SMAD versus non-SMAD signaling is determined by lateral mobility of bone morphogenetic protein (BMP) receptors

Abstract

Bone (or body) morphogenetic proteins (BMPs) belong to the TGFβ superfamily and are crucial for embryonic patterning and organogenesis as well as for adult tissue homeostasis and repair. Activation of BMP receptors by their ligands leads to induction of several signaling cascades. Using fluorescence recovery after photobleaching, FRET, and single particle tracking microscopy, we demonstrate that BMP receptor type I and II (BMPRI and BMPRII) have distinct lateral mobility properties within the plasma membrane, which is mandatory for their involvement in different signaling pathways. Before ligand binding, BMPRI and a subpopulation of BMPRII exhibit confined motion, reflecting preassembled heteromeric receptor complexes. A second free diffusing BMPRII population only becomes restricted after ligand addition. This paper visualizes time-resolved BMP receptor complex formation and demonstrates that the lateral mobility of BMPRI has a major impact in stabilizing heteromeric BMPRI-BMPRII receptor complexes to differentially stimulate SMAD versus non-SMAD signaling.

FIGURE 1.

BMP receptors type I and II have different lateral mobilities. a, FRAP analysis of transiently expressed YFP-BMPRIa and YFP-BMPRII. Fluorescence intensities normalized to the value before photobleaching are plotted versus time. b, c, and e, SPT traces and analysis of BMPRIa, BMPRIb, and BMPRII receptor mobility on cell surface. C2C12 cells expressing HA-tagged BMPRIa, BMPRIb, or BMPRII were QDot-labeled via tag-specific antibodies and subjected to SPT microscopy followed by mobility analysis. Representative movement trajectories of individual receptor molecules from five independent experiments demonstrate the confinement of receptor mobility to a certain area. Scale bar, 100 nm. d and f, average motion of receptors was determined by MSD analysis. MSD for each receptor type was plotted versus time lag (t_lag), and the resulting functions were fitted to models of confined and free diffusion. Data points from five independent experiments for each receptor type were pooled. Error bars, S.E. of each MSD data point for each particular lag time.

FIGURE 2.

Lateral mobility of BMPRII is reduced upon ligand stimulation. a, FRAP analysis of BMPRIa and BMPRII lateral diffusion upon BMP-2 stimulation. YFP-BMPRIa and YFP-BMPRII constructs were transiently expressed and imaged as in Fig. 1a. Fluorescence intensities normalized to the value before photobleaching are plotted versus time upon stimulation with BMP-2. The low recovery of BMPRII reflects the increased amount of immobilized BMPRII on the cell surface. b, c, and e, SPT traces of BMPRIa, BMPRIb, and BMPRII after 5-min BMP-2 stimulation. C2C12 cells expressing HA-tagged BMPRIa, BMPRIb, or BMPRII were QDot-labeled and imaged upon BMP-2 stimulation as described in the legend to Fig. 1b. Representative trajectories (from five independent experiments) of BMPRIa, BMPRIb, and BMPRII show confined movement for all receptor types after ligand stimulation. Scale bar, 100 nm. d and f, analysis of BMPRIa, BMPRIb, and BMPRII MSD versus time shows a comparable average mobility for all receptor types after BMP-2 stimulation. Data points from five independent experiments were pooled. Error bars, S.E. of each MSD data point for each particular lag time.

FIGURE 3.

