GRAM domain proteins specialize functionally distinct ER-PM contact sites in human cells
- University of California, Davis, United States
- School of Medicine, University of California, Davis, United States
- EPFL, Switzerland
Figures
Figure 1 with 5 supplements
Defining a new family of human ER MCS proteins.
(A) Human proteins GRAMD1a-c and GRAMD2a and b are members of a protein family with similarity to the yeast Ltc1/Lam proteins, which all possess an unstructured N-terminus, a GRAM domain, and an …
Figure 1—figure supplement 1
GRAMD1a and GRAMD2a localize to ER-PM contact sites.
(A) Maximum Likelihood phylogenetic tree (bootstrapped 1000 times) of proteins possessing GRAM domains and PH domains from Homo sapiens, Saccharomyces cerevisiae, and Drosophila melanogaster. …
Figure 1—video 1
GRAMD1a-eGFP with mCherry-Sec61β and lyn-mCherry Z-stack, sample images are displayed in Figure 1C
https://doi.org/10.7554/eLife.31019.004
Figure 1—video 2
GRAMD2a-eGFP with mCherry-Sec61β and lyn-mCherry Z-stack, sample images are displayed in Figure 1D
https://doi.org/10.7554/eLife.31019.005
Figure 1—video 3
3D rendering of GRAMD1a-eGFP with mCherry-Sec61β and lyn-mCherry: orthogonal view, sample images displayed in Figure 1—figure supplement 1B
https://doi.org/10.7554/eLife.31019.006
Figure 1—video 4
3D rendering of GRAMD2a-eGFP with mCherry-Sec61β and lyn-mCherry: orthogonal view, sample images displayed in Figure 1—figure supplement 1C
https://doi.org/10.7554/eLife.31019.007
Figure 2 with 1 supplement
GRAMD2a and GRAMD1a mark distinct ER-PM contact sites.
(A) Analysis of the relative localization of GRAMD1a-eGFP and GRAMD2a-mCherry in Cos7 cells. Two sample cells are shown. Bottom panel is quantification of the amount of overlapping total …
-
Figure 2—source data 1
Figure 2D Co-localization analysis: Top table is pixel overlap of GRAMD2a and GRAMD1a with E-Syt2 and E-Syt3; standard Error shown.
Bottom table is corresponding two-tailed t-test values.
- https://doi.org/10.7554/eLife.31019.010
Figure 2—figure supplement 1
Relationship of GRAMD1a and GRAMD2a to E-Syt2/3.
(A–B) TIRF imaging of Cos7 cells expressing GRAMD2a-eGFP with mCherry-E-Syt2 (A) or mCherry-E-Syt3 (B) and BFP-Sec61β. (C) Localization of GRAMD2a-eGFP in wild type HeLa cells and HeLa E-Syt1/2/3 …
Figure 3 with 1 supplement
Gene set enrichment analysis of GRAMD1a and GRAMD2a indicated distinct physiological functions.
(A) Comparison of enrichment results between GRAMD1a and GRAMD2a in transcriptome data of liver samples from 193 female human individuals. Normalized enrichment score (NES) of GRAMD1a and GRAMD2a …
Figure 3—figure supplement 1
Gene set enrichment analysis of GRAMD1a and GRAMD2a in males.
(A) Comparison of enrichment results between GRAMD1a and GRAMD2a in transcriptome data of liver samples from 234 male humans. Normalized enrichment score (NES) of GRAMD1a and GRAMD2a are used to …
Figure 4 with 1 supplement
GRAMD2a is targeted to the PM via PI(4,5)P2.
(A–B) Examination of the PI(4,5)P2-dependent behavior of PM-associated GRAMD2a-eGFP (A) and GRAMD1a-eGFP (B). PM PI(4,5)P2 was reversibly depleted by 10 s addition (middle panel) and removal (left …
-
Figure 4—source data 1
Figure 4D Quantification of liposome binding assays with recombinant His6-GRAMD2aΔTM: liposome assays were repeated four times; standard Error shown.
