BMP2-induced chemotaxis requires PI3K p55γ/p110α-dependent phosphatidylinositol (3,4,5)-triphosphate production and LL5β recruitment at the cytocortex
- Christian Hiepen1,
- Andreas Benn†1,
- Agnieszka Denkis†1,
- Ilya Lukonin1,
- Christoph Weise1,
- Jan H Boergermann1 and
- Petra Knaus1Email author
© Hiepen et al.; licensee BioMed Central Ltd. 2014
Received: 10 March 2014
Accepted: 13 May 2014
Published: 30 May 2014
BMP-induced chemotaxis of mesenchymal progenitors is fundamental for vertebrate development, disease and tissue repair. BMP2 induces Smad and non-Smad signalling. Whereas signal transduction via Smads lead to transcriptional responses, non-Smad signalling induces both, transcriptional and immediate/early non-transcriptional responses. However, the molecular mechanisms by which BMP2 facilitates planar cell polarity, cortical actin rearrangements, lamellipodia formation and chemotaxis of mesenchymal progenitors are poorly understood. Our aim was to uncover the molecular mechanism by which BMP2 facilitates chemotaxis via the BMP2-dependent activation of PI3K and spatiotemporal control of PIP3 production important for actin rearrangements at the mesenchymal cell cytocortex.
We unveiled the molecular mechanism by which BMP2 induces non-Smad signalling by PI3K and the role of the second messenger PIP3 in BMP2-induced planar cell polarity, cortical actin reorganisation and lamellipodia formation. By using protein interaction studies, we identified the class Ia PI3K regulatory subunit p55γ to act as a specific and non-redundant binding partner for BMP receptor type II (BMPRII) in concert with the catalytic subunit p110α. We mapped the PI3K interaction to a region within the BMPRII kinase. Either BMP2 stimulation or increasing amounts of BMPRI facilitated p55γ association with BMPRII, but BMPRII kinase activity was not required for the interaction. We visualised BMP2-dependent PIP3 production via PI3K p55γ/p110α and were able to localise PIP3 to the leading edge of intact cells during the process of BMP2-induced planar cell polarity and actin dependent lamellipodia formation. Using mass spectrometry, we found the highly PIP3-sensitive PH-domain protein LL5β to act as a novel BMP2 effector in orchestrating cortical actin rearrangements. By use of live cell imaging we found that knock-down of p55γ or LL5β or pharmacological inhibition of PI3K impaired BMP2-induced migratory responses.
Our results provide evidence for an important contribution of the BMP2-PI3K (p55γ/p110α)- PIP3-LL5β signalling axis in mesenchymal progenitor cell chemotaxis. We demonstrate molecular insights into BMP2-induced PI3K signalling on the level of actin reorganisation at the leading edge cytocortex. These findings are important to better understand BMP2–induced cytoskeletal reorganisation and chemotaxis of mesenchymal progenitors in different physiological or pathophysiological contexts.
KeywordsActin BMP BMP receptor Chemotaxis LL5beta Migration p110alpha p55gamma PHLDB2 PIK3R3
Gradients of bone morphogenetic proteins (BMPs) act as mesenchymal guidance cues during development, disease and tissue repair by molecular mechanisms that remain poorly defined . In particular, the directional migration (chemotaxis) of neural crest cells, bone marrow stromal cells and endothelial cells along gradients of BMP2 has been reported [2–5]. BMPs signal through binding to cell surface hetero-oligomeric receptor complexes comprising type I (BMPRI) and type II (BMPRII) receptors . Activated BMP receptor complexes induce canonical-Smad and non-Smad signalling cascades . Activation of the type I receptor kinase by the type II receptor kinase induces phosphorylation and thus nuclear translocation of Smad1/5/8, leading to transcription of Smad-dependent target genes .
