Calcium-stimulated disassembly of focal adhesions mediated by an ORP3/IQSec1 complex
- Department of Cell Biology, University of Virginia Health System, United States
- Department of Molecular Biology, University of Texas Southwest Medical Center, United States
- Howard Hughes Medical Institute, United States
- Department of Chemical Engineering, University of Virginia, United States
- Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, United States
Figures
Figure 1 with 1 supplement
IQSec1 promotes focal adhesion disassembly in MDA-MB-231 cells.
(A) Bright field images of control and IQSec1 depleted cells, Bar = 20 µm. (B) Control and IQSec1 depleted cells, fixed and stained for β1-integrin (green), FAK (red) and actin (blue), Bar = 10 µm. …
Figure 1—figure supplement 1
IQSec1 splice variants, expression in MDA-MB-231 cells and knockdown efficiency.
(A) Domain organization of IQSec1 splice variants. Red arrow indicates splice variants experimentally validated by RT-PCR analysis to be present in MDA-MB-231 cells. Note the approximate molecular …
Figure 2 with 1 supplement
IQSec1 activates ARF5 to promote FA disassembly.
(A) Arf5 depletion leads to increased focal adhesion size. Control (left) and Arf5-depleted cells (right) were stained for endogenous β1-integrin (green) and FAK (red). Bar = 10 µm. (B) Expression …
Figure 2—figure supplement 1
Knockdown of Arf5 but not Arf6 phenocopies IQSec1 depletion.
(A) Quantitation of endogenous Arf6 protein from MDA-MB-231 cells treated with shRNA targeting Arf6. (B) Quantitation of Arf5 knockdown. Due to the poor quality of antibodies, cells were transfected …
Figure 3 with 1 supplement
IQSec1 associates with ORP3 and ORP3 knockdown phenocopies IQSec1 knockdown.
(A) Lysates from cells co-expressing mCherry-IQSec1 and either unfused GFP or GFP-ORP3 (left) were immunoprecipitated with GFP-TRAP beads (Chromotek) and probed by western blotting for GFP and …
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Figure 3—source data 1
Mass spectroscopy of IQSec1-interacting proteins- data underlying Figure 3A.
Please refer to Materials and methods for details.
- https://cdn.elifesciences.org/articles/54113/elife-54113-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Knockdown of ORP3 also increases focal adhesion area in HT1080 cells.
(A) Quantitation of FA size from control or ORP3-depleted HT1080 cells, using vinculin as a marker for FA. N for control = 758, ORP3 KD hairpin1 = 853 ORP3 KD hairpin2 = 617 adhesions. Data were …
Figure 4 with 1 supplement
ORP3 is recruited to STIM1-positive ER/PM contacts in response to calcium influx.
(A) Still images from live cells (Video 5) co-expressing GFP-ORP3 and mCherry-STIM1, before and after thapsigargin (1 µM) treatment. Bar = 10 µm. Magnified view of the boxed area ia shown at right. …
Figure 4—figure supplement 1
Recruitment of ORP3 to the plasma membrane.
(A) Esyt-1 and ORP3 are co-recruited in response to calcium influx. MDA-MB-231 cells expressing GFP-E-Syt1 and mCherry-ORP3 were treated with thapsigargin (1 µM) and imaged live for 10 min. Panels …
Figure 5 with 1 supplement
ORP3 requires PI(4,5)P2 for recruitment to the PM, and acts upstream of IQSec1 to stimulate Arf5 activation.
(A) Live cells expressing GFP-ORP3, mCherry-CRY2-5-PtaseOCRL and CINB-CAAX (two components of the blue light system) were stimulated with blue light to deplete PI(4,5)P2 and then treated with PMA …
Figure 5—figure supplement 1
Mapping the binding site for ORP3 on IQSec1.
(A) Domain organization of truncation mutants of IQSec1. (B) Interaction of WT ORP3 with truncation mutants of IQSec1. Lysates of cells co-expressing WT GFP-ORP3 and various truncation mutants of …
Figure 6 with 2 supplements
Phosphoregulation of ORP3 recruitment to the plasma membrane.
(A) MDA-MB-231 cells expressing GFP-ORP3 were mock treated with DMSO or with the pan-PKC inhibitor Gö6983 (20 µM) for 2 hr. Both groups of cells were then stimulated with thapsigargin (1 µM) and …
Figure 6—figure supplement 1
Mapping the interaction between ORP3 and IQSec1.
(A) Predictive model of IQSec1, generated using Raptor-X. SEC7-PH domain tandem (based on the existing crystal structure) is at the right, predicted N-terminal helices are numbered 1–4. Arrow …
Figure 6—figure supplement 2
Imaging of focal adhesions upon inhibition of PKC or PI4KIIIα.
(A) Inhibition of either PKC or PI4KIIIa results in larger FAs. Representative images of MDA-MB-231 cells mock treated or treated with Gö6983 or GSK-A1, fixed and stained for endogenous vinculin. (B)…
Figure 7 with 1 supplement
ORP3 extracts PI4P from the plasma membrane in exchange for PC.