- https://doi.org/10.7554/eLife.31019.015
Figure 4—figure supplement 1
Schematic depiction of liposome binding assays.
A. Recombinant GRAMD2aΔTM was incubated with liposomes and ultracentrifugation was used to separate liposome-protein complexes from free protein.
Figure 5 with 3 supplements
GRAMD2a pre-marks ER-PM membrane contact sites used for STIM1 recruitment during SOCE.
(A) Cortical ER as visualized with ER marker GFP-Sec61β in cells overexpressing GRAMD2a-mCherry (top panel) or mCherry-STIM1 (bottom panel). (B) Behavior of mCherry-STIM1 at resting Ca2+ and upon …
-
Figure 5—source data 1
Quantification of the percentage of co-localized total fluorescent pixels of GRAMD2a-GFP with mCherry-STIM1 or mCherry-STIM1 with GRAMD2a-GFP as a function of time after TG addition.
1 μM TG is added at T = 30 s. Standard Error shown. Analysis was performed on n = 12 cells from three independent experiments.
- https://doi.org/10.7554/eLife.31019.018
-
Figure 5—source data 2
Figure 5B representative line graph: Total pixels of GRAMD2a-eGFP and mCherry-STIM1 fluorescence during TG-treatment experiments (1 μM TG is added at T = 30 s) for sample cell.
Quantification of 12 cells shown in Figure 5—source data 1. Figure 5—figure supplement 1D Bar Graph: Top table is pixel overlap of GRAMD2a and GRAMD1a with STIM1; standard Error shown. Bottom table is corresponding two-tailed t-test values.
- https://doi.org/10.7554/eLife.31019.019
Figure 5—figure supplement 1
Supporting evidence for STIM1 recruitment to GRAMD2a-marked ER-PM contact sites.
(A) Western Blot analysis of GRAMD2a-eGFP and GRAMD1a-eGFP (α-GFP) in Cos7 and U2OS cells transfected with low (0.1 μg) and high (1 μg) amounts of plasmids expressing GRAMD2a-eGFP and GRAMD1a-eGFP. …
Figure 5—video 1
GRAMD2a + STIM1 during TG treatment time-lapse for top cell displayed in Figure 5B
https://doi.org/10.7554/eLife.31019.020
Figure 5—video 2
GRAMD2a + STIM1 during TG treatment time-lapse for bottom cell displayed in Figure 5B
https://doi.org/10.7554/eLife.31019.021
Figure 6 with 1 supplement
STIM1ΔK is defective for targeting to PI(4,5)P2-enriched ER-PM contact sites marked by GRAMD2a.
(A–C) Fluorescence images of Cos7 cells expressing mCherry-STIM1ΔK, a PI(4,5)P2-insensitive mutant, and either (A) GRAMD2a-eGFP or (C) GRAMD1a-eGFP before and after TG treatment along with (B) …
-
Figure 6—source data 1
Figure 5B Bar Graph: Top table is pixel overlap of GRAMD2a and GRAMD1a with STIM1 and STIM1ΔK; standard Error shown.
Bottom table is corresponding two-tailed t-test values.
- https://doi.org/10.7554/eLife.31019.024
Figure 6—figure supplement 1
STIM1ΔK does not respond to TG in HeLa cells.
(A) HeLa cells expressing mCherry-STIM1 (top panel) and mCherry-STIM1ΔK (bottom panel) before and after TG treatment to deplete ER Ca2+.
Figure 7 with 3 supplements
GRAMD2a organizes ER-PM domains that selectively function in calcium homeostasis.
(A) Kinetics and intensity of mCherry-STIM1 recruitment to PM before, during, and after 1 uM TG treatment in wildtype U2OS cells, GRAMD2a knock out (KO) cells, and GRAMD2a KO with GRAMD2a-eGFP …
Figure 7—figure supplement 1
STIM1 recruitment is altered in GRAMD2a KO cells.