Heteromeric BMPRIa-BMPRIb complexes before and after ligand stimulation. a, ligand-independent BMPRIa-BMPRII complexes were analyzed by FRET acceptor photobleaching of transiently expressed YFP-BMPRIa and CFP-BMPRII. Quantification shows normalized fluorescence intensity in arbitrary units (a.u.) before and after photobleaching. The increase in CFP intensity upon YFP (acceptor) photobleaching indicates the existence of BMPRI-BMPRII complexes without ligand stimulation. b, time-resolved FRET on individual cells expressing YFP-BMPRIa and CFP-BMPRII constructs indicated an increase of the FRET ratio after ligand stimulation. Cells were co-transfected with constructs encoding YFP-BMPRIa and CFP-BMPRII. Stimulation with BMP-2 (arrow) caused an immediate increase in the FRET ratio (bottom) with a two-exponential rise with constants of about 300 and 600 s with a corresponding increase in the YFP (yellow) and a decrease in the CFP (cyan) channel (top). The maximal FRET ratio increase was about 20%. c, enhanced DRM association of endogenous BMPRII upon BMP-2 stimulation as assessed by co-fractionation with the DRM marker Caveolin1. C2C12 cells were stimulated with BMP-2 for 5 min. After cell surface biotinylation, cells were used for sucrose gradient fractionation. Biotinylated proteins were precipitated and subjected to Western blot analysis (WB). d, quantification of Western blot analysis demonstrates the increased proportion of BMPRII in DRM fractions after BMP-2 addition.

FIGURE 4.

Ligand-induced stabilization of heteromeric BMP receptor complex. Co-localization dynamics of individual QDot585-labeled HA-BMPRIb and QDot655-labeled Myc-BMPRII receptors (co-expressed in C2C12 cells), as assessed by simultaneous two-color SPT. Five independent experiments were performed. a, a representative BMPRIb (green)-BMPRII (red) receptor pair displays in the absence of ligand several events of recombination between receptors, changing between merged (yellow) and separated states. b, time-resolved distance analysis of the representative BMPRIb-BMPRII receptor pair depicted in a. The numbers indicated at specific times on the graph correspond to the images in a. The distance between the centers of fluorescence intensity of BMPRIb and BMPRII was measured for this (and for each) interacting complex and plotted sequentially versus time. A distance of 400 nm was set as a threshold (dotted line; for more details, see supplemental material) to mark the border between the merged and separated states. This analysis revealed that BMPRIb WT-BMPRII complexes typically separate and recombine several times during the lifetime of an individual heteromeric receptor complex. c, diagram of dynamic co-localization shows that BMP-2 addition changes the co-localization pattern of an individual representative BMPRIb-BMPRII receptor pair. The merged state (tight complex) is visualized by black arrows, and the separated state (loose complex) is shown by gray arrows. The numbers indicate the duration of the respective state. d–f, average duration of merged state (d), number of recombination events (e), and total complex lifetime (f) in receptor pairs consisting of BMPRII and BMPRIb WT with or without BMP-2 stimulation. Quantification of these co-localization parameters was performed with data from five independent experiments and an equal number of potential complex pairs. Ligand stimulation increased the values of all three analyzed parameters in complexes consisting of BMPRII and BMPRIb WT.

FIGURE 5.

Confined mobility of BMPRIb requires both specialized membrane domains and cytoskeletal components. a, short term effects of cholesterol depletion by MβCD on BMPRIb mobility as assessed by SPT. C2C12 cells expressing HA-tagged BMPRIb were QDot-labeled and imaged in a time series 0–10 min after MβCD application. Five independent experiments were performed. Trajectories and respective MSD graphs of a single representative receptor before and 5 and 10 min after MβCD application demonstrate gradual mobility increase of BMPRIb over time. Scale bar, 100 nm. b, MSD analysis from five independent experiments demonstrates MβCD-induced increase of average BMPRIb mobility starting at 5-min treatment duration. c and d, representative trajectories (c) and MSD analysis (d) of BMPRIb mobility after 60 min MβCD pretreatment show long traces and free diffusion-like motion. Scale bar, 100 nm. e, control of siRNA-mediated Caveolin1 knock-down efficiency in C2C12 cells using Western blot. f and g, role of Caveolin1 scaffolds for BMPRIb mobility as assessed by SPT. C2C12 cells expressing BMPRIb were subjected to transient siRNA-mediated Caveolin1 knockdown. SPT microscopy and analysis of BMPRIb mobility were performed as described. Representative trajectories (f) and MSD analysis (g) demonstrate increased BMPRIb lateral mobility under Caveolin1 knockdown. Scale bar, 100 nm. h–k, role of cytoskeleton for BMPRIb mobility as assessed by SPT. C2C12 cells expressing BMPRIb were treated with nocodazole for 0, 20, and 40 min (h and i) and 90 min (j and k) to induce microtubule disruption before QDot labeling and SPT (as described). Representative trajectories (h and j) and MSD analysis (i and k) of BMPRIb mobility under these conditions show increased BMPRIb mobility upon cytoskeletal perturbation. Scale bar, 100 nm. Error bars, S.E. of each MSD data point for each particular lag time.