Whereas the molecular basis of canonical Smad signalling and its role in gene transcription is well explored, the molecular activation mechanism and the cellular functions of the non-Smad pathways, which rather act directly and independently of gene transcription, are poorly understood. In particular, the molecular mechanism of BMP-induced phosphatidylinositol 3-kinase (PI3K) activation, its signalling route and cellular function are poorly characterised. In recent years, several studies unveiled a requirement of PI3K for BMP2-induced migration of various cell types with mesenchymal origin by yet unknown mechanisms [9–11].
Here, for the first time, we addressed the molecular activation mechanism of BMP2-induced PI3K signalling in undifferentiated mesenchymal progenitor cells and the role of the lipid-product of PI3K, the membrane-bound second messenger PtdIns-3, 4, 5-triphosphate (PI (3, 4, 5) P3; hereafter referred to as PIP3) in BMP2-induced actin reorganisation.
Class Ia PI3Ks are dimeric lipid kinases composed of one out of five possible regulatory subunits encoded by Pik3r1 (encoding splice isoforms p85α, p55α and p50α), Pik3r2 (p85β) or Pik3r3 (p55γ) [12, 13]. The regulatory subunit is bound by one of three catalytic subunits, termed p110, encoded by Pik3ca (p110α), Pik3cb (p110β) or Pik3cd (p110δ) . Catalytic activity is initiated upon regulatory subunit Src homology 2 (SH2) domain binding to phospho-tyrosine (pTyr) residues within a specific peptide context . Thereafter, activated PI3K phosphorylates the 3-hydroxyl group of PtdIns-4, 5-bisphosphate (PIP2) to produce the second messenger PIP3. PIP3 recruits Pleckstrin homology (PH) domain-containing regulators to the inner plasma membrane. One main PI3K effector is protein kinase B (PKB/Akt) . Besides Akt, PH-domain-containing cytoskeletal regulators sense PIP3 and mediate cortical actin dynamics at the so-called leading edge cytocortex. As such, the PH-like domain family B member 2 (Phldb2, hereafter referred to as LL5β) acts as a sensitive PIP3 effector during the establishment of planar cell polarity (PCP), lamellipodia formation, protrusion and subsequent chemotaxis . LL5β orchestrates actin rearrangements through tethering actin cross-linkers of the filamin family to PIP3-rich plasma membranes [17–19].
In this study, we identified that the PI3K regulatory subunit p55γ functions as a novel BMPRII-interacting protein. It acts in concert with p110α to mediate BMP2-induced PIP3 production and hence cortical actin rearrangements. We visualised that BMP2-induced PI3K activity produces PIP3 at the cytocortex, which subsequently recruits LL5β to orchestrate cortical actin crosslinking. Either knock-down of p55γ or LL5β or pharmacological inhibition of PI3K impaired BMP2-induced directional cell migration. Hence our study presents the first insights into the molecular activation and regulation mechanism by which BMP2 facilitates PI3K activity and the cytocortical signalling events leading to cortical actin reorganisation, PCP and chemotaxis. These molecular details are important to better understand BMP2-induced chemotaxis of mesenchymal progenitor cells during vertebrate development, tissue repair or disease.