(A) Alignment of GRAMD2a CRISPR knock-out (KO) and wild type sequence. (B) Samples images of wildtype U2OS, GRAMD2a KO, and GRAMD2a KO with GRAMD2a-eGFP transiently transfected cells that were …
Figure 7—figure supplement 2
PM PI(4,5)P2, cholesterol and caveolin-1 are not apparently altered in GRAMD2a KO cells.
(A) TIRF imaging of BFP-Sec61β and lyn-mCherry in WT U2OS cells and GRAMD2a KO cells. Total pixels of cortical ER were divided by total area of cells, judged by total staining of lyn-mCherry, to …
Figure 7—figure supplement 3
E-Syt1 localization is altered in GRAMD2a KO cells.
(A) mCherry-E- Syt1 distribution in three additional representative wild type U2OS cells and GRAMD2a KO cells at T=0 (pre-TG treatment) and T=250 (220 sec after TG treatment). (B) mCherry-E- Syt1 …
Figure 8 with 1 supplement
Model of GRAMD2a function.
GRAMD2a is a constitutive PI(4,5)P2-dependent ER-PM tether that localizes to a subset of E-Syt2/3 contacts. GRAMD1a marks distinct ER-PM contacts in a PI(4,5)P2-independent manner. GRAMD2a pre-marks …
Figure 8—figure supplement 1
GRAMD1a localization is cell line dependent.
(A) Localization of GRAMD1a-eGFP in COS7, U2OS, and Arpe19 cells relative to ER-marker BFP-Sec61β. Representative images shown from at least 10 cells that were obtained from three biological …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers |
|---|---|---|---|
| 6s-His Tag monoclonal antibody | Western Blot antibody | ThermoFisher (His.H8) | RRID:AB_557403 |
| STIM1 monoclonal antibody | Western Blot antibody | ThermoFisher (CDN3H4) | RRID:AB_2197884 |
| GAPDH polyclonal antibody | Western Blot antibody | SigmaAldrich (G9545) | RRID:AB_796208 |
| anti-mCherry polyclonal antibody | Western Blot antibody | ThermoFisher (PA5-34974) | RRID:AB_2552323 |
| Goat anti-mouse or anti-rabbit antibodies | Western Blot antibody (DyLight 800 and DyLight 680) | ThermoFisher | |
| Oxotremorine M | Non-selective muscarinic acetylcholine receptor agonist | SigmaAldrich | |
| Thapsigargin | Non-competative SERCA inhibitor | Invitrogen/Life-technologies | |
| Fura-PE3 AM | Ratiometric cytosolic calcium indicator | Teflabs | |
| Lipofectamine2000 | Tissue culture transfection reagent | ThermoFisher | |
| Lipids | All lipids used for liposome binding assays | Avanti Lipids | |
| COS7 cells | Cercopithecus aethiops kidney cell line | G. Voeltz (U of Colorado, Boulder) | RRID:CVCL_0224 |
| U2OS cells | Human bone osteosarcoma epithelial cell line | G. Voeltz (U of Colorado, Boulder) | RRID:CVCL_0042 |
| HeLa cells | Homo sapiens cervix adenocarcinoma cell line | P. De Camilli (Yale U) | RRID:CVCL_0058 |
| ARPE19 cells | Human retinal pigment epithelial cell line | ATCC (CRL-2302) | RRID:CVCL_0145 |
| HEK293 cells | Homo sapiens embryonic kidney cell line | E. J. Dickson collection | RRID:CVCL_0045 |
| Fiji (ImageJ) software | Software used to analyse all microscopy images | ImageJ | RRID:SCR_002285 |
| ImageStudioLight | Software used to analyse all Western Blots | LI-COR BioSciences | RRID:SCR_014211 |
| Mega7 | Software used to analyse all Western Blots | MEGA Software |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.31019.031