FIGURE 5.

Confined mobility of BMPRIb requires both specialized membrane domains and cytoskeletal components. a, short term effects of cholesterol depletion by MβCD on BMPRIb mobility as assessed by SPT. C2C12 cells expressing HA-tagged BMPRIb were QDot-labeled and imaged in a time series 0–10 min after MβCD application. Five independent experiments were performed. Trajectories and respective MSD graphs of a single representative receptor before and 5 and 10 min after MβCD application demonstrate gradual mobility increase of BMPRIb over time. Scale bar, 100 nm. b, MSD analysis from five independent experiments demonstrates MβCD-induced increase of average BMPRIb mobility starting at 5-min treatment duration. c and d, representative trajectories (c) and MSD analysis (d) of BMPRIb mobility after 60 min MβCD pretreatment show long traces and free diffusion-like motion. Scale bar, 100 nm. e, control of siRNA-mediated Caveolin1 knock-down efficiency in C2C12 cells using Western blot. f and g, role of Caveolin1 scaffolds for BMPRIb mobility as assessed by SPT. C2C12 cells expressing BMPRIb were subjected to transient siRNA-mediated Caveolin1 knockdown. SPT microscopy and analysis of BMPRIb mobility were performed as described. Representative trajectories (f) and MSD analysis (g) demonstrate increased BMPRIb lateral mobility under Caveolin1 knockdown. Scale bar, 100 nm. h–k, role of cytoskeleton for BMPRIb mobility as assessed by SPT. C2C12 cells expressing BMPRIb were treated with nocodazole for 0, 20, and 40 min (h and i) and 90 min (j and k) to induce microtubule disruption before QDot labeling and SPT (as described). Representative trajectories (h and j) and MSD analysis (i and k) of BMPRIb mobility under these conditions show increased BMPRIb mobility upon cytoskeletal perturbation. Scale bar, 100 nm. Error bars, S.E. of each MSD data point for each particular lag time.

FIGURE 6.

The transmembrane region of BMPRIb is a determinant of the receptor's lateral mobility. a, mutagenesis approach for identification of structural elements responsible for the immobility of BMPRIb. The transmembrane domain of BMPRIb was subjected to targeted mutagenesis by a polyalanine scan. The WT sequence of the transmembrane domain is depicted with the mutated amino acids shown in boldface letters. The respective constructs were stably transduced into C2C12 cells, and the generated stable cell lines were named C2C12-BMPRIb WT, -TM2, -TM5, and -PKT, respectively. b and c, reduced DRM association of BMPRIb transmembrane domain mutants as assessed by co-fractionation of receptor with Caveolin1. C2C12-BMPRIb WT, -PKT, -TM2, and -TM5 cells were used for sucrose gradient fractionation, and fractions were subjected to Western blot analysis (WB) (b). The histogram (c) depicts the percentage of receptor present in DRM fractions obtained by quantification of the Western blot analysis. d–i, mutations of transmembrane domain increase lateral mobility of BMPRIb. C2C12-PKT, -TM2, and -TM5 cells were used for antibody-mediated QDot labeling of receptor and subjected to SPT. Representative trajectories and MSD analysis show a strong increase either in confinement size for the PKT (d and e) or in lateral mobility for TM2 (f and g) and TM5 (h and i) in comparison with BMPRIb WT. For MSD analysis, data points from five independent experiments were pooled. Scale bar, 100 nm. Error bars, S.E. of each MSD data point for each particular lag time.

