Supplementary Figure 7: Golgi rheology experimental set-up and controls. | Nature Cell Biology

Supplementary Figure 7: Golgi rheology experimental set-up and controls.

From: Extracellular matrix mechanical cues regulate lipid metabolism through Lipin-1 and SREBP

Supplementary Figure 7

a, Simplified diagram illustrating the technique to measure Golgi rheology. Left: an internalized bead (red) is immobilized in proximity to the Golgi membranes (green) by a laser optical trap (yellow cones). Right: when the stage and thus the whole cell is moved towards the bead (blue arrow), the Golgi apparatus displaces the bead away from the trap center (red arrow). Since the Golgi microenvironment has visco-elastic properties, the bead position partially relaxes in time attracted towards the trap center. b, Golgi rheology was measured in RPE1 cells freely spreading on fibronectin-coated glass (Large, n = 39 cells) or plated on micropatterned fibronectin islands restraining cell area and F-actin contractility (Small, 960 μm2 n = 28 cells; 490 μm2 n = 21 cells). GFP-Rab6-positive Golgi membranes were pushed towards a cytoplasmic bead, previously immobilized by an optical trap, by moving the microscope stage in a series of five 0.5µm steps in 1 min. The graph shows the averaged displacements of the bead as a function of time during the whole duration of the experiment. The graph corresponding to the first step (0<t<15) is shown at higher magnification in Fig. 7b. Gray shadows: s.e.m. error bars. c, Analysis of the relaxation curves obtained by moving the bead away from the Golgi (not shown), which serve as negative control. Large n = 28 cells; 960 μm2 n = 29 cells; 490 μm2 n = 26 cells. All n values are pooled across independent experiments. Data are mean and s.e.m.; two-tailed unpaired Student’s t-tests. Source data in Supplementary Table 3.

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