BMP2-induced PI3K signalling is required for chemotaxis
PI3K regulatory subunit p55γ interacts with the long and short forms of BMPRII
BMPRII becomes tyrosine phosphorylated in a BMP2-dependent manner
Class Ia PI3Ks interact with activated growth factor receptors via pTyr motifs recognised by the SH2 domains of the regulatory subunit . BMPRII is a serine/threonine kinase and its tyrosine phosphorylation has not been investigated to our knowledge. The cytosolic part of BMPRII-LF contains 24 tyrosines; the majority of tyrosines are located within the kinase domain, a few in the C-terminal tail and none in the juxtamembrane region preceding the kinase domain (Figure 2B). An in silico alignment of the BMPRII cytosolic domain with known SH2 domain-binding peptides (Figure 2B, marked with *)  and analysis using ScanSite oriented peptide library technique (marked with **)  identified five potential tyrosines that could act as SH2 domain docking sites (black lines indicate locations of all other BMPRII tyrosines in cytosolic domains). To first analyse BMP2-dependent tyrosine phosphorylation of BMPRII, we transfected HEK293T cells with HA-tagged BMPRII-LF, followed by immunoprecipitation using anti-HA antibody. BMPRII tyrosine phosphorylation was investigated using an anti-pTyr antibody. We found basal Tyr phosphorylation of BMPRII-LF in starved cells (Figure 2F, lower panel, lane 1), which increased upon 15 to 60 minutes stimulation with BMP2 (lanes 3 to 6). This kinetic profile resembles Smad1/5/8 phosphorylation by activated receptor complexes (Figure 2F, upper panel). A BMP2-dependent Tyr phosphorylation of endogenous BMPRII was also confirmed using C2C12 cells upon pull-down of endogenous BMPRII after 60 minutes’ BMP2 stimulation compared to non-stimulated control (Additional file 1: Figure S1C,D,E). A vice versa approach by performing a pTyr pull-down upon BMP2 stimulation on BMPRII-LF-HA transfected HEK293T cells and subsequent blotting using anti- HA antibody also confirmed the tyrosine phosphorylation of BMPRII (Additional file 1: Figure S1D). The pTyr specificity of the antibody was proven by sodium orthovanadate treatment of cells and additionally by dephosphorylation using Antarctic phosphatase treatment of the membrane after western blotting with pTyr antibody (Additional file 1: Figure S1E). To identify particular phosphorylated tyrosine residues on BMPRII, respective mass spectrometry approaches have to be performed in the future. Together, these results confirm that BMPRII is tyrosine phosphorylated in a BMP2-dependent manner and provides the required features to associate with p55γ.
BMPRII-kinase activity is dispensable but the presence of BMPRI enhances BMPRII-p55γ interaction
BMP2-induced PI3K signalling is specifically mediated via p55γ
BMP2-induced PIP3 production is dependent on p55γ
PIP3 and PIP3 effectors localise to BMP2-induced cortical actin-rich lamellipodia
Additional file 6: Movie.(AVI 16 MB)
The PIP3-binding protein LL5β localises to BMP2-induced cortical actin-rich lamellipodia
PI3K p55γ/p110α and LL5β are required for BMP2-induced migration and chemotaxis
Since the initial discovery that BMPs act as chemotactic guidance cues , the molecular mechanism of how BMPs initiate cell migration and chemotaxis has remained poorly understood. However, an important role for BMP-induced cell migration has been demonstrated in several excellent developmental [2, 3, 33], repair and disease studies [9, 34]. Here, we aimed to close a gap in the mechanistic molecular understanding of how BMPs in general activate PI3K signalling in progenitor cells at the molecular level and how this influences actin reorganisation at the cytocortex and, hence, lamellipodia formation. We uncovered major and crucial aspects of the molecular mechanism by which BMP2 initiates and extends PI3K-signalling at the plasma membrane, visualised and localised BMP2-induced PIP3 for the first time in intact cells, and confirmed the requirement of p55γ and LL5β for BMP2-induced migration and chemotaxis of mesenchymal progenitor cells.