(A–F) MDA-MB-231 cells expressing the PI4P probe GFP-P4M2X and either mCherry alone (A) mCherry-ORP3 (C) or mCherry-ORP31-555 (E) were imaged live. Images show stills before (Frame 1) and after …
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Figure 7—source data 1
Mass spectroscopy of lipids bound to ORP3 – data underlying Figure 7I.
Cells expressing either unfused GFP or GFP-ORP3 were treated (or not) with thapsigargin as described in Methods. Samples were immunoprecipitated with anti-GFP antibody, bound lipids were extracted and analyzed by mass spectroscopy.
- https://cdn.elifesciences.org/articles/54113/elife-54113-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
Model summarizing the finding of this study.
Calcium influx via STIM1/Orai1 channels adjacent to focal adhesions at the trailing edge stimulates PKC-dependent phosphorylation of ORP3, exposing its interaction sites. The PH domain binds …
Author response image 1
ORP3 fails to co-IP with HA-R-Ras (left), and depletion of neither ORP3 nor IQSec1 impacts R-Ras activity in a pulldown assay (A).
Quantitation in panel B is from 3 independent experiments.
Videos
Video 1
MDA-MB-231 cell expressing GFP-paxillin, imaged for 60 min at a rate of 1 frame every 15s.
Bar = 10 µm.
Video 2
MDA-MB-231 cell depleted of IQSec1 and expressing GFP-paxillin, imaged for 60 min at a rate of 1 frame every 15 s.
Bar = 10 µm.
Video 3
Movie showing GFP-STIM1 making contact with Ds-Red-paxillin-labelled focal adhesions.
Cell was imaged live for 30 min at a rate of 1 frame every 15 s. Bar = 2.5 µm.
Video 4
MDA-MB-231 cell expressing GFP-ORP3 and Ds-Red-Paxillin was imaged live for 30 min at a rate of 1 frame every 15 s.
Notice the accumulation of ORP3 puncta around FAs at retracting edges of the cell. Bar = 10 µm.
Video 5
MDA-MB-231 cell expressing GFP-ORP3 and mCherry-STIM1, imaged live for 30 min at a rate of 1 frame every 15 s.
Thapsigargin was added 2.5 min after the start of the movie. Note the co-recruitment of ORP3 and STIM1 to the PM after thapsigargin treatment. These changes have been quantified in Figure 4B. Bar = 1…
Video 6
MDA-MB-231 cell expressing GFP-P4M2X (probe for phosphatidylinositol-4-phosphate/PI4P) and unfused mCherry was imaged live for 30 min at a rate of 1 frame every 15 s.
Thapsigargin was added 2.5 min after the start of the movie. Bar = 10 µm. Note that PM PI4P levels remain unchanged after addition of thapsigargin (quantified in Figure 7B).
Video 7
MDA-MB-231 cell expressing GFP-P4M2X and mCherry-ORP3 was imaged live for 30 min at a rate of 1 frame every 15 s.
Thapsigargin was added 2.5 min after the start of the movie. Notice the loss of GFP-P4M2X from the PM with the concomitant recruitment of mCherry-ORP3 (quantified in Figure 7D). The reduction in …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (H. sapiens) | MDA-MB-231 | ATCC | CRM-HTB-26 | Human breast cancer cell line |
| Cell line (H. sapiens) | HEK293 | ATCC | CRL-1573 | Human embryonic kidney cell line |
| Cell line (H. sapiens) | HT1080 | ATCC | CCL-121 | Human fibrosarcoma cell line |
| Transfected construct (H. sapiens) | FLAG-IQSec1 (NP_001127854.1) | Genscript, Piscataway, NJ | Plasmid to transiently express IQSec1 | |
| Transfected construct (H. sapiens) | mCherry-ORP3 | Vesa Olkonnen Lehto et al., 2005 | Plasmid to transiently express mCherry-ORP3 | |
| Transfected construct (H. sapiens) | Xpress tagged FFAT double mutant of ORP3 | Vesa Olkonnen Lehto et al., 2005 | Plasmid to transiently express ORP3∆FFAT | |
| Transfected construct (H. sapiens) | Xpress tagged PH domain mutant of ORP3 | Vesa Olkonnen Lehto et al., 2005 | Plasmid to transiently express ORP3∆PH | |
| Transfected construct (H. sapiens) | Xpress tagged ORP31-445 | Vesa Olkonnen Lehto et al., 2005 | Plasmid to transiently express ORP3 truncation mutant 1–445 | |
| Transfected construct (H. sapiens) | Xpress tagged ORP3398-886 | Vesa Olkonnen Lehto et al., 2005 | Plasmid to transiently express ORP3 truncation mutant 398–886 | |
| Transfected construct (H. sapiens) | Xpress tagged ORP31-555 | Vesa Olkonnen Lehto et al., 2005 | Plasmid to transiently express ORP3 truncation mutant 1–555 | |
