUT4 within the clusters was characterized by step size distribution (c); shape of clusters was analyzed by (d) fitting with ellipses (Dmin, Dmax – diameters along minor and major axis), and (e) by calculating circularity as 4pi*(area/perimeterˆ2). (f) EM micrograph depicting immuno-gold labeling of plasma membrane with rabbit anti-GLUT4 and 5 nm gold-conjugated goat-anti-rabbit antibodies.
Figure 4.
GLUT4 Interaction with Pre-existing Clusters.
GLUT4-EOS association with (a) (see also Movie S5) and dissociation from (b) (see also Movie S6) pre-existing GLUT4 clusters. Activated GLUT4-EOS molecule, detected in the red channel, colocalizes with the cluster visible in green channel (non-activated GLUT4-EOS). The last frame is shown for an association event (a), and the first frame is shown for dissociation event (b). Trajectory of the activated GLUT4-EOS molecule is shown in white; white circles depict the site of the cluster. All bars, 2 µm. (c) and (d) show graphs of the Mean Square Displacement (MSD) for association and dissociation events depicted in (a) and (b) correspondingly.
Figure 5.
GLUT4 Dwell Time at the Clusters: Rates of Dissociation and Endocytosis.
(a) and (b) - histograms of dwell time for individual GLUT4-EOS molecules localized within the clusters in control cells (gray) and cells treated with KCN (red). Fat cells treated with 2 mM KCN for 15 min showed complete inhibition of GLUT4 endocytosis due to ATP depletion (Kono, Robinson et al. 1977; Quon, Guerre-Millo et al. 1994). Dwell time data was pooled from at least 15 cells for each condition. The dwell time of GLUT4-EOS molecule was measured as the time between appearance or activation of GLUT4-EOS at the cluster site and the time when the molecule was lost for more than three consecutive frames. Exponential fit of the histogram data was used to calculate the rates of disappearance under different illumination protocols for at least 5 cells in each condition (basal, insulin, with and without KCN). The combined rate of disappearance was considered to be a sum of three independent processes (bleaching, lateral dissociation from cluster and endocytosis): K(p) = p*Kb+Kd+Ke, where Kb – rate constant of bleaching, p – relative exposure to excitation light, Kd and Ke– rate constants for dissociation and endocytosis from clusters respectively. To control for bleaching, we measured the rates of disappearance at four different illumination protocols with constant exposure texp = 200 ms but with different intervals between acquisitions (tint = 0.2, 0.5, 1.0, and 2 sec), which correspond to the relative exposure to excitation light p = texp/tint : 1.0; 0.4; 0.2; and 0.1. (c) and (d) show graphs of combined rate of disappearance K(p) as a function of the relative exposure for basal and insulin stimulated conditions. Red circles correspond to cells pre-treated with KCN for 15 min. The combined rate of disappearance for KCN-treated cells was assumed to be K(p) = p*Kb+Kd, with the rate constant of endocytosis (Ke) being essentially zero. The rate constants of dissociation Kd and endocytosis Ke from clusters were determined from intersection of linear fit of the data with ordinate axis.
Figure 6.
Formation of the Clusters via GLUT4-Specific Retention During Exocytosis.
Sequences of consecutive time-lapse frames showing examples of GLUT4 vesicle fusion resulting in complete dispersal of GLUT4 molecules “fusion-with-dispersal” (a) (see also Movie S7) and formation of GLUT4 cluster “fusion-with-retention” (b) (see Movie S8). Lower panels show corresponding graphs for time-lapse fluorescence changes at the site of fusion. Mean fluorescence intensity was calculated for circular regions of 1 µm radius. Red channel corresponds to activated GLUT4-EOS molecules; green channel corresponds to IRAP-pHluorin. IRAP-pHluorin fluorescence spikes correspond to luminal pH equilibration upon fusion pore opening (black arrows). Note that IRAP-pHluorin leaves the site of fusion when GLUT4 forms a cluster. Frames are shown immediately before and after fusion with 200 ms interval. All bars, 1 µm. (c) and (d) show cartoon depictions of GLUT4 molecules leaving site of fusion during “fusion-with-dispersal” and forming a cluster during “fusion-with-retention”.