The role of the BMP receptor complex in activating PI3K signalling
Here, we describe the specific association of the class Ia PI3K regulatory subunit p55γ with BMPRII for the first time. This interaction is enhanced by either BMP2 stimulation or the presence of BMPRI whereas the kinase activity of BMPRII seems dispensable. This observation may reflect the same mechanism by which BMPRII is incorporated into BISCs upon stimulation with BMP2 , where the high affinity receptor for BMP2 (BMPRI) recruits BMPRII into the complex upon BMP2 binding. Moreover, we showed previously that BISC-mediated signalling and BMPRII recruitment towards BMPRI is required for non-Smad signalling [25, 26]. We therefore speculate that the BMPRI kinase is required for PI3K activation whereas BMPRII acts as a scaffolding hub to provide PI3K for BMPRI-dependent activation mechanisms that have not yet been defined. This hypothesis is underlined by our previous findings of reduced BMP2-induced Akt phosphorylation upon pharmacological inhibition of BMPRI kinase activity  (see also Additional file 5: Figure S5A). BMPRI activity seems crucial in mediating the association of p55γ with BMPRII and, thus, PI3K activity. Research on the related TGF-β pathway identified that the high affinity TGF-β receptor type II associated constitutively with p85α, whereas the low affinity TGF-β type I receptor only associated with p85α in a ligand-dependent manner . However, it should be considered that BMPRI is the high affinity and BMPRII the low affinity receptor for BMP2. This would therefore represent a mirror-image scenario of PI3K regulatory subunit interaction in BMP versus TGF-β receptors. Tyrosine phosphorylation of BMPRII is essential for an association with class Ia PI3K p55γ. Despite its classification as a tyrosine-like kinase , a BMPRII dual kinase activity in vivo is still speculative and needs to be proven. Our experiments have shown that BMP2 stimulation rapidly induces BMPRII tyrosine phosphorylation in vitro, comparable to the kinetics of Smad1/5/8 phosphorylation via a yet unknown mechanism. Moreover, we identified BMPRII tyrosine residues that could act as direct putative SH2 domain docking sites. Since the interaction site for p55γ could be mapped to the BMPRII kinase, we speculate that pTyr motifs in the BMPRII kinase domain are required for its interaction. However, with the techniques applied here, we cannot comment on potential intermediate adaptor proteins or additional tyrosine kinases facilitating p55γ interaction and BMP2-dependent BMPRII tyrosine phosphorylation respectively. Along the same line, studies on the related activin pathway have already suggested the involvement of additional adaptor proteins that facilitate the interaction of PI3K regulatory subunits to the activin receptor ActRII . The tyrosine kinases TrkC  and Src  also interact with BMPRII and could thus facilitate or mediate its tyrosine phosphorylation at sites required for the interaction to p55γ. Taken together, the BMP2-dependent tyrosine phosphorylation of BMPRII provides the required features for interaction with p55γ, but further research will be required to unravel the contribution of yet unknown tyrosine kinases and adaptor proteins that may be involved in this interaction.
Exclusive role for p55γ in BMP2-induced PI3K signalling
To date, data regarding unique functions of p55γ are poor, mainly because it is speculated that the five different PI3K regulatory subunits have redundant functions and may compensate for each other. The data presented here show that p55γ provides specific functions during BMP2-induced PI3K signalling. This is underlined by its exclusive association with BMPRII, its BMP2-dependent phosphorylation in the iSH2 domain, and the effects on Akt phosphorylation and cell migration when knock-down of p55γ was performed. We have confirmed that, besides p55γ, all other class Ia regulatory subunits, namely p85α (including splice isoforms p55α and p50α) and p85β, are detectable at the mRNA level in undifferentiated multipotent C2C12 cells (data not shown). A prominent role for PI3K regulatory subunits during cytoskeletal rearrangements has already been described, especially in the context of actin reorganisation . Interestingly, some studies have proposed that PI3K regulatory subunits provide non-redundant signalling functions dependent on their sub-cellular localisation within a cell [42, 43]. This is in line with our data, showing that p55γ, but not p85α, interacts and co-localises with BMPRII, predominantly at the cell periphery. It still remains unclear how BMPRII selectivity for p55γ over p85α is achieved. The p55γ high-resolution crystal structure has not been determined and the SH2 and iSH2 domains of human p85α and p55γ share about 81.1% sequence identity. Based on the data presented here, we now propose two possible mechanisms by which BMPRII selectivity for p55γ could occur. First, our research revealed BMP2-dependent phosphorylation of the conserved Tyr199 within iSH2 of p55γ, but not p85α. Phosphorylation of p55γ iSH2 could induce structural changes, favouring an association of p55γ with BMPRII over that of the p85α SH2 domain. Second, the N-terminal 34 residues of p55γ bind to tubulin . Because the p55γ N-terminal sequence is unique and not present in p85α, it was proposed that this interaction specifically recruits p55γ to the cell periphery . During onset of cortical actin rearrangements, microtubule plus ends penetrate the leading edge cytocortex together with actin nucleating factors . The binding of p55γ to microtubules, especially at the very tip, could thus provide a sub-cellular pool of p55γ for signalling involved in cortical actin-driven lamellipodia formation.