| Transfected construct (H. sapiens) | GFP-Paxillin | Rick Horwitz (Allen Research Institute, Seattle, WA) | Plasmid to transiently express GFP-paxillin | |
| Transfected construct (H. sapiens) | DsRed-Paxillin | Rick Horwitz (Allen Research Institute, Seattle, WA) | Plasmid to transiently express DsRed-paxillin | |
| Transfected construct (H. sapiens) | mCherry-STIM1 and | Isabelle Derre (University of Virginia) | Plasmid to transiently express mCherry-STIM1 | |
| Transfected construct (H. sapiens) | YFP-VAP-A | Isabelle Derre (University of Virginia) | Plasmid to transiently express YFP-VAP-A | |
| Transfected construct (H. sapiens) | mCherry-CRY2-5-PtaseOCRL and | Olaf Idevall-Hagren (Uppsala University, Sweden) Idevall-Hagren et al., 2012 | Plasmid for optogenetic activation of OCRL | |
| Transfected construct (H. sapiens) | CINB-CAAX | Olaf Idevall-Hagren Idevall-Hagren et al., 2012 | Plasmid for optogenetic activation of OCRL | |
| Transfected construct (L. pneumophila) | GFP-P4M2X | Tamas Balla (NIH, Bethesda, Maryland) Hammond et al., 2014 | Biosensor for PI4P | |
| Transfected construct (H. sapiens) | GFP- E-Syt1 | Pietro De Camilli (Yale Univ. New Haven, CT) | Plasmid to transiently express GFP-E-Syt1 | |
| Transfected construct (H. sapiens) | GFP-PLCδ | David Castle (Univ. Virginia) | Biosensor for PI(4,5)P2 | |
| Antibody | mouse monoclonal antibody to HA (16B12) | Biolegend (San Diego, CA) | WB (1:1000) | |
| Antibody | rabbit polyclonal to GFP | ThermoFisher Scientific (Rockford, Il) | (A10262) | WB (1:2000) |
| Antibody | Mouse monoclonal anti-Xpress | ThermoFisher Scientific (Rockford, Il) | (R910-25) | WB (1:1000) |
| Antibody | Rabbit polyclonal antibody to mCherry | Biovision (San Francisco, CA) | (5993) | WB (1:1000) |
| Antibody | Mouse monoclonal antibody to tubulin | Sigma-Aldrich | (T9026) | WB(1:10,000) |
| Antibody | mouse monoclonal antibody to vinculin | Sigma-Aldrich | (V9131) | IIF (1:400) |
| Antibody | Mouse monoclonal antibody to human β1-integrin | BD Biosciences (Mississauga, ON, Canada) | (610467) | IIF (1:250) |
| Antibody | mouse antibody to GFP | Santa Cruz | (sc-9996) | WB (1:1000) |
| Antibody | mouse antibody to ORP3 | Santa Cruz | (sc-514097) | WB (1:1000) |
| Antibody | rabbit antibody to FAK | Santa Cruz | (sc-557) | IIF (1:100) |
| Antibody | Alexa Fluor 488 donkey anti-mouse secondary antibody | ThermoFisher Scientific (Rockford, Il) | (A10037) | IIF (1:500) |
| Antibody | Alexa Fluor 568 donkey anti-mouse secondary antibody | ThermoFisher Scientific (Rockford, Il) | (A21202) | IIF (1:500) |
| Antibody | Alexa Fluor 488 donkey anti-rabbit secondary antibody | ThermoFisher Scientific (Rockford, Il) | (A10042) | IIF (1:500) |
| Antibody | Alexa Fluor 568 donkey anti-rabbit secondary antibody | ThermoFisher Scientific (Rockford, Il) | (A21206) | IIF (1:500) |
| Antibody | IRDye 800CW Secondary Antibody | Li-COR | WB (1:10,000) | |
| Antibody | IRDye 650 Secondary Antibody | Li-COR | WB (1:10,000) | |
| Chemical compound, drug | Alexa Flour 647 phalloidin | ThermoFisher Scientific (Rockford, Il) | (A22287) | Dilution of 1:1000 |
| Chemical compound, drug | Thapsigargin | Tocris (Bristol, UK) | (1138) | 1 µM |
| Chemical compound, drug | Phorbol 12-myristate 13-acetate (PMA) | Tocris (Bristol, UK) | (1201/1) | 2.5 µM |
| Chemical compound, drug | YM 58483 (BTP2) | Tocris (Bristol, UK) | (3939/10) | 25 µM |
| Chemical compound, drug | Gö6983 | Abcam (Cambridge, UK) | (ab144414) | 20 µM |
| Commercial assay or kit | Odyssey PBS Blocking Buffer | Li-COR | For immunoblotting 1:1 diluted in PBST (0.1% tween-20) | |
| Commercial assay or kit | Q5 Site-Directed Mutagenesis Kit | NEB | ||
| Software, algorithm | NEBaseChanger | NEB | Software to design primers for site-directed mutagenesis | |
| Other | GFP-TRAP_A beads | Chromotek (Germany) | gta-10 | For precipitation of GFP-tagged proteins (25 µl of beads) |
| Other | Protein A Beads | (Cell Signalling, Danvers, MA) | (9863) | For immunoprecipitation (25 µl of bead) |
