Fig 1: Manipulation of ER-localized Rac1 affects ERES ACo-occurrence of mCherry-Rac1 and GFP-Sec61ß at the endoplasmic reticulum (ER) in HeLa cells stably expressing mCherry-Rac1 and transiently expressing GFP-Sec61ß as monitored by confocal microscopy. SRRF imaging mode was used in Andor DragonFly spinning disk confocal microscope for obtaining the image. Scale bar: 5 and 1 µm (inset). See also Movie EV3.BLine profile of two marked spots from (A) showing the overlap between mCherry-Rac1 and GFP-Sec61ß signal at the ER.CSide-by-side comparison of cells with endogenously tagged GFP-Rac1. The image on the left shows a confocal image and the image on the right was performed using SRRF mode. Scale bar: 5 µm.DCells with endogenous GFP-Rac1 were fixed and stained against Sec31A to label ERES. Arrows indicate Rac1-puncta colocalizing with ERES. Magnified view of two spots (numbered 1 and 2 in merged channel) is shown. Scale bar: 5 and 1 µm (inset).ERepresentative images showing Sec31A marked ERES in HeLa cells transfected with indicated plasmids. HeLa cells were transfected with the plasmids 16 h before immunolabeling with Sec31A. Scale bar: 5 µm.FQuantification of ERES number in HeLa cells from the experiment described in C. Between 90 and 100 cells were quantified from four independent experiments. Average ERES per cell ± standard deviation is shown. Each dot represents average ERES/cell from an experiment. A one-way ANOVA was used test for statistical significance. *P-value <0.05, ns = nonsignificant.GImages represent ERES in HeLa cells that were transfected with the indicated plasmids and cultured on the crossbow micropattern for 4 h. Cells were transfected with the plasmids 16 h before they were trypsinized and seeded on the micropatterns for ERES staining. Scale bar: 5 µm.HThe dot plot shows ERES count in individual cells from (G). Mean ± standard deviation is shown for each condition. At least 58 cells were used for counting ERES number. Independent experiments were performed three times. Statistical significance was tested using one-way ANOVA, *P-value <0.05, ns = nonsignificant.IMerged confocal images show distribution of Rac1-specific GEF TIAM (magenta) in HeLa cells before (0), and after optogenetic recruitment at indicated time points. CIBN-Sec61ß (green) was used to recruit Cry2-TIAM to the ER with 488 nm laser. Scale bar: 5 µm.JGraph shows the number of SNAP-Sec16A marked ERES in HeLa cells before (0) and after (30, 60, and 90 s) recruitment of TIAM to the ER from two independent experiments. Whiskers indicate min-max range and central band indicates the median of all data. Number of cells = 9. A paired t-test was run to compare t0 and t90. *P-value <0.05.