FIGURE 7.

BMPRIb mutants with enhanced lateral mobility fail to induce non-SMAD signaling and osteoblastic differentiation, whereas the SMAD pathway is not altered. a, Western blot analysis of SMAD1/5/8 in C2C12-BMPRIb WT, -TM2, -TM5, and -PKT cells. Cells were stimulated with BMP-2 or GDF-5 for 30 min. Cell lysates were subjected to Western blot analysis of the respective phosphorylated proteins. GAPDH was used as loading control. A representative experiment from three independent experiments is shown. Quantification of Western blot (b) depicts relative intensity of phospho-specific signals normalized to respective GAPDH. c, analysis of SMAD-mediated transcriptional activation by BMP-responsive reporter (BRE-LUC) in C2C12-BMPRIb WT, -TM2, -TM5, and -PKT cells. The respective cell lines were transiently transfected with BRE-LUC and RLTK and stimulated with GDF-5 for 6 h, and relative luciferase activity was measured. (Reporter gene assays stimulated with BMP-2 are shown in supplemental Fig. S3a.) A representative experiment from three independent experiments is shown. All measurements were conducted in triplicate. d, Western blot analysis of p38 and AKT phosphorylation in C2C12-BMPRIb WT, -TM2, -TM5, and -PKT cells. Cells were stimulated with BMP-2 or GDF-5 for 30–60 min. Cell lysates were subjected to Western blot analysis of the respective phosphorylated proteins. GAPDH was used as loading control. A representative experiment from three experiments is shown. Quantification of the Western blot (e) depicts relative intensity of phospho-specific signals normalized to respective GAPDH. f, ALP activity assay. C2C12-BMPRIb WT, -TM2, -TM5, and -PKT cells were stimulated with the indicated concentrations of GDF-5 for 72 h. ALP production indicative of osteoblastic differentiation was assessed by a colorimetric assay of enzymatic activity of ALP. (ALP assays stimulated with BMP-2 are shown in supplemental Fig. S3b.) A representative experiment from three experiments is shown. All measurements were conducted in triplicate. g, expression analysis of osteoblastic differentiation markers in C2C12-BMPRIb WT or mutant cells. C2C12-BMPRIb WT, -TM2, -TM5, and -PKT cells were starved, stimulated with GDF-5 for 12 h to measure expression of Runx2 and Osterix or for 48 h to measure expression of ALP and OCN, and used for RNA isolation. Quantitative RT-PCR was performed to analyze gene expression of Runx2, ALP, Osterix, and OCN. Gene expression was normalized to HPRT expression and plotted as mean normalized expression (MNE). (Expression analysis upon BMP-2 stimulation is shown in supplemental Fig. S3c.) All measurements were conducted in triplicate. Error bars, S.E.

FIGURE 8.

Enhanced lateral mobility of BMPRIb fails to stabilize the BMPRI-BMPRII signaling receptor complex. Shown are co-localization dynamics of individual QDot585-labeled HA-BMPRIb mutants and QDot655-labeled Myc-BMPRII receptors (co-expressed in C2C12 cells) as assessed by simultaneous two-color SPT (for details, see Fig. 4). Data from five independent experiments were pooled. a–c, average duration of merged state (a), number of recombination events (b), and the total complex lifetime (c) in receptor pairs consisting of BMPRII and the indicated BMPRIb mutants with or without BMP-2 stimulation. Quantification of these co-localization parameters was performed with data from five independent experiments and an equal number of potential complex pairs. Enhanced lateral mobility of BMPRIb still binds BMPRII but fails to stabilize the heteromeric BMPRI-BMPRII complex. Bars of BMPRIb-WT are identical to those in Fig. 4d and are depicted to facilitate comparability. Error bars, S.E. of either merged state, number of recombination events or of complex life-time with indicated receptors.