Besides specific functions of the class Ia PI3K regulatory subunits, class I catalytic subunits also attract increasing attention to provide non-redundant signalling functions . The catalytic subunit p110α has been implicated in BMP2-induced PI3K signalling and cell migration by others using a pharmacological targeting approach . In line with those observations, we found that p110α is in complex with p55γ and BMPRII. Moreover, this complex produced PIP3 in a BMP2-dependent fashion. Thus, we propose that BMP2-induced PI3K signalling is transduced specifically by the p55γ/p110α class Ia PI3K complex. This could be of particular importance for cancer therapy because activating mutations in p110α are frequently found in human cancers, and p55γ is differentially up-regulated in several tumours, which is sufficient to stimulate tumour angiogenesis . This, together with the crucial role of BMP2 in oncogenic transformation and tumour angiogenesis [46–48], suggests that the p55γ/p110α complex positively regulates BMP2-induced motility, chemotaxis, and invasion of endothelial and cancer cells [9, 49, 50]. Whether the PI3K p55γ/p110α dimer indeed represents an attractive molecular target to interfere with BMP2-related cancers will require intense investigations in future.
BMP2-induced PIP3 acts as a cellular compass at the leading edge and recruits LL5β
Numerous cellular activities have been reported to depend on BMP2-induced PI3K signalling [9–11, 51–56]. Most previous studies focused on the role of PI3K-induced Akt activity with Akt being the major PI3K effector. In the present study, we investigated the role and function of PIP3 beyond Akt activation and focused on PIP3 localisation and recruitment of cytoskeletal regulators. We visualised BMP2-dependent PIP3 production in a spatiotemporal manner to gain further insight into its function. We found PIP3 became quickly enriched in BMP2-induced lamellipodia at the cytocortex, especially in cells that displayed strong PCP, suggesting that PIP3 acts as a cellular compass at the leading edge of migrating cells. PIP3 recruits PH-domain-containing proteins that facilitate rearrangements of the actin cytoskeleton . With this knowledge, we aimed to identify PH-domain proteins that link BMP2-induced PIP3 to actin regulators. The BMP2-induced lamellipodia are tightly cross-linked F-actin networks located at the cytocortex of the leading edge. During maturation and protrusion, these actin-rich lamellipodia form broad lamella that allow for the formation of new adhesion sites . In agreement with our observations, we identified a specific interaction of PH-domain protein LL5β with PIP3. LL5β acts as a highly sensitive PIP3 effector during epidermal growth factor-induced chemotaxis and lamellipodia formation . It regulates the actin cytoskeleton through interaction with and co-recruitment of filamin C  and filamin A . Filamins orchestrate cortical actin into three-dimensional structures by cross-linking of F-actin filaments . Interestingly, besides tethering filamins, LL5β also tethers Cytoplasmic linker associated proteins (CLASPs) to the leading edge [17, 18]. CLASPs attach microtubule tips to the cell cortex, which is important for microtubule stabilisation and thus PCP. Therefore, our findings provide evidence that LL5β acts as a BMP2-dependent multifunctional PIP3-sensing scaffold that eventually also orchestrates microtubule stabilisation at the cytocortex and thus links BMP2-dependent actin rearrangements to microtubule stabilisation.
p55γ and LL5β are required for BMP2-induced migration and chemotaxis
The potency of BMP2 in stimulating migration of cells with mesenchymal origin is well known. Here, we raised the question of whether our findings contribute in particular to BMP2-induced cortical actin rearrangements, PCP and chemotaxis. We demonstrated that loss of p55γ prevents cells from efficient PCP establishment during wound healing and that knock-down of p55γ or LL5β resulted in impaired BMP2-induced chemotaxis. We therefore conclude that the pro-migratory effects of BMP2 are driven by PI3K signalling leading to PIP3-dependent cytoskeletal actin rearrangements, and result mainly in directional migration explained by the ‘compass’ function of PIP3.
