Fig 1.
The fluorogen-activating protein (FAP) biosensor system.
CXCR4 is expressed as a fusion protein with α-FAP and US27 is expressed as a fusion protein with β-FAP, which each bind with high affinity to distinct membrane impermeable fluorogen dyes. When extracellular fluorogen is not bound to the FAP, neither component exhibits fluorescence, and intracellularly localized FAP-tagged receptors are not exposed to membrane impermeable fluorogens. Upon addition of fluorogens to cell expressing FAP-tagged receptors, binding occurs rapidly and fluorescence can be measured using flow cytometry or fluorescence microscopy. Endocytosed receptors retain the bound fluorogen labels they acquired while present on the cell surface, allowing tracking of receptors from the surface to the cell’s interior.
Fig 2.
NIH-3T3 cells express FAP-tagged CXCR4 and US27 receptors.
(A) NIH-3T3 mouse fibroblasts were stably transfected with plasmids encoding αFAP-tagged CXCR4 and βFAP-tagged US27. Cells were labeled with membrane impermeable α and β fluorogens, and surface expression of each receptor measured via flow cytometry. The red histogram represents CXCR4, blue histogram is US27, and black histogram is unstained cells. (B) Cells were seeded in a glass-bottom dish, labeled with membrane impermeable α and β fluorogens, and imaged immediately using a Zeiss LSM700 laser scanning confocal microscope. Scale bar, 20 μm. (C) Cells were labeled with Fluo-4 AM calcium indicator dye, then stimulated with either PBS or 100 ng/ml CXCL12/SDF and fluorescence intensity was measured with flow cytometry. RFU = relative fluorescence units. Error bars represent standard error of three replicate experiments.
Fig 3.
US27 increases CXCL12-induced endocytosis of CXCR4.
(A) Confocal images of 3T3-CXCR4 and 3T3-CXCR4/US27 cells grown in glass-bottom dishes and labeled with 100 nM membrane impermeable αRED fluorogen after treatment with 100 ng/ml CXCL12. Scale bar, 10 μm. (B) Total intracellular fluorescence of cells quantified from images taken every 5 minutes (a subset of which is shown in A) with Image J software. Error bars are standard error from average measurement of five cells. * indicates p<0.05, Student t test.
Fig 4.
US27 is rapidly and constitutively internalized independent of CXCR4 or CXCL12.
(A) Live cell imaging after labeling with 100 nM membrane impermeable βRED and treatment with either PBS or 100 ng/ml CXCL12. Scale bar, 20 μm. (B) Quantification of US27 intracellular fluorescence levels in images acquired every five minutes in 3T3-US27 (gray) or 3T3-CXCR4/US27 cells (black). (C) Comparison of US27 (gray) and CXCR4 (black) internalization rates in 3T3-CXCR4/US27 cells. Error bars are standard error from average measurement of five cells.
Fig 5.
US27 colocalizes with CXCR4 after CXCL12 treatment but also internalizes independently.
3T3-CXCR4/US27 cells were seeded in glass-bottom dishes, labeled with 100nM membrane impermeable αRED (CXCR4) and βGREEN (US27) fluorogens, and then treated with (A) PBS or (B) 100 ng/ml CXCL12. Images were acquired every five minutes with representative images shown. Scale bar, 20 μm. White arrows indicate discrete yellow punctae indicating overlapping red and green signal.
Fig 6.
US27 delays CXCR4 surface recovery.
(A) 3T3-CXCR4 and (B) 3T3-CXCR4/US27 cells were treated with 100 ng/ml CXCL12 or PBS for the indicated time points, harvested, and then labeled with membrane impermeable αRED fluorogen and fluorescence intensity was measured via flow cytometry. (C) Percent of total initial surface levels for CXCR4 at each time interval. Error bars indicate standard error among three replicate experiments.
Fig 7.
US27 surface levels remain constant over time.
3T3-CXCR4/US27 cells were treated with 100 ng/ml CXCL12 or PBS for the indicated times, harvested, and then labeled with membrane impermeable fluorogen and analyzed via flow cytometry. (A) αRED fluorescence labeling of surface US27. (B) Fluorescence intensity pre-treatment was set as 100% and intensity at each time point expressed as % initial level. (C) αRED labeling of surface CXCR4 and (D) percent total initial surface levels calculated as above. Error bars represent standard error among three independent replicate experiments.