Our molecular findings provide a basis for explaining the important mechanism of BMP2-induced cortical actin rearrangements and chemotaxis, which we have graphically summarised (Figure 8). The novel in vitro data presented here close gaps in our current understanding of how BMP2 gradients influence the cellular cytoskeleton and hence mesenchymal progenitor cell chemotaxis. Interestingly, PIP3 production increases the efficacy of cells in detecting and processing shallow chemokine gradients . This suggests that the molecular mechanism identified here is important for mesenchymal progenitor cells that respond to BMP2 gradients in vivo where they might originate from distant locations. To visualise this in vivo in the context of our novel molecular findings will be the future goal and a translation of this knowledge towards the fields of developmental biology and regenerative medicine is expected.
Chemicals, recombinant growth factors and inhibitors
All chemicals were purchased from Sigma Aldrich unless stated otherwise. Recombinant human BMP2 was kindly provided by Walter Sebald (University of Würzburg, Würzburg, Germany). The inhibitor LDN-193189 was a kind gift from Paul Yu (Harvard Medical School, Boston, MA, USA) and described elsewhere . LY294002 was purchased from Cell Signaling Technology (Cell Signaling Technology Inc., Danvers, MA, USA) and PI103 was purchased from Echelon Bioscience (Echelon Bioscience Inc., Salt Lake City, USA).
Phospho-specific antibodies, as well as protein- and tag-specific antibodies, were used and applied as recommended by the manufacturer. A detailed list of all antibodies used in this study is provided in Additional file 7.
C2C12 cells and HEK293T cells (both from American Type Culture Collection) were cultivated in Dulbecco’s modified Eagle’s Medium (DMEM) (Biochrom GmbH, Berlin, Germany) supplemented with 10% (v/v) foetal calf serum and 100 U/ml penicillin/streptomycin. To maintain highest plasticity, C2C12 cells were kept undifferentiated and competent for BMP-induced signalling by subculture conditions that maintained a low density corresponding to approximately 150,000 cells per 182 cm2. Cells were split every other day when reaching 30% to 40% confluency and not used at passages higher than 20. Seeding in higher densities such as required for scratch wound healing was performed 12 hours prior to the experiment. C2C12 cells were transfected 48 hours prior to seeding in six-well plates with 0.5 to 3 μg plasmid DNA or 50nM siRNA (Dharmacon, GE Healthcare, Lafayette, CO, USA) (see Additional file 8: Table T1) using Lipofectamine2000 and Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions. HEK293T cells were transfected using polyethyleneimine and expanded in high glucose (4,500 mg/l glucose) DMEM, 48 hours prior to experiment. All experiments requiring BMP2 stimulation were conducted after 6 hours starvation in DMEM without serum. Cells were grown on uncoated cell culture plastic unless stated otherwise.
The plasmids encoding human BMPRII-LF-HA or mouse BMPRIb-HA were described previously [20, 62, 63]. Single point mutations used to generate kinase dead receptors were generated by cyclic mutagenesis PCR as described in . The construct encoding N-terminal flag-tagged p55γ was generated by cloning the full-length open reading frame of mouse p55γ into the TOPO-TA vector (Invitrogen, Carlsbad, CA, USA) before ligation via EcoRI/NotI into pcDNA3.1 basic. Cloning primers used in this paper are available upon request. The construct encoding HA-tagged p85α was a kind gift from Bart Vanhaesebroeck (QMUL, London, UK). The construct encoding GFP-tagged PH-domain of Akt was a kind gift from Kerstin Danker (Charité Berlin, Germany). All constructs were verified by DNA sequencing.
Immunoprecipitation of expressed proteins from HEK293T cells was performed using a modified radio-immunoprecipitation assay buffer containing 0.5% (w/v) sodium dodecyl sulphate and 0.1% Nonidet P-40. Immunoprecipitation from C2C12 cell extracts was performed using a modified radio-immunoprecipitation assay with 0.1% sodium dodecyl sulphate and 0.5% Nonidet P-40. A detailed description of the immunoprecipitation and immunoblotting procedures can be found in Additional file 7. PIP bead assay was purchased from Echelon Bioscience and precipitation was performed according to manufacturer’s instructions.
Identification of p55γ binding to GST-BMPRII was performed as described in . PIP bead-binding proteins were identified by matrix-assisted laser desorption ionisation-time of flight mass spectrometry-based peptide mass fingerprinting as described previously .
Scratch wound healing
The scratch wound healing assay was performed using cell culture inserts (ibidi GmbH) according to the manufacturer’s instructions on uncoated tissue culture plastic. A detailed description of the procedure can be found in Additional file 7. The rate of cell migration was measured by quantifying the intensity translocation values for three independent biological replicates per condition using a selective mask filter (Slidebook).
Boyden chamber assay
Two-dimensional chemotaxis was assayed using the μ-slide chemotaxis chamber system (ibidi GmbH, Martinsried, Germany) according to accompanying instructions with the following modifications: 1 day prior to seeding, chambers were coated with 0.5% gelatin solution in humidified atmosphere washed for 1 hour and dried at 37°C. Pictures were taken using a 4× objective in bright field modus. Measurements were performed using an automated sample table mounted on an Axiovert 200 M (Carl Zeiss, Jena, Germany) in combination with Axiovision Mark&Find tool. Manual cell tracking was performed using the open source ImageJ plugin Manual tracking v2.0.
Immunofluorescence and live cell imaging
For detection of fluorescent signals, we used the Alexa-conjugated secondary antibody system (Invitrogen, Carlsbad, CA, USA) and an inverted fluorescence Axiovert 200 microscope (Carl Zeiss, Jena, Germany) equipped with a live cell imaging heating and CO2 chamber mounted to a CoolSnapHQ CCD camera (Roper Scientific, Martinsried, Germany). Confocal images were taken using a Zeiss LSM519 laser scanning confocal using 63× magnification Plan Apochromat objective. A detailed description is provided in Additional file 7.
Statistics and bioinformatics
Detailed information and description of statistical analysis on co-localisation studies, intensity translocation values, western blot quantification, used databases and artwork programmes is provided in Additional file 7.
We provide an inventory of supplemental information, supplemental experimental procedures, supplemental information and supplemental references (Additional file 7).
BMP-induced signalling complex
Bone morphogenetic protein 2
Bone morphogenetic protein receptor type I/II
BMP receptor type II-long form
BMP receptor type II-short form
Cytoplasmic linker associated proteins
Fluorescent lipophilic cationic indocarbocyanine dye I
Fluorescent lipophilic cationic indocarbocyanine dye O
Human influenza hemagglutinin-tag
Inter-Src homology 2 domain
p110 catalytic subunit p110 alpha
PI3K regulatory subunit p55 gamma
Planar cell polarity
Pleckstrin homology domain
- PHLDB2 (also known as LL5β):
Pleckstrin homology-like domain family B member 2
Src homology 2 domain
Transforming growth factor beta
This work was supported by the Berlin Brandenburg School for Regenerative Therapies (DFG graduate school 203, fellowship to CH and AD) and by SFB958 (to PK) as well as Sonnenfeld-Stiftung (to CH) and funding from the Berlin School of Integrative Oncology (to AB). We thank Prof. Dr Sebald (Würzburg, Germany) for recombinant BMP2 and Prof. Dr Vanhaesebroeck (UCL, London, UK) for DNA constructs. We thank Gisela Wendel and Johanna Scholz for excellent technical support. We are grateful to Dr Mariona Graupera, Prof. Dr Anne Ridley and Dr David Yadin for valuable